Articles de revues : « FVIII clearance »

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Swystun, Laura L., Colleen Notley, Kate Sponagle, Paula D. James et David Lillicrap. « Regulation Of Factor VIII Clearance By Mannose-Binding Lectins ». Blood 122, n^o 21 (15 novembre 2013) : 2340. http://dx.doi.org/10.1182/blood.v122.21.2340.2340.

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Abstract The coagulation factors von Willebrand factor (FVIII) and factor VIII (FVIII) circulate in the plasma in a non-covalent complex. VWF acts as a carrier for FVIII and protects it from proteolysis and accelerated clearance. However, in the absence of VWF, the molecular mechanisms that regulate FVIII clearance are largely uncharacterized. The glycosylation of FVIII may regulate its interaction with lectin clearance receptors such as the asialoglycoprotein receptor and siglec-5. The FVIII polypeptide is modified by the addition of 24 N-linked glycans and 7 O-linked glycans. The majority of N-linked glycans on FVIII are complex type and predominantly localized to the B-domain. However, two high mannose glycans are found on the A1 and C1 domains. In these studies, we characterized the ability of mannose binding lectins to interact with FVIII and mediate its clearance from the plasma. We have previously reported that the endothelial mannose binding lectin CLEC4M is a novel clearance receptor for FVIII. Removal of N-linked glycans, or pre-incubation of recombinant CLEC4M with the mannose polymer mannan attenuated binding of CLEC4M to FVIII. Additionally, the pre-incubation of CLEC4M-expressing cells with mannan significantly decreased binding and internalization of FVIII. In these studies, high mannose glycans on recombinant human FVIII were removed using the endoglycosidase endoH. Effective removal of FVIII high mannose glycans by endoH was confirmed via a lectin binding assay to Galanthus Nivalis lectin (50%, p=0.006). The binding of recombinant CLEC4M-Fc to endoH-treated FVIII was measured using a solid phase binding assay. Removal of high mannose glycans attenuated the interaction between FVIII and CLEC4M-Fc by 41% (p=0.005). A 49% decrease in the internalization of endoH-treated FVIII was observed on CLEC4M-expressing cells relative to control (p=0.046). We next sought to confirm the contribution of mannose binding lectins to the clearance of FVIII in a murine model. Although there is no murine ortholog to CLEC4M, mice express a series of CLEC4M homologs termed SIGNRs. Similar to CLEC4M, SIGNR1 is expressed on liver sinusoidal endothelial cells. While significant structural differences exist between CLEC4M and SIGNR1, they retain 59% amino acid identity between their carbohydrate recognition domains, and SIGNR1 has been shown to bind mannosylated as well as fucosylated glycoproteins. We assessed the contribution of SIGNR1 to VWF and FVIII clearance. Plasma levels of VWF:Ag, and FVIII:C were quantified in SIGNR1 deficient mice by ELISA and chromogenic assay respectively. Endogenous VWF (88% of wild type, p=0.233, n=35) and FVIII (110% of wild type, p=0.435, n=35) in SIGNR1 deficient mice were not different compared to wild type mice. SIGNR1/VWF deficient mice were bred on a C57Bl/6 background; mice received tail vein infusions of recombinant human FVIII (200 U/kg) or plasma derived VWF-FVIII complex (200 U/kg). Blood was sampled via retro-orbital puncture. VWF:Ag and FVIII:Ag levels were measured with ELISA and elimination half life was calculated using one phase exponential decay. Half-lives of infused recombinant human FVIII (VWF-/- t½=8.74 minutes, SIGNR1-/-/VWF-/- t½=10.03 minutes, p=0.898) and human plasma-derived VWF-FVIII complex (VWF-/- t½=46.09 minutes, SIGNR1-/-/VWF-/- t½=49.83 minutes, p=0.934) were not significantly different. We finally sought to characterize the contribution of other mannose-binding lectins to FVIII clearance in mice. VWF deficient mice received tail vein infusions of mannan (6 mg/kg) or saline followed after 3 minutes by tail vein infusions of recombinant human FVIII (200 U/kg). Pre-infusion of mannan into VWF deficient mice did not modify the half-life of FVIII relative to the control (control t½= 10.56 minutes, mannan t½= 9.38 minutes. p=0.33). We also assessed the clearance of endoH-treated FVIII in VWF deficient mice, and did not observe a change relative to the control (control t½=12.28 minutes, endoH FVIII t½=11.23 minutes, p=0.812). These studies suggest that CLEC4M interacts with FVIII in part through its high mannose glycans in vitro. However, our in vivo studies were unable to reveal a significant role for mannose-binding lectins in FVIII clearance in mouse models. Disclosures: James: CSL Behring: Honoraria, Research Funding; Octapharma: Honoraria, Research Funding; Baxter: Honoraria; Bayer: Honoraria.

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Makogonenko, Evgeny M., Andrey G. Sarafanov, Natalya M. Ananyeva, Klaus-Peter Radtke, Dudley K. Strickland et Evgueni L. Saenko. « B Domain of Coagulation Factor VIII Regulates Exposure of Its Heparin-Binding Site. » Blood 106, n^o 11 (16 novembre 2005) : 1014. http://dx.doi.org/10.1182/blood.v106.11.1014.1014.

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Abstract B domain of coagulation factor VIII (fVIII) was previously considered to be dispensable for fVIII function. Recently, it was found that the B domain is important for fVIII intracellular interaction with its chaperon and likely involved in fVIII clearance via asialoglycoprotein receptor. At the same time, the major clearance mechanism of fVIII involves initial interaction with heparan sulfate proteoglycans (HSPGs) followed by internalization via low-density lipoprotein receptor-related protein (LRP), member of low-density lipoprotein receptor (LDLR) family (Saenko et al, 1999; Sarafanov et al, 2001). It is possible that recently discovered clearance of fVIII via LDLR (Bovenschen et al, 2005) occurs in the same way. Since it was previously shown that fVIII binding sites for LRP are not located within B domain, we investigated if the latter regulates fVIII interaction with HSPGs. To explore this role of B domain, we compared the binding of plasma-derived fVIII (pd-fVIII) and recombinant B domain-deleted fVIII (BDD-fVIII) to immobilized LRP and heparin (a model of HSPGs) in surface plasmon resonance-based assay. The corresponding affinities were assessed by processing the binding signals obtained for five different concentrations of each analyte. Both pd-fVIII and BDD-fVIII showed similar affinities for LRP (KD 42–60 nM). The LRP-binding site of BDD-fVIII was partially blocked by pre-incubation with its carrier protein von Willebrand factor (vWf) indicating that it is only partially accessible within fVIII/vWf complex. This was further confirmed by the finding that monoclonal antibody 413, which recognizes a high-affinity LRP-binding site within the fVIII A2 domain, interacted with ~25% of BDD-fVIII molecules bound to immobilized vWf. The affinities of pd-fVIII and recombinant BDD-fVIII for immobilized heparin were similar (KD ~20 nM) and 2-fold higher than that for purified A2 domain (KD ~46 nM). Noteworthy, the maximal binding level (Rmax) proved to be 10-fold lower for pd-fVIII in comparison with BDD-fVIII indicating that in the circulation intact fVIII may have limited interaction with HSPGs. Importantly, pre-incubation with vWf did not interfere with the interaction between BDD-fVIII and heparin (KD ~ 19.5 and 21.8 nM, Rmax ~ 194 and 354 RU for BDD-fVIII and BDD-fVIII/vWf, respectively) thus revealing that heparin-binding site of fVIII is completely exposed in BDD-fVIII/vWf complex. These findings suggest that the presence of B domain in circulating fVIII/vWf complex may regulate fVIII clearance by preventing its interaction with HSPGs. The absence of B domain leads to exposure of heparin-binding site within fVIII and binding of fVIII/vWf complex to HSPGs. This binding may be a driving force in fVIII clearance which involves subsequent exposure of LRP (LDLR)-binding site(s) and internalization of fVIII from its complex with vWf via these receptors.

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Bukkems, Laura H., Jessica M. Heijdra, Nico C. B. de Jager, Hendrika C. A. M. Hazendonk, Karin Fijnvandraat, Karina Meijer, Jeroen C. J. Eikenboom et al. « Population pharmacokinetics of the von Willebrand factor–factor VIII interaction in patients with von Willebrand disease ». Blood Advances 5, n^o 5 (8 mars 2021) : 1513–22. http://dx.doi.org/10.1182/bloodadvances.2020003891.

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Abstract Recent studies have reported that patients with von Willebrand disease treated perioperatively with a von Willebrand factor (VWF)/factor VIII (FVIII) concentrate with a ratio of 2.4:1 (Humate P/Haemate P) often present with VWF and/or FVIII levels outside of prespecified target levels necessary to prevent bleeding. Pharmacokinetic (PK)-guided dosing may resolve this problem. As clinical guidelines increasingly recommend aiming for certain target levels of both VWF and FVIII, application of an integrated population PK model describing both VWF activity (VWF:Act) and FVIII levels may improve dosing and quality of care. In total, 695 VWF:Act and 894 FVIII level measurements from 118 patients (174 surgeries) who were treated perioperatively with the VWF/FVIII concentrate were used to develop this population PK model using nonlinear mixed-effects modeling. VWF:Act and FVIII levels were analyzed simultaneously using a turnover model. The protective effect of VWF:Act on FVIII clearance was described with an inhibitory maximum effect function. An average perioperative VWF:Act level of 1.23 IU/mL decreased FVIII clearance from 460 mL/h to 264 mL/h, and increased FVIII half-life from 6.6 to 11.4 hours. Clearly, in the presence of VWF, FVIII clearance decreased with a concomitant increase of FVIII half-life, clarifying the higher FVIII levels observed after repetitive dosing with this concentrate. VWF:Act and FVIII levels during perioperative treatment were described adequately by this newly developed integrated population PK model. Clinical application of this model may facilitate more accurate targeting of VWF:Act and FVIII levels during perioperative treatment with this specific VWF/FVIII concentrate (Humate P/Haemate P).

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Pipe, Steven W., Robert R. Montgomery, Kathleen P. Pratt, Peter J. Lenting et David Lillicrap. « Life in the shadow of a dominant partner : the FVIII-VWF association and its clinical implications for hemophilia A ». Blood 128, n^o 16 (20 octobre 2016) : 2007–16. http://dx.doi.org/10.1182/blood-2016-04-713289.

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Abstract A normal hemostatic response to vascular injury requires both factor VIII (FVIII) and von Willebrand factor (VWF). In plasma, VWF and FVIII normally circulate as a noncovalent complex, and each has a critical function in the maintenance of hemostasis. Furthermore, the interaction between VWF and FVIII plays a crucial role in FVIII function, immunogenicity, and clearance, with VWF essentially serving as a chaperone for FVIII. Several novel recombinant FVIII (rFVIII) therapies for hemophilia A have been in clinical development, which aim to increase the half-life of FVIII (∼12 hours) and reduce dosing frequency by utilizing bioengineering techniques including PEGylation, Fc fusion, and single-chain design. However, these approaches have achieved only moderate increases in half-life of 1.5- to 2-fold compared with marketed FVIII products. Clearance of PEGylated rFVIII, rFVIIIFc, and rVIII-SingleChain is still regulated to a large extent by interaction with VWF. Therefore, the half-life of VWF (∼15 hours) appears to be the limiting factor that has confounded attempts to extend the half-life of rFVIII. A greater understanding of the interaction between FVIII and VWF is required to drive novel bioengineering strategies for products that either prolong the survival of VWF or limit VWF-mediated clearance of FVIII.

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Blasko, Eric, Lilley Leong, Derek S. Sim, Liang Tang, Elena Ho, Jim Wu, Katalin Zsofia Kauser et Babu Subramanyam. « Reduced Polyethylene Glycol-Conjugated B-Domain–Deleted Factor VIII (PEG-BDD-FVIII) Clearance : Selective Peg Steric Modulation without Affecting Potency ». Blood 124, n^o 21 (6 décembre 2014) : 1471. http://dx.doi.org/10.1182/blood.v124.21.1471.1471.

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Abstract Prophylactic factor VIII (FVIII) replacement therapy in hemophilia A requires intravenous administration up to every other day due to the short half-life of FVIII in plasma. Plasma half-life extension of FVIII by polyethylene glycol (PEG) conjugation is thought to be mediated by decreasing hepatic clearance of FVIII. BAY 94-9027 is a rationally designed B-domain–deleted (BDD) FVIII molecule, in which a single 60-kDa PEG molecule was attached to a specific amino acid (1804) to increase its circulating half-life and reduce the exposure to epitopes reported to cause immunogenicity in the A3 domain while preserving full biological function. BAY 94-9027 is currently in clinical trials and has prolonged half-life and improved efficacy in animal models and humans. As a first step in determining whether the half-life extension with BAY 94-9027 is related to steric hindrance exerted by PEG, we investigated whether PEG impacts BAY 94-9027 binding interactions. Direct binding of HKB11-derived FVIII, BAY 94-9027 or BDD-FVIII, was assessed by measuring the ability of a panel of immobilized monoclonal antibodies directed toward different FVIII domains to capture FVIII. Interactions with more physiologic partners were indirectly assessed by thrombin generation assay (TGA) and by an in vitro hepatocyte clearance assay. TGA monitored FVIII-dependent thrombin generation, while the hepatocyte clearance assay assessed the ability of primary human hepatocytes to remove FVIII from the incubation medium. Our results indicate that the presence of the A3-directed PEG reduced BAY 94-9027 capture by immobilized antibodies directed toward the FVIII regions at or near the site of conjugation. Capture by antibodies directed toward the A3 and C2 domains were most impacted, while those directed toward A1 and A2 still bound BAY 94-9027. The A3-specific C7F7 antibody showed ~50% lower capture of BAY 94-9027 vs BDD-FVIII at 20 ng/mL of FVIII. C7F7 capture of PEG-BDD-FVIII was further reduced when a di-PEG conjugate of BDD-FVIII was subjected to the same assay, again confirming that PEG sterically modulates PEG-BDD-FVIII reactivity to the antibody. To determine whether the steric effects observed with PEG may impact FVIII function globally, TGA was performed with BAY 94-9027 spiked into FVIII-deficient plasma and subjected to 1 pM tissue factor initiation. By TGA, both BDD-FVIII and BAY 94-9027 generated comparable peak thrombin levels, with EC50 values of 3.9 and 3.2 nM for BDD-FVIII and BAY 94-9027, respectively. As thrombin generation is a consequence of activated FVIII amplification of factor X activation by activated factor IX, these results indicate that the PEG did not disrupt activated PEG-BDD-FVIII interactions with its partners in the factor Xase enzyme complex, consistent with published PEG-BDD-FVIII efficacy. By hepatocyte clearance assay, PEG-BDD-FVIII clearance was reduced ~30-40% compared with BDD-FVIII, regardless of whether von Willebrand factor was present. This reduction in hepatocyte clearance is likely to contribute to the prolonged plasma half-life reported for BAY 94-9027 (Mei B, et al. Blood. 2010;116(2):270-279; Coyle TE, et al. Journal of Thrombosis and Haemostasis. 2014;12(4):488-496). Disclosures Blasko: Bayer Healthcare: Employment. Leong:Bayer Healthcare: Employment. Sim:Bayer Healthcare: Employment. Tang:Bayer Healthcare: Employment. Ho:Bayer Healthcare: Employment. Wu:Bayer Healthcare: Employment. Kauser:Bayer Healthcare: Employment. Subramanyam:Bayer Healthcare: Employment.

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James, Andra H., Peter Kouides, Barbara A. Konkle et Claire S. Philipp. « Transient Decrease in Factor VIII Following Delivery : Clearance or Consumption ? » Blood 118, n^o 21 (18 novembre 2011) : 2286. http://dx.doi.org/10.1182/blood.v118.21.2286.2286.

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Abstract Abstract 2286 Background: Von Willebrand factor (VWF) and factor VIII (FVIII) levels increase during pregnancy and return to baseline by one month postpartum (PP). Understanding the normal levels during this period has implications for the management of women with bleeding disorders. As part of a larger study of von Willebrand disease (VWD) postpartum, we obtained VWF ristocetin cofactor (VWF:RCo), VWF antigen (VWF:Ag) and FVIII levels on 26 women without a known bleeding disorder to establish normal ranges during the PP period. Methods: Subjects were enrolled during the last trimester of pregnancy from obstetric clinics and physician practices affiliated with 4 university medical centers. VWF:RCo, VWF:Ag, and FVIII were obtained at enrollment, on admission to the hospital for childbirth, and at 4 hrs, 12 hrs, 24 hrs, 48 hrs, 72 hrs, and at 7, 14, 21, 28, and 42 days PP (6 weeks PP). Specimens were processed within an hour of venipuncture and centrally analyzed at the Duke Clinical Coagulation Laboratory. Means, standard deviations and 95% confidence intervals (CI) were established for each assay at each time point. Results: In the third trimester, mean VWF levels (VWF:RCo = 134 IU/dL [95% CI 113, 154]; VWF:Ag = 182 IU/dL [156, 209]) were 60–80% higher than the baseline levels at 6 weeks PP (VWF:RCo = 85 IU/dL [65, 105]; VWF:Ag = 103 IU/dL [87, 119]). On admission for childbirth, mean VWF levels were another 10–20% higher than the third trimester values (VWF:RCo = 147 IU/dL [108, 186]; VWF:Ag = 213 IU/dL [171, 255]). VWF levels peaked at 12 hrs PP (VWF:RCo = 214 IU/dL [171, 256]; VWF:Ag = 248 IU/dL [204, 293]) which was 140–160% above baseline (6 weeks PP). VWF levels gradually declined to 10% below baseline (VWF:RCo = 74 IU/dL [56, 93]; VWF:Ag = 97 IU/dL [80, 114]) at 3 weeks PP before returning to baseline at 6 weeks PP. This is in contrast to FVIII levels. In the third trimester, mean FVIII levels (FVIII = 127 IU/dL [111, 143]) were 40–50% higher than baseline (FVIII = 86 IU/dL [75, 97]), but on admission for childbirth, FVIII levels were 5% lower (FVIII = 121IU/dL [94, 148]) than the third trimester values. By 24 hrs PP, FVIII levels had dropped another 15% (FVIII = 103 IU/dL [91, 114]) before rising 30% to 134 IU/dL (101, 167). They gradually declined to 10% below baseline at 3 weeks PP (FVIII = 80 IU/dL [69, 91]; see figure) before returning to baseline at 6 weeks PP. These patterns were consistent despite age, race/ethnicity, parity or mode of delivery, although in women who underwent cesarean delivery, the initial nadir in FVIII levels occurred sooner than in women with vaginal delivery (4–12 hrs PP as opposed to 24 hours) and the subsequent peak in levels occurred sooner than in women with vaginal delivery (2 days as opposed to 3 days PP). Conclusions: Unlike VWF levels which increase after delivery, FVIII levels drop 15% after delivery before rising again over the next 1–2 days, and then decline gradually. The explanation for this drop in FVIII is not, clear, but may be due to altered clearance or consumption of FVIII at delivery. Disclosures: James: CSL Behring: Membership on an entity's Board of Directors or advisory committees, Research Funding. Kouides:CSL Behring: Membership on an entity's Board of Directors or advisory committees, Research Funding. Konkle:CSL Behring: Membership on an entity's Board of Directors or advisory committees. Philipp:CSL Behring: Membership on an entity's Board of Directors or advisory committees, Research Funding.

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Ogiwara, Kenichi, Laura L. Swystun, Ilinca Georgescu, Christine Brown, Angie Tuttle, Shawn Tinlin, Jayne Leggo et al. « Clearance and Genetic Variability of Von Willebrand Factor Are Major Determinants of the Pharmacokinetic Behavior of Factor VIII Concentrates in the Treatment of Pediatric Hemophilia A ». Blood 124, n^o 21 (6 décembre 2014) : 473. http://dx.doi.org/10.1182/blood.v124.21.473.473.

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Abstract Background: Although Factor VIII (FVIII) concentrates are now routinely used for the prophylactic treatment of hemophilia A (HA), the optimal doses and intervals between administrations are difficult to predict because of variable pharmacokinetics of FVIII (FVIII-PK) between patients. Previous studies in HA have revealed a close relationship between FVIII-PK and the FVIII carrier protein, von Willebrand factor (VWF). A large genome-wide association study from the CHARGE consortium highlighted several novel loci associated with plasma levels of VWF and FVIII in normal subjects, and the five genetic loci associated with FVIII levels coincided with those influencing VWF levels (Smith, 2010). Objective: To investigate the effects of VWF synthesis, clearance and genetic variability on FVIII-PK in young HA patients. We hypothesized that 1) plasma VWF:Ag levels (VWF secretion and clearance), 2) polymorphic variants within the FVIII binding region of VWF, and 3) the glycosylation pattern of VWF (N-linked and ABO blood group antigen) would influence FVIII-PK. Methods: HA males recruited at two large academic pediatric hemophilia centers (The Hospital for Sick Children in Toronto and the Medical University of Vienna) were enrolled. Blood was collected at 5 time points (pre, post FVIII-infusion: 1, 9, 24, and 48 h), and FVIII-PK parameters, clearance (CL), volume of distribution (VD) and half-life (HL), were calculated based on a Bayesian model. Plasma levels of VWF (VWF:Ag), VWF propeptide (VWFpp) and FVIII binding ability of VWF (VWF:FVIIIB) were also evaluated. Genetic analysis of the FVIII-binding region and glycosylation sites of VWF was performed. Results: Samples from 33 boys [median age 10.9 years (range 6.5-17.9)] with severe HA were evaluated. Median values of FVIII-CL, VD and HL were 0.032 dl/h/kg (range 0.018-0.062), 0.47 dl/kg (0.29-0.78), and 10.2 h (6.7-16.8), respectively. VWF:Ag, VWFpp, VWFpp/VWF:Ag ratio and VWF:FVIIIB were 86.6 IU/dl (39.9-141.6), 88.2 U/dl (43.5-156.6), 1.09 (0.33-1.71) and 70.3% (41.2-101.9), respectively. FVIII-CL (r=-0.41, p<0.05) and HL (r=0.56, p<0.001) correlated with VWF:Ag. Interestingly, while VWFpp levels did not correlate with FVIII-HL, the VWFpp/VWF:Ag ratio (a measure of VWF clearance) was negatively correlated (r=-0.47, p<0.01), suggesting that plasma VWF:Ag, especially its rate of clearance, can significantly influence FVIII-HL. Comparing the patients with the longest (13.3-16.8 h, n=8) and shortest (6.7-9.4 h, n=8) FVIII-HLs, significant differences (p<0.05) were observed for VWF:Ag and VWFpp/VWF:Ag ratio, but not for age, VWFpp, and VWF:FVIIIB. Sequencing of the VWF D’D3 region associated with FVIII-binding identified 9 SNPs (5 non-synonymous and 4 synonymous) in exons 17-21 and 24-27. Two SNPs in exon 18 (rs1063856: c.2365A>G, Thr789Ala, and rs1063857: c.2385T>C, Tyr795=) previously identified in the CHARGE study, segregated as a haplotype. In the longest FVIII-HL group there was a 43.8% prevalence of the infrequent alleles and two patients in this group were homozygous for these alleles. In contrast, the shortest FVIII-HL group had no homozygous subjects and only a 12.5% prevalence of the infrequent haplotype. Since c.2365A>C (Thr789Pro) has been reported as a type 2N VWD variant, we studied the VWF:FVIIIB properties of the Thr789Ala variant. The two patients homozygous for the Thr789Ala showed VWF:FVIIIB levels of 85.8% and 85.5% of normal in their plasmas. Recombinant Thr789Ala and Thr789Pro variants derived from 293T cells showed lower (p<0.01) VWF:FVIIIB levels of 67% and <20%, respectively, when compared to recombinant wild-type VWF. This data indicates that the VWF SNP haplotype, rs1063856/rs1063857, appears to be associated with a longer FVIII-HL without increasing FVIII-binding ability. As ABO blood group is associated with VWF glycosylation and its subsequent clearance we analyzed the distribution of blood groups in the patients with the longest and shortest FVIII-HL and found that there were no blood group O patients in the longest FVIII-HL group while 63% of patients in the shortest FVIII-HL group were blood group O (p<0.05). Sequencing of consensus N-glycosylation sites in VWF showed no variants. Conclusion: In this pediatric HA population, FVIII-PK was significantly influenced by VWF:Ag, rate of VWF clearance and blood group, and a VWF SNP haplotype in the FVIII binding region might also modify FVIII-PK. Disclosures No relevant conflicts of interest to declare.

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Lenting, Peter J., Vincent Muczynski, Gabriel Aymé, Cecile V. Denis et Olivier D. Christophe. « Von Willebrand Factor Interaction with FVIII : Development of Long Acting FVIII Therapies ». Blood 128, n^o 22 (2 décembre 2016) : SCI—8—SCI—8. http://dx.doi.org/10.1182/blood.v128.22.sci-8.sci-8.

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Abstract Coagulation factor VIII (FVIII) and von Willebrand factor (VWF) both play a centrol role in hemostasis, illustrated by the severe bleeding disorders associated with their functional absence. Despite their different functionalities in hemostasis and being products from two different genes, both proteins circulate in a tight, non-covalently linked complex. The physiological concequences of complex formation are many, including stabilization of FVIII heterodimeric structure, protection of FVIII from protelytic degradation, and modulation of FVIII immunogenicity. Another relevant issue relates to the chaperone function of VWF, allowing FVIII to survive in the circulation. FVIII levels are markedly reduced in patients with no detectable VWF protein or with a defect in VWF-FVIII complex formation, indicating that VWF prevents FVIII from premature clearance. Moreover, evidence points to FVIII actually being predominantly cleared as part of the VWF-FVIII complex rather than as a separate protein. First, it is possible to predict FVIII half-life fairly accurately by knowing antigen levels of VWF and its propeptide in combination with blood group. Second, when FVIII and VWF are co-injected in Vwf-deficient mice, FVIII is targeted to the same macrophages as is VWF. Since the end of the 1990s, our knowledge on the clearance mechanism of FVIII and VWF has started to emerge, and multiple clearance receptors for both proteins have now been identified. Interestingly, there exists a large overlap in receptor-repertoire between FVIII and VWF. These findings have taught us that it will be difficult to design single-mutant FVIII or VWF variants that have prolonged half-lives. How then to prolong the half-life of FVIII to improve treatment of hemophilia A? Several novel bioengineered FVIII variants have been developed, including PEGylation, Fc fusion and single-chain design, aiming to increase FVIII half-life. These approaches have so far achieved only moderate increases in half-life (1.5- to 2-fold compared to marketed FVIII products), significantly less than when similar modifications are being applied to factor IX. Indeed, it seems as if in designing these FVIII variants, the role of the significant other in the complex has been overlooked, since FVIII clearance is principally determined by VWF. Could we instead use VWF as a tool to prolong half-life of FVIII? This option is actually limited by the nature of the interaction between VWF and FVIII. Although of high affinity, the interaction is characterized by high association- and dissociation-rates. Infusing FVIII in combination with long-acting VWF variants will therefore result in a rapid redistribution of FVIII to endogenous VWF, as has elegantly been shown by the group of Ginsburg. To overcome this limitation, we have designed a FVIII variant (FVIII-KB013bv) in which we have replaced the B-domain by a single-domain, llama-derived antibody fragment (nanobody) that recognizes the D'D3-region of VWF. Consequently, the dissociation-rate of the VWF/FVIII complex is reduced 100-fold. Preliminary studies revealed that FVIII-KB013bv has a two-fold prolonged half-life compared to FVIII, likely due to improved VWF binding properties. Combination of the FVIII-nanobody fusion protein with long-acting VWF variants is anticipated to prolong its half-life well beyond the limit of the current long-acting FVIII variants. Disclosures Lenting: NovoNordisk: Consultancy, Research Funding.

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Schambeck, C. M. « Das Janusgesicht der Einzelfaktoren ». Hämostaseologie 27, n^o 04 (2007) : 268–72. http://dx.doi.org/10.1055/s-0037-1617092.

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ZusammenfassungDas Wissen um eine Blutungsneigung infolge Einzelfaktorenmangel ist Allgemeingut. Das Gegenteil – eine Thromboseneigung infolge hoher Einzelfaktorenspiegel – scheint nicht überraschend, doch erst in jüngster Zeit wurde ein Zusammenhang zwischen dem Spiegel von Einzelfaktoren und dem Risiko für venöse Thromboembolien beschrieben. Gut dokumentiert ist die Rolle hoher Faktor- VIII(FVIII)-Spiegel. Das Risiko für ein erstmaliges Thromboseereignis ist ähnlich hoch wie das Risiko infolge einer APC-Resistenz. Ein familiärer Hintergrund wurde für hohe FVIII-Spiegel beschrieben. Veränderungen innerhalb des FVIII- oder von-Willebrand-Faktor(VWF)-Gens scheinen nicht verantwortlich zu sein, da Polymorphismen dieser Gene nicht mit venösen Thromboembolien assoziiert sind. Eine erhöhte FVIII/VWF-Ratio deutet auf eine verminderte FVIII-Clearance hin. Möglicherweise ist das Low-density-lipoprotein- receptor-related-Protein – dieser Rezeptor vermittelt die hepatische Clearance des FVIII-VWF-Komplexes – involviert. Der Prothrombin-G20210A-Polymorphismus ist mit hohen Prothrombinspiegeln assoziiert, die vielleicht über eine Fibrinolyseresistenz des Gerinnsels zum Thromboserisiko beitragen.

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Garcia-Martínez, Iris, Nina Borràs, Marta Martorell, Rafael Parra, Carme Altisent, Lorena Ramírez, Maria Teresa Álvarez-Román et al. « Common Genetic Variants in ABO and CLEC4M Modulate the Pharmacokinetics of Recombinant FVIII in Severe Hemophilia A Patients ». Thrombosis and Haemostasis 120, n^o 10 (29 juillet 2020) : 1395–406. http://dx.doi.org/10.1055/s-0040-1714214.

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AbstractThe pharmacokinetic (PK) response of severe hemophilia A (HA) patients to infused factor VIII (FVIII) shows substantial variability. Several environmental and genetic factors are associated with changes in FVIII plasma levels and infused FVIII PK. Based on the hypothesis that factors influencing endogenous FVIII can affect FVIII PK, the contribution of single-nucleotide variants (SNVs) in candidate genes was investigated in 51 severe HA patients. The effects of blood group, F8 variant type, von Willebrand factor antigen and activity levels, age, and weight were also explored. The myPKFiT device was used to estimate individual PK parameters, and SNVs and clinically reportable F8 variants were simultaneously analyzed in an Illumina MiSeq instrument, using the microfluidics-based Fluidigm Access Array system. The contribution of SNVs to FVIII half-life and clearance was addressed by robust regression modeling, taking into account other modulators. In line with previous studies, we provide robust evidence that age, body weight, and blood group, as well as SNVs in ABO and CLEC4M, participate in the variability of FVIII PK in HA patients. Main results: each copy of the rs7853989 (ABO) allele increases FVIII half-life by 1.4 hours (p = 0.0131) and decreases clearance by 0.5 mL/h/kg (p = 5.57E-03), whereas each additional rs868875 (CLEC4M) allele reduces FVIII half-life by 1.1 hours (p = 2.90E-05) and increases clearance by 0.3 mL/h/kg (p = 1.01E-03). These results contribute to advancing efforts to improve FVIII replacement therapies by adjusting to each patient's PK profile based on pharmacogenomic data. This personalized medicine will decrease the burden of treatment and maximize the benefits obtained.

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Swystun, Laura L., Kenichi Ogiwara, Orla Rawley, Christine Brown, Ilinca Georgescu, Wilma Hopman, Veerle Labarque et al. « Genetic determinants of VWF clearance and FVIII binding modify FVIII pharmacokinetics in pediatric hemophilia A patients ». Blood 134, n^o 11 (12 septembre 2019) : 880–91. http://dx.doi.org/10.1182/blood.2019000190.

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Abstract Factor VIII (FVIII) pharmacokinetic (PK) properties show high interpatient variability in hemophilia A patients. Although previous studies have determined that age, body mass index, von Willebrand factor antigen (VWF:Ag) levels, and ABO blood group status can influence FVIII PK, they do not account for all observed variability. In this study, we aim to describe the genetic determinants that modify the FVIII PK profile in a population of 43 pediatric hemophilia A patients. We observed that VWF:Ag and VWF propeptide (VWFpp)/VWF:Ag, but not VWFpp, were associated with FVIII half-life. VWFpp/VWF:Ag negatively correlated with FVIII half-life in patients with non-O blood type, but no correlation was observed for type O patients, suggesting that von Willebrand factor (VWF) half-life, as modified by the ABO blood group, is a strong regulator of FVIII PK. The FVIII-binding activity of VWF positively correlated with FVIII half-life, and the rare or low-frequency nonsynonymous VWF variants p.(Arg826Lys) and p.(Arg852Glu) were identified in patients with reduced VWF:FVIIIB but not VWF:Ag. Common variants at the VWF, CLEC4M, and STAB2 loci, which have been previously associated with plasma levels of VWF and FVIII, were associated with the FVIII PK profile. Together, these studies characterize the mechanistic basis by which VWF clearance and ABO glycosylation modify FVIII PK in a pediatric population. Moreover, this study is the first to identify non-VWF and non-ABO variants that modify FVIII PK in pediatric hemophilia A patients.

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Mertens, Koen, Niels Bovenschen, Louis M. Havekes et Bart J. M. van Vlijmen. « Role of Low Density Lipoprotein Receptor in the Clearance of Coagulation Factor VIII In Vivo. » Blood 104, n^o 11 (16 novembre 2004) : 1925. http://dx.doi.org/10.1182/blood.v104.11.1925.1925.

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Abstract Low Density Lipoprotein Receptor (LDLR) is the archetype of the family of endocytic receptors that also includes Low-density lipoprotein Receptor-related Protein (LRP), Megalin, and Very Low Density Lipoprotein Receptor (VLDLR). While most of these receptors bind a variety of ligands, LDLR has restricted specifity. Its known ligands are apolipoprotein E (apoE) and apolipoprotein B100 (apoB100). Ligand binding to the LDL-receptor family is inhibited by the Receptor-Associated Protein (RAP). We have previously reported that RAP overexpression elevates plasma levels of factor VIII (FVIII) in both normal mice and mice with hepatic LRP deficiency [Bovenschen et al. (2003) Blood 101, 3933–3939]. This implies that LRP, but also another RAP-sensitive mechanism contributes to the regulation of FVIII in vivo. This study addresses the question whether LDLR, despite its restricted ligand specificity, binds FVIII and contributes to its clearance. In vitro binding was studied using a recombinant LDLR fragment spanning the extracellular domain of complement-type repeats 1–7. The purified fragment efficiently bound to immobilized ApoE and apoB-100 containing LDL. The immobilized LDLR fragment also bound human FVIII, with half-maximal binding at 156 nM FVIII, and binding was inhibited by RAP. Human von Willebrand Factor (VWF) or non-activated factor IX did not bind to the LDLR fragment. The relevance of the FVIII-LDLR interaction was assessed in vivo employing LDLR−/− mice, cre/loxP-mediated conditional LRP-deficient mice (LRP−), and mice with the combined deficiency. Plasma FVIII levels of controls, LDLR−/− and LRP− mice were 1.1, 0.9 and 1.8 U/ml, respectively. This suggests that LRP, but not LDLR regulates FVIII in plasma. Surprisingly, however, mice that combined LDLR deficiency with hepatic LRP deficiency displayed much higher FVIII levels (median value 4.6 U/ml) than mice lacking LRP alone. This suggests that LDLR does have the potential of regulating FVIII levels. LDLR−/− LRP− mice further displayed elevated levels of VWF (median value 3.3 U/ml), but not of factor V or factor IX. The possibility was considered that FVIII levels were elevated secondary to the profound changes in lipoprotein profiles. To this end, we also examined ApoE deficient mice, which have reduced LDL, and mice that overexpress ApoC1, which is associated with elevated levels of cholesterol- and triglyceride VLDL. ApoE−/− LDLR−/−LRP− mice had a median FVIII level of 4.2 U/ml, which is close to that of LDLR−/−LRP− mice. Mice that overexpressed human ApoC1 had elevated levels of cholesterol and triglycerides, but 0.5 U/ml FVIII. This demonstrates that elevated FVIII levels were independent of lipoprotein levels. The role of LDLR and LRP in FVIII clearance were further studied by analyzing the pharmacokinetics of human FVIII. In normal mice the Mean Residence Time (MRT) was 160 min [68% confidence intervals (CI) 117–218 min]. MRT was 200 [CI 154–259] min in LDLR−/− mice, and 263 [CI 206–336] min in LRP− mice. This confirms the previously described role of LRP in FVIII clearance. Strikingly, in LDLR−/−LRP− mice the MRT of FVIII was 760 [691–826] min, which is approximately 5-fold longer than in control mice. These data demonstrate that LRP and LDLR act in concert in regulating FVIII levels in plasma. In the absence of LDLR, LRP maintains normal FVIII levels, while hepatic LRP deficiency is largely compensated by LDLR. This regulatory role of LDLR represents a novel link between LDLR and the hemostatic system.

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Yee, Andrew, Austin N. Oleskie, Robert D. Gildersleeve, Colin A. Kretz, Min Su, Beth M. McGee, Keisha M. Carr, Georgios Skiniotis et David Ginsburg. « Partial in Vivo FVIII Stabilization by VWF Fragments ». Blood 120, n^o 21 (16 novembre 2012) : 15. http://dx.doi.org/10.1182/blood.v120.21.15.15.

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Abstract Abstract 15 Plasma factor VIII (fVIII) circulates in complex with von Willebrand factor (VWF) and is rapidly cleared in the absence of VWF. Previous in vitro studies have 1) localized the fVIII-binding region of VWF to the N-terminus, comprised of the contiguous D' and D3 domains and 2) observed reduced affinity for fVIII upon alterations to the tertiary structure of VWF. To gain insight into the structure-function of VWF for fVIII stabilization, we tested VWF fragments for in vivo fVIII stabilization and investigated the architecture of a VWF fragment in complex with fVIII. For the in vivo study, fragments of murine Vwf cDNA were cloned into the hepatic-specific expression vector, pLIVE, and modified to fuse tandem E and FLAG tags to the C-terminus. The following VWF fragments were expressed in vivo by hydrodynamic tail vein injection into Vwf−/− mice: 1) a monomer of the VWF D'D3 domains (monoD'D3; M1-C22, S764-P1274); 2) a truncation of monomeric D'D3 (truncD'D3; M1-C22, S764-R1035); 3) dimers of D'D3 (diD'D3; M1-P1274); 4) multimers of D'D3 (multiD'D3; M1-P1274, G2713-K2813); 5) dimers of mature VWF subunits (DPro, M1-C22, S764-K2813); or 6) full length, multimeric VWF (wtVWF, M1-K2813). Expression of all VWF fragments persisted throughout the period of observation (4 weeks) with peak antigenic levels at 1 or 3 days post-injection. Prolonged elevation of plasma fVIII activity (fVIIIa) from ∼10% to ∼50–200% were observed (100% defined as the fVIIIa level of pooled platelet poor plasma from 10 wild type C57BL/6 mice) for all but the truncated monomer of D'D3 (Figure 1). Significantly increased fVIIIa levels (p<0.05, relative to pre-injection) were first observed at 1 day, peaked at 3 days, and persisted for the duration of observation. A minimal VWF fragment (S764-R1035, truncD'D3) reported to bind fVIII in vitro significantly increased plasma fVIIIa to 34% only at 3 days post-injection. Clearance of VWF fragments from circulation were determined from injections of pooled platelet poor plasma containing recombinant VWF fragments derived from hydrodynamically injected mice into naïve Vwf−/− mice. Nonlinear regression estimated the half-life for monoD'D3 (3.4hr), diD'D3 (2.1hr), multiD'D3 (2.3hr), DPro (2.8hr), and wtVWF (3.5hr). To examine how dimers of D'D3 bind fVIII, diD'D3 from HepG2 conditioned media was purified either alone or with recombinant fVIII, and negative stained samples were visualized by electron microscopy (EM). Single-particle EM analysis revealed that each subunit of the dimer binds 1 fVIII molecule. 3D EM reconstructions indicate that the light chain of fVIII directly interacts with, and potentially induces torsion in the flexible D'D3 domains of VWF. Together, these results emphasize the importance of VWF's tertiary structure in fVIII stabilization and that the N-terminal D'D3 alone is sufficient to support fVIII survival in vivo. These findings could lead to improved methods of recombinant fVIII production and the development of novel approaches to treatment for hemophilia and von Willebrand disease. Figure 1. fVIIIa of hydrodynamically injected mice at indicated time points. Figure 1. fVIIIa of hydrodynamically injected mice at indicated time points. Disclosures: Ginsburg: Shire Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Portola Pharmaceuticals: Consultancy; Catalyst Biosciences: Consultancy; Baxter Pharmaceuticals: benefit from payments to Children's Hosptial, Boston, and the University of Michigan Patents & Royalties; Merck Pharmaceuticals: Consultancy.

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Kurasawa, James H., Svetlana A. Shestopal, Elena Karnaukhova, Evi B. Struble, Timothy K. Lee et Andrey G. Sarafanov. « Characterization of Molecular Interaction of Blood Coagulation Factor VIII and Its Clearance Receptor, Low-Density Lipoprotein Receptor. » Blood 120, n^o 21 (16 novembre 2012) : 2206. http://dx.doi.org/10.1182/blood.v120.21.2206.2206.

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Abstract Abstract 2206 Background. Clearance of blood coagulation factor VIII (FVIII) is mediated by several receptors (Saenko E. et al, 1999; Lenting P. et al, 1999 and Sarafanov A. et al, 2001) and one of them was proposed to be low-density lipoprotein receptor (LDLR) (Bovenschen N. et al, 2005). Besides FVIII, the major ligands of LDLR are plasma lipoproteins. The ligand-binding portion of LDLR is represented by a cluster of seven complement-type repeats. Each repeat forms a structurally autonomous domain, which are connected with flexible linkers. Two and five adjacent CRs were shown to form sites for the lipoproteins ApoE and ApoB, respectively, and the site for FVIII has not been determined. Objective. As a part of elucidation of mechanisms of FVIII clearance, our goal was to identify the FVIII-binding region on LDLR. Experimental Approach. Our experimental strategy was based on generation of recombinant CR-doublets that systematically overlap the CR cluster in LDLR (Fig. 1) and testing their interactions with FVIII. The proteins were expressed in insect cells using a baculovirus system and tested for structural integrity by circular dichroism. The functional properties of the fragments were assessed by their binding with alpha-2-macroglobulin receptor-associated protein (RAP), a universal ligand of a family of receptors that LDLR belongs to. The binding studies were performed using surface plasmon resonance technique. Using this assay, the LDLR fragments were further tested for interaction with different FVIII preparations, such as plasma-derived FVIII and recombinant FVIII: Advate (full-size FVIII) and Xyntha (B-domain deleted FVIII). The specificity of these interactions was tested in a competitive binding assay using an anti-FVIII single-chain variable antibody fragment KM33 known to inhibit binding FVIII to LDLR (Limburg V. et al, 2005). Also, the specificity of the interaction of LDLR fragments and FVIII was tested upon mutating the fragments by targeting conservative tryptophanes, one residue per selected CR. Upon expressing the mutants, their structural adequacy to the respective wild-type forms was confirmed by circular dichroism. Results. Three overlapping CR doublets were found active for binding with all FVIII preparations (Fig. 2). In presence of increased concentrations of KM33 and upon site-directed mutagenesis of the LDLR fragments, this binding was diminished confirming its specificity. Conclusions. The binding site of LDLR for FVIII is formed by a region spanned by the complement-type repeats from second to fifth. We proposed a tentative model of the interaction (Fig. 3), in which this region of the receptor contacts the A3 and C1 domains of FVIII. These portions of FVIII were previously shown to be involved in interaction with another FVIII clearance receptor known as LDLR-related protein (Bovenschen N. et al, 2003 and Meems H. et al, 2011). Disclosures: No relevant conflicts of interest to declare.

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Bovenschen, Niels, Koen Mertens, Lihui Hu, Louis M. Havekes et Bart J. M. van Vlijmen. « LDL receptor cooperates with LDL receptor–related protein in regulating plasma levels of coagulation factor VIII in vivo ». Blood 106, n^o 3 (1 août 2005) : 906–12. http://dx.doi.org/10.1182/blood-2004-11-4230.

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Abstract Low-density lipoprotein (LDL) receptor (LDLR) and LDLR-related protein (LRP) are members of the LDLR family of endocytic receptors. LRP recognizes a wide spectrum of structurally and functionally unrelated ligands, including coagulation factor VIII (FVIII). In contrast, the ligand specificity of LDLR is restricted to apolipoproteins E and B-100. Ligand binding to the LDLR family is inhibited by receptor-associated protein (RAP). We have previously reported that, apart from LRP, other RAP-sensitive mechanisms contribute to the regulation of FVIII in vivo. In the present study, we showed that the extracellular ligand-binding domain of LDLR interacts with FVIII in vitro and that binding was inhibited by RAP. The physiologic relevance of the FVIII–LDLR interaction was addressed using mouse models of LDLR or hepatic LRP deficiency. In the absence of hepatic LRP, LDLR played a dominant role in the regulation and clearance of FVIII in vivo. Furthermore, FVIII clearance was accelerated after adenovirus-mediated gene transfer of LDLR. The role of LDLR in FVIII catabolism was not secondary to increased plasma lipoproteins or to changes in lipoprotein profiles. We propose that LDLR acts in concert with LRP in regulating plasma levels of FVIII in vivo. This represents a previously unrecognized link between LDLR and hemostasis.

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Haberichter, Sandra L., Pamela A. Christopherson, Veronica H. Flood, Joan Cox Gill, Kenneth D. Friedman et Robert R. Montgomery. « Von Willebrand Factor (VWF) Propeptide and Factor VIII (FVIII) Levels Identify the Contribution of Decreased Synthesis and/or Increased Clearance Mechanisms in the Pathogenesis of Type 1 Von Willebrand Disease (VWD) in the Zimmerman Program ». Blood 128, n^o 22 (2 décembre 2016) : 874. http://dx.doi.org/10.1182/blood.v128.22.874.874.

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Abstract Laboratory diagnosis of VWD is challenging, with multiple tests required to obtain an accurate assessment. Plasma VWF level represents a balance between synthesis, secretion, and clearance. Although synthesized together, VWFpp and VWF circulate in plasma independently with differing half-lives. Plasma VWFpp level is used to assess synthesis/secretion, VWFpp/VWF:Ag ratio to indicate clearance of VWF, and FVIII/VWF:Ag ratio to assess VWF synthesis and clearance. We sought to identify the underlying VWD pathophysiology in subjects enrolled in the Zimmerman Program including 245 healthy controls, 175 "low VWF" (VWF:Ag 30-50), 69 type 1 (VWF:Ag<30), 57 type 1C (VWF:Ag<30 and VWFpp/VWF:Ag>3), and 9 type 1-severe (1S) (VWF:Ag<5). FVIII levels were significantly reduced (p < 0.0001) in VWD subjects compared to controls. The mean FVIII/VWF:Ag ratio in "low VWF" (1.5), types 1 (2.4), 1C (2.9), and 1S (8.7) were significantly different from controls (p < 0.0001). VWFpp levels in all subjects were significantly lower than controls with type 1S subjects demonstrating the most reduced levels. The mean VWFpp/VWF:Ag was significantly increased compared to controls in type 1 (1.9) and 1C (8.4) subjects. VWFpp level, VWFpp/VWF:Ag and FVIII/VWF:Ag were used to define the underlying pathophysiology in VWD subtypes. A VWFpp/VWF:Ag > 3.0 indicates increased VWF clearance. VWFpp level < 50 IU/dL or FVIII/VWF:Ag > 2.0 indicate reduced VWF synthesis/secretion. A combination of increased clearance/reduced secretion may be identified or neither mechanism. Only 9% of "low VWF" subjects had increased FVIII/VWF:Ag, while 15% had decreased VWFpp suggesting reduced secretion. Other unidentified mechanisms may explain the majority of these "low VWF" cases. In type 1, 38 had increased FVIII/VWF:Ag and 48 had reduced VWFpp indicating reduced secretion in 55-70% of cases. In type 1C, all had increased VWFpp/VWF:Ag, 44 had increased FVIII/VWF:Ag and 19 reduced VWFpp. Reduced secretion may play a role in 33 -77% of these subjects, in addition to increased VWF clearance. All 9 type 1S had reduced VWFpp and 8/9 had increased FVIII/VWF, indicating a reduced secretion mechanism in nearly all cases. To assess the influence of sequence variant (SV) location on VWF synthesis/secretion, levels were analyzed by presence or absence of SV and SV location by VWF domain. 62% of the VWD cohort had SV identified (23% with >1 SV) while 38% had no SV. Of those with SV, 52% had increased FVIII/VWF:Ag and 48% had decreased VWFpp, suggesting reduced secretion; 31% had increased VWFpp/VWF:Ag indicative of increased clearance; and 32% had neither mechanism. In the group with no SV, only 7% had increased FVIII/VWF:Ag and 17% had decreased VWFpp while 77% had neither mechanism identified. Of those subjects with reduced secretion as indicated by increased FVIII/VWF:Ag, the majority of SV were found in A1 (29%), D3 (22%) and D1 (13%) which was similar to those with reduced secretion indicated by decreased VWFpp level in A1 (25%), D3 (21%) and D1 (15%). An increased VWFpp/VWF:Ag ratio predicting increased clearance was found in patients with SV in D3, A1 and D4 domains. Patients with both increased clearance and reduced secretion had SV in A1 (53%), D3 (19%) and D4 (11%). Subjects who had neither mechanism identified had SV in A2 (25%), C1-C6 (18%) and D2 (16%), suggesting these SV are associated with other, yet unidentified mechanisms. Although VWFpp level and FVIII/VWF:Ag are both thought to indicate VWF synthesis/secretion, some discrepancies were observed. VWFpp level may be the more specific marker, as VWFpp and VWF share a common precursor protein. Reduced secretion plays a role in nearly all type 1S, 70% of type 1, and 15% of "low VWF" subjects. Additionally, SV in A1, D3, and D4 may be associated with decreased secretion and/or increased clearance while those in D1 may be associated with decreased secretion alone. No SV were found in 85% of "low VWF" subjects that is consistent with the observation that the majority of these cases (81%) had neither decreased secretion nor increased clearance mechanisms identified. The mechanistic cause of bleeding in these patients remains undefined. Assay of VWFpp and corresponding VWF:Ag ratios may help to define the underlying mechanism in VWD subjects and identify true type 1C VWD patients, which is clinically important for therapeutic treatment. Disclosures Flood: CSL Behring: Consultancy; Baxalta: Consultancy. Friedman:Shire: Consultancy; NovoNordisk: Consultancy; CSL Behring: Consultancy; Alexion: Speakers Bureau.

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Muczynski, Vincent, Caterina Casari, François Moreau, Gabriel Aymé, Charlotte Kawecki, Paulette Legendre, Valerie Proulle, Olivier D. Christophe, Cécile V. Denis et Peter J. Lenting. « A factor VIII–nanobody fusion protein forming an ultrastable complex with VWF : effect on clearance and antibody formation ». Blood 132, n^o 11 (13 septembre 2018) : 1193–97. http://dx.doi.org/10.1182/blood-2018-01-829523.

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Key Points The fusion between FVIII and anti-VWF nanobodies increases affinity for VWF 25-fold without compromising FVIII activity. Stabilized VWF binding results in a twofold enhanced circulatory survival of FVIII and reduced anti-FVIII antibody formation.

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Batsuli, Glaivy, Wei Deng, John F. Healey, Ernest T. Parker, W. Hunter Baldwin, Courtney Cox, Brenda Nguyen et al. « High-affinity, noninhibitory pathogenic C1 domain antibodies are present in patients with hemophilia A and inhibitors ». Blood 128, n^o 16 (20 octobre 2016) : 2055–67. http://dx.doi.org/10.1182/blood-2016-02-701805.

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Key Points C1 domain antibodies with low inhibitor titers by the Bethesda assay are pathogenic in mice due to increased fVIII clearance. Monoclonal and patient-derived polyclonal anti-fVIII C1 domain antibodies recognize similar B-cell epitopes.

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Planque, Stephanie, Miguel Antonio Escobar, Yasuhiro Nishiyama et Sudhir Paul. « A Protease Resistant Factor VIII Analog for Blockade of Inhibitory Antibodies ». Blood 116, n^o 21 (19 novembre 2010) : 3361. http://dx.doi.org/10.1182/blood.v116.21.3361.3361.

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Abstract Abstract 3361 High titer inhibitory antibodies (Abs) to Factor VIII (FVIII) generated in response to FVIII replacement therapy can render the therapy ineffective in hemophilia A (HA) patients. Abs to FVIII contain serine protease-like nucleophilic sites that react covalently with electrophilic phosphonate diester groups placed on the Lys side chains of FVIII (E-FVIII), resulting in restoration of the ability of exogenous FVIII to act as a cofactor for blood coagulation (Planque et al, J Biol Chem 2008; 283, p11876-11886). Irreversible inactivation of Abs by E-FVIII could potentially permit administration of FVIII for treatment of acute bleeding episodes in HA patients with high titer inhibitory antibodies. Another favorable effect of E-FVIII is its ability to block the production of anti-E-FVIII Abs by cultured memory B cells (Smith et al., J Thromb Haemost 2007; 5 Supplement 2: O-M-063). Here we report the resistance of E-FVIII to proteolytic clearance, a property predicted to enable prolonged blockade of pathogenic anti-FVIII immunity. E-FVIII was degraded by the serine proteases thrombin, trypsin and chymotrypsin at rates 100, 29 and 23-fold less rapidly, respectively, compared to FVIII, determined by electrophoretic generation of protein fragments. The effect of E-FVIII on thrombin catalyzed VAL-PRO-ARG-aminomethylcoumarinamide hydrolysis followed classical irreversible inhibitory kinetics, consistent with covalent bonding of the phosphonate group to the thrombin active site. Thrombin affinity (Ki) for E-FVIII and the rate of covalent bond formation (k2), respectively, were 1331 nM and 1.8 min-1. E-FVIII resistance to thrombin should reduce its consumption in the coagulation pathway, increasing its availability for the reaction with anti-FVIII Abs. FXa, an enzyme that contributes in the metabolic clearance of FVIII from blood, also degraded E-FVIII more slowly compared to FVIII. The half-life of E-FVIII in human serum and human plasma in vitro was substantially superior to that of FVIII. Only weak binding of E-FVIII to human von Willebrand Factor (VWF) was detected, suggesting that stabilizing interactions with VWF are not the reason for increased E-FVIII resistance to the proteases. E-FVIII did not prolong the time to clot formation determined by the APTT test and did not inhibit FXa generation in the chromogenic Coamatic test. Therefore, E-FVIII does not cause a global inhibition of the serine protease pathways required for blood coagulation. The reaction model entails covalent bonding of the E-FVIII phosphonate if productive noncovalent contact with the enzyme is established. As the E-FVIII is present at limiting concentrations (<Ki), generalized protease inhibition does not occur. These data suggest that clearance of E-FVIII in vivo by blood-borne proteases should be limited, potentially affording a longer half-life and prolonged blockade of pathogenic antibody effects. Reduced E-FVIII digestion by intracellular proteases may also attenuate the presentation of pathogenic FVIII antigenic epitopes to T cells, thereby, reducing the production of inhibitory anti-FVIII antibodies. Disclosures: Planque: Covalent Immunology Products: Consultancy, Equity Ownership, Patents & Royalties, Research Funding. Escobar:Covalent Immunology Products: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Nishiyama:Covalent Immunology Products: Consultancy, Equity Ownership, Patents & Royalties. Paul:Covalent Immunology Products: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties.

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Rottensteiner, Hanspeter, Peter L. Turecek, Rona Pendu, Alexander B. Meijer, Peter Lenting, Koen Mertens, Eva-Maria Muchitsch, Hartmut Ehrlich et Hans Peter Schwarz. « PEGylation or Polysialylation Reduces FVIII Binding to LRP Resulting in Prolonged Half-Life in Murine Models. » Blood 110, n^o 11 (16 novembre 2007) : 3150. http://dx.doi.org/10.1182/blood.v110.11.3150.3150.

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Abstract The low-density lipoprotein (LDL)-receptor-related protein 1 (LRP1) binds FVIII and is involved in the in vivo clearance and regulation of FVIII plasma levels. The significance of LRP in the regulation of FVIII metabolism has been demonstrated both in conditional LRP1 knock-out mice resulting in increased endogenous levels and prolonged survival of FVIII and by blocking LRP in hemophilic mice leading to prolonged half-life of infused human FVIII. In the current study we investigated whether random chemical modification of lysine residues of recombinant FVIII with branched polyethylene glycol (PEG) or polysialic acid (PSA) polymers interferes with LRP interaction. The modified FVIII variants, both of which gave rise to an increased half-life in mouse PK studies, were compared with rFVIII for binding to LRP1 by surface plasmon resonance (SPR) methodology. The typical binding of FVIII to LRP1 was more reduced by polysialylation than by PEGylation. To further delineate the region within the binding domain of LRP1 that recognizes FVIII, the isolated clusters II and IV were analyzed for FVIII interaction in an ELISA-based assay. Interestingly, FVIII bound to both clusters with comparable affinity, indicating that the CR of cluster II and IV have the same ligand-binding specificity. The PEGylated FVIII showed a reduced binding to both immobilized clusters, but also for the modified protein there was no clear preference for one of the two domains. The specificity of FVIII binding to cluster II was further analyzed by competition experiments. Increasing concentrations of cluster II diminished the signal for both FVIII and the PEGylated variant with an IC50 of about 1 mM. Also heparin had a weak competitive effect, suggesting that this inherently anticoagulant compound could contribute to increase the half-life of FVIII. While the LRP1 binding site in the light chain of FVIII is accessible also in non-activated FVIII, that within the heavy chain of FVIII is only exposed after its activation by thrombin. It was therefore additionally tested whether limited thrombin treatment would increase the affinity of modified FVIII for LRP1. While a 40% increase in binding of rFVIII was observed, this was not the case for modified FVIII, indicating that the attached polymers also interfere with this interaction site of FVIII. The presented data suggest that drastically reduced binding to LRP1 contributes to the increase in half-life of modified FVIII. Because polysialylation and PEGylation both involve surface-exposed lysine residues, the findings further support the view that lysine-containing surface loops in FVIII are key elements in the interaction with its clearance receptor LRP. The in vivo significance of inhibiting the FVIII receptor interaction was demonstrated in VWF-FVIII double knock-out mice. While FVIII, co-administered with VWF, had a half-life of 2.1 h, infusion of PEG-rFVIII alone in these animals lacking both VWF and FVIII led to a dramatic increase of FVIII half-life to 8.3 h. The prolonged half-life of PEG-rFVIII in the absence of VWF suggests that PEG-rFVIII interferes with clearance mechanisms and may substitute for the necessary VWF-FVIII complex formation and several of the physiological VWF-FVIII protective mechanisms. Hence, PEG-rFVIII has potential as the treatment of choice in patients with type Normandy von Willebrand’s disease.

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van Moort, Iris, Laura H. Bukkems, Jessica M. Heijdra, Roger E. G. Schutgens, Britta A. P. Laros-van Gorkom, Laurens Nieuwenhuizen, Felix J. M. van der Meer et al. « von Willebrand Factor and Factor VIII Clearance in Perioperative Hemophilia A Patients ». Thrombosis and Haemostasis 120, n^o 07 (1 juin 2020) : 1056–65. http://dx.doi.org/10.1055/s-0040-1710591.

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Abstract Background von Willebrand factor (VWF) is crucial for optimal dosing of factor VIII (FVIII) concentrate in hemophilia A patients as it protects FVIII from premature clearance. To date, it is unknown how VWF behaves and what its impact is on FVIII clearance in the perioperative setting. Aim To investigate VWF kinetics (VWF antigen [VWF:Ag]), VWF glycoprotein Ib binding (VWF:GPIbM), and VWF propeptide (VWFpp) in severe and moderate perioperative hemophilia A patients included in the randomized controlled perioperative OPTI-CLOT trial. Methods Linear mixed effects modeling was applied to analyze VWF kinetics. One-way and two-way analyses of variance were used to investigate perioperative VWFpp/VWF:Ag ratios and associations with surgical bleeding. Results Fifty-nine patients with median age of 48.8 years (interquartile range: 34.8–60.0) were included. VWF:Ag and VWF:GPIbM increased significantly postoperatively. Blood type non-O or medium risk surgery were associated with higher VWF:Ag and VWF:GPIbM levels compared with blood type O and low risk surgery. VWFpp/VWF:Ag was significantly higher immediately after surgery than 32 to 57 hours after surgery (p < 0.001). Lowest VWF:Ag quartile (0.43–0.92 IU/mL) was associated with an increase of FVIII concentrate clearance of 26 mL/h (95% confidence interval: 2–50 mL/h) compared with highest VWF antigen quartile (1.70–3.84 IU/mL). VWF levels were not associated with perioperative bleeding F(4,227) = 0.54, p = 0.710. Conclusion VWF:Ag and VWF:GPIbM levels increase postoperatively, most significantly in patients with blood type non-O or medium risk surgery. Lower VWF antigen levels did not lead to clinically relevant higher FVIII clearance. VWF:Ag or VWF:GPIbM levels were not associated with perioperative hemorrhage.

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Lunghi, Barbara, Massimo Morfini, Nicola Martinelli, Silvia Linari, Giancarlo Castaman et Francesco Bernardi. « Combination of CLEC4M rs868875 G-Carriership and ABO O Genotypes May Predict Faster Decay of FVIII Infused in Hemophilia A Patients ». Journal of Clinical Medicine 11, n^o 3 (29 janvier 2022) : 733. http://dx.doi.org/10.3390/jcm11030733.

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The C-type lectin CLEC4M binds and internalizes factor VIII (FVIII). Common CLEC4M variants have been associated with FVIII pharmacokinetic (PK) profiles in hemophilia A (HA) patients. The two-compartment PK analysis of plasma-derived (pd-) and full length recombinant FVIII concentrates was conducted in twenty-six patients (FVIII:C ≤ 2 IU/dL). F8, ABO blood-groups, and the CLEC4M rs868875A/G polymorphism were genotyped. CLEC4M genotype groups differed for the elimination rate constant K 1-0 (p < 0.001), half-life (K 1-0 HL), and the Beta rate constant. Patients treated with pd-FVIII also differed in the Alpha phase. In linear regression models, the contribution of the CLEC4M genotypes to FVIII PK parameters remained significant after correction for ABO, age, and VWF antigen levels at PK. Combined CLEC4M rs868875A/G and ABO genotypes displayed significant interaction (K 1-0, p = 0.014). Compared to other combined genotypes, the G-carriers/O genotypes showed half-reduced K 1-0 HL (p = 0.008), and faster FVIII clearance (mean 7.1 ± 2.2 mL/h/kg SE) than in the G-carriers/non-O (mean 2.4 ± 0.3 mL/h/kg SE), (p = 0.038). Comparison in HA patients recruited in several countries suggests that CLEC4M genotypes coherently influence infused FVIII half-life and clearance. Our analysis supports substantially faster FVIII decay associated with the rs868875 G-carrier/ABO O genotypes, which has potential implications for genetically tailored substitutive HA treatment.

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Seth Chhabra, Ekta, Nancy Moore, Chris Furcht, Amy M. Holthaus, Jiayun Liu, Tongyao Liu, Volker Schellenberger, John Kulman, Joe Salas et Robert Peters. « Evaluation of Enhanced in Vitro Plasma Stability of a Novel Long Acting Recombinant FVIIIFc-VWF-XTEN Fusion Protein ». Blood 126, n^o 23 (3 décembre 2015) : 2279. http://dx.doi.org/10.1182/blood.v126.23.2279.2279.

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Abstract INTRODUCTION More than 95% of circulating clotting factor VIII (FVIII) exists in a non-covalent complex with von Willebrand Factor (VWF). While VWF stabilizes and protects FVIII from its clearance pathways, it also subjects FVIII to VWF-mediated clearance. Thus, interaction with VWF imposes a limitation on the extent of FVIII half-life extension achieved by current technologies (Fc fusion, PEGylation etc.). Recombinant FVIIIFc-VWF-XTEN (rFVIIIFc-VWF-XTEN) is a novel fusion protein, consisting of the FVIII binding D'D3 domains of VWF fused to a single chain rFVIIIFc (scFVIIIFc). Appending the domains of VWF to FVIII provides the protection and stability of endogenous VWF, while avoiding the limitation imposed by VWF clearance. Besides D'D3 domains, it also contains two XTEN linkers. XTEN is an unstructured polypeptide consisting of six amino acids repeats (Gly, Ala, Pro, Glu, Ser, Thr). Fusion of XTEN to a protein reduces the rate of clearance and degradation of the fusion protein. In rFVIIIFc-VWF-XTEN, one XTEN linker replaces the B-domain of FVIII and other is attached to the D'D3 domains. In preclinical studies, this protein has shown >4-fold prolonged half-life and similar in vivo acute efficacy compared to rFVIII. In the current study, we examined the impact of various modifications on the in vitro plasma stability of rFVIIIFc-VWF-XTEN protein. MATERIALS AND METHODS rFVIIIFc-VWF-XTEN is a fusion protein which is expressed as a dual chain molecule. One chain expresses the D'D3 domains linked to a Fc monomer through an XTEN linker. This polypeptide is co-expressed with a single chain rFVIIIFc monomer to generate a dimer, via the disulfide-bond between the Fc domains. To assess the in vitro plasma stability, fusion proteins were expressed in HEK293 cells, purified and incubated with plasma from FVIII KO (Hem A) or FVIII/VWF DKO mice, for various time periods at 37 degree centigrade. After the desired incubation time, plasma stability of the recombinant proteins was determined by FVIII chromogenic activity assay. Results and Conclusions rFVIIIFc-VWF-XTEN fusion protein showed significantly enhanced in vitro plasma stability compared to rFVIII. In FVIII KO plasma, rFVIII started losing activity by 4 hours, and by 24 hours it lost more than 80% of its activity. The decline in activity was more pronounced and rapid when rFVIII was incubated with FVIII/VWF DKO plasma, mainly due to the absence of protection provided by VWF. Conversely, in the case of rFVIIIFc-VWF-XTEN, there was no significant drop in activity even after 6 hours (in both FVIII KO and DKO plasma). By 24 hours, only 10-15% activity reduction was observed in FVIII KO plasma and about a 35% decrease in DKO plasma. Further studies were conducted to evaluate various parameters which contributed to the improved stability of this fusion protein. Our results suggest that there are multiple factors which contribute to the overall stability of rFVIII-VWF-XTEN protein. These include: presence of covalently attached D'D3 domains, enhanced stability of single chain FVIII isoform used in the fusion protein and presence of the XTEN linker in the B-domain of FVIII. These data suggest that superior plasma stability of this novel fusion protein might be a contributing factor to its prolonged in vivo half-life and efficacy. Disclosures Seth Chhabra: Biogen: Employment, Equity Ownership. Moore:Biogen: Employment, Equity Ownership. Furcht:Biogen: Employment, Equity Ownership. Holthaus:Biogen: Employment. Liu:Biogen: Employment, Equity Ownership. Liu:Biogen: Employment, Equity Ownership. Schellenberger:Amunix Operating Inc: Employment. Kulman:Biogen: Employment. Salas:Biogen: Employment, Equity Ownership. Peters:Biogen: Employment.

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Chun, Haarin, John R. Pettersson, Svetlana A. Shestopal, Wells W. Wu, Ekaterina S. Marakasova, Philip Olivares, Stepan S. Surov et al. « Characterization of Protein Unable to Bind Von Willebrand Factor in Recombinant Factor VIII Products : Can We Reduce Their Immunogenicity ? » Blood 136, Supplement 1 (5 novembre 2020) : 25–26. http://dx.doi.org/10.1182/blood-2020-133286.

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Introduction Therapeutic products with blood coagulation factor VIII (FVIII) have a wide range of protein contents per activity unit (or IU/mg, specific activity), available from previous studies (Lin et al, 2004; Butenas et al, 2009) and the prescribing information. The wide range of the specific activities seems to be more pronounced in recombinant FVIII products (rFVIII) compared to plasma-derived FVIII, implying presence of protein with altered structure and biochemical properties. In particular, rFVIII products were reported to contain a protein fraction (FVIII*), unable to bind von Willebrand factor (VWF) without functional activity (Lin et al, 2004;Ofosu et al 2012). Furthermore, FVIII* may represent a risk factor for development of FVIII inhibitors in Hemophilia A patients, as the binding to VWF was shown to reduce FVIII immunogenicity in a tissue culture and mice model systems (Gangadharan et al, 2017; Muczynski et al, 2018). Due to these reasons, FVIII* can be defined as an impurity, which needs to be understood and controlled in rFVIII products. Study objective To develop a methodology to isolate FVIII* from rFVIII samples and to characterize this fraction in various rFVIII products. Experimental design Using immobilized VWF affinity chromatography (IVAC) for analysis of FVIII samples, protein fractions collected from (i) the column flow-through (FVIIIFT, corresponding to FVIII*) and (ii) the column-bound and eluted fraction (FVIIIEL) were characterized using polyacrylamide gel electrophoresis (PAGE) gels followed by silver-staining and immunoblotting, FVIII activity test, surface plasmon resonance, mass spectrometry, and for plasma clearance in mice. Results A robust IVAC methodology was developed. Using this method, we isolated the FVIIIFT and FVIIIEL from all ten third-generation rFVIII products marketed in the USA by study time. These products represent all current pharmaceutical variants of rFVIII including full-size FVIII, B-domain deleted (truncated) FVIII, Fc-fused FVIII, single-chained FVIII, and PEGylated protein, including those with the extended plasma half-life. FVIIIFT was found in all rFVIII products at levels up to 22% of total protein. Compared to FVIIIEL, FVIIIFT had similar pattern of polypeptide bands by PAGE, but lower functional activity, significantly reduced sulfation at Tyr1680 important for VWF binding, moderately decreased interaction with recombinant cluster II of a low-density lipoprotein receptor related protein 1 (a major clearance receptor of FVIII), and approximately 3-times faster clearance in mice (Figure 1). Conclusions Our results show that the FVIII* structure is generally similar to that of the major fraction of rFVIII, while it differs by microheterogeneity in post-translational modifications and possibly local misfolding. The data suggest that upon administration of a rFVIII product in patients, its FVIII* fraction is rapidly removed from the circulation, resulting in a decrease of the effective dosage. Our findings demonstrate a potential of IVAC to control FVIII* fraction in rFVIII products, including its removal from the products during their manufacture. These applications may lead to achieving better quality and efficacy of rFVIII products including reduction of their immunogenicity for improving the care of Hemophilia A. Disclosures No relevant conflicts of interest to declare.

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Morfini, M., P. M. Mannucci, P. M. Tenconi, G. Longo, M. G. Mazzucconi, F. Rodeghiero, N. Ciavarella, V. De Rosa et A. Arter. « Pharmacokinetics of Monoclonally-Purified and Recombinant Factor VIII in Patients with Severe von Willebrand Disease ». Thrombosis and Haemostasis 70, n^o 02 (1993) : 270–72. http://dx.doi.org/10.1055/s-0038-1649564.

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SummaryA monoclonally-purified factor VIII (FVIII) concentrate, containing little von Willebrand factor (vWF), was infused to 11 patients with severe von Willebrand disease and unmeasurable levels of plasma vWF. In comparison with the historical data obtained infusing hemophiliacs in the same conditions, monoclonally-purified FVIII had a significantly shorter half-life and faster clearance from plasma but similar in vivo recovery and volume of distribution. Two additional patients with severe von Willebrand disease were also infused with recombinant FVIII totally devoid of vWF. Half-life was very short and in vivo recovery low, with a larger volume of distribution than for monoclonally-purified FVIII. We conclude that in patients with severe von Willebrand disease the small amounts of vWF contained in the monoclonally-purified FVIII concentrate are not sufficient to stabilize infused FVIII, nor to support the normal circulation of endogenous FVIII that these patients produce at a normal rate.

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Meijer, Alexander B., Sigrid D. Roosendaal, Bas de Laat, Maartje van den Biggelaar, Vincent Limburg, Carmen van der Zwaan, Kees W. Rodenburg et Koen Mertens. « Effective Factor VIII Endocytosis Is under Conformational Control of von Willebrand Factor. » Blood 110, n^o 11 (16 novembre 2007) : 1767. http://dx.doi.org/10.1182/blood.v110.11.1767.1767.

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Abstract Von Willebrand Factor (VWF) protects Factor VIII (FVIII) from rapid clearance of the cofactor from the circulations. This notion may agree with the observation that VWF blocks FVIII binding to LDL receptor (LDLR) and LDL receptor-related protein (LRP) in vitro. In spite of the presence of VWF, however, we have previously demonstrated that LRP and LDLR are involved in the catabolism of FVIII in vivo. This suggests that dissociation of the FVIII-VWF complex in plasma or at the cell surface drives the LRP/LDLR dependent clearance of FVIII. We therefore first assessed the effect of VWF on the binding of FVIII to the cell surface employing confocal microscopy. To this end, a functional FVIII derivative containing the yellow fluorescent protein (FVIIIYFP) was incubated with LRP-expressing or LDLR-expressing CHO cells at 4 °C in the presence and absence of VWF. The result showed that there was a distinct yellow fluorescent staining of the cell surface irrespective of the presence of VWF. When the same experiment was performed at 37 °C, however, yellow fluorescent focal spots rapidly appeared inside the cells but only in the absence of VWF. Co-localization studies demonstrated that these spots originated from FVIIIYFP present in early endosomes. These findings suggest that VWF blocks the transfer of FVIII to its endocytic receptors but not the binding of FVIII to the cell surface. Intriguingly, in the presence of ristocetin, FVIIIYFP was localized inside the cells not only in the absence of VWF but also in its presence. In agreement with this finding, flow cytometric analysis confirmed that VWF was no longer able to prevent endocytosis of FVIIIYFP in the presence of ristocetin. These results suggest that switching VWF in the VWF-FVIIIYFP complex into its active conformation triggers the endocytic uptake of FVIII. The restored endocytosis of FVIII was not the consequence of a reduced affinity of “active-VWF” for FVIII. The latter was concluded from solid phase binding studies that showed that FVIII-VWF complex formation is indistinguishable in the presence and absence of ristocetin. We propose that switching VWF in the VWF-FVIII complex into its active conformation initiates at the cell surface a sequence of molecular events, which ultimately lead to the endocytic uptake of FVIII by cells expressing LRP or LDLR.

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Montgomery, Robert R., Scot A. Fahs, Jeremy G. Mattson, Hartmut Weiler et Qizhen Shi. « A Murine Model of Type 2N VWD Was Developed By CRISPR/Cas9 Gene Editing and Recapitulates Human Type 2N VWD ». Blood 132, Supplement 1 (29 novembre 2018) : 221. http://dx.doi.org/10.1182/blood-2018-99-110324.

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Abstract Von Willebrand Disease (VWD) is caused by either an inherited deficiency of von Willebrand factor (VWF) protein or synthesis of a dysfunctional von Willebrand factor (VWF) that is referred to as a type 2 variant. One of the important functions of VWF is to serve as a carrier protein for FVIII - prolonging FVIII's plasma half-life from <2 hrs without VWF to 10-12 hrs with VWF. A variant form of VWF was identified in 1982 in which VWF did not bind FVIII resulting in FVIII's rapid clearance and a phenotype similar to moderate hemophilia and currently referred to as type 2N VWD. Type 2N VWD is caused by an autosomal recessive variant of VWF in which mutations in the D'D3 region of VWF cause decreased or absent binding of FVIII to VWF resulting in rapid FVIII clearance similar to that seen in the absence of VWF. These individuals may have a type 2N mutation on each of their VWF alleles or, more commonly, have 1 allele with a 2N mutation and their 2nd allele contains a null mutation (producing no VWF). In each of these scenarios, only functionally abnormal VWF is synthesized, resulting in reduced or absent FVIII binding, rapid FVIII clearance, and a marked reduction in plasma FVIII. While Lillicrap and coworkers have demonstrated 2N VWF expression by hydrodynamic transfection in a VWF-/- mouse, their approach represents a model of plasma 2N VWF dysfunction but does not recapitulate human 2N VWD where the abnormal VWF is not only in plasma, but also in endothelial cells and platelets. We selected 8 different human type 2N mutations, and mutated murine VWF at these positions and expressed the mutant VWF in HEK293T cells in vitro. Binding of each of the 2N mutants of mouse VWF to both murine and human FVIII was tested. The G785E 2N mutation had the most severe defect with negligible binding to both human (rhF8) and mouse FVIII (mF8) and was therefore chosen for further study. Using CRISPR/Cas9 gene editing, we generated two lines of VWF2N mice (termed VWF2N1/2N1 and VWF2N2/2N2) each with a 2N causative 2354G>A (G785E) mutation on the C57BL6 background. Plasma VWF levels were measured by ELISA and FVIII was measured by chromogenic assay. VWF binding to FVIII was determined by FVIII binding to monoclonal captured VWF or by VWF binding to monoclonal captured FVIII. For the remainder of this abstract, we will only discuss the VWF2N1/2N1 mice and refer to them as wt VWF+/+, heterozygous VWF2N/+ or homozygous VWF2N/2N mice. The plasma VWF in VWF+/+, VWD2N/+ or VWF2N/2N mice was 109±31, 100±27, and 115±29 U/dL. The plasma FVIII levels in homozygous VWF2N/2N mice were markedly reduced to 4.9±2.2 U/dL compared to either wt (84±21U/dL) or heterozygous (82±18U/dL) controls. VWF binding to rhF8 was reduced to <2% in VWF2N/2N mice and 57±9% in VWF2N/+ mice when compared to wt controls. To assess the clinical phenotype of VWF2N/2N mice, tail bleeding times were determined demonstrating prolongation to 3.1±0.4 hrs in VWF2N/2N mice in contrast to 2.4±0.4 in VWF2N/+ and 2.1±1.0 hrs in wt controls. ROTEM assessment of whole blood clotting time (wbCT) and whole blood thrombin generation assays (wbTGA) were performed. The wbCT was 10.7±1.1 min in VWF2N/2N mice compared to 8.3±0.5 min in VWF2N/+ heterozygotes. In wbTGA, the Lag Time and Peak Time in the VWF2N/2N group were 18.53±5.57 and 34.15±8.37 min, respectively, which were significantly longer than those obtained in the VWF2N/+ group (8.3±0.5 and 17.83±2.33 min, respectively). Peak Thrombin, Endogenous Thrombin Potential, and Thrombin Generation rate in the VWF2N/2N group were significantly lower than those obtained in the VWF2N/+ group. In human type 2N VWD, FVIII synthesis is normal but plasma levels are reduced because of increased FVIII clearance in the absence of VWF binding. Infusion of rhVWF (no FVIII) into the VWF2N/2N mice demonstrated rhVWF peaking by 30 minutes with a rescue of the endogenous mouse FVIII (44±1.7 U/dL) at 4-6 hrs post infusion. This demonstrates improved FVIII survival in the presence of normal VWF. In summary, we have developed a novel mouse model by gene editing with both the pathophysiology and clinical phenotype found in Type 2N patients. Plasma levels of VWF are normal in our VWF2N/2N mice. This VWF is incapable of binding FVIII at neutral pH, but is otherwise fully functional. This is a unique model of 2N VWD that can be used to investigate the biological properties of VWF/FVIII association. Disclosures Montgomery: BCW: Patents & Royalties: GPIbM assay patent to the BloodCenter of Wisconsin.

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Landskroner, Kyle A., Zhi-Hua Cui, James Newgren, Michael Fournel, Glenn F. Pierce et Gary J. Jesmok. « Evaluation of PEG-FVIII Molecules with Prolonged Half-Lives in a Murine FVIII-Dependent Bleeding Model. » Blood 108, n^o 11 (16 novembre 2006) : 1624. http://dx.doi.org/10.1182/blood.v108.11.1624.1624.

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Abstract Current treatment of patients with hemophilia A often requires the frequent infusion of Factor VIII (FVIII) due to its short circulating half-life. A longer-acting FVIII molecule could profoundly impact patients’ lives by extending bleeding protection with a reduced frequency of infusions. Several strategies to prolong plasma concentrations of FVIII have been attempted. In particular, targeting domains on FVIII that bind to LRP, the putative clearance receptor, has been a popular strategy. We have investigated the use of site-directed pegylation of B-domain deleted (BDD) FVIII to evaluate the utility of PEG as a method to decrease FVIII clearance through steric hindrance of LRP binding, or other unknown clearance mechanisms, while minimizing decreases in vWF binding and in vivo activity. The evaluation of novel constructs required the development of in vivo pharmacokinetic models and a FVIII-dependent bleed model. We describe the development of an acute bleed model following uniform tail transection in the hemophilia A mouse that is FVIII dependent and allows the evaluation of the acute pharmacologic effects of FVIII or variants in vivo. Pharmacokinetic analysis of recombinant FVIII (rFVIII) and its variants was performed in rabbits over 32-hours and rFVIII or variants were measured using a modified Coatest® to differentiate endogenous rabbit FVIII from the administered human FVIII. For efficacy evaluations, hemophilia A mice were anesthetized with isoflurane and their pre-warmed tail was cut by a scalpel and placed into a new tube of warmed saline (37–40°C). Blood was collected over 40 minutes and blood loss was measured gravimetrically. Three modes of treatment were evaluated: prevention of bleeding (drug was administered 5 minutes before injury), treatment of an acute bleeding event (drug was administered 5 minutes after injury), and a delayed injury model (tail cut occurred at 20 or 24 hours after the drug administration). Over the course of 40 minutes control (C57BL6) mice demonstrated negligible bleeding (approximately 41 ± 8 μL) compared to 919 ± 26 μL in hemophilia A mice. A dose response curve was constructed for doses ranging from 0.1 to 5.0 IU of human rFVIII per mouse. Hemophilia A mice treated with 200 IU/kg of human rFVIII (5 IU/mouse) lost a similar volume of blood as control mice. The protective effect was rFVIII dose dependent over a range of 4–200 IU/kg (0.1–5 IU/mouse). In contrast, more rFVIII was required to stop an acute bleeding event when administered after the injury. In the delayed injury model, mice injured 24 hours after drug administration had a significantly larger mean blood volume loss compared to mice injured 20 hours post drug administration. Pegylated rFVIII constructs with longer half-lives also had increased activity over time compared to non-pegylated rFVIII in this mouse model. These results describe a superior hemophilia A tail bleed model that demonstrates FVIII-dependent bleeding reduction in response to acute hemorrhage over a 40 minute time course. This is the first demonstration of a hemophilia A mouse model in which all untreated animals uniformly bleed and all control animals demonstrate negligible bleeding. This model was used to evaluate the in vivo hemostatic efficacy of new rFVIII molecules that were designed to have superior pharmacologic and/or pharmacokinetic properties compared to rFVIII.

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Shestopal, Svetlana A., Leonid A. Parunov, Mikhail V. Ovanesov, Timothy K. Lee et Andrey G. Sarafanov. « Characterization of Interaction of Factor VIII with Engineered Variants of a Single-Chain Variable Antibody Fragment ». Blood 132, Supplement 1 (29 novembre 2018) : 1170. http://dx.doi.org/10.1182/blood-2018-99-115455.

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Abstract Introduction Replacement therapy for Hemophilia A requires frequent infusions of Factor VIII (FVIII) due to its relatively short half-life of ~12 h in plasma. Previous attempts to extend this half-life by genetic and chemical modification of FVIII met the barrier of ~20 h, which is a half-life of von Willebrand factor (VWF), a carrier of FVIII in plasma. A single-chain variable antibody fragment (scFv) KM33 was shown to inhibit FVIII activity, and interactions with VWF and the low-density lipoprotein receptor-related protein 1 (LRP), the major clearance receptor of FVIII (Bovenschen et al, 2005, Blood, 106:906-12). A study indicated that scFv KM33 may prolong the half-life of FVIII in mice to the level exceeding that of VWF half-life (Mertens et al, US Patent 2008, 20080219983A1). This would make scFv KM33 a promising tool for new designs of the longer-acting FVIII products. Study objective We aimed to generate a scFv KM33 variant that can delay FVIII clearance but can be removed from FVIII during its activation by thrombin. Such antibody fragment may extend the half-life of FVIII above that of VWF. Experimental design We generated three scFv KM33 variants with different linkers connecting the subunits VL and VH of the antibody fragment. The linkers contained variants of thrombin cleavage sites identical to those on FVIII. The proteins were expressed using a baculovirus system, purified by Ni-affinity and size exclusion chromatography (SEC), and tested for their properties. Results The engineered scFv variants, along with the unmodified KM33, were tested for binding to FVIII by surface plasmon resonance (SPR). All scFv versions demonstrated similar affinity for FVIII (~1 nM). In addition, a selected variant of scFv inhibited FVIII binding to LRP. These showed that the modifications of scFv did not affect its binding to FVIII. Thrombin treatment of the engineered scFv variants resulted in dissociation of their VL and VH domains, verified by SEC. However, the respective rates of thrombin cleavage were slower than that of FVIII. The preparation of a thrombin-cleaved scFv still inhibited the interaction of FVIII with LRP by SPR, similarly to that observed for the unmodified KM33. All variants of scFv inhibited FVIII activity in a thrombin generation assay suggesting that their moiety remained in complex with FVIII upon its activation. Conclusions The rate of thrombin cleavage of sites within FVIII is higher than that of the identical sites within the scFv. This suggests that additional determinants of FVIII (e. g. sulfated tyrosines adjacent to the sites) contribute to the higher rate of cleavage. The cleavage of the linker between the VL and VH subunits of scFv KM33 results in dissociation of the subunits and breakdown of the antibody fragment. This mechanism is likely applicable to any scFv, and may be useful in a broad range of applications involving such ligands. Both subunits of thrombin-cleaved scFv KM33, most likely, re-assemble on FVIII and form a tertiary complex FVIII/VL/VH. In turn, thrombin cleavage of the scFv, complexed with FVIII, does not result in its dissociation from FVIII. These indicate that in such design, an scFv should have lower affinity for FVIII to ensure its release from the complex. Disclosures No relevant conflicts of interest to declare.

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Grossmann, Ralf, Sarah Zonnur, Mario Berger, Kathleen Teuchert, Alois Spahn, Ulrich Walter et Christian Schambeck. « High factor VIII (FVIII) levels in venous thromboembolism : role of unbound FVIII ». Thrombosis and Haemostasis 92, n^o 07 (2004) : 42–46. http://dx.doi.org/10.1160/th04-02-0063.

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SummaryTheoretically, von Willebrand factor (VWF) should be capable of binding all factor VIII (FVIII), but an unbound FVIII (uFVIII) plasma fraction remains. In patients’ status post deep-vein thrombosis (DVT), an altered uFVIII fraction and high FVIII levels might be indicative of dysfunctional FVIII regulation. Out of 928 consecutive DVT patients, 321 were found to have high FVIII levels. After excluding 183 patients with known causes for high FVIII levels, plasma samples with unexplainably high FVIII levels were available from 84 patients. To capture the FVIIIVWF-complex, superparamagnetic polystyrene beads with covalently attached streptavidin were coated with biotinylated anti-rabbit Ig and incubated with rabbit anti-human VWF-Ig. Slowly thawed plasma samples were added to cooled beads, which were then separated by a magnetic particle concentrator. The uFVIII fraction was calculated by dividing the FVIII activity in the supernatant of the FVIII-VWF-complex-free sample by the FVIII activity in the supernatant of the control sample. Additionally, the VWF residuum in the supernatant was determined. Compared to age- and sex-matched blood donors, thrombosis patients showed a significantly higher plasma FVIII/VWF ratio (median: 1.3 vs. 1.0, p<0.001). uFVIII fraction data were adjusted for VWF residuum. After forward stepwise logistic regression, uFVIII had an odds ratio of 0.48 (95% CI 0.34 – 0.65), i. e. the uFVIII fraction was reduced in thrombosis patients. Analysis of covariance confirmed these results: In thrombosis patients, the estimated mean of the uFVIII fraction was significantly lower (6.34% vs. 7.58%, p<0.001). In conclusion, thrombosis patients with high FVIII levels showed a higher FVIII/VWF ratio, similar to mice with defective FVIII clearance.The clearly reduced uFVIII fraction lends further support to the hypothesis of a modified FVIII clearance.

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Fijnvandraat, K., E. Berntorp, J. W. ten Cate, H. Johnsson, M. Peters, G. Savidge, L. Tengborn, J. Spira et C. Stahl. « Recombinant, B-domain Deleted Factor VIII (r-VIII SQ) : Pharmacokinetics and Initial Safety Aspects in Hemophilia A Patients ». Thrombosis and Haemostasis 77, n^o 02 (1997) : 298–302. http://dx.doi.org/10.1055/s-0038-1655957.

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SummaryThe pharmacokinetics of a second-generation recombinant B-domain deleted factor VIII (FVIII) preparation (r-VIII SQ) were studied in 36 patients with severe hemophilia A. In contrast to full-length recombinant FVIII, no albumin needs to be added to stabilize the final formulation of this B-domain deleted FVIII preparation.The in vivo recovery and half-life of r-VIII SQ were similar to those of plasma-derived (pd) FVIII (mean half-life of r-VIII SQ, 11.7 h). The volume of distribution and clearance were slightly, but significantly, higher for r-VIII SQ than for pdFVIII (p<0.05). Peak plasma levels of FVIII were consistently related to the administered dose of r-VIII SQ (r = 0.94, p<0.0001). The pharmacokinetic profile of r-VIII SQ remained essentially unchanged in a dose range of 25-100 IU/kg body weight and could be reproduced after repeated doses. r-VIII SQ was well tolerated.In conclusion, deletion of the B-domain of FVIII does not influence its in vivo pharmacokinetics.

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Mei, Baisong, Clark Pan, Haiyan Jiang, Hendri Tjandra, Jonathan Strauss, Yaoqi Chen, Tongyao Liu et al. « Rational design of a fully active, long-acting PEGylated factor VIII for hemophilia A treatment ». Blood 116, n^o 2 (15 juillet 2010) : 270–79. http://dx.doi.org/10.1182/blood-2009-11-254755.

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Abstract A long-acting factor VIII (FVIII) as a replacement therapy for hemophilia A would significantly improve treatment options for patients with hemophilia A. To develop a FVIII with an extended circulating half-life, but without a reduction in activity, we have engineered 23 FVIII variants with introduced surface-exposed cysteines to which a polyethylene glycol (PEG) polymer was specifically conjugated. Screening of variant expression level, PEGylation yield, and functional assay identified several conjugates retaining full in vitro coagulation activity and von Willebrand factor (VWF) binding.PEGylated FVIII variants exhibited improved pharmacokinetics in hemophilic mice and rabbits. In addition, pharmacokinetic studies in VWF knockout mice indicated that larger molecular weight PEG may substitute for VWF in protecting PEGylated FVIII from clearance in vivo. In bleeding models of hemophilic mice, PEGylated FVIII not only exhibited prolonged efficacy that is consistent with the improved pharmacokinetics but also showed efficacy in stopping acute bleeds comparable with that of unmodified rFVIII. In summary site-specifically PEGylated FVIII has the potential to be a long-acting prophylactic treatment while being fully efficacious for on-demand treatment for patients with hemophilia A.

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Liu, Tongyao, David Lillicrap, Xin Zhang, Andrea Labelle, Sandra Powell, Baisong Mei, John E. Murphy, Glenn F. Pierce et Haiyan Jiang. « Site-Specific PEGylation of Factor VIII (PEG-FVIII) Preserves Full Clotting Activity and Extends Therapeutic Efficacy in HemophiliaA Dogs ». Blood 112, n^o 11 (16 novembre 2008) : 511. http://dx.doi.org/10.1182/blood.v112.11.511.511.

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Abstract To improve the effectiveness of Factor VIII replacement therapy for Hemophilia A, we sought to develop a PEGylated Factor VIII that would effectively treat bleeding episodes, while reducing the frequency of intravenous injections required for prophylaxis. Previously, we found that the site-specific PEGylation of Factor VIII (PEG-FVIII) preserves full clotting activity, prolongs circulating half-life and extends therapeutic efficacy in a number of bleeding models in hemophilic mice. To further characterize its activity, four naïve Hemophilia A dogs were treated with either PEG-FVIII or unmodified BDD-FVIII in a cross-over study design. All treated dogs showed normalized Whole Blood Clotting Time (WBCT), whole blood Thromboelastograph (TEG) profile, and Cuticle Bleeding Time within 30 min from dosing. Pharmacokinetic analysis of the decay of plasma FVIII activity and antigen levels showed that PEG-FVIII achieved 2-fold longer half-life and reduced clearance and volume of distribution relative to BDD-FVIII. Consistently, PEG-FVIII also demonstrated significantly prolonged efficacy relative to BDD-FVIII by measurement of WBCT and TEG. Both BDD-FVIII and PEG-FVIII were well tolerated in naïve HemA dogs, normal hematology and serum chemistry values were observed following administration. However, two naive dogs that received BDD-FVIII and one naive dog that received PEG-FVIII developed detectable neutralizing antibodies to human FVIII as early as on day 9 post-treatment. In summary, consistent with our previously reported findings in hemophilic mice, in comparison to BDD-FVIII, PEG-FVIII demonstrated superior half-life, full activity in stopping acute bleeding and prolonged efficacy in hemophilia A dogs. Taken together, the results support the use of site-specific PEGylation to create a homogeneous therapeutic for both prophylactic and on-demand treatment of patients with Hemophilia A.

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Bovenschen, Niels, Joachim Herz, Jos M. Grimbergen, Peter J. Lenting, Louis M. Havekes, Koen Mertens et Bart J. M. van Vlijmen. « Elevated plasma factor VIII in a mouse model of low-density lipoprotein receptor–related protein deficiency ». Blood 101, n^o 10 (15 mai 2003) : 3933–39. http://dx.doi.org/10.1182/blood-2002-07-2081.

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Abstract It has been established that low-density lipoprotein receptor-related protein (LRP) is involved in the cellular uptake and degradation of coagulation factor VIII (FVIII) in vitro. To address the physiologic role of LRP in regulating plasma FVIII in vivo, we used cre/loxP–mediated conditional LRP- deficient mice (MX1cre+LRPflox/flox). Upon inactivation of the LRP gene, MX1cre+LRPflox/flox mice had significantly higher plasma FVIII as compared with control LRPflox/floxmice (3.4 and 2.0 U/mL, respectively; P < .001). Elevated plasma FVIII levels in MX1cre+LRPflox/flox mice coincided with increased plasma von Willebrand factor (VWF) (2.0 and 1.6 U/mL for MX1cre+LRPflox/flox and control LRPflox/flox mice, respectively; P < .05). Elevation of plasma FVIII and VWF persisted for at least 6 weeks after inactivation of the LRP gene. Upon comparing plasma FVIII and VWF in individual mice, we observed an increase of the FVIII/VWF ratio in MX1cre+LRPflox/flox mice as compared with control LRPflox/flox mice. Administration of either a vasopressin analog or an endotoxin resulted in increased plasma VWF, but not FVIII. In clearance experiments, MX1cre+LRPflox/flox mice displayed a 1.5-fold prolongation of FVIII mean residence time. Adenovirus-mediated overexpression of the 39-kDa receptor–associated protein (RAP) in normal mice resulted in a 3.5-fold increase of plasma FVIII. These data confirm that the regulation of plasma FVIII in vivo involves a RAP-sensitive mechanism. Surprisingly, plasma FVIII in MX1cre+LRPflox/flox mice increased 2-fold after RAP gene transfer. We propose that RAP-sensitive determinants other than hepatic LRP contribute to the regulation of plasma FVIII in vivo.

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Swystun, Laura L., Colleen Notley, Ilinca Georgescu, Paula D. James et David Lillicrap. « The Endothelial Lectin Receptor CLEC4M Internalizes Factor VIII and Von Willebrand Factor Via a Clathrin-Coated Pit-Dependent Mechanism ». Blood 122, n^o 21 (15 novembre 2013) : 1091. http://dx.doi.org/10.1182/blood.v122.21.1091.1091.

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Abstract Regulation of plasma levels of the coagulation factors von Willebrand factor (VWF) and factor VIII (FVIII) involves a dynamic balance between biosynthesis, secretion, and clearance. Clearance of VWF and FVIII occurs through receptor-mediated endocytosis, with both LDL receptor gene family (eg. LRP1), and lectin receptors (eg. asialoglycoprotein receptor, siglec-5) contributing to this process. We have previously characterized the endothelial lectin CLEC4M as an endocytic receptor for FVIII and VWF. These proteins represent the first endogenous glycoprotein ligands indentified for CLEC4M. Previous studies have characterized CLEC4M as an adhesive receptor capable of facilitating viral infection in trans. Thus, the mechanism by which CLEC4M internalizes VWF and FVIII, and the subsequent fate of these ligands, is uncharacterized. We hypothesize that CLEC4M is able to endocytose VWF and FVIII via a clathrin-coated pit-dependent mechanism. We further hypothesize that endocytosis of FVIII and VWF by CLEC4M-expressing cells targets FVIII and VWF to lysosomes for degradation. As CLEC4M is expressed exclusively on the endothelial cells of the hepatic sinusoids and lymph nodes, and commercially prepared liver sinusoidal endothelial cells do not retain CLEC4M expression, we generated a HEK 293 cell line that stably expresses CLEC4M (>90% positive by flow cytometry). We first inhibited the CLEC4M-mediated endocytic pathways by preincubating cells with methyl-β-cyclodextrin to deplete cell membrane cholesterol, dynasore hydrate to inhibit dynamin GTPase activity, and pitstop-2 to block the N-terminus of clathrin. Cells were then incubated with recombinant human FVIII, or plasma derived VWF-FVIII complex for 1 hour and internalization was visualized by immunofluorescence. A quantitative analysis of VWF or FVIII-positive objects was performed using Image Pro software. CLEC4M-expressing 293 cells internalized both FVIII as well as the VWF-FVIII complex. Binding and internalization of VWF was reduced by methyl-β-cyclodextran (65%, p=0.067), by dynasore hydrate (92%, p=0.055), and by pitstop-2 (83%, p=0.032). Binding and internalization of FVIII was similarly reduced by methyl-β-cyclodextran (50%, p=0.0050), by dynasore hydrate (60%, p=0.0225), and by pitstop-2 (90%, p=0.0086). This suggests that CLEC4M mediates endocytosis of VWF and FVIII via a clathrin-coated pit-dependent mechanism that involves lipid rafts. To visualize the endocytic pathway of VWF and FVIII, CLEC4M-expressing 293 cells were incubated with FVIII or the VWF-FVIII complex for 15, 30, or 60 minutes. Colocalization of FVIII, VWF and/or CLEC4M with markers for early endosomes (early endosomal antigen-1), late endosomes (Rab9), and lysosomes (LAMP1) was observed using immunofluorescence. For all conditions, CLEC4M-negative and isotype controls were performed. We have previously confirmed that FVIII and VWF are internalized by CLEC4M expressing cells, and that VWF colocalizes with a marker for early endosomes. When CLEC4M-expressing cells were exposed to the VWF-FVIII complex, we observed a partial colocalization of VWF and FVIII, confirming that CLEC4M is able to internalize the VWF-FVIII complex. When CLEC4M-expressing cells were exposed to FVIII, we observed colocalization between FVIII and early endosomal antigen 1 within 15 minutes, confirming that upon internalization by CLEC4M, VWF and FVIII are targeted to early endosomes. We next observed the transport of VWF and FVIII to late endosomes and lysosomes. When CLEC4M-expressing cells were incubated with VWF and FVIII for 30 minutes, we observed colocalization of both proteins with Rab9, a marker for late endosomes. When CLEC4M-expressing cells were incubated with FVIII for 1 hour, we observed colocalization of FVIII with LAMP1, a lysosomal marker, suggesting that the internalization of FVIII by CLEC4M leads to lysosome-mediated degradation of FVIII. In contrast, incubation of CLEC4M-expressing cells with VWF for up to 2 hours did not result in co-localization with LAMP1. These studies confirm that, in the context of the stable cell system used in these experiments, the cell surface lectin receptor CLEC4M mediates endocytosis of FVIII and VWF through a clathrin-coated pit-dependent pathway. Internalization of VWF and FVIII by CLEC4M targets these proteins to early and late endosomes; FVIII is subsequently targeted to lysosomes for degradation. Disclosures: James: CSL Behring: Honoraria, Research Funding; Octapharma: Honoraria, Research Funding; Baxter: Honoraria; Bayer: Honoraria.

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Seth Chhabra, Ekta, Tongyao Liu, John Kulman, Susannah Patarroyo-White, Buyue Yang, Qi Lu, Douglas Drager et al. « BIVV001, a new class of factor VIII replacement for hemophilia A that is independent of von Willebrand factor in primates and mice ». Blood 135, n^o 17 (23 avril 2020) : 1484–96. http://dx.doi.org/10.1182/blood.2019001292.

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Abstract Factor VIII (FVIII) replacement products enable comprehensive care in hemophilia A. Treatment goals in severe hemophilia A are expanding beyond low annualized bleed rates to include long-term outcomes associated with high sustained FVIII levels. Endogenous von Willebrand factor (VWF) stabilizes and protects FVIII from degradation and clearance, but it also subjects FVIII to a half-life ceiling of ∼15 to 19 hours. Increasing recombinant FVIII (rFVIII) half-life further is ultimately dependent upon uncoupling rFVIII from endogenous VWF. We have developed a new class of FVIII replacement, rFVIIIFc-VWF-XTEN (BIVV001), that is physically decoupled from endogenous VWF and has enhanced pharmacokinetic properties compared with all previous FVIII products. BIVV001 was bioengineered as a unique fusion protein consisting of a VWF-DʹD3 domain fused to rFVIII via immunoglobulin-G1 Fc domains and 2 XTEN polypeptides (Amunix Pharmaceuticals, Inc, Mountain View, CA). Plasma FVIII half-life after BIVV001 administration in mice and monkeys was 25 to 31 hours and 33 to 34 hours, respectively, representing a three- to fourfold increase in FVIII half-life. Our results showed that multifaceted protein engineering, far beyond a few amino acid substitutions, could significantly improve rFVIII pharmacokinetic properties while maintaining hemostatic function. BIVV001 is the first rFVIII with the potential to significantly change the treatment paradigm for severe hemophilia A by providing optimal protection against all bleed types, with less frequent doses. The protein engineering methods described herein can also be applied to other complex proteins.

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Sarafanov, Andrei G., Natalya M. Ananyeva, Midori Shima et Evgueni L. Saenko. « Cell Surface Heparan Sulfate Proteoglycans Participate in Factor VIII Catabolism Mediated by Low Density Lipoprotein Receptor-related Protein ». Journal of Biological Chemistry 276, n^o 15 (12 janvier 2001) : 11970–79. http://dx.doi.org/10.1074/jbc.m008046200.

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We have demonstrated previously that catabolism of a coagulation factor VIII (fVIII) from its complex with von Willebrand factor (vWf) is mediated by low density lipoprotein receptor-related protein (LRP) (Saenko, E. L., Yakhyaev, A. V., Mikhailenko, I., Strickland, D. K., and Sarafanov, A. G. (1999)J. Biol. Chem.274, 37685–37692). In the present study, we found that this process is facilitated by cell surface heparan sulfate proteoglycans (HSPGs). This was demonstrated by simultaneous blocking of LRP and HSPGs in model cells, which completely prevented fVIII internalization and degradation from its complex with vWf. In contrast, the selective blocking of either receptor had a lesser effect.In vivostudies of clearance of125I-fVIII-vWf complex in mice also demonstrated that the simultaneous blocking of HSPGs and LRP led to a more significant prolongation of fVIII half-life (5.5-fold) than blocking of LRP alone (3.5-fold). The cell culture andin vivoexperiments revealed that HSPGs are also involved in another, LRP-independent pathway of fVIII catabolism. In both pathways, HSPGs act as receptors providing the initial binding of fVIII-vWf complex to cells. We demonstrated that this binding occurs via the A2 domain of fVIII, since A2, but not other portions of fVIII or isolated vWf, strongly inhibited cell surface binding of fVIII-vWf complex, and the affinities of A2 and fVIII-vWf complex for the cells were similar. The A2 site involved in binding to heparin was localized to the region 558–565, based on the ability of the corresponding synthetic peptide to inhibit A2 binding to heparin, used as a model for HSPGs.

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Hoebarth, Gerald, Susan Kubik, Martin Wolfsegger, John-Philip Lawo, Alfred Weber, Herbert Gritsch, Werner Hoellriegl et al. « Pharmacokinetics of Baxter’s Longer Acting rFVIII (BAX 855) in Factor VIII Ko Mice, Rats and Cynomolgus Monkeys ». Blood 118, n^o 21 (18 novembre 2011) : 4346. http://dx.doi.org/10.1182/blood.v118.21.4346.4346.

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Abstract Abstract 4346 The pharmacokinetic profile of BAX 855, a longer acting PEGylated variant of Baxter’s recombinant FVIII based on the ADVATE™ manufacturing process, was assessed in comparison to ADVATE™ after a single intravenous bolus injection at a target dose of 200 IU/kg BW in mice and rats and 350 IU/kg BW in cynomolgus monkeys. Mean residence time (MRT), terminal half-life (HL), total clearance standardized per kg body mass (Cl), the AUC0-tlast (the area under the concentration vs. time curve from 0 to the last measured time point), the in vivo recovery (IVR) and volume of distribution at steady state (Vss) for FVIII activity (mice and cynomolgus monkey), FVIII antigen (rats) and FVIII-bound PEG were evaluated in all three models. Blood was sampled at baseline and each of the time points after a single intravenous bolus injection of BAX 855 or ADVATE™. A serial sacrifice design was used for the PK in mice. Sixteen FVIII ko mice (B6;129S4-F8tm2Kaz; m/f) for BAX 855 and eight FVIII ko mice for ADVATE™ per time point were bled by cardiac puncture under anesthesia for blood sampling 5 minutes – 48 hours after a single intravenous bolus injection. A single treatment design was used for the single dose PK in Sprague Dawley rats: 8m + 8f for BAX 855 and 4m + 4f for ADVATE™. A single treatment design was also used for the cynomolgus monkeys: 4m + 4f for BAX 855 and 2m + 2f for ADVATE™. Blood samples were drawn from rats and cynomolgus monkeys for citrated plasma (for analysis of baseline FVIII levels) before administration and 5 minutes - 48 hours (rats) and 5 minutes to 96 hours (cynomolgus monkeys) after administration. The citrated plasma samples were analyzed for FVIII activity (chromogenic assay) in mice and cynomolgus monkeys, for FVIII–bound PEG (using a PEG-FVIII ELISA) in all models and FVIII antigen (using a FVIII ELISA) in rats. In all three models a prolongation in MRT of Baxter’s and Nektar’s new BAX 855 compared with ADVATE™ could be demonstrated. FVIII activity analysis showed an increase of MRT in mice from 4.9 to 7.9 hours and in cynomlogus monkeys from 7.5 to 11.5 hours. This prolongation was also reflected in the terminal half-lives (4.3 to 5.9 hours in mice and 5.7 to 9.4 hours in cynomolgus monkeys). According to this prolongation a lower clearance [mL/h/kg] could be observed for BAX 855 than for ADVATE™ (22.1 to 12.2 in mice and 8.1 to 4.9 in monkeys). Similar levels in all PK parameters could be shown when measuring FVIII-bound PEG in all three preclinical models and FVIII antigen analysis in rats. These PK data provide evidence that PEGylation of human rFVIII increases the circulation time. Disclosures: Hoebarth: Baxter Innovations GmbH: Employment. Kubik:Baxter Innovations GmbH: Employment. Wolfsegger:Baxter Innovations GmbH: Employment. Lawo:Baxter Innovations GmbH: Employment. Weber:Baxter Innovations GmbH: Employment. Gritsch:Baxter Innovations GmbH: Employment. Hoellriegl:Baxter Innovations GmbH: Employment. Schiviz:Baxter Innovations GmbH: Employment. Ehrlich:Baxter Innovations GmbH: Employment. Scheiflinger:Baxter Innovations GmbH: Employment. Turecek:Baxter Innovations GmbH: Employment. Schwarz:Baxter BioScience: Employment. Muchitsch:Baxter Innovations GmbH: Employment.

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Boross, Peter, Cafer Yildiz, Kamran Bakhtiari, Joost C. M. Meijers et C. Erik Hack. « Towards Restoring Coagulation in Hemophilia By Antibody-Mediated Inhibition of Antithrombin ». Blood 126, n^o 23 (3 décembre 2015) : 550. http://dx.doi.org/10.1182/blood.v126.23.550.550.

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Abstract Currently hemophilia A is mainly treated with factor VIII (FVIII). However, 20-25% of the hemophiliacs develop inhibiting antibodies (inhibitors) against FVIII. Hemophiliacs with inhibitors are treated with high dose FVIII or with therapies that bypass the need for FVIII such as activated FVII (rFVIIa, NovoSeven) or activated prothrombin concentrate. These have some disadvantages including rapid clearance, price and a risk of thrombo-embolism. In hemophilia, the balance between coagulation and anticoagulation is shifted towards the latter. This balance can be restored by administration of clotting factors but also by reducing anticoagulation mechanisms. Antithrombin (AT) constitutes a main anticoagulant mechanism as it inhibits thrombin, FXa and other activated clotting factors. In silico studies as well as observations that AT deficiency reduces bleeding in hemophilic mice and humans strongly support the concept that lowering functional AT levels bypasses the need for FVIII to generate thrombin. A Phase 1 clinical trial by Alnylam Pharmaceuticals showed that lowering AT activity by RNA-interference can boost thrombin generation in hemophilia. However, RNA-interference is an experimental therapy with unknown long term effects and is not suitable for acute treatment of bleeding as it takes weeks to become effective. We hypothesize that monoclonal antibodies (mAbs) that block AT activity can be used as FVIII bypass therapy. Such a mAb-based therapy is suitable for on demand and for prophylactic therapy, is relatively cheap and does not require administration of activated clotting factors. We have generated a panel of mouse anti-AT mAbs exhibiting varying binding characteristics to human AT. To assess the inhibitory activity of anti-AT mAbs, FXa activity assay was performed after FXa was incubated with normal plasma supplemented with the different anti-AT mAbs. Five anti-AT mAbs were identified that inhibit AT activity at around equimolar ratio. Inhibitory anti-AT mAbs were able to dose-dependently enhance thrombin generation in normal and hemophilic plasma. Heparin caused a marked prolongation of aPTT of normal and FVIII-deficient plasma, which was almost completely normalized by addition of the inhibitory mAb anti-AT 5G1.1, but not by anti-AT 6E7.2, indicating that these mAbs exert their inhibitory activity via different mechanisms. These data provide initial in vitro proof-of-concept that inhibitory anti-AT mAbs can be used to improve coagulation and may be used as FVIII bypass therapy. Disclosures Boross: Prothix B.V.: Employment. Hack:Prothix B.V.: Equity Ownership, Patents & Royalties.

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Björkman, Sven, MyungShin Oh, Gerald Spotts, Phillip Schroth, Sandor Fritsch, Bruce M. Ewenstein, Kathleen Casey, Kathelijn Fischer, Victor S. Blanchette et Peter W. Collins. « Population pharmacokinetics of recombinant factor VIII : the relationships of pharmacokinetics to age and body weight ». Blood 119, n^o 2 (12 janvier 2012) : 612–18. http://dx.doi.org/10.1182/blood-2011-07-360594.

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Comparison of the pharmacokinetics (PK) of a coagulation factor between groups of patients can be biased by differences in study protocols, in particular between blood sampling schedules. This could affect clinical dose tailoring, especially in children. The aim of this study was to describe the relationships of the PK of factor VIII (FVIII) with age and body weight by a population PK model. The potential to reduce blood sampling was also explored. A model was built for FVIII PK from 236 infusions of recombinant FVIII in 152 patients (1-65 years of age) with severe hemophilia A. The PK of FVIII over the entire age range was well described by a 2-compartment model and a previously reported problem, resulting from differences in blood sampling, to compare findings from children and adults was practically abolished. The decline in FVIII clearance and increase in half-life with age could be described as continuous functions. Retrospective reduction of blood sampling from 11 to 5 samples made no important difference to the estimates of PK parameters. The obtained findings can be used as a basis for PK-based dose tailoring of FVIII in clinical practice, in all age groups, with minimal blood sampling.

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van Schooten, Carina J., Shirin Shahbazi, Evelyn Groot, Beatrijs D. Oortwijn, H. Marijke van den Berg, Cécile V. Denis et Peter J. Lenting. « Macrophages contribute to the cellular uptake of von Willebrand factor and factor VIII in vivo ». Blood 112, n^o 5 (1 septembre 2008) : 1704–12. http://dx.doi.org/10.1182/blood-2008-01-133181.

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Abstract Von Willebrand factor (VWF) and factor VIII (FVIII) circulate in a tight noncovalent complex. At present, the cells that contribute to the removal of FVIII and VWF are of unknown identity. Here, we analyzed spleen and liver tissue sections of VWF-deficient mice infused with recombinant VWF or recombinant FVIII. This analysis revealed that both proteins were targeted to cells of macrophage origin. When applied as a complex, both proteins were codirected to the same macrophages. Chemical inactivation of macrophages using gadolinium chloride resulted in doubling of endogenous FVIII levels in VWF-null mice, and of VWF levels in wild-type mice. Moreover, the survival of infused VWF was prolonged almost 2-fold in VWF-deficient mice after gadolinium chloride treatment. VWF and FVIII also bound to primary human macrophages in in vitro tests. In addition, radiolabeled VWF bound to human THP1 macrophages in a dose-dependent, specific, and saturable manner (half-maximal binding at 0.014 mg/mL). Binding to macrophages was followed by a rapid uptake and subsequent degradation of the internalized protein. This process was also visualized using a VWF–green fluorescent protein fusion protein. In conclusion, our data strongly indicate that macrophages play a prominent role in the clearance of the VWF/FVIII complex.

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Cooke, Esther J., Chanond A. Nasamran, Kathleen M. Fisch et Annette von Drygalski. « Mechanisms of Iron Clearance from the Joint in FVIII-Deficient Mice after Induced Hemarthrosis ». Blood 134, Supplement_1 (13 novembre 2019) : 157. http://dx.doi.org/10.1182/blood-2019-131576.

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Introduction Hemarthrosis in hemophilia causes toxic iron accumulation in the joint, which contributes to synovitis. This study aimed to explore mechanisms and timing of iron clearance from the joint space in mouse models of induced hemarthrosis. Methods Hemarthrosis was induced by sub-patellar puncture in FVIII-deficient mice and in hypocoagulable BALB/c (HypoBALB/c) mice treated with 10 µg/ml warfarin for 7 days and 0.25 mg/kg anti-FVIII antibody 2 hours before knee puncture. Warfarin was reversed on day 2 post-injury with 100 IU/Kg 4-factor prothrombinase complex concentrate and the hematocrit determined in all mice as a measure of joint bleeding. Joint tissue was harvested at baseline and 2 or 4 weeks post-injury for analysis by histology. Ferric iron (Fe3+) was detected by Prussian Blue staining either during or after joint decalcification. Macrophages and macrophage-like synoviocytes were detected in joint tissue by immunohistochemistry with an anti-CD68 antibody. Synovial tissue was harvested from FVIII-deficient mice on day 3 and 2 weeks post-injury for gene expression studies by RNA sequencing (single-end; 75 bp) on an Illumina NextSeq500 platform. The limma-voom method (R BioConductor) was used for differential expression analyses. Results Knee injury caused substantial and comparable hemarthrosis in FVIII-deficient and HypoBALB/c mice (mean day 2 hematocrit: 27 % and 28 %, respectively). Post-decalcification Prussian Blue staining detected ferric iron accumulation in FVIII-deficient mice at week 4 only (5.3-fold increase compared to baseline, p=0.003). No ferric iron was detected in HypoBALB/c mice despite similar bleed volumes. In FVIII-deficient mice, Prussian Blue staining during decalcification was more sensitive and preserved detection of extracellular ferric iron, revealing a significant increase in ferric iron at 2 weeks post-injury relative to baseline (38-fold, p=0.005), which persisted at 4 weeks (23-fold, p=0.03). These findings coincided with increased CD68 staining at 2 weeks (36-fold, p=0.0002) and 4 weeks (8-fold, p=0.1). CD68-positive cells were dispersed throughout synovium at 2 weeks but appeared more clustered at 4 weeks and co-localized with iron staining, suggesting migration and iron uptake between 2 and 4 weeks post-injury. In HypoBALB/c mice, CD68 staining increased at 2 weeks (11-fold, p=0.008) but to a lesser extent than in FVIII-deficient mice, and was comparable to baseline at 4 weeks. Together, this suggests an altered mechanism of iron clearance in hemophilia. RNA sequencing revealed differential expression of 11/57 genes relating to iron transport in synovium on day 3, persisting somewhat at 2 weeks. Upregulated genes on day 3 included heme-oxygenase-1 (heme-degrading enzyme; 31-fold, p=3x10-6), lipocalin-2 (iron-binding protein; 10-fold, p=0.001) and solute carrier family (slc) 11 member 1 (macrophage iron transporter; 3-fold, p=0.0004). Down-regulated genes on day 3 included ceruloplasmin (efflux of cellular iron; 17-fold, p=5x10-5) and its homolog hephaestin (5-fold, p=0.002), CD163 (macrophage scavenging receptor for hemoglobin; 5-fold, p=0.03) and slc22 member 17 (lipocalin-2 receptor; 2-fold, p=0.03). Gene expression changes revealed key players involved in scavenging, degradation and transport of iron in synovium after hemarthrosis, and may expose mechanisms of impaired iron clearance in hemophilia pending further studies. Conclusions Iron handling after hemarthrosis, including uptake and transport in synovium and/or delivery to plasma transferrin, may be impaired in hemophilia and contribute to the evolution of hemophilic arthropathy. Unbiased RNA sequencing created several hypotheses that can be tested to further to elucidate mechanisms and timing of aberrant iron handling. Disclosures von Drygalski: Hematherix Inc.: Membership on an entity's Board of Directors or advisory committees, Other: Founder; University of California San Diego: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties; UniQure, Bayer, Bioverativ/Sanofi, Pfizer, Novo Nordisk, Biomarin, Shire, CSL Behring: Consultancy.

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Bossard, M. J., M. Graninger, H. Gritsch, W. Höllriegl, M. Kaliwoda, P. Matthiessen, A. Mitterer et al. « BAX 855, a PEGylated rFVIII product with prolonged half-life ». Hämostaseologie 32, S 01 (2012) : S29—S38. http://dx.doi.org/10.1055/s-0037-1619772.

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SummaryA longer acting recombinant FVIII is expected to serve patients’ demand for a more convenient prophylactic therapy. We have developed BAX 855, a PEGylated form of Baxter’s rFVIII product ADVATE™ based on the ADVATE™ manufacturing process. The conjugation process for preparing BAX 855 uses a novel PEG reagent. The production process was adjusted to yield a rFVIII conjugate with a low PEGylation degree of about 2 moles PEG per FVIII molecule. This optimised modification degree resulted in an improved PK profile for rFVIII without compromising its specific activity. PEGylation sites were identified by employing various HPLC- and MS-based methods. These studies not only indicated that about 60% of the PEG chains are localised to the B-domain, which is cleaved off upon physiological activation during the coagulation process, but also demonstrated an excellent lot to lot consistency with regard to PEGylation site distribution. Detailed biochemical characterization further showed that PEGylated FVIII retained all the physiological functions of the FVIII molecule with the exception of binding to the LRP clearance receptor which was reduced for BAX 855 compared to ADVATE™. This might provide an explanation for the prolonged circulation time of BAX 855 as reduced receptor binding might slow-down clearance. Preclinical studies showed improved pharmacokinetic behaviour and clinically relevant prolonged efficacy compared to ADVATE™ without any signs of toxicity or elevated immunogenicity. The comprehensive preclinical data package formed the basis for approval of the phase 1 clinical study by European authorities which started in 2011.

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Schiviz, Alexandra, Gerald Hoebarth, Martin Wolfsegger, Paolo Rossato, Alfred Weber, Herbert Gritsch, Hanspeter Rottensteiner et al. « Pharmacokinetics of BAX 826, a Polysialylated Full-Length rFVIII, in Hemophilia a Mice, Rats, and Cynomolgus Monkeys ». Blood 126, n^o 23 (3 décembre 2015) : 1073. http://dx.doi.org/10.1182/blood.v126.23.1073.1073.

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Abstract Factor VIII (FVIII) is a critical component of the intrinsic coagulation pathway. Plasma-derived or recombinant (r) FVIII concentrates are used in patients with hemophilia A to provide a FVIII level sufficient to treat and prevent bleeding episodes. Prophylactic FVIII levels can only be maintained by administering several infusions per week. Extended FVIII circulation times would reduce the frequency of infusions, increase patient compliance, reduce the number of bleeds, and offer the possibility to achieve higher trough levels of FVIII. Prolonged circulation can be achieved by modifying the FVIII molecule with hydrophilic polymers, for example with polysialic acid (PSA). BAX 826, Baxalta's polysialylated FVIII is based on ADVATE, a full length recombinant FVIII molecule with an established extensive safety and efficacy profile. The aim of the presented studies was to assess the pharmacokinetic profile of BAX 826 in hemophilia A mice, wild-type rats, and cynomolgus monkeys. Unmodified rFVIII (ADVATE) was used as the reference compound. Test and reference compounds were administered at the same dose. Hemophilia A mice and Sprague Dawley rats were intravenously injected with BAX 826 at a target dose of 200 IU/kg rFVIII, and blood was sampled pre-dose and 5 min to 48 h after administration. Cynomolgus monkeys received a target dose of 350 IU/kg rFVIII and blood sampled 5 min to 120 h after administration. Citrated plasma was prepared and analyzed for FVIII activity (chromogenic), FVIII antigen (ELISA), and PSA-rFVIII concentration. The primary endpoint was area under the curve from administration time to the last quantifiable time point (AUC0-tlast). Mean residence time (MRT) and systemic clearance (CLs) were also assessed. Unless stated otherwise, results for FVIII activity (mice, monkeys) and FVIII antigen (rats) are presented. In mice, the AUC0-tlast for BAX 826 was 20.6 h*IU/mL, which was 2.4 times larger than for ADVATE (8.71 h*IU/mL); MRT was 10.6 h for BAX 826 and 5.8 h for ADVATE. Clearance was lower for BAX 826 (9.4 vs. 22.3 mL/h/kg). In rats, the AUC0-tlast for BAX 826 was 18.0 h*IU/mL, which was 1.8 times larger than for ADVATE (9.9 h*IU/mL). MRT was 12.5 h for BAX 826 and 4.0 h for ADVATE, and CLs was 5.3 and 28.1 mL/h/kg. In monkeys, the geometric mean of AUC0-tlast was 189.0 h*IU/mL for BAX 826 and 39.6 h*IU/mL for ADVATE. MRT was 23.4 and 10.1 h, and CLs was 2.25 and 6.72 mL/h/kg for BAX 826 and ADVATE, respectively. In monkeys, a baseline FVIII activity level was detected and adequately taken into account in calculating pharmacokinetic parameters. Nevertheless, to better follow the pharmacokinetic profile of BAX 826, the polysialylated rFVIII concentration was also assessed using a PSA specific assay. PSA-FVIII was measured in all animals after administration of BAX 826. In summary, pharmacokinetics studies in three animal species provided evidence that modification of ADVATE with PSA increases circulation time and exposure compared with the unmodified protein. Disclosures Schiviz: Baxalta Innovations GmbH: Employment. Hoebarth:Baxalta Innovations GmbH: Employment. Wolfsegger:Baxalta Innovations GmbH: Employment. Rossato:Baxalta Innovations GmbH: Employment. Weber:Baxalta Innovations GmbH: Employment. Gritsch:Baxalta Innovations GmbH: Employment. Rottensteiner:Baxalta Innovations GmbH: Employment. Turecek:Baxalta Innovations GmbH: Employment. Scheiflinger:Baxalta Innovations GmbH: Employment. Hoellriegl:Baxalta Innovations GmbH: Employment. Putz:Baxalta Innovations GmbH: Employment.

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Hoebarth, Gerald, Susan Kubik, Martin Wolfsegger, John-Philip Lawo, Alfred Weber, Herbert Gritsch, Hartmut J. Ehrlich, Friedrich Scheiflinger, Hans Peter Schwarz et Eva-Maria Muchitsch. « Single Dose Pharmacokinetics of a Recombinant FVIIa In Factor VIII KO Mice, Rats, and Cynomolgus Monkeys ». Blood 116, n^o 21 (19 novembre 2010) : 4655. http://dx.doi.org/10.1182/blood.v116.21.4655.4655.

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Abstract Abstract 4655 The purpose of these PK studies was to assess the pharmacokinetic profile of Baxter's rFVIIa in comparison with a commercially available rFVIIa after a single intravenous bolus injection at a target dose of 0.6 mg/kg BW in mice and rats and 2.7 mg/kg BW in cynomolgus monkeys. The AUC0-t last (the area under the concentration vs. time curve from 0 to the last measured time point) was evaluated as primary endpoint. Secondary endpoints were terminal half-life (HL), mean residence time (MRT) and total clearance standardized per kg body mass (Cl) for FVIIa protein and clotting activity. In cynomolgus monkeys only FVIIa clotting activity was evaluated. Blood was sampled at baseline and each of the time points after a single intravenous bolus injection of Baxter's rFVIIa or the commercially available rFVIIa. The citrated plasma samples were analyzed for FVIIa activity and FVII protein (antigen). A serial sacrifice design was used for the PK in mice. Ten FVIII ko mice (B6;129S4-F8tm1Kaz; 5m/5f) per time point were bled by cardiac puncture under anesthesia for blood sampling 5 – 200 minutes after a single intravenous bolus injection. A single animals design was used for the single dose PK in Sprague Dawley rats (5m/5f) and cynomolgus monkeys (2m/2f). In rats and cynomolgus monkeys blood samples for citrated plasma were drawn before administration (for analysis of baseline FVIIa levels) and 5 – 270 minutes (rats) or 5 minutes to 15 hours (cynomolgus monkeys) after administration. Plasma samples were analyzed for FVIIa protein (antigen) using a FVII ELISA calibrated for FVIIa measurement and for FVIIa activity using a FVIIa clotting assay. In all three species bioequivalence of Baxter's rFVIIa and the reference item could be shown for the primary endpoint (AUC0-tlast) for FVIIa activity and antigen (the latter was only tested in mice and rats). Additionally, secondary endpoints of FVIIa activity (terminal half-life, mean residence time and total clearance) were similar for Baxter's rFVIIa and the reference item. In mice, HL was 0.64 h, MRT was 0.80 h and Cl was 245 ml/kg/h for Baxter's new rFVIIa. Values for the secondary endpoints in rats were 1.17 h for HL, 1.29 h for MRT and 102.6 mL/kg/h for Cl. In cynomolgus monkeys, HL was 2.00 h, MRT was 2.45 h and Cl was 33.6 mL/kg/h. In summary, the pharmacokinetic profiles of Baxter's rFVIIa and the commercially available rFVIIa were similar in all species studied. Disclosures: Hoebarth: Baxter Innovations GmbH: Employment. Kubik:Baxter Innovations GmbH: Employment. Wolfsegger:Baxter Innovations GmbH: Employment. Lawo:Baxter Innovations GmbH: Employment. Weber:Baxter Innovations GmbH: Employment. Gritsch:Baxter Innovations GmbH: Employment. Ehrlich:Baxter Innovations GmbH: Employment. Scheiflinger:Baxter Innovations GmbH: Employment. Schwarz:Baxter Innovations GmbH: Employment. Muchitsch:Baxter Innovations GmbH: Employment.

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Flier, Arjan van der, Zhan Liu, David R. Light et Haiyan Jiang. « Immunohistochemical Staining of rFVIIIFc and rFVIII in Liver Cells Differentiates Between a VWF-Dependent and Independent Clearance Pathway in Mice ». Blood 122, n^o 21 (15 novembre 2013) : 2331. http://dx.doi.org/10.1182/blood.v122.21.2331.2331.

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Abstract Introduction rFVIIIFc is a fully recombinant fusion protein consisting of a single B domain-deleted human FVIII covalently attached to the dimeric Fc domain of human IgG1. rFVIIIFc has a 1.5-fold extended half-life and decreased clearance compared to rFVIII in patients with hemophilia A (Powell, Blood 2012). The Fc region of rFVIIIFc binds to the neonatal Fc receptor (FcRn), which is part of a naturally occurring pathway that cycles IgG back into circulation, delaying lysosomal degradation. Our previous studies with FcRn-chimeric mice showed that the decreased clearance of rFVIIIFc is mediated by FcRn expressed in somatic cells and not in hematopoietic cells. Biodistribution studies with 125I-rFVIIIFc identified liver as the major organ for rFVIIIFc disposition. Furthermore, qPCR studies showed that three different cell types in liver all express FcRn: hepatocytes (HC), liver sinusoidal endothelial cells (LSEC) and Kupffer cells (KC). In primary liver cell co-cultures, rFVIIIFc and rFVIII were both internalized by somatic LSEC and HC, but not KC, in the absence of von Willebrand factor (VWF). It has been reported that in vivo, 95% of FVIII circulates as a non-covalent complex with VWF (Lenting, JTH 2007) and we found that VWF delays liver uptake of FVIII and improves the circulating half-life of rFVIIIFc in mice. Aim Compare the cellular localization of rFVIII and rFVIIIFc in the murine liver, in the presence and absence of VWF, in order to identify the cells responsible for the prolonged half-life of rFVIIIFc. Methods FVIII KO (HemA) and FVIII/VWF DKO mice were dosed with rFVIII, rFVIIIFc or the mutants rFVIIIFc-IHH and rFVIIIFc-N297A that are deficient in interacting with FcRn and FcgR, respectively. The cellular localization in the liver of rFVIII, rFVIIIFc and mutants was investigated by immunohistochemistry, using a panel of anti-human FVIII monoclonal antibodies or anti-human-IgG (Fc) for detection, along with markers for LSEC, KC, and VWF. Results In HemA mice with circulating endogenous VWF, the majority of both rFVIII and rFVIIIFc signal is found in KC, which co-stain for VWF. In contrast, neither rFVIII nor rFVIIIFc is detected in the endothelial cells of the large vessels that stained for VWF in the Weibel-Palade bodies. Closer examination shows faint vesicular staining by rFVIII in HC in contrast to a diffuse staining by rFVIIIFc in the liver sinusoid. However in FVIII/VWF DKO mice lacking VWF, neither rFVIII nor rFVIIIFc is detected in KC consistent with the notion that internalization of FVIII by KC is mediated by VWF. The majority of rFVIII is found in HC, whereas rFVIIIFc again appears as a diffused liver sinusoidal staining pattern that is more intense than that observed in the HemA mice expressing VWF. These findings suggest free rFVIII is internalized and cleared by HC, while rFVIIIFc is cycled out of the HC and localizes in the Space of Disse, hence its sinusoidal localization. Alternately, in the absence of VWF, rFVIIIFc may cycle through LSEC rather than HC. In order to distinguish these two pathways, an rFVIIIFc variant, rFVIIIFc-IHH, which is not competent to bind FcRn, was tested. In a similar manner as rFVIII, the rFVIIIFc-IHH mutant localizes into HC in DKO mice and is found predominantly in KC in HemA with endogenous VWF. In contrast, rFVIIIFc-N297A, which is not competent to bind FcgR, localizes similarly to rFVIIIFc in DKO mice, excluding a role for FcgR. Conclusions Our current immunohistochemical studies and previous biodistribution and PK studies in mice indicate that there are two parallel, linked clearance pathways for rFVIII and rFVIIIFc. rFVIII or rFVIIIFc complexed with endogenous VWF is internalized predominantly by macrophages, including KC. However, because the VWF-FVIII complex is in constant equilibrium, a fraction of free rFVIII or rFVIIIFc is available for clearance by HC. We propose that this free fraction of rFVIIIFc entering HC is then cycled by FcRn back into the liver sinusoid and into circulation, in contrast to the free rFVIII entering the HC. Staining of the rFVIIIFc-IHH mutant in HC suggests that these cells play a dominant role in vivo, however LSEC may also contribute to cycling of VWF-free rFVIIIFc. The fate of the VWF-bound rFVIIIFc fraction internalized by KC is less clear, however data using FcRn-chimeric mice suggest that FcRn expressed in hematopoietic cells, including KC contributes only marginally to the delayed clearance of rFVIIIFc. Disclosures: van der Flier: Biogen Idec: Employment, Equity Ownership. Liu:Biogen Idec: Employment, Equity Ownership. Light:Biogen Idec: Employment, Equity Ownership. Jiang:Biogen Idec: Employment, Equity Ownership.

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Holmberg, Heidi L., Marianne Kjalke, Ditte Karpf, Ida Hilden, Hermann Pelzer, Mikael Koefoed-Hansen, Laust Johnsen et al. « High Affinity Binding of FVIII to VWF Is Not Required for the Haemostatic Effect of FVIII In Vivo ». Blood 118, n^o 21 (18 novembre 2011) : 1182. http://dx.doi.org/10.1182/blood.v118.21.1182.1182.

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Abstract Abstract 1182 VWF protects FVIII from clearance in the circulation and is believed to ensure location of platelets to the site of injury. However, it is unknown if binding of FVIII to VWF has a role in localizing and thereby also facilitating the effect of FVIII in vivo. In the present study, a FVIII variant, FVIII-Y1680F, lacking the high affinity binding to VWF (Leyet et al. JCB 1991; 15; 740) was used to evaluate the binding of FVIII to VWF in clot formation hemophilia A mice in vivo. Binding of the FVIII variant to immobilized VWF was evaluated by surface plasmon resonance showing a 50–25 fold reduction in the Kd for FVIII-Y1680F compared to wt FVIII. Furthermore, pharmacokinetic studies in hemophilia A mice indicated that FVIII-Y1680F is basically devoid of VWF binding in vivo. The circulating half-life decreased from 7–8 hours for wt FVIII to 0.5 hours for FVIII-Y1680F (see figure) which is comparable to the half-life of wt FVIII in VWF knockout mice (0.5 hours). Using a chromogenic assay the specific activity of FVIII-Y1680F was 9200 IU/mg similar to that of wt FVIII confirming normal activity FVIII-Y1680F after cleavage with thrombin and removal of VWF. As the short half-life may influence the haemostatic effect of FVIII-Y1680F in vivo, a 40 kDa PEG moiety was attached to the O-glycan in the B-domain of the FVIII variant. This re-establishes the circulating half-life (6.1 hours) to that of wt FVIII without affecting the specific activity in vitro. The haemostatic effect of 40K-O-PEG FVIII-Y1680F was subsequently used to investigate if high affinity VWF binding of FVIII influences its haemostatic effect in vivo. The acute haemostatic effect of 40K-O-PEG-FVIII-Y1680F was compared to wt FVIII (Advate®) in the tail bleeding model in hemophilia A mice at doses equivalent to the 50% of the maximal effect and at maximal efficacy (20 and 280 IU/kg; Elm et al., Hemophilia 2011 epub). The blood loss was significantly reduced at both doses with comparable effect of 40K-O-PEG-FVIII-Y1680F and wt FVIII (see see figure, * indicates significant differences compared to vehicle treated hemophilia A mice). This indicates that the lack of VWF binding does not interfere with the haemostatic properties of FVIII in this particular model. To further support these data, the haemostatic effect of 40K-O-PEG-FVIII-Y1680F was tested in the FeCl3 injury model (2.5, 5 and 10 IU/kg) in hemophilia A mice. No clot formation was observed in vehicle mice and 40K-O-PEG-FVIII-Y1680F normalized dose dependently the clot formation time comparable to wt FVIII. In conclusion, the current data suggest that the haemostatic effect of FVIII in vivo is not dependent on high affinity binding of FVIII to VWF. Disclosures: Holmberg: Novo Nordisk A/S: Employment. Kjalke:Novo Nordisk A/S: Employment. Karpf:Novo NOrdisk A/S: Employment. Hilden:Novo Nordisk A/S: Employment. Pelzer:Novo Nordisk A/S: Employment. Koefoed-Hansen:Novo Nordisk A/S: Employment. Johnsen:Novo Nordisk A/S: Employment. Thim:Novo Nordisk A/S: Employment. Karlsson:Novo Nordisk A/S: Employment. Jespersgaard:Novo Nordisk A/S: Employment. Bolt:Novo Nordisk: Employment. Stennicke:Novo Nordisk A/S: Employment.

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Atiq, Ferdows, Lisette M. Schütte, Agnes E. M. Looijen, Johan Boender, Moniek P. M. de Maat, Marieke J. H. A. Kruip et Frank W. G. Leebeek. « VWF and FVIII Levels after Desmopressin Are Associated with the Bleeding Phenotype in Type 1 VWD ». Blood 134, Supplement_1 (13 novembre 2019) : 1116. http://dx.doi.org/10.1182/blood-2019-123634.

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Introduction The bleeding phenotype of type 1 von Willebrand disease (VWD) patients is very heterogeneous. We hypothesized that part of this heterogeneity is explained by variability in response of von Willebrand factor (VWF) and factor VIII (FVIII) levels to stress during hemostatic challenges. Patients who have a strong increase in VWF and FVIII levels during hemostatic challenges, may have less frequent or less severe bleeding episodes compared to patients who have a smaller increase in VWF and FVIII levels. Therefore, we investigated whether VWF and FVIII levels after desmopressin, which mimic in vivo hemostatic response during hemostatic challenges, are associated with the bleeding score, and therefore may explain part of the heterogeneity in bleeding phenotype of type 1 VWD patients. Methods In this retrospective cohort study, we included type 1 VWD patients of all ages who were diagnosed and treated in the Erasmus University Medical Center. All patients had a hemorrhagic diathesis or family history of VWD and historically lowest VWF antigen (VWF:Ag) and/or VWF Ristocetin Cofactor activity (VWF:RCo) ≤ 0.30 IU/mL with VWF activity/ VWF antigen ratio above 0.60. All patients participated in the Willebrand in the Netherlands (WiN) study, in which a self-administered Tosetto bleeding score was assessed. Results We identified 173 eligible type 1 VWD patients in our treatment center who participated in the WiN study. After exclusion of 50 patients who did not receive a desmopressin test dose, and 1 patient who did not have a bleeding score assessment, we included 122 type 1 VWD patients. Table 1 shows the patient characteristics. Higher FVIII activity during the complete time course (i.e. one, three and 5-6 hours) after desmopressin, and higher VWF and FVIII levels combined at three hours after desmopressin, were associated with a lower bleeding score, respectively β=-0.9 (95% CI: -1.7; -0.1) and β=-1.2 (-1.9; -0.5), both adjusted for age, sex, BMI and comorbidities. FVIII:C at three hours after desmopressin was significantly associated with a lower bleeding score: β=-1.5 (-3.0; -0.1), whereas VWF levels were not associated (Figure 1). Moreover, patients with VWF and FVIII levels in the highest quartile three hours after desmopressin administration had a lower bleeding score compared to other patients (Figure 2). The largest difference was found for FVIII:C: β=-5.1 (-8.4; -1.8), adjusted for age, sex, BMI, comorbidities and baseline FVIII:C. These patients also less often had an abnormal bleeding score: OR=0.1 (0.0-0.5), adjusted for age, sex, BMI, comorbidities and baseline FVIII:C. Lastly, patients with an increased clearance of VWF, defined as VWF propeptide/VWF:Ag ratio >2.2, had lower VWF:Ag (β=-0.3, -0.6; -0.1), VWF:Act (β=-0.5, -0.7; -0.2), VWF:CB (β=-0.5, -1.0; -0.1) and FVIII:C (β=-0.7, -1.0; -0.4) at three hours after desmopressin administration compared to other type 1 VWD patients, all adjusted for age, sex, BMI and comorbidities. An increased clearance of VWF was also associated with a higher bleeding score: β=3.2 (0.2; 6.2), adjusted for age, sex, BMI, comorbidities and baseline VWF and FVIII levels. Discussion In type 1 VWD patients, the desmopressin induced increase of VWF and FVIII levels is associated with a lower bleeding score. Especially higher VWF and FVIII levels at three hours after desmopressin were associated with a milder bleeding phenotype, suggesting that sustained VWF and FVIII levels after a hemostatic challenge may be important to prevent bleeding. This was strengthened by an association between increased clearance of VWF and a higher bleeding score. Furthermore, patients with VWF and FVIII levels in the highest quartile three hours after desmopressin had 5 points lower bleeding score, and patients with FVIII:C in the highest quartile had 10 times less often an abnormal bleeding score. These results indicate that a good hemostatic response may have important and clinically relevant consequences on the bleeding phenotype, and may compensate for the low baseline VWF and FVIII levels in type 1 VWD patients. In conclusion, VWF and FVIII levels after desmopressin administration, which mimic in vivo hemostatic response to hemostatic challenges, are associated with the bleeding phenotype in type 1 VWD patients. This may partly explain the well-known variability in bleeding phenotype of type 1 VWD patients. Disclosures Atiq: Sobi: Other: Travel grant (ISTH 2019); Professor Heimburger Award 2018 (CSL Behring): Research Funding. Schütte:CSL Behring: Other: Travel grant. Boender:Sobi: Employment; Professor Heimburger Award 2016 (CSL Behring): Research Funding. Kruip:Bayer: Research Funding; Pfizer: Research Funding; Daiichy Sankyo: Research Funding; Boehringer Ingelheim: Research Funding. Leebeek:Shire/Takeda: Consultancy; UniQure: Consultancy; Novo Nordisk: Consultancy; Shire/Takeda: Research Funding; Sobi: Other: Travel grant; CSL Behring: Research Funding.

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Castaman, Giancarlo. « CHANGES OF VON WILLEBRAND FACTOR DURING PREGNANCY IN WOMEN WITHOUT AND WITH VON WILLEBRAND DISEASE ». Mediterranean Journal of Hematology and Infectious Diseases 5, n^o 1 (16 juillet 2013) : e2013052. http://dx.doi.org/10.4084/mjhid.2013.052.

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Delivery in von Willebrand disease (VWD) represents a significant hemostatic challenge because of the variable pattern of changes observed during pregnancy of von Willebrand factor (VWF) and factor VIII (FVIII), the protein carried by VWF. The wide heterogeneity of phenotypes and of the underlying pathophysiological mechanisms associated with this disorder prompt a careful evaluation of pregnant women with VWD to plan the most appropriate treatment at time of parturition. VWF and FVIII increase significantly during pregnancy in normal women, already within the first trimester, reaching levels by far > 100 U/dL by the time of parturition. In women with VWD, levels at baseline of VWF and FVIII > 30 U/dL are usually associated with a high likelihood to achieve normal levels at the end of pregnancy and specific anti-hemorrhagic prophylaxis is seldom required. Women with basal level < 20 U/dL usually have a poor increase since most of these women carry mutations associated with increased VWF clearance or are compound heterozygous for different VWF mutations which prevent the achievement of satisfactory hemostatic levels. While women with mutations associated with increased clearance show a full, albeit transitory correction of their hemostatic deficiency after desmopressin administration, compound heterozygous need replacement therapy because they do not respond well to this agent. Patients with abnormal VWF:RCo/VWF:Ag ratio at baseline (e.g. < 0.6), typically associated with type 2 VWD, maintain the abnormality throughout pregnancy and VWF:RCo usually does not attain safe levels ³ 50 U/dL. These women require replacement therapy with VWF-FVIII concentrates. Delayed post-partum bleeding may occur when replacement therapy is not continued for some days. Tranexamic acid may be useful at discharge to avoid excessive lochia.

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Batsuli, Glaivy, Seema R. Patel, Courtney Cox, Wallace H. Baldwin, John S. Lollar et Shannon L. Meeks. « Epitope Dependent Augmentation of the Immune Response in Hemophilia Α Mice Immunized with Factor VIII/Antibody Immune Complexes ». Blood 134, Supplement_1 (13 novembre 2019) : 2387. http://dx.doi.org/10.1182/blood-2019-127436.

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Introduction: The immune response to factor VIII (fVIII) is a CD4+ T cell dependent process initiated by fVIII recognition and processing by antigen presenting cells. The C1 and C2 domains have been proposed as the primary domains that mediate fVIII internalization by dendritic cells. Our prior studies demonstrate that fVIII pre-bound to anti-C1 and C2 domain monoclonal antibodies (MAbs) reduces fVIII internalization by murine derived dendritic cells. However, anti-A1 and A3 domain antibodies increase fVIII endocytosis by dendritic cells. In this study, we analyzed the antibody titers of hemophilia A mice immunized with binary fVIII and MAb complexes to determine whether changes in fVIII internalization observed in vitro correspond to changes in the immune response to fVIII in vivo. Methods: Exon16 (E16) knockout mice deficient in fVIII were immunized with B-domain deleted fVIII in the presence of either anti-fVIII MAb 2-116 (anti-A1, IgG2a), 4A4 (anti-A2, IgG2a), 2-113 (anti-A3, IgG1), B136 (anti-C1, IgG2a), or 3D12 (anti-C2, IgG2b) versus fVIII alone. Mice were immunized by weekly retro-orbital injections of 0.1 µg fVIII incubated with 1 µg anti-fVIII MAb for 4 weeks followed by a boost dose of 0.2 µg fVIII and 2 µg anti-fVIII MAb one week later at week 5. Anti-fVIII ELISA titers (in arbitrary units, AU) were analyzed from plasma samples collected at week 7 to account for IgG half-life. A separate cohort of mice immunized with anti-fVIII MAbs alone served as controls to normalize ELISA titers in each fVIII/MAb group to account for residual injected MAbs in the plasma samples. Results: Hemophilia A mice immunized with fVIII/2-116 and fVIII/4A4 complexes significantly increased anti-fVIII ELISA titers compared to mice immunized with fVIII alone (figure 1). Mice immunized with fVIII/2-116 and fVIII/4A4 had median ELISA titers of 7,401 AU (interquartile range, IQR, 2,654 - 11,910 AU) and 3,620 AU (IQR 1,062 - 7,969 AU), respectively, compared to mice immunized with fVIII (median titer 1,063 AU, IQR 402 - 2,476 AU). MAb 2-116 is a non-inhibitory antibody with a titer of <1 Bethesda Unit (BU)/mg IgG, while 4A4 is a highly inhibitory antibody with a titer of 40,000 BU/mg IgG. Neither MAb interferes with fVIII binding to von Willebrand factor (VWF) or phospholipid vesicles. Mice immunized with fVIII/2-113 (median titer 2,210 AU, IQR 402 - 8,318 AU), fVIII/B136 (median titer 123 AU, IQR 0 - 9,709 AU), and fVIII/3D12 (median titer 3,244 AU, IQR 0 - 5,180 AU) did not have significantly different anti-fVIII ELISA titers compared to mice immunized with fVIII. However there was a trend towards reduced anti-fVIII titers with fVIII/B136 and fVIII/3D12 injections. MAbs B136 and 3D12 inhibit fVIII binding to VWF and have been shown to significantly increased fVIII clearance in hemophilia A mice compared to fVIII in a VWF-dependent manner. Median titers for mice immunized with MAbs alone to account for residual injected MAbs were 13 AU (2-116), 12 AU (4A4), 18 AU (2-113), 16 AU (B136), and 4 AU (3D12). Conclusions: Immunization of hemophilia A mice with fVIII/MAb complexes, specifically anti-A1 MAb 2-116 and anti-A2 MAb 4A4, enhance the immune response to fVIII. MAb 2-116 significantly increased anti-fVIII antibody titers in vivo, which correlates with increased fVIII internalization by immature dendritic cells observed in vitro. A better understanding of the effect of anti-fVIII antibodies on fVIII conformational changes could provide insight into whether these changes alter fVIII recognition by immune cells and subsequently propagate the immune response to fVIII at the onset of inhibitor formation or during immune tolerance induction. Disclosures Batsuli: Genentech: Other: Advisory board participant; Bayer: Other: Advisory board participant; Octapharma: Other: Advisory board participant. Meeks:HEMA Biologics: Membership on an entity's Board of Directors or advisory committees; Novo Nordisk: Membership on an entity's Board of Directors or advisory committees; Genentech: Membership on an entity's Board of Directors or advisory committees; Bioverativ: Membership on an entity's Board of Directors or advisory committees; Takeda-Shire: Membership on an entity's Board of Directors or advisory committees; Bayer: Membership on an entity's Board of Directors or advisory committees.

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