Littérature scientifique sur le sujet « FVIII clearance »

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.

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.

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).

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.

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.

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.

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.

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.

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.

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.

Background Genetic variants in the LDL receptor (LDLR) gene have been associated with higher LDL cholesterol levels, premature atherosclerotic cardiovascular disease (ASCVD) and increased cardiovascular risk. However, the increased arterial thrombotic risk observed in LDLR carriers is partly independent from the lipoprotein levels and could be related to the LDLR modulation of FVIII levels. Indeed, a role of LDLR in FVIII clearance has been shown and LDLR variants have been associated with higher FVIII levels. To date, it is not clear which are the LDLR variants involved in FVIII clearance and the underlying mechanism. With this background and gap of knowledge, the present study aims to: - identify the LDLR variants associated with high FVIII coagulant activity (FVIII:C≥150 IU/dL) in a cohort of DVT (Deep Veen Thrombosis) patients and healthy control subjects; - functionally and biochemically characterize the LDLR variants (identified in this study and described in literature) associated with high FVIII levels in order to understand their ability to modulate the FVIII clearance, in terms of FVIII binding and/or uptake. Methods We selected 596 Italian subjects (298 DVT patients and 298 controls) enrolled and sequenced in the frame of the DVT-Milan study. FVIII:C was measured by one-stage clotting assay. A multiplexed next generation sequencing was performed. In order to select the variants to be characterized in vitro, we analyzed the association between LDLR variants and high FVIII:C (FVIII:C≥150 IU/dL). For common and low-frequency LDLR variants a logistic regression model was performed. Rare LDLR variants have been analyzed using cumulative association tests. Wild type (WT) and selected mutant LDLR vectors were transiently transfected in LDLR-deficient Chinese hamster ovary (CHOldlA7) cells with chemical methods. The LDLR expression in total cell lysates was assessed by western blotting (WB). Immunofluorescence (IF) was performed to evaluate total, surface and intracellular expression of LDLR proteins. Results In our cohort of DVT patients and healthy control subjects we identified the missense LDLR variant rs45508991 associated with high FVIII levels (OR=5.47, p=0.06). We selected 4 LDLR variants to be characterized in vitro: the variant rs45508991 and 3 synonymous variants (rs688, rs2228671 and rs5925), previously reported to be associated with higher FVIII levels or increased risk of ischemic stroke and/or coronary artery disease. Among the 4 LDLR variants expressed in vitro, we observed, by both WB and IF, a significant reduced expression of total LDLR protein carrying the rs45508991 variant, compared to the WT LDLR. Moreover, by IF, we observed that the surface LDLR expression was significantly reduced in cells expressing the variant rs45508991. Conclusions and perspectives Our hypothesis is that a reduced expression of LDLR, total and on cell surface, caused by the identified variant rs45508991, could impair FVIII binding and clearance, leading to an increase in FVIII levels. In order to test this hypothesis, we will perform the fluorescence-activated cell sorting (FACS) with the goal to confirm IF findings concerning the cell surface LDLR expression in all samples and to evaluate the LDLR-mediated binding of FVIII.

The antiphospholipid antibodies (aPL) are commonly associated with thrombotic events and obstetric complications. However, apart from the bleeding complications of antithrombotic therapy, the acquired coagulopathy caused by the aPL, particularly by lupus anticoagulant and anticardiolipin antibodies, might be occasionally manifested as a hemorrhagic syndrome with various clinical severity. Bleeding symptoms vary from mild (mucocutaneous) up to life-threatening (gastrointestinal, intracranial). The bleeding may be the first manifestation of aPL or appear concomitantly with thrombosis. The underlying hemostatic changes include thrombocytopenia, platelet function disorders, and coagulation factor inhibitors or deficiencies, namely prothrombin, FVII, FVIII, FX, and FXI. Thrombocytopenia is the most common finding, seen in up to 53% of patients with aPL, although it is usually mild to moderate and associated with significant bleeding only in a minority of cases. Of interest, patients with severe thrombocytopenia appear to be less likely to suffer from thrombotic events. The involved pathophysiological mechanisms are heterogeneous. Non-neutralizing antibodies against coagulation factors resulting in increased clearance, specific antibodies against platelet membrane glycoproteins, increasing platelet activation and aggregation with subsequent consumption, and immune-mediated platelet clearance are among those identified. Immunosuppression, preferably with corticosteroids, represents the first-choice therapeutic approach. Plasmapheresis is efficient in the case of catastrophic antiphospholipid syndrome. Antithrombotic therapy can be challenging, but its administration should continue as much as possible.

Coagulation abnormalities are among the number of potential risk factors toinitiate the bleeding episodes from gastrcr-esophageal varices in liver cirrhosis. The impairment of liver clearance of activated coagulation factors, the release of thromboplastin-like activity from the necrotic liver cells and the hemodynamic changes due to expanded bollaterals may all contribute to activate the coagulation cascade.However, little is known about the mechanisms leading to this activation. Activated Factor VII (FVIIa) is known totrigger both intrinsec and extrinsec coagulation pathway. Therefore, we measured FVIIa in a group of 33 cirrhotic patients in order to see if a difference between bleeders and non-bleeders patients would correlate with Factor VII activation. The patients were divided in two groups according to the presence or the absence of major bleeding from gastroesophageal varices; haemorragic episodes were confirmed by a gastroscopic examination performed during or immediately after bleeding episodes. Factor VII coagulant assay (VII:C - one stage clotting method) and Factor VII coupled amidolytic assay (VII:CHR) were performed and a factor VII activity ratio (VIIa) was calculated as VII:C/VII:CHR. The results (mean ± S.E.) are summarized in this table:No difference in Vila distribution was seen when the patients were divided on the base of liver impairment (according to Child’s criteria) .Our study shows that FVII activation is related to bleeding from esophageal varices but not to the degree of liver impairment and strongly suggests the existence of an hypercoagulable state in liver cirrhosis, probably related to major bleeding from gastroesophageal varices in cirrhotic patients.