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1
Yada, Koji, Kenichi Ogiwara, Masaru Shibata, Midori Shima, and Keiji Nogami. "Effects of anti-factor VIII inhibitor antibodies on factor VIIa/tissue factor-catalysed activation and inactivation of factor VIII." Thrombosis and Haemostasis 105, no. 06 (2011): 989–98. http://dx.doi.org/10.1160/th10-12-0781.
Анотація:
SummaryFactor (F)VIIa/tissue factor (TF) rapidly activates FVIII activity by proteolysis at Arg372 and Arg740, and subsequently inactivates FVIIIa activity by proteolysis at Arg336, although this activation is weaker than that by thrombin. The effects of anti-FVIII inhibitor antibodies on these reactions remain unknown, however. In this study, 13 of anti-FVIII inhibitor antibodies recognising the A2 or C2 domain were prepared. None of them, irrespective of epitope specificity, significantly affected FVIIa/TFcatalysed FVIII activation in one-stage clotting assays. Anti-A2 and anti-C2 type 2 antibodies had little effect on the inactivation phase. Anti-C2 type 1 antibodies, however, modulated inactivation by 40–60% of that seen with control IgG, suggesting that the activity of FVIIIa generated by FVIIa/TF persisted in the presence of this specific type of inhibitor. SDS-PAGE analysis demonstrated that all antibodies had little effect on FVIIa/TF-catalyzed proteolysis at Arg372 and Arg740. Anti-C2 type 1, however, significantly delayed cleavage at Arg336 in dose-dependent manners. Neither anti-A2 nor anti-C2 type 2 affected this reaction, and the findings were consistent with the results of the functional assays. In addition, anti-C2 monoclonal antibodies with type 1 and 2 demonstrated similar patterns of reaction as the anti-C2 polyclonal antibodies in FVIIa/TF-mediated FVIII mechanisms. We demonstrated that FVIIa/TF activated FVIII even in the presence of anti-FVIII antibodies, but inactivation patterns appeared to depend on inhibitor type. It could be important to determine the characteristic of these inhibitor antibodies for prediction of their effects on FVIIa-related FVIII reactions, and the results could have significant therapeutic implications.Note: An account of this work was presented at the 51st annual meeting of the American Society of Hematology, 2009, New Orleans, LA, USA. This work was supported by grants for MEXT KAKENHI 21591370 in Japan and Bayer Hemophilia Award program.
2
Yada, Koji, Keiji Nogami, Kenichi Ogiwara, Katsumi Nishiya, Masahiro Takeyama, and Midori Shim. "Effects of Anti-FVIII Inhibitors On Factor VIIa/Tissue Factor-Catalyzed Activation and Inactivation of Factor VIII." Blood 114, no. 22 (November 20, 2009): 3169. http://dx.doi.org/10.1182/blood.v114.22.3169.3169.
Анотація:
Abstract Abstract 3169 Poster Board III-110 Factor (F)VIIa with tissue factor (TF) is a primary trigger of blood coagulation. We have recently demonstrated that FVIIa/TF rapidly activated FVIII by proteolysis of the heavy chain (HCh), and served physiologically as a potent activator for up-regulation of FVIII activity in very early-timed phase (ASH #1036, 2008). FVIII inhibitors develop as alloantibodies in multi-transfused patients with hemophilia A and also arise as autoantibodies in normal individuals. FVIII inactivation by inhibitors is associated with impairment of FVIII(a) cofactor function through the binding to functional crucial epitopes in FVIII. Anti-C2 inhibitors prevent FVIII binding to phospholipid, von Willebrand factor, and FXa. Anti-A2 inhibitors prevent FVIII binding to FIXa and thrombin. However, effects of these inhibitors on FVIIa action for FVIII have remained to be studied. In this study, we prepared 13 of anti-FVIII inhibitor IgGs (2 of anti-A2, 7 of anti-C2 with type 1 behavior, and 4 of anti-C2 with type 2). We first examined FVIIa/TF-catalyzed FVIII activation in the presence of anti-FVIII inhibitors in one-stage clotting assay. The levels of FVIII activity (10 nM) elevated rapidly by ∼2.0-fold within 30 sec after adding of FVIIa/TF (1 nM), and subsequently decreased to the initial level within 20 min. The presence of anti-FVIII inhibitors did not significantly affect FVIIa/TF-catalyzed FVIII activation (by 1.7∼2.2-fold) compared to normal IgG. This action was independent of the difference of inhibitor epitopes. In addition, FVIIa-catalyzed FVIIIa inactivation with anti-A2 or anti-C2 with type 2 inhibitors was little affected, similar to that with normal IgG. However, of note, all of anti-C2 with type 1 significantly inhibited FVIIa-catalyzed inactivation of FVIIIa. Inactivation rates of FVIIa with anti-C2 with type 1 (k ∼0.15) was ∼40% less than that with control IgG (k ∼0.24), supporting that the presence of anti-C2 with type 1 might persist the activity of FVIIIa generated by FVIIa. To clarify this inhibitory mechanism of anti-C2 with type 1, we performed FVIIa-catalyzed FVIII cleavage in Western blotting. FVIIa/TF (1 nM) proteolyzed the HCh of FVIII (10 nM) rapidly by cleavages at Arg372 (and Arg740), whilst cleavage at Arg336 in the A1 domain was appeared at ∼2.5 min, supporting that cleavages at Arg372 and Arg336 by FVIIa contribute to the up- and down-regulation of FVIII(a) activity, respectively. All inhibitors, independent of recognizing epitopes, did not affect FVIIa-catalyzed cleavage at Arg372. However, the presence of anti-C2 type 1 delayed the cleavage at Arg336 in timed- and dose-dependent manners, whilst either anti-A2 or anti-C2 type 2 did not affect, consistent with the functional inactivation results. FVIIa binds to the A2, A3, and C2 domains in FVIII. Based on our findings, FVIIa-interactive sites on FVIII unlikely overlapped with anti-A2 and -C2 inhibitor epitopes, and inhibition of Arg336 cleavage may be due to conformational change caused by antibody binding. Furthermore, FVIIa indeed activates FVIII even in the presence of anti-FVIII inhibitors, different from thrombin, FXa, etc, and it would be important to predict the effect of FVIIa for FVIII to determine the characteristics of anti-FVIII inhibitors. Disclosures No relevant conflicts of interest to declare.
3
Nakajima, Yuto, Koji Yada, Keiji Nogami, and Midori Shima. "A Novel Mechanism of Factor VIIa/Tissue Factor (TF)-Catalyzed Activation and Inactivation of B-Domain-Deleted Factor VIII in the Early Initiation Phases of Coagulation." Blood 132, Supplement 1 (November 29, 2018): 1162. http://dx.doi.org/10.1182/blood-2018-99-115645.
Анотація:
Abstract We have reported that factor (F)VIII was rapidly activated by FVIIa/tissue factor (TF) in vitro by limited proteolysis of the heavy chain (HCh) at Arg372 and Arg740 in the very early-timed coagulation phase and inactivated by proteolysis at Arg336 (JTH 2010). Furthermore, the activation could be observed even in the presence of anti-FVIII inhibitors irrespective of their type of kinetics and the epitope recognized, whilst the inactivation was moderated by anti-C2 inhibitor with type 1 kinetics (Thromb Haemost 2011). A role of FVIII B-domain on FVIIa/TF-catalyzed activation and inactivation remain unknown, however. In this study, focusing on the roles of the B-domain of FVIII, we investigated the mechanism(s) of FVIIa/TF-catalyzed FVIIIa activation and inactivation by utilizing B-domain deleted (BDD)-FVIII as well as full-length (FL)-FVIII. We firstly examined FVIIa/TF-catalyzed activation and inactivation of FL- or BDD-FVIII(a) by a one-stage clotting assay. The FVIII activity (FVIII:C) of FL-FVIII (10nM) rapidly increased by ~1.7-fold within 0.5 min after addition of FVIIa (1nM)/TF (0.1nM), and subsequently decreased to the initial levels within 15 min (k = ~0.03). Interestingly, FVIII:C of BDD-FVIII (10nM), which increased up to ~1.7-fold of the initial level within 0.5 min after addition of FVIIa (1nM)/TF (0.1nM) similar to that of FL-FVIII, demonstrated a slower reduction to the initial level within 30 min (k = ~0.015) than that of FL-FVIII. In order to explore these inhibitory mechanisms of FVIIa/TF-catalyzed inactivation of BDD-FVIIIa, we investigated FVIIa/TF-catalyzed proteolytic cleavage of both BDD-FVIII and FL-FVIII by using SDS-PAGE. A rapid proteolysis in the heavy chain (Hch) of FL-FVIII within 0.5 min after addition of FVIIa/TF was observed by the cleavage at Arg740, followed by the cleavage at Arg372 and the subsequent cleavage at Arg336, consistent with our previous study. In contrast, it was of surprise that the proteolysis in the Hch of BDD-FVIII by cleavage at Arg372 was little observed after addition of FVIIa/TF, whilst that by the cleavage at Arg336 was observed within 0.5 min preceding the elevation of FVIII:C. The initial velocity of Arg336 cleavage at 0.5 min for BDD-FVIII (4.4/min) was ~3.3-times higher than that for FL-FVIII (1.4/min) by a densitometry. To the next, we examined the spontaneous dissociation of A2-domain from FVIIa/TF-catalyzed FL- or BDD-FVIIIa by a one stage clotting assay. In the presence of excess amount of A2-subunit (400nM), more than 50% of FVIIa/TF-catalyzed FVIIIa inactivation was inhibited compared to that in its absence, but no significant difference was observed between FL- and BDD-FVIII, suggesting that the spontaneous dissociation of A2-domain little affected the inhibition of the FVIIa/TF-catalyzed inactivation of BDD-FVIIIa. To further clarify the mechanism of FVIIa/TF-catalyzed BDD-FVIII activation/inactivation, we prepared and stably expressed recombinant BDD-FVIII mutants, R336A and R372A. FVIIa(1nM)/TF(0.1nM)-catalyzed activation and inactivation of R336A and R372A (10nM) was examined by a one stage clotting assay. FVIII:C of R336A and R372A rapidly increased by ~2.0-fold of the initial level within 0.5 min after addition of FVIIa/TF, similarly to that of wild type BDD-FVIII, and that of R336A subsequently decreased to the initial level within 30min (k = ~0.04), whilst little reduction of FVIII:C was observed for R372A (k = ~0.004). Evaluated by SDS-PAGE, FVIIa/TF-catalyzed proteolytic cleavage at Arg336 was predominantly observed for R372A within 0.5min after addition of FVIIa/TF, whilst cleavage at Arg372 was conversely observed for R336A. Taken together, FVIIa/TF-catalyzed activation of BDD-FVIII could be predominantly initiated by the cleavage at Arg336 or secondarily at Arg372 and resistance to the cleavage at Arg372 would hamper the subsequent inactivation. In conclusion, the B-domain of FVIII would regulate the FVIIa/TF-catalyzed activation and inactivation of FVIII by controlling the order of proteolytic cleavage at Arg336 and Arg372. We believe that our findings should also contribute to the development of more effective combination therapy of FVIIa and BDD-FVIII for hemophilia A with inhibitor. Disclosures Yada: Shire Japan Co., Ltd.: Other: Teacher at a endowed course. Nogami:Chugai Pharmaceutical Co., Ltd: Consultancy, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties: Anti-FIXa/X bispecific antibodies , Research Funding, Speakers Bureau. Shima:Chugai Pharmaceutical Co., Ltd: Consultancy, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties: Anti-FIXa/X bispecific antibodies , Research Funding, Speakers Bureau; F. Hoffmann-La Roche Ltd: Membership on an entity's Board of Directors or advisory committees.
4
Ogiwara, Kenichi, Keiji Nogami, Masahiro Okuda, Katsumi Nishiya, Masahiro Takeyama, and Midori Shima. "Interactions of Factor VIII with Tissue Factor Contributes to the Acceleration of Factor Xa Generation in the Initiation Phase of Blood Coagulation." Blood 114, no. 22 (November 20, 2009): 3177. http://dx.doi.org/10.1182/blood.v114.22.3177.3177.
Анотація:
Abstract Abstract 3177 Poster Board III-116 Activated factor (F)VII complex with tissue factor (FVIIa/TF) initiates the blood coagulation by generating FXa as extrinsic Xase complex (ex-Xase). Although FVIIa/TF also activates FIX, FIXa little functions without its cofactor, FVIIIa. A tiny amount of thrombin generated by FXa activates FV and FVIII, followed by forming of intrinsic Xase complex (in-Xase) and prothrombinase complex, respectively. These formations result in ‘thrombin burst’ and successful hemostasis. Although thrombin is thought to be a unique potent activator of FVIII in vivo, FXa and FVIIa/TF also activate FVIII in vitro. We have recently reported the detailed mechanism by which FVIIa/TF activated FVIII more rapidly in early timed-phase than thrombin (Blood Abst.1036, 2008). In this study, we further developed to examine whether TF affected FVIII(a) function. (1) FVIIa/TF rapidly increased FVIII activity by 4.7-fold of initial in the presence of Ca2+ and phospholipid (PL), following by inactivation, in one-stage clotting assay. However, since even in the presence of TF alone, FVIII activity elevated by 1.8-fold of initial, actual increase of FVIII activity by FVIIa/TF was 2.6-fold. A possibility that TF might bind to FVIIa contained in FVIII-deficient plasmas used, was negligible, since FVIIa-inhibitor used blocked an ex-Xase effect >95%. In the presence of FVIIa-inhibitor, residue FVIII activity with TF was ∼50%, thus TF alone affected FVIII cofactor activity independently of FVIIa. (2) Using SDS-PAGE, the addition of TF accelerated FVIII cleavage by FVIIa, whilst decelerated that by thrombin and FXa. (3) Surface plasmon resonance-based assays showed that FVIII(a) directly bound to TF with high affinity (Kd; ∼3 nM). (4) The effect of FVIIa/TF on in-Xase was evaluated in FXa generation assay. 0.1 nM FVIIa/TF, 1 nM FVIII, 90 nM FIX and 20 μM PL were reacted with 150 nM FX at various combinations. FVIIa/TF and FVIIa/TF/FVIII/FIX generated FXa with 3.9 and 10.4 nM/min, respectively. When FVIIa-inhibitor was added prior to addition of FX, FXa generated by FVIIa/TF and FVIIa/TF/FVIII/FIX were 5% and 46% (0.2 and 4.8 nM/min) of those without FVIIa-inhibitor, respectively. The latter was considered as FXa generated by in-Xase. Therefore, FXa derived from in-Xase was ∼40% of total FXa in this condition. (5) FVIIIa/FIXa (1 nM/2 nM)-dependent FXa generation in the presence of TF was evaluated. FXa generation in the presence of TF (0.02 and 0.3 nM) increased by ∼2 and ∼6-folds, respectively, of that in its absence. Furthermore, the functional affinity of FVIIIa for in-Xase complex in the presence of TF (0.1 nM), showed an ∼1.5-fold greater than that in its absence (Km; 4.9 ± 0.4 and 7.1 ± 0.9 nM, respectively). In conclusion, FVIIa/TF can generate FVIIIa in early timed-phase in vitro as well as FXa and FIXa, and possess potential of forming in-Xase. In addition, TF directly binds to FVIII(a), and functions in-Xase complex more efficiently by enhancing the affinity of FVIIIa for in-Xase. Although TF-dependent these reactions may be terminated rapidly via anticoagulant systems such as tissue factor pathway inhibitor, our data suggest that interactions of FVIII with TF might contribute to the acceleration of FXa generation in the initiation phase of blood coagulation. Disclosures Okuda: Sysmex Corporation: Employment.
5
Ogiwara, Kenichi, Keiji Nogami, Tetsuhiro Soeda, Tomoko Matsumoto, Katsumi Nishiya, and Midori Shima. "Mechanisms of Factor VIII Activation by Recombinant Factor VIIa Analog through Tissue Factor-Independent Manner." Blood 112, no. 11 (November 16, 2008): 1028. http://dx.doi.org/10.1182/blood.v112.11.1028.1028.
Анотація:
Abstract Factor VIIa (FVIIa), complexed with tissue factor (TF), is a trigger of blood coagulation. Analog of recombinant FVIIa (rFVIIa), NN1731 (V158D/E296V/M298Q) possesses a greater hemostatic effect than rFVIIa and has been expected in clinical application. Factor X activation rate of NN1731 compared to rFVIIa was 1.2-fold in the presence of TF (TF(+)), and was 30-fold on activated platelets in its absence (TF(−))(Allen, Arterioscler Thromb Vasc Biol.2007; 27: 683). This TF-independent mechanism likely attributes to excellent effects by NN1731. More recently, we reported the physiological role of FVIIa/TF-dependent FVIII activation in the early phase of blood coagulation. Therefore, we were tempted to investigate the action of NN1731 in FVIII activation. Time-dependent change in FVIII activity after the addition of rFVIIa/NN1731 was examined by one-stage clotting assay under the presence of phospholipids (PS:PC:PE=1:6:3), CaCl2 and TF(+)/TF(−). NN1731 raised FVIII activity up to peak level rapidly within 30 sec (TF(+)), following by inactivation. Peak level of FVIII activity by NN1731 in TF(−) reached to the same peak level of that in TF(+) within 5 min, and this peak level persisted for ~30 min. Whilst, peak FVIII level by rFVIIa in TF(−) showed only ~35% of that in TF(+) even at 30 min. FVIII activating rate of NN1731 was observed to be 1.2-fold (TF(+)) and 3.8-fold (TF(−)) of rFVIIa-catalyzed activation. Kinetics by the Xa generation assay showed the Km values of NN1731 in FVIII activation were ~1.5-fold lower than those of rFVIIa (NN1731/rFVIIa; TF(+) 27.3/49.2 nM and TF(−) 50.5/68.1 nM). Vmax values of NN1731 in FVIII activation, however, showed the obvious difference between TF(+) (2.3-fold; NN1731/rFVIIa 70.0/30.4 nM•min−1) and TF(−) (7.9-fold; 92.5/11.7 nM•min−1), compared to rFVIIa. Inactivation of FVIIIa by NN1731 was somewhat faster than that by rFVIIa. FVIII cleavages by NN1731 were analyzed using SDS-PAGE/Western blotting. The heavy chain of FVIII was proteolyzed at Arg740 (A2-B junction), Arg372 (A1-A2 junction) and Arg336 (within the A1), faster by NN1731 than by rFVIIa. These predominant cleavages by NN1731 were more evident in TF(−). However, little cleavage of the light chain of FVIII was observed by both proteases. FVIII cleavages were correlated with the observations of FVIII activation and/or inactivation. To further localize the binding region for NN1731, we evaluated the interactions between FVIII and Glu-Gly-Arg-active site modified (EGR-) NN1731, lacking enzymatic activity, in a surface plasmon resonance-based assay. The Kd value of EGR-NN1731 with FVIII was similar to that of EGR-rFVIIa (6.3 and 7.8 nM, respectively). Binding was particularly evident with the A2, A3, and C2 domains, whilst the A1 domain failed to bind, similar to the results obtained by rFVIIa. We demonstrated that NN1731 possesses higher potential as an activator for up-regulation of FVIII activity than rFVIIa. Furthermore, catalytic activity of NN1731 in TF(−), rather than binding affinity, likely attributes to this potential of its analog. We concluded that the analog has another novel mechanism in its potent hemostatic effect through FVIII activation in TF-independent manner.
6
Meeks, Shannon, Ernest T. Parker, Amy L. Dunn, John F. Healey, and Pete Lollar. "Proteolytically Inactivatable Factor VIII is Less Immunogenic than Factor VIII in a Murine Hemophilia A Model." Blood 114, no. 22 (November 20, 2009): 27. http://dx.doi.org/10.1182/blood.v114.22.27.27.
Анотація:
Abstract Abstract 27 Patients with hemophilia A have a congenital deficiency of the factor VIII (fVIII) protein due to a mutation in the fVIII gene that frequently leads to absence of detectable expression of fVIII. Accordingly, the therapeutic replacement fVIII protein potentially is recognized as non-self by the immune system. Thirty percent of patients with severe hemophilia A develop detectable inhibitory anti-fVIII antibodies (inhibitors). Additionally, greater than 90 percent of hemophilia A mice treated with human fVIII develop inhibitors using dosing schedule that mimics use in humans. Because fVIII is an immunologically foreign protein, it might be expected that a hemophilia A patient would make a fVIII inhibitor. However, intravenous injection of soluble proteins in either humans or rodents usually results in tolerance rather than a humoral immune response. One major difference between fVIII and other proteins is that it is released from its large carrier protein von Willebrand factor (VWF) and is potentially exposed to the immune system at sites of active hemostasis and inflammation. Heat-inactivated, denatured fVIII, which maintains all T-cell epitopes but lacks several B-cell epitopes, is less immunogenic than native fVIII, suggesting that fVIII-dependent thrombin generation along the intrinsic pathway of blood coagulation may provide co-stimulatory signals necessary for the immune response (Skupsky BS, Zhang A, Scott DW Blood 2008; 112:1220a). We constructed a B domain-deleted human fVIII mutant, designated fVIIIi, which contains alanine substitutions at two critical thrombin cleavage sites, Arg372 and Arg1689, and purified it to homogeneity. FVIIIi does not develop procoagulant activity and is not released from VWF in response to thrombin. Therefore fVIIIi is less likely than wild-type fVIII to be exposed to the immune system at sites of active hemostasis and inflammation. Additionally, VWF binds to the immunodominant fVIII C2 domain and potentially hides part of fVIII from the immune system. FVIIIi was antigenically intact judging from intact binding to a panel of11 mouse anti-fVIII monoclonal antibodies whose epitope specificity was represented by all five domains of BDD fVIII. The immunogenicity of wild-type fVIII and fVIIIi was compared in a murine hemophilia A model in which groups of 25 mice received 8 weekly injections of physiologic doses of fVIII. Plasma was collected weekly for total anti-fVIII antibody titers by ELISA and one week following the last injection for total anti-fVIII antibody titers, inhibitor titers by Bethesda assay and for epitope mapping. Mice treated with fVIIIi had significantly lower levels of inhibitory as well as total anti-fVIII antibodies than mice treated with wild-type fVIII. Domain mapping using single human domain hybrid human/porcine molecules as ELISA antigens revealed that hemophilia A mice broadly recognized all fVIII domains in response to either wild-type or fVIIIi, although fVIIIi produced less anti-light chain antibodies. Mice in both the wild-type fVIII and fVIIIi groups produced antibodies that recognized the phospholipid-binding site of the C2 domain, even though this site overlaps the VWF binding site on fVIII. There was no difference in the isotype spectrum of the antibodies made to fVIII or fVIIIi. This study indicates that inactivatable fVIII is less immunogenic than native fVIII and suggests that the immunogenicity of fVIII is related either to its interaction with VWF or to events triggered by activation of the coagulation mechanism. Disclosures: No relevant conflicts of interest to declare.
7
Soeda, Tetsuhiro, Keiji Nogami, Tomoko Matsumoto, Kenichi Ogiwara, Katsumi Nishiya, and Midori Shima. "Tissue Factor-Dependent Activation of Factor VIII by Factor VIIa in the Early Phase of Blood Coagulation." Blood 112, no. 11 (November 16, 2008): 1036. http://dx.doi.org/10.1182/blood.v112.11.1036.1036.
Анотація:
Abstract Factor VIIa (FVIIa), complexed with tissue factor (TF), is a trigger of blood coagulation through activation of factor X in the initiation phase. FVIIa can catalyze intrinsic clotting factors such as not only factor IX, but also factor VIII (FVIII). However the role and the mechanisms of the FVIIa-catalyzed FVIII are poorly understood. We first examined FVIIa-catalyzed FVIII activation in the presence of phospholipid (PL) using a one-stage clotting assay. The levels of FVIII activity elevated rapidly by ~4-fold within 30 sec after the addition of FVIIa (1 nM)-TF (1 nM)complex, and subsequently decreased to the initial level within 20 min. This time-dependent reaction was enhanced by the presence of TF and PL in dose-dependent manners, but was moderately inhibited (~50%) in the presence of von Willebrand factor at physiological concentration of 10 μg/mL. FVIII cleavage was evaluated using western blotting immediately after the addition of FVIIa-TF complex. The heavy chain of FVIII was proteolyzed more rapidly (at 15 sec) by cleavages at Arg740 (A2-B junction) and Arg372 (A1-A2 junction) by FVIIa-TF complex, whilst the cleavage at Arg336 in the A1 domain was appeared at ~2.5 min. However little cleavage of the light chain of FVIII was observed, supporting that cleavages at Arg740/Arg372 and Arg336 by FVIIa-TF complex contribute to the up- and down-regulation of FVIII(a) activity, respectively. Of interest, no proteolysis of isolated intact heavy chain was observed, indicating that the proteolysis of the heavy chain was governed by the presence of the light chain. Compared to FVIII activation by thrombin (0.1–1 nM), the activation by FVIIa (0.1–1 nM)-TF (1 nM) complex was observed more rapidly. The activation rate observed by the addition of FVIIa-TF complex was ~50-fold greater than that by thrombin. Kinetics by the chromogenic Xa generation assay showed the catalytic efficiency (kcat/Km; 8.9 min−1/32.8 nM) on FVIIa-TF complex-catalyzed FVIII activation showed ~4-fold greater than that on thrombin-catalyzed activation (kcat/Km; 7.5 min−1/86.4 nM). Furthermore, the catalytic efficiencies on cleavages at Arg740 and Arg372 of FVIII by FVIIa-TF complex were ~3- and ~20-fold greater compared to those by thrombin, respectively. These findings suggested that FVIIa-TF complex was a greater FVIII activator than thrombin in very early phase. In order to localize the binding region for FVIIa, we evaluated the interactions between FVIII subunit and Glu-Gly-Arg-active site modified FVIIa, lacking enzymatic activity, in a surface plasmon resonance-based assay. The heavy chain of FVIII bound to EGR-FVIIa with higher affinity than the light chain (Kd; 2.1 and 45 nM, respectively). Binding was particularly evident with the A2, A3, and C2 domains (Kd; 34, 37, and 44 nM, respectively), whilst the A1 domain failed to bind. In conclusion, we demonstrated that FVIIa-TF complex rapidly activated FVIII by proteolysis of the heavy chain and the activation was governed by the presence of the light chain. Furthermore, present results suggested the role of TF-dependent FVIII activation by FVIIa which is responsible for the initiation phase of blood coagulation.
8
Messer, Amanda S., Barbara Ulmasov, Yogesh Kumar, Kanagasabai Vadivel, Degang Zhong, Philip Fay, and S. Paul Bajaj. "Epitope Mapping of a Monoclonal Antibody to Factor VIII That Inhibits Factor IXa:Factor VIIIa Interaction and Thrombin Activation of Factor VIII." Blood 118, no. 21 (November 18, 2011): 1177. http://dx.doi.org/10.1182/blood.v118.21.1177.1177.
Анотація:
Abstract Abstract 1177 Factor VIII (FVIII) circulates in plasma as a noncovalent heterodimer consisting of a heavy chain (HC, A1-a1-A2-a2-B domains) and a light chain (LC, a3-A3-C1-C2 domains) in a noncovalent complex with von Willebrand factor (wVF). Thrombin (IIa) cleaves FVIII between the A1-a1/A2 domains at Arg372, A2-a2/B domains at Arg740 and B-a3/A3 domains at Arg1689 generating FVIIIa that consists of an A1-a1/A2-a2/A3-C1-C2 heterotrimer. FVIIIa increases the efficiency of Factor IXa (FIXa) catalyzed activation of Factor X (FX) in a Ca2+ and phospholipid (PL) dependent manner. The A3-C1-C2 segment of FVIIIa plays an important role in FIXa:FVIIIa interaction. Here, we describe a series of experiments to map the epitope of a monoclonal antibody (mAb) that is reported to inhibit FVIII clotting activity in a one stage clotting assay (Brown et al; J Lab Clin Med, 101: 793–805, 1983). The binding of mAb to FVIII, B-domain deleted FVIII and isolated LC was assessed using surface plasmon resonance. In these experiments, mAb captured on a protein A/G coupled CM5 sensor chip served as the ligand, and FVIII and its isolated fragments served as the analytes. The Kd of binding of LC (∼40 nM) was similar to FVIII and the B-domain deleted FVIII. No binding was observed for isolated A1 and A2 domains. Further, in plasma based inhibition assays, the Kd of binding of mAb to FVIII-vWF complex and to FVIII was ∼30 nM. This suggests that the mAb epitope does not significantly overlap with the vWF binding site in the acidic a3 region of LC. Western blot analysis confirmed that the mAb is specific for the LC of FVIII. Moreover, IIa-cleaved LC starting at residue 1690 gave only a weak signal and FXa-cleaved LC starting at residue 1721 did not react with the mAb in Western blots. These data suggest that the epitope for this mAb spans the IIa-cleavage site in the LC. Consistent with these observations, the A3-C1-C2 fragment but not the C1-C2 fragment expressed in COS cells reacted with the mAb. To further define a part of the epitope in the IIa-cleaved LC, twelve A3 domain deletion fragments were constructed and expressed in E. coli. Western blot analysis of these fragments restricted the partial epitope to 1690–1710 residues of the IIa-cleaved LC. In additional experiments, the mAb did not inhibit mouse, rabbit or canine plasma FVIII in a one stage clotting assay. It did however inhibit porcine plasma FVIII with ∼40 nM Kd, sheep plasma FVIII with ∼ 68 nM Kd, and bovine plasma FVIII with ∼300 nM Kd. Analysis of the sequence alignment of residues 1680 to 1710 of FVIII from each species indicated that residues 1681 to 1694 of human FVIII most likely constitute the epitope of this mAb. The dissimilarity and the charge differences in amino acids suggest that residues Asp1681, Glu1684, Asn1685, and Ser1687 on the N terminal side and Lys1693 on the C terminal side of the IIa-cleavage site Arg1689-Ser1690 may be important for this epitope. Fluorescence energy transfer (FRET) experiments indicated that the mAb inhibits FIXa interaction with the IIa-cleaved LC consisting of A3-C1-C2 domains. In these experiments, A3-C1-C2 subunit was labeled with acrylodan (fluorescence donor) and FIXa was labeled with fluorescein-Phe-Phe-Arg-chloromethylketone (fluorescence acceptor). In the presence of FIXa, the acrylodan fluorescence was quenched indicating a biomolecular complex formation. Addition of 1.2 μM mAb abolished the acrylodan fluorescence quenching suggesting inhibition of the FIXa:LC interaction. Notably, the mAb did not inhibit activation of FX by FIXa/Ca2+/PL and FXa-cleaved FVIIIa (instead of IIa-cleaved FVIIIa). This suggests that the mAb inhibits FIXa:LC interaction by a steric hindrance and not by a direct blockage of the FIXa:LC interactive sites. In summary, the mAb inhibits clotting by preventing FVIII activation by IIa. The epitope of the mAb appears to be restricted to residues 1681–1694 of FVIII. Notably, in some of the hemophilia A patients, the epitope of the inhibitory antibodies is confined to the IIa-cleavage site including the a3 acidic domain of LC. To locate the epitope for such antibodies, one of the approaches used was to construct porcine and human FVIII hybrids. Our strategy may represent a simplified approach to locate the epitope of similar antibodies in hemophilia A patients. Such antibodies may bind strongly to LC and weakly to IIa-cleaved LC. Further, these antibodies may not bind to FXa-cleaved LC or A1/A2 subunits. Disclosures: No relevant conflicts of interest to declare.
9
Zakas, Philip, Kristopher Knight, Ernest T. Parker, H. Trent Spencer, Eric Gaucher, and Christopher B. Doering. "Bioengineering Coagulation Factor VIII through Ancestral Protein Reconstruction." Blood 126, no. 23 (December 3, 2015): 123. http://dx.doi.org/10.1182/blood.v126.23.123.123.
Анотація:
Abstract The development of transformative hemophilia A therapeutics has been hindered by the size, instability, immunogenicity and biosynthetic inefficiency of coagulation factor VIII (FVIII). Through the study of FVIII orthologs from existing vertebrate species, we discovered unique molecular, cellular and biochemical properties that can overcome the limitations of human FVIII. This approach facilitated the development of recombinant porcine FVIII for acquired hemophilia A and has enabled low resolution mapping and bioengineering of functional sequence determinants into human FVIII. To further extend this bioengineering approach, we employed a novel methodology termed ancestral protein reconstruction that provides certain advantages over 'rational design' approaches including a priori confidence that each ancestral FVIII is hemostatically functional. First, a mammalian FVIII phylogenetic tree with corresponding ancestral node (An) sequences was constructed through Bayesian inference using both DNA and amino acid-based models in PAML Version 4.1 (Figure 1). The limited availability of non-mammalian sequences precluded accurate ancestor prediction outside of this class. Initially, we selected 14 An-FVIII sequences for reconstruction and subsequent molecular, cellular, biochemical and immunological characterization. Each An-FVIII displayed activity in coagulation assays utilizing human hemophilia A plasma as a substrate thus demonstrating evolutionary mammalian compatibility. Infusion of highly purified preparations of several An-FVIIIs into hemophilia A mice also corrected the bleeding phenotype following a tail transection bleeding challenge confirming in vivo functionality. To study biosynthetic efficiency, secreted FVIII activity and mRNA transcript levels were analyzed following transfection of An-FVIII plasmids into HEK293 and BHK-M cell lines. An-53, common ancestor to rodents and primates, and An-68, ancestor to a subset of current rodents, displayed the highest FVIII biosynthetic efficiencies that were 12 and 15 fold greater than human FVIII, respectively (P = 0.002; Mann Whitney U test). These two An-FVIII sequences share 95 and 87% amino acid identity to human FVIII, respectively. In contrast, intermediate ancestors between An-53 and human FVIII, designated An-55, -56 and -57, do not display enhanced biosynthetic efficiency suggesting that the functional sequence determinant of high expression was lost during primate evolution. Predicting that high expression ancestral FVIIIs would be enabling to gene therapy approaches, An-53, An-68 and human FVIII cDNAs were placed in an AAV expression cassette under the control of a potent liver-specific promoter and the resulting plasmid DNA was infused hydrodynamically into hemophilia A mice. An-53 and An-68, but not human FVIII vector treated animals, achieved sustained, therapeutic plasma FVIII activity levels over 4 weeks (0.1 - 0.6 IU/ml versus <0.01 IU/ml, respectively). Recombinant An-FVIIIs were expressed, purified and biochemically characterized by SDS-PAGE, specific activity, decay following thrombin activation and inhibitor recognition. Early mammalian and all primate lineage thrombin-activated An-FVIII(a) displayed half-lives between 1.5 - 2.2 min that were not distinguishable from human FVIII. We have shown previously that modern murine, porcine, and ovine FVIIIa display significantly longer half-lives and thus this property may have evolved under positive selection. Supporting this conclusion, An-68 and An-78 display prolonged half-lives of 16 and 7 min, respectively. Lastly, the immune recognition of An-FVIIIs by a panel of A2 and C2 domain targeting inhibitory murine monoclonal antibodies as well as hemophilia A inhibitor patient plasmas was examined and many examples of reduced reactivity were revealed, which may enable the development of less immunogenic FVIII products. Herein, we report molecular discoveries that enhance our understanding of FVIII structure/function and provide a blueprint for bioengineering novel FVIII molecules with enhanced properties. These studies also show 'proof of concept' for ancestral protein reconstruction as a powerful approach to studying the biochemistry, molecular biology and evolution of the vertebrate coagulation system, which should enable identification of other new hematological drug targets and candidate biotherapeutics. Figure 1. Figure 1. Disclosures Spencer: Expression Therapeutics: Equity Ownership. Doering:Expression Therapeutics: Equity Ownership; Bayer Healthcare: Consultancy, Honoraria, Research Funding.
10
Radtke, Klaus-Peter, Dean Chamberlain, John H. Griffin, and Andrew J. Gale. "Whole Blood Thromboelastogram Assays Demonstrate Prolonged Factor VIIIa Potency for Recombinant Disulfide Bond-Stabilized Factor VIII Variants." Blood 104, no. 11 (November 16, 2004): 2976. http://dx.doi.org/10.1182/blood.v104.11.2976.2976.
Анотація:
Abstract Following proteolytic activation of factor VIII (FVIII) by thrombin, the FVIIIa A2 domain, A3 domain and light chain (A3-C1-C2 domains) form a non-covalent hetero-trimer. Because spontaneous A2 subunit dissociation causes loss of FVIIIa activity, we previously made two mutants each with two new Cys to form a disulfide bond linking residues 662 (A2) and 1828 (A3) (FVIIIC662-C1828) or residues 664 (A2) and 1826 (A3) (FVIIIC664-C1826). Following thrombin activation, each FVIIIa mutant was stabile compared to wild type (wt) B-domain-deleted (BDD) FVIII. Previous SDS-PAGE data showed that the A2 domain was disulfide linked to the light chain. To show that this is true for undenatured FVIIIa, here we used surface plasmon resonance (SPR) to monitor A2 dissociation from thrombin-activated wild type and variant FVIII species that were bound to the sensor surface via a monoclonal antibody. Following passage of thrombin over sensor-bound FVIII, only wt FVIII showed a characteristic decrease of SPR reflecting A2 subunit dissociation and thrombin-treated FVIIIC662-C1828 and FVIIIC664-C1826 showed only minor decreases in SPR. Thus, SPR data directly demonstrate that engineered inter-domain disulfide bridges between the A2 and A3 domains prevent A2 domain dissociation from FVIIIa. In contrast to simple plasma coagulation assays of FVIIIa, rotational thromboelastogram (RoTEG) assays of whole blood provide multiple parameters reflecting clot formation, clot quality, and clot dissolution. RoTEG assays using fresh severe hemophilia A whole blood that was reconstituted with either wt FVIII, or FVIIIC662-C1828 or FVIIIC664-C1826 were performed to test the hypothesis that the disulfide-stabilized FVIIIa mutants would show improved potency for thrombin generation. After recalcification of hemophilia A blood with added FVIII, we measured the clotting time (CT), the rate of clot-formation, the clot-firmness time (CFT), defined as the time required to reach a specified clot firmness, and the clot firmness at 5 min (CF-A5), defined as the clot firmness at 5 min after the observed CT. Samples reconstituted with disulfide-bridge-stabilized FVIII mutants or wt-FVIII had comparable CTs at similar concentrations. However, in comparison to wild type BDD-FVIII, comparable rates of clot-formation, CFTs and CF-A5 were observed for up to 10-fold lower concentrations of each disulfide-bridge-stabilized FVIII mutant. The differences between wt and FVIII mutants were especially pronounced at very low FVIII concentrations whereas at FVIII concentrations >0.01 U/mL the differences were less apparent. Because clot formation occurs early relative to overall thrombin generation which is better reflected by CFT and CF-A5 values, we interpret these data to indicate that the disulfide-stabilized FVIIIa variants provide sustained thrombin generation in whole blood compared to wt FVIII and speculate that these FVIII variants may prove superior to wt FVIII for stabilizing a hemostatic plug by providing sustained thrombin generation capacity.
11
Ogiwara, Kenichi, Midori Shima, and Keiji Nogami. "Factor VIII activation by factor VIIa analog (V158D/E296V/M298Q) in tissue factor-independent mechanisms." Thrombosis and Haemostasis 106, no. 10 (2011): 665–74. http://dx.doi.org/10.1160/th11-04-0264.
Анотація:
SummaryFactor (F)VIIa with tissue factor (TF) is a primary trigger of blood coagulation. The recombinant (r)FVIIa analog, NN1731 (V158D/E296V/ M298Q) containing a thrombin/FIXa-mimicking catalytic domain, is ~30-fold more effective on activated platelets without TF, but ~1.2-fold with TF, than rFVIIa for FX activation. We have recently demonstrated the FVIIa/TF-dependent FVIII activation in the early coagulation phase. We assessed the action of NN1731 on FVIII activation. NN1731/TF increased FVIII activity ~2.9-fold within 30 seconds, followed by rapid inactivation, and was slightly more active than rFVIIa/TF. NN1731-catalysed activation, however, was enhanced ~6-fold at 5 minutes (min), and its peak level persisted for ~30 min. NN1731/TF proteolysed FVIII at Arg740, Arg372, and Arg336, similar to rFVIIa/TF, but cleavage by NN1731 alone was much slower at Arg336 than at Arg740 and Arg372. The Km and Vmax for NN1731/TF-catalysed activation were ~1.8-fold lower and ~2.3-fold greater than rFVIIa/TF. The Km for NN1731 alone was ~1.3-fold lower than rFVIIa, whilst the Vmax was ~7.9-fold greater, indicating that the efficiency of FVIII activation by NN1731 and NN1731/TF was ~11- and ~4-fold greater, respectively, than equivalent reactions with rFVIIa. In SPR-based assays, NN1731 bound to FVIII and the heavy chain (Kd; 0.62 and 1.9 nM) with ~1.4- and ~3.1-fold higher affinity than rFVIIa, and the A2 domain contributed to this increase. Von Willebrand factor moderated NN1731-catalysed activation more significantly than NN1731/TF. In conclusion, NN1731 was a greater potential than rFVIIa in up-regulating FVIII activity, and the TF-independent FVIII activation might represent a potential extra mode of its enhanced haemostatic effect.
12
Takeyama, Masahiro, Keiji Nogami, Tomoko Matsumoto, Tetsuhiro Soeda, Tsukasa Suzuki, Kunihiro Hattori, and Midori Shima. "Characterisation of an antibody specific for coagulation factor VIII that enhances factor VIII activity." Thrombosis and Haemostasis 103, no. 01 (2010): 94–102. http://dx.doi.org/10.1160/th09-05-0338.
Анотація:
SummaryMany reports have identified factor (F)VIII inhibitory antibodies with epitopes located in all subunits of the FVIII molecule. Antibodies that promote FVIII activity do not appear to have been reported. We characterised, for the first time, a unique anti-FVIII monoclonal antibody, mAb216, that enhanced FVIII coagulant activity. The mAb216 shortened the activated partial thromboplastin time and specifically increased FVIII activity by ~1.5-fold dose-dependently. FXa generation and thrombin generation were similarly increased by ~1.4- and ~2.5-fold, respectively. An A2 epitope, not overlapping the common A2 epitope, was identified and the antibody was shown to enhance thrombin (and FXa)-catalysed activation of FVIII by modestly accelerating cleavage at Arg372. The presence of mAb216 mediated an ~1.5-fold decrease in Km for the FVIII-thrombin interaction. Enhanced FVIII activity was evident to an equal degree, even the presence of anti-FVIII neutralising antibodies with epitopes in each subunit. In addition, mAb216 depressed the rates of heat-denatured loss of FVIII activity and FVIIIa decay by 2 to ~2.5-fold. We have developed an anti-A2, FVIII mAb216 that augmented procoagulant activity. This enhancing effect could be attributed to an increase in thrombin-induced activation of FVIII, mediated by cleavage at Arg372 and a tighter interaction of thrombin with the A2 domain. The findings may cast new light on new principles for improving the treatment of haemophilia A patients.
13
Shoko, Furukawa, Keiji Nogami, Kenichi Ogiwara, and Midori Shima. "Mechanism of Tissue Factor (TF) Enhancing Factor (F)VIII Activity on FXa Generation in Initial Phase of Coagulation and Interaction Between TF and FVIII C2 Domain." Blood 128, no. 22 (December 2, 2016): 1391. http://dx.doi.org/10.1182/blood.v128.22.1391.1391.
Анотація:
Abstract In the cell-based coagulation model, factor (F)VIIa complex with tissue factor (TF) initiates the blood coagulation by generating FXa as the extrinsic tenase complex and activates FIX which composes the intrinsic tenase complex. We demonstrated that FVIIa/TF directly activated FVIII in an early coagulation phase (Soeda, JTH, 2010), and TF enhanced the intrinsic tenase activity via possible interaction with FVIIIa (Ogiwara, ASH, 2010). In this study, we clarified the enhancing mechanism of intrinsic tenase activity in the TF-related up-regulation of FVIII, and identified the TF-interactive region on FVIII. To explore the enhancing mechanism of TF for FVIII regulation, we performed the FXa generation assay with various amounts of FVIII or thrombin-mediated FVIIIa, constant FIXa (1 nM), FX (300 nM) and phospholipid vesicles (PL; 20 µM) in the presence of recombinant lipidated TF (rTF, Innovin®). The Km value for FVIII in the presence of rTF was ~2.4-fold lower than that its absence (Km; 5.6±0.5, 13.3±3.7 nM, respectively, p<0.05). Similarly, the Km for FVIIIa in the presence of TF was ~1.5-fold lower than that in its absence (Km; 6.3±0.6 nM, 9.7±1.6 nM, respectively, p<0.05), supporting that the presence of TF could promote the FXa-catalyzed activation of FVIII and FVIIIa-dependent generation of intrinsic FXa. To further evaluate the effect of TF on FVIII-dependent FXa generation, the FXa generation assay with FVIIa/TF-activated FVIIIa (FVIIIa-VIIa/TF) was also performed. The initial velocity on FXa generation with FVIIIa-VIIa/TF was 22.6 nM/min. However, the initial velocity on FXa generation with FVIIIa-VIIa/TF by addition of FVIIa-inhibitor (E-76), not to generate FVIIa/TF-dependent FXa, was 3.4 nM/min, and that with FVIII alone was 0.05 nM/min. In addition, the initial velocity with FVIIa/TF alone was 10.4 nM/min. These findings supported that the TF increased FXa generation greater than the additive effect of FVIII-dependent and FVIIa/TF-dependent FXa generation in early initiation phase of coagulation prior to thrombin generation. Since tissue factor pathway inhibitor (TFPI) is present in physiological circulating whole blood, a similar experiment on FXa generation assay was repeated under the presence of TFPI, estimated to be present at 0.5 nM in the pre-coagulant state or at 15 nM in the coagulant state in circulating blood. The initial velocity on FXa generation with FVIIIa-VIIa/TF was reduced by the presence of 0.5 or 15 nM TFPI (17.9 and 12.6 nM/min, respectively). By contrast, the initial velocity on FVIIIa-VIIa/TF-dependent FXa generation with addition of FVIIa inhibitor was little reduced by 0.5 nM TFPI, whilst was reduced by 15 nM TFPI (3.5 and 2.5 nM/min, respectively). These findings supported that the TFPI possibly didn't inhibit TF on the enhanced intrinsic tenase on association with FVIII in the pre-coagulant state. We further reported that TF enabled FXa to activate FVIII, irrespective of von Willebrand factor (VWF), and the direct association of rTF and non-lipidated TF with FVIII (Furukawa, ISTH, 2015). Since TF is transmembrane protein, however, we performed a surface plasmon resonance (SPR)-based assay (BIAcore®) and solid phase-based ELISA to identify the interactive region(s) on FVIII to recombinant soluble TF (sTF; Altor BioScience), a portion of TF outside of the PL membrane. An SPR-based assay revealed the direct binding of intact FVIII, LCh (a3-A3C1C2, A3C1C2) subunit, C2 domain to immobilized sTF (Kd; 2.3±0.6, 5.8±1.0, 10.5±3.5, 11.8±0.5 nM, respectively). The intact HCh, A1 or A2 domain to sTF failed to bind, however. A non-equilibrium ELISA also revealed that sTF bound to immobilized C2 domain with moderate affinity (Kdapp; 16.9±2.2 nM), and the interaction was dependent on ionic strength and Ca2+. In addition, the presence of VWF significantly competitively inhibited the C2 and sTF binding by ~90% (IC50; 5.7 µg/ml) at the maximal concentration employed, suggesting that the C2 domain-TF interaction could activate FVIII by FXa even in the presence of VWF. We concluded that it might be possible that TF enhanced the FVIII-mediated FXa generation by not only FVIIa but also FXa, additionally this enhancing mechanism might not be suppressed by TFPI in the initiation phase of coagulation. Furthermore, TF might function to FVIII activation, irrespective of presence of VWF, by the binding to C2 domain through the competition with VWF. Disclosures Nogami: Sysmex Corporation: Patents & Royalties, Research Funding; F. Hoffmann-La Roche Ltd.: Honoraria, Membership on an entity's Board of Directors or advisory committees; Chugai Pharmaceutical Co., Ltd.: Honoraria, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding. Shima:Sysmex Corporation: Patents & Royalties, Research Funding; F. Hoffmann-La Roche Ltd.: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Chugai Pharmaceutical Co., Ltd.: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding.
14
Wilhelm, Amelia R., Nicole A. Parsons, Benjamin J. Samelson-Jones, Robert J. Davidson, Charles T. Esmon, Rodney M. Camire, and Lindsey A. George. "Activated protein C has a regulatory role in factor VIII function." Blood 137, no. 18 (May 6, 2021): 2532–43. http://dx.doi.org/10.1182/blood.2020007562.
Анотація:
Abstract Mechanisms thought to regulate activated factor VIII (FVIIIa) cofactor function include A2-domain dissociation and activated protein C (APC) cleavage. Unlike A2-domain dissociation, there is no known phenotype associated with altered APC cleavage of FVIII, and biochemical studies have suggested APC plays a marginal role in FVIIIa regulation. However, the in vivo contribution of FVIIIa inactivation by APC is unexplored. Here we compared wild-type B-domainless FVIII (FVIII-WT) recombinant protein with an APC-resistant FVIII variant (FVIII-R336Q/R562Q; FVIII-QQ). FVIII-QQ demonstrated expected APC resistance without other changes in procoagulant function or A2-domain dissociation. In plasma-based studies, FVIII-WT/FVIIIa-WT demonstrated dose-dependent sensitivity to APC with or without protein S, whereas FVIII-QQ/FVIIIa-QQ did not. Importantly, FVIII-QQ demonstrated approximately fivefold increased procoagulant function relative to FVIII-WT in the tail clip and ferric chloride injury models in hemophilia A (HA) mice. To minimize the contribution of FV inactivation by APC in vivo, a tail clip assay was performed in homozygous HA/FV Leiden (FVL) mice infused with FVIII-QQ or FVIII-WT in the presence or absence of monoclonal antibody 1609, an antibody that blocks murine PC/APC hemostatic function. FVIII-QQ again demonstrated enhanced hemostatic function in HA/FVL mice; however, FVIII-QQ and FVIII-WT performed analogously in the presence of the PC/APC inhibitory antibody, indicating the increased hemostatic effect of FVIII-QQ was APC specific. Our data demonstrate APC contributes to the in vivo regulation of FVIIIa, which has the potential to be exploited to develop novel HA therapeutics.
15
Rozenshteyn, Diana, and Andrew J. Gale. "Cathepsin G, a leukocyte protease, activates coagulation factor VIII." Thrombosis and Haemostasis 99, no. 01 (2008): 44–51. http://dx.doi.org/10.1160/th07-08-0495.
Анотація:
SummaryNeutrophils and monocytes express cathepsin G and can also bind to activated platelets, thus they can be localized to the site of active coagulation. Previous studies have suggested that cathepsin G inactivated coagulation factorVIII (FVIII) and was thus anticoagulant. But other studies have indicated procoagulant functions for cathepsin G in activation of coagulation factorV or activation of platelets among other possible mechanisms. Therefore, it remains unclear if cathepsin G is anticoagulant or procoagulant. We investigated the effects of human neutrophil cathepsin G on FVIII/VIIIa. Cathepsin G activates FVIII to a partially active form while having only a minor inactivating effect on thrombin- activated FVIIIa. This inactivation is mostly due to decreased stability of FVIIIa since a disulfide bond that prevents A2 subunit dissociation from FVIIIa prevents any loss of activity due to cathepsin G proteolysis. FVIII that has been cleaved by cathepsin G can still be activated by thrombin if A2 subunit dissociation is prevented. Cathepsin G cleavages of FVIII are limited to a few specific sites that are mostly located near known activating and inactivating cleavage sites. Cathepsin G cleavage sites near to thrombin cleavage sites likely contribute to the partial activation of FVIII. Therefore, it is possible that cathepsin G from neutrophils and monocytes may provide some pro-coagulant effect by activating FVIII.
16
Hausl, Christina, Rafi U. Ahmad, Maria Sasgary, Christopher B. Doering, Pete Lollar, Günter Richter, Hans Peter Schwarz, Peter L. Turecek, and Birgit M. Reipert. "High-dose factor VIII inhibits factor VIII–specific memory B cells in hemophilia A with factor VIII inhibitors." Blood 106, no. 10 (November 15, 2005): 3415–22. http://dx.doi.org/10.1182/blood-2005-03-1182.
Анотація:
AbstractHemophilia A in its severe form is a life-threatening hemorrhagic disease that is caused by mutations in the factor VIII (FVIII) gene (symbol F8). About 25% of patients who receive replacement therapy develop neutralizing antibodies that inhibit the function of substituted FVIII. Long-term application of high doses of FVIII has evolved as an effective therapy to eradicate the antibodies and to induce long-lasting immune tolerance. Little is known, however, about the immunologic mechanisms that cause the down-modulation of anti-FVIII antibodies by high doses of FVIII. We report that high doses of FVIII inhibit the restimulation of FVIII-specific memory B cells and their differentiation into antibody-secreting plasma cells in vitro and in vivo in a murine model of hemophilia A. The inhibition of memory B-cell responses is irreversible and not mediated by FVIII-specific T cells. Furthermore, it seems to involve the activation of caspases. We conclude that the inhibition of FVIII-specific memory B cells might be an early event in the down-modulation of anti-FVIII antibodies in patients with hemophilia A who receive high doses of FVIII.
17
Doshi, Bhavya, Courtney Cox, Bagirath Gangadharan, Christopher B. Doering, and Shannon L. Meeks. "Factor VIII Supplementation Improves Recombinant VIIa Initiated Thrombin Generation in Hemophilia A Inhibitor Patient Plasmas." Blood 118, no. 21 (November 18, 2011): 28. http://dx.doi.org/10.1182/blood.v118.21.28.28.
Анотація:
Abstract Abstract 28 Hemophilia A is an X-linked recessive disorder that is caused by a deficiency or defect of factor VIII (fVIII) coagulant protein. Approximately 20–30% of patients with severe hemophilia A develop antibodies (Abs) against fVIII (inhibitors) following fVIII replacement therapy, which makes bleeding episodes more difficult to control. Patients with inhibitors are treated with fVIII-bypassing agents such as recombinant factor VIIa (rfVIIa) or activated prothrombin-complex concentrate. However for unknown reasons, some patients display poor hemostatic response to bypass therapy and improved treatment options are needed. Thrombin generation assays provide an in vitro methodology for monitoring bypass therapy in hemophilia (Turecek PL et al. Pathophysiol Haemost Thromb 2003; Varadi K et al. Haemophilia 2004). Recently, it was demonstrated by us and others that combination of fVIII and by-passing agents potentiates in vitro thrombin production in hemophilia A inhibitor plasma (Klintman J et al. Br J Haematol 2010). In our study we investigated the potentiation fVIII confers to fVIIa initiated in vitro thrombin generation using a panel of anti-fVIII Abs with known epitopes. We showed that kinetics of inhibition and Ab epitope were the dominant factors influencing ability of fVIII to potentiate in vitro thrombin production. Specifically, monoclonal Abs targeting only 2 of 11 epitopes, 1 of 3 non-overlapping A2 epitopes and 1 of 2 non-overlapping C2 epitopes, inhibited thrombin generation in a manner that could not be recovered by fVIII supplementation. Here, we analyzed in vitro thrombin generation in epitope-mapped plasmas from 10 patients with hemophilia A and long-standing inhibitors after addition of fVIIa alone or in conjunction with fVIII. Methods: FVIII inhibitor plasmas from 10 patients with hemophilia A were obtained as part of an IRB approved study at the Emory Comprehensive Hemophilia Center. FVIII inhibitor titers and inhibitor kinetics were determined using a modified Bethesda assay. Samples were classified as having type II inhibitors if undiluted plasma resulted in incomplete inhibition of residual fVIII activity (Meeks SL et al. Blood 2007). Thrombin generation assays were carried out in the presence of 2.25 μg/ml recombinant fVIIa in the presence or absence of 1 U/ml recombinant full-length fVIII using reagents purchased from DiaPharma (West Chester, OH). The parameters analyzed include endogenous thrombin potential (area under thrombin generation curve), peak thrombin concentration, time to peak thrombin, lag time (time to 1/6th of peak thrombin) and index velocity (Vi-peak thrombin divided by time to peak minus lag time). Domain specific epitope mapping was carried out using direct ELISA and human/porcine domain hybrid fVIII proteins. Results: Domain mapping of the Abs in the plasmas identified 2 plasmas with predominantly anti-A2 Abs, 4 with predominantly anti-C2 Abs, 2 with both anti-A2 and anti-C2 Abs, and 2 with antibodies that were porcine fVIII cross-reactive (see Table). Plasmas with inhibitor titers less than 25 BU/ml were more responsive to fVIII supplementation with 6 of 7 having increased thrombin generation. Plasmas harboring even trace anti-A2 Abs were more resistant to increased thrombin generation with fVIII supplementation than plasmas with anti-C2 Abs alone. Conclusion: This study suggests a more favorable response to fVIII supplementation of rfVIIa may be predicted by the presence of anti-C2 Abs or inhibitory titers less than 25 BU/ml. In conjunction with our previous monoclonal Ab data, further mapping of epitopes within the fVIII A2 and C2 domains may help improve the ability to predict positive responses to fVIII supplementation of by-passing agents.PatientInhibitor Titer (BU/ml)DomainFVIII InhibitorThrombin Generation (fVIII + fVIIa vs. fVIIa)122A2Type IIIncreased242A2Type IIEqual384C2, small A2Type IEqual47C2Type IIncreased58C2Type IIIncreased620C2Type IEqual78C2Type IIncreased842C2, small A2Type IEqual922Porcine cross-reactiveType IIIncreased105.2Porcine cross-reactiveType IIncreased Disclosures: No relevant conflicts of interest to declare.
18
Zhong, Degang, Evgueni L. Saenko, Midori Shima, Matthew Felch, and Dorothea Scandella. "Some Human Inhibitor Antibodies Interfere With Factor VIII Binding to Factor IX." Blood 92, no. 1 (July 1, 1998): 136–42. http://dx.doi.org/10.1182/blood.v92.1.136.413k35_136_142.
Анотація:
Factor VIII (fVIII) functions as a cofactor of factor IXa in the intrinsic pathway of blood coagulation. Its absence or abnormality causes the bleeding disorder hemophilia A. About 23% of hemophiliacs who receive therapeutic fVIII infusions develop antibodies that inhibit its activity. We previously showed by inhibitor neutralization assays that the fVIII A2 and C2 domain polypeptides contain common inhibitor epitopes. Often hemophilic inhibitor plasmas were partially neutralized by C2 and more completely neutralized by fVIII light chain (A3-C1-C2), suggesting the presence of an additional major inhibitor epitope(s) within the A3-C1 domains. In immunoprecipitation assays, 17 of 18 inhibitor IgGs bound to recombinant 35S-A3-C1. Amino acids 1811-1818 of the A3 domain comprise a binding site for factors IX and IXa. Three inhibitor IgGs prevented binding of factor IXa to fVIII light chain, and the binding of each IgG to light chain was competed by A3 peptide 1804-1819. The generation of factor Xa by the fVIIIa/fIXa complex in a chromogenic assay was prevented by these inhibitors. Therefore, we propose that another important mechanism of fVIII inactivation by human inhibitors is the prevention of fVIIIa/fIXa association.
19
Ponder, Katherine P. "FIXing Factor VIII inhibitors." Blood 119, no. 2 (January 12, 2012): 325–26. http://dx.doi.org/10.1182/blood-2011-11-389486.
Анотація:
In this issue of Blood, Milanov and colleagues demonstrate that a Factor IX (FIX) variant that does not require activated Factor VIII (FVIIIa) for activity induces coagulation in hemophilia A mice with FVIII inhibitors.1 This protein might be developed as a bypass agent.
20
Peerlinck, Kathelijne, Marc G. Jacquemin, Jef Arnout, Marc F. Hoylaerts, Jean Guy G. Gilles, Renaud Lavend’homme, Karen M. Johnson, et al. "Antifactor VIII Antibody Inhibiting Allogeneic but not Autologous Factor VIII in Patients With Mild Hemophilia A." Blood 93, no. 7 (April 1, 1999): 2267–73. http://dx.doi.org/10.1182/blood.v93.7.2267.
Анотація:
Abstract Two unrelated patients with the same Arg2150His mutation in the factor VIII (FVIII) C1 domain, a residual FVIII activity of 0.09 IU/mL, and inhibitor titres of 300 and 6 Bethesda Units, respectively, were studied. Further analysis of patient LE, with the highest inhibitor titer, showed that (1) plasma or polyclonal IgG antibodies prepared from LE plasma inhibited the activity of allogeneic (wild-type) but not of self FVIII; (2) the presence of von Willebrand factor (vWF) increased by over 10-fold the inhibitory activity on wild-type FVIII; (3) the kinetics of FVIII inhibition followed a type II pattern, but in contrast to previously described type II inhibitors, LE IgG was potentiated by the presence of vWF instead of being in competition with it; (4) polyclonal LE IgG recognized the FVIII light chain in enzyme-linked immunosorbent assay and the recombinant A3-C1 domains in an immunoprecipitation assay, indicating that at least part of LE antibodies reacted with the FVIII domain encompassing the mutation site; and (5) LE IgG inhibited FVIII activity by decreasing the rate of FVIIIa release from vWF, but LE IgG recognized an epitope distinct from ESH8, a murine monoclonal antibody exhibiting the same property. We conclude that the present inhibitors are unique in that they clearly distinguish wild-type from self, mutated FVIII. The inhibition of wild-type FVIII by LE antibody is enhanced by vWF and is associated with an antibody-dependent reduced rate of FVIIIa release from vWF.
21
Peerlinck, Kathelijne, Marc G. Jacquemin, Jef Arnout, Marc F. Hoylaerts, Jean Guy G. Gilles, Renaud Lavend’homme, Karen M. Johnson, et al. "Antifactor VIII Antibody Inhibiting Allogeneic but not Autologous Factor VIII in Patients With Mild Hemophilia A." Blood 93, no. 7 (April 1, 1999): 2267–73. http://dx.doi.org/10.1182/blood.v93.7.2267.407k21_2267_2273.
Анотація:
Two unrelated patients with the same Arg2150His mutation in the factor VIII (FVIII) C1 domain, a residual FVIII activity of 0.09 IU/mL, and inhibitor titres of 300 and 6 Bethesda Units, respectively, were studied. Further analysis of patient LE, with the highest inhibitor titer, showed that (1) plasma or polyclonal IgG antibodies prepared from LE plasma inhibited the activity of allogeneic (wild-type) but not of self FVIII; (2) the presence of von Willebrand factor (vWF) increased by over 10-fold the inhibitory activity on wild-type FVIII; (3) the kinetics of FVIII inhibition followed a type II pattern, but in contrast to previously described type II inhibitors, LE IgG was potentiated by the presence of vWF instead of being in competition with it; (4) polyclonal LE IgG recognized the FVIII light chain in enzyme-linked immunosorbent assay and the recombinant A3-C1 domains in an immunoprecipitation assay, indicating that at least part of LE antibodies reacted with the FVIII domain encompassing the mutation site; and (5) LE IgG inhibited FVIII activity by decreasing the rate of FVIIIa release from vWF, but LE IgG recognized an epitope distinct from ESH8, a murine monoclonal antibody exhibiting the same property. We conclude that the present inhibitors are unique in that they clearly distinguish wild-type from self, mutated FVIII. The inhibition of wild-type FVIII by LE antibody is enhanced by vWF and is associated with an antibody-dependent reduced rate of FVIIIa release from vWF.
22
Takeyama, Masahiro, Keiji Nogami, Tetsuhiro Soeda, Tsukasa Suzuki, Kunihiro Hattori, Kohei Tatsumi, Yuri Fujita, Ichiro Tanaka, Akira Yoshioka, and Midori Shima. "Characterization of an Anti-Factor VIII Monoclonal Antibody with A1 and A3 Epitopes Which Increases Factor VIII Activity." Blood 108, no. 11 (November 16, 2006): 1702. http://dx.doi.org/10.1182/blood.v108.11.1702.1702.
Анотація:
Abstract Epitopes of anti-factor VIII (FVIII) neutralizing antibodies distribute all of the FVIII domains. They inhibit FVIII for instance by blocking the FVIII interaction with factor IXa, factor X/factor Xa (FX/FXa), phospholipid, von Willebrand factor, or thrombin. Therefore, localization of such FVIII inhibitory effect gives us useful information for understanding of FVIII structure and function. We expect FVIII enhancing antibody also may be a useful tool. In this study, we found an anti-FVIII monoclonal antibody (named by moAb216) that increased the FVIII cofactor activity. The addition of moAb216 increased FVIII activity by ~1.6-fold dose-dependently in one-stage clotting assay. The increase of FVIII activity in the presence of moAb216 correlated with that of generated thrombin or factor Xa in thrombin or FXa generation based-assay. Blotting analysis revealed that this antibody reacted with only intact FVIII molecule, whilst failed to react with either SDS-treated FVIII, FVIIIa (active-form), or isolated each A1, A2, and A3-C1-C2 subunit. Individual monoclonal antibody, with an epitope of the A1 or A3 acidic region in FVIII, competitively inhibited FVIII binding to moAb216 as well as the increase of FVIII activity by its antibody. However, anti-A2 or anti-C2 monoclonal antibody did not affect. These results supported that this unique antibody, with a discontinuous epitope spanning both acidic regions in the A1 and A3 domains, recognized the native conformation of FVIII. To examine the mechanism(s) of increasing effect by moAb216, we focused on the activation and/or inactivation of FVIII. Rate constants on thrombin- and FXa-catalyzed activation of FVIII in the presence of moAb216 were ~2 and 3-fold greater, respectively, in dose-dependent manners compared with that of FVIII in its absence. On the other hand, the antibody inhibited the activated protein C (APC)-catalyzed FVIII inactivation with ~10-fold lower of inactivation rate constant. SDS-PAGE analysis revealed that moAb216 accelerated the cleavage at Arg372 in the A1-A2 junction by thrombin and FXa, whilst decelerated the cleavage at Arg336 within the A1 domain by APC. In addition, FVIII activity in the presence of moAb216 was more stable following heat denaturation analysis than that in its absence. We demonstrated that the increasing effect of FVIII activity by moAb216 was attributed to the change of cleavage at Arg372 and/or Arg336 in the heavy chain caused by interaction with the A1/A3 domains. Furthermore, moAb216 would be useful as a new replacement therapy for hemophilia A patients, since this increases and stabilizes FVIII activity, and can function even in the presence of anti-FVIII (A2 and C2) inhibitors.
23
Gale, Andrew J., Diana Rozenshteyn, and Justin Riceberg. "The Neutrophil Proteases, Elastase and Cathepsin G, May Modulate the Activity of Factor VIII." Blood 108, no. 11 (November 16, 2006): 1716. http://dx.doi.org/10.1182/blood.v108.11.1716.1716.
Анотація:
Abstract Neutrophils and monocytes express cathepsin G and elastase and also can bind to activated platelets, thus they can be localized to the site of active coagulation. Early studies suggested that cathepsin G and elastase inactivated factor VIII (FVIII) and were thus anticoagulant. But other studies have suggested procoagulant functions for cathepsin G and elastase in activation of factor V or activation of platelets among other possible mechanisms. Therefore, we investigated the effects of human neutrophil elastase and human neutrophil cathepsin G on FVIII/VIIIa. Elastase does inactivate both FVIII and FVIIIa but cathepsin G activates FVIII while having very little effect on FVIIIa. Cathepsin G activation of FVIII is enhanced by phospholipid vesicles, apparently due to enhanced rate of cleavage and stabilization of the resulting molecule. The maximum level of activation is less than that of thrombin, but it is still four-fold as measured in an APTT assay. Cleavage sites for both proteases in FVIII were identified by Edman degradation and gel analysis. FVIII cleavages are limited to a few specific sites that are mostly located near known activating and inactivating cleavage sites. A notable exception is a cleavage site for elastase after valine 26 in the A1 domain. Cathepsin G cleavage sites near to thrombin cleavage sites likely contribute to the partial activation of FVIII. The unique elastase cleavage site at valine 26 likely contributes to the inactivation of FVIII and FVIIIa. Therefore, it is possible that neutrophils and monocytes may provide some pro-coagulant effect by activating FVIII and may also provide negative feedback by inactivating FVIIIa as well.
24
Nogami, Keiji, Katsumi Nishiya, Yoshihiko Sakurai, Ichiro Tanaka, John Giddings, Evgueni Saenko, Akira Yoshioka, and Midori Shima. "Human Factor VIII Inhibitor Alloantibodies with a C2 Epitope Inhibit Factor Xa-catalyzed Factor VIII Activation: A new Anti-factor VIII Inhibitory Mechanism." Thrombosis and Haemostasis 87, no. 03 (2002): 459–65. http://dx.doi.org/10.1055/s-0037-1613026.
Анотація:
SummaryFactor VIII (FVIII) inhibitor alloantibodies react with the A2, C2, or A3-C1 domains of FVIII and inactivate FVIII activity. We recently demonstrated that an anti-C2 monoclonal antibody with a Val2248Gly2285 epitope, inhibited factor Xa (FXa)-catalyzed FVIII activation, and that a FXa binding site for FVIII was located within residues Thr2253-Gln2270. In this study, we investigated whether anti-C2 alloantibodies inhibit FXa-catalyzed FVIII activation. Anti-C2 alloantibodies from four patients inhibited FVIII activation by FXa in onestage clotting assay. Furthermore, analysis by SDS-PAGE showed that all alloantibodies inhibited FVIII proteolytic cleavage by FXa independently of phospholipid. To confirm direct inhibition of FVIII and FXa interaction, we examined the effect of alloantibodies on FVIII binding to anhydro-FXa, a catalytically inactive FXa, in ELISA. All alloantibodies and C2-affinity purified F(ab)’2 preparations inhibited FVIII binding to anhydro-FXa dose-dependently. Our results revealed a new inhibitory mechanism of FVIII, mediated by inhibition of FXa in the presence of anti-C2 alloantibodies.
25
Okayama, Yusuke, Masato Bingo, Kazuki Sakatoku, Hiroshi Okamura, Satoru Nanno, Mitsutaka Nishimoto, Yasuhiro Nakashima, Hideo Koh, Masayuki Hino, and Hirohisa Nakamae. "The safety of the combination therapy of recombinant factor VIIa and plasma-derived factor VIIa and factor X for refractory hemorrhage in acquired hemophilia A." Blood Coagulation & Fibrinolysis 34, no. 6 (July 19, 2023): 419–22. http://dx.doi.org/10.1097/mbc.0000000000001243.
Анотація:
Acquired hemophilia A (AHA) is a rare, life-threatening hemorrhagic disease caused by autoantibodies against factor VIII (FVIII), and bypassing agents (BPA) are used to control bleeding. However, some cases need a change of BPA or BPAs given sequentially or in combination for refractory bleeding. A 71-year-old man was admitted with subcutaneous hemorrhage. Laboratory investigations showed prolongation of activated partial thromboplastin time (APTT) and low-coagulation FVIII activity and FVIII inhibitor; we, therefore, diagnosed AHA. He was treated with recombinant factor VIIa (rFVIIa) BPA and prednisolone. However, his symptoms did not improve sufficiently, thus we switched BPA to activated prothrombin complex concentrate. Unfortunately, this was not effective and he suffered hemorrhagic shock. Therefore, we selected rFVIIa, with plasma-derived FVIIa and factor X (pd-FVIIa/FX) as combination therapy, and hemostasis was achieved without thrombosis. This case suggests that the combination of rFVIIa and pd-FVIIa/FX short-term can be well tolerated for refractory hemorrhage in AHA.
26
Takeyama, Masahiro, Keiji Nogami, Tomoko Matsumoto, and Midori Shima. "Factor VIII A2 Domain Contains a Binding Site for Factor X." Blood 124, no. 21 (December 6, 2014): 4226. http://dx.doi.org/10.1182/blood.v124.21.4226.4226.
Анотація:
Abstract Acquired hemophilia A (AHA) is a rare hemorrhagic disease in which autoantibodies against coagulation factor (F) VIII impair the coagulation system. The inhibitors developed in AHA are polyclonal autoantibodies and the majority of FVIII inhibitors bind to the A2, A3, or C2 domains. Depending on the location of the epitope, different mechanisms of action for the anti-FVIII antibodies have been reported. Anti-A3 antibodies neutralize the procoagulant activity of FVIII by preventing its interaction with FIXa. Anti-C2 antibodies inhibit the binding of FVIII to phospholipid membrane and/or von Willebrand factor, whereas A2 and A3 inhibitors block the binding of FVIII to FIXa and FX, respectively, and obstruct the formation of the Xase complex. We have a case of AHA whose inhibitor recognizes only A2 domain and attempted several approaches to determine the mechanism of neutralizing FVIII. Thrombin and plasmin generation assay using patient’s plasma showed that the thrombin and plasmin generation in this AHA patient were decreased compared with that in congenital severe hemophilia A patient. Furthermore, FX generation (Coatest) in this AHA was also decreased compared with that in congenital severe hemophilia A patient (p<0.05). These results indicated that this inhibitor impaired the generation of Xase complex and might cause the severe bleeding disorder in this patient. The IgG subclass of inhibitor in our case was IgG1 and IgG4. Western blotting analysis using FVIIIa revealed that the inhibitor IgG recognized only A2 domain. Furthermore, western blotting analysis using FVIII A2 fragment, digested by activated protein C, showed that the inhibitor IgG bound to FVIII A2N (residue 372-562) fragment. It is known that FVIII A2 domain contains FIXa and thrombin binding sites. Western blotting analysis revealed that the inhibitor IgG inhibited Arg336 cleavage in FVIIIa by FIXa and Arg372 cleavage in FVIII by thrombin. However, the FXa-catalyzed cleavage at Arg372 in FVIII was inhibited by this inhibitor IgG. ELISA-based assay showed that the inhibitor IgG inhibited FX binding to FVIII A2. These results suggest that FX(a) binds to FVIII A2 domain. Therefore, to determine the direct binding of FX and FVIII A2 domain, ELISA-based assay was employed to assess this interaction. ELISA-based assay showed that FVIII A2 fragment bound FX in a dose-dependent manner with moderate affinity (Kd = 338 nM). FX inhibited FVIII A2 fragment binding to immobilized FX up to 70% with an inhibition constant (Ki = 254 nM) similar to the affinity constant. It is known that the residue 484-509 in the A2 domain interacts with FIXa. We hypothesized that FX binding site in the A2 domain might be in the opposite side of FIXa binding site in the A2 domain. According to the 3-D model of FVIII molecule, we prepared synthetic peptides corresponding to FVIII A2 residues 400-409, 409-419, and 420-429. To determine the specificity of these sequences for FX interaction, we examined the effects of these peptides on FVIII A2 binding to FX using ELISA-based assay. The 400-409 peptide inhibited the A2 and FX interaction up to 70%. In contrast, the 410-419 and the 420-429 peptides inhibited the interaction up to 30%. Covalent cross-linking was observed between the 400-409 peptide and FX following reaction with EDC using SDS-PAGE. These results indicate that FVIII A2 domain contains the binding site for FX(a), and the 400-409 region in the FVIII A2 domain contributes to a unique FX(a)-interactive site. Disclosures No relevant conflicts of interest to declare.
27
Neuenschwander, Pierre F. "Evidence of Abnormal Coagulation: Production of Factor VIII α-Fragment In Vivo." Blood 106, № 11 (16 листопада 2005): 1020. http://dx.doi.org/10.1182/blood.v106.11.1020.1020.
Анотація:
Abstract Treatment of thrombosis typically involves the administration of coagulation inhibitors that must be carefully monitored and balanced so as to reduce unwanted coagulation (thrombosis) while maintaining normal or near-normal hemostasis. This balancing is necessary since the anticoagulants used alter enzymatic activities that are involved in both processes. This therapeutic strategy is based entirely on the view that thrombosis occurs by the same general pathways as normal hemostasis. While the enzymatic cascade of blood coagulation is well described and well accepted, numerous other minor reactions have been shown to occur in vitro but have not been examined in great detail due to the belief that they do not occur significantly during normal coagulation in vivo. We postulate that in certain pathological environments some of these minor procoagulant reactions may in fact become significant and lead to thrombogenic situations. If true, this could potentially allow novel targets for anticoagulation to be identified. In addition, the inhibition of these abnormal reactions could attenuate pathological coagulation whilst having limited or no effect on normal hemostatic reactions. One candidate reaction is the proteolysis of factor VIII (fVIII) by the factor VIIa-tissue factor (fVIIa-TF) complex, which results in a mixture of active and inactive fVIII molecules. We have previously shown that this reaction occurs in vitro using purified plasma components and in situ in a plasma-based system. Both of these systems produce a low level of fVIII activation with sustained (albeit low) fVIIIa activity. While it remains possible that this reaction is important in early hemostasis the elevated levels of TF in many pathological situations raises the possibility that this reaction may be more pronounced under certain circumstances in disease states. Examination of the importance of this reaction in vivo is an extremely important issue, but very difficult to address due to the inability to ascertain if fVIII activity or fragments found in vivo derive from fVIIa-TF proteolysis or proteolysis by other enzymes such as thrombin, factor Xa, or activated protein C. With this in mind we have developed an antibody reagent that can specifically detect a fVIII fragment that is a unique product of fVIII proteolysis by the fVIIa-TF complex. This antibody detects only fVIIa-TF proteolyzed fVIII (fVIII cleaved at Arg336) and its major product (α-fragment) on Western blots but not intact (unactivated) fVIII or thrombin-activated fVIII. Using this antibody we screened samples of pulmonary lavage and pleural fluid from normal patients as well as patients with acute respiratory distress syndrome, interstitial lung disease, pneumonia and lung cancer—all of which have associated procoagulant pathologies. Sandwich ELISAs of patient samples showed variably elevated levels of α-fragment (from 100 – 2000 pM) compared to normal controls (~5 pM). Western blots of lavage samples confirmed the presence of α-fragment in samples as well as the elevated levels compared to normals. These data strongly support the notion that alternative “abnormal” coagulation products can be and are generated in vivo in certain pathological settings. The data are also strongly suggestive that the fVIIa-TF complex is the most likely source of fVIII α-fragment. Although it remains unclear if fVIII α-fragment is one of the causative agents in the procoagulant pathologies of these disorders or merely an indicator of the abnormal procoagulant state, its presence in vivo indicates that the role of abnormal coagulation reactions should be further investigated.
28
York, Elizabeth S., Jasmine Ito, and Glaivy Batsuli. "Investigating persistent factor VIII-specific IgM in the humoral immune response to factor VIII." Journal of Immunology 210, no. 1_Supplement (May 1, 2023): 229.12. http://dx.doi.org/10.4049/jimmunol.210.supp.229.12.
Анотація:
Abstract Hemophilia A is an X-linked bleeding disorder resulting in bleeding events due to insufficient levels of factor VIII (FVIII). The most significant complication in the management of hemophilia A is the formation of polyclonal neutralizing IgG (i.e., inhibitors) which impede FVIII procoagulant activity. In a hemophilia A (FVIII KO) murine model injected with multiple doses of FVIII, FVIII-specific IgM were found to persist despite an established IgG response. In attempt to characterize the role of IgM in FVIII immunity, 18 IgMs were purified from immortal FVIII-specific hybridomas and FVIII binding was confirmed via ELISA. IgMs demonstrated weak binding to FVIII in a static ELISA model with mean optical densities (OD) at 405 nm of 0.33 ± 0.43 compared to anti-FVIII IgG with a mean OD of 1.85 ± 0.02. A novel fluid-phase ELISA, developed in our lab, utilizing N-hydroxysuccinimide (NHS)-activated magnetic beads indirectly conjugated to FVIII increased IgM-FVIII binding with mean OD of 1.15 ± 0.60. IgM binding also demonstrated porcine FVIII cross-reactivity. To evaluate the effect of IgM on antibody titers in vivo, FVIII KO mice received 5 weekly injections of either FVIII or 4 separate immune complexes (IC) of IgM-FVIII. Mice injected with IgM-FVIII IC showed slightly higher median IgG ELISA titers than FVIII alone. These findings suggest a potential role for persistent anti-FVIII IgM in propagating FVIII inhibitor development. Further studies determining the effect of IgM-FVIII IC on complement activation, as well as anti-FVIII IgM epitope specificity and clonality utilizing computational methods of RNA-sequencing, may provide additional mechanistic insight into the role of persistent IgM in the humoral immune response to FVIII. Hemostasis and Thrombosis Research Society- Student Research Award (EY), NIH K99HL150595 (GB), and Hemophilia of Georgia Clinical Scientist Development Grant (GB).
29
Yada, Koji, Keiji Nogami, Kenichi Ogiwara, and Midori Shima. "A novel mechanism of Enhancing the Haemostatic Effect in the Combination with Recombinant Factor VIII and Activated Prothrombin Complex Concentrate(APCC) in Hemophilia A Patients with Inhibitor." Blood 118, no. 21 (November 18, 2011): 1178. http://dx.doi.org/10.1182/blood.v118.21.1178.1178.
Анотація:
Abstract Abstract 1178 We have reported that factor (F)VIIa/tissue factor (TF) rapidly activated FVIII by proteolysis of the heavy chain (HCh), and that appeared to serve physiologically as an activator for up-regulation of FVIII activity in very early-timed coagulation phase (J Thromb Haemost. 2010;8:2494). Furthermore, the activation phase could be observed even in the presence of anti-FVIII inhibitors, independently of their types of kinetic and epitope of inhibitors, whilst the inactivation was moderated by anti-C2 with type 1 behavior (Thromb Haemost. 2011;105:989). More recently, the other group has reported that the combination of FVIII and bypassing agent, APCC, also potentiated the thrombin generation in hemophilia A plasmas with inhibitors (Klintman et al. Br J Haematol. 2010;151:381), but the mechanisms have remained unknown. In this study, we investigated the hemostatic effect of the combination therapy with APCC and FVIII. We first examined FVIII activation catalyzed by APCC in one-stage clotting assay. The activity level of FVIII (10 nM) elevated rapidly by ∼3-fold within 1 min after addition of APCC (0.05 U/mL)/TF (0.5 nM), and subsequently decreased to the initial level within 10 min. However, the addition of APCC without TF little affected FVIII activity within 10 min, but after then gradually elevated its activity. The presence of E-76, FVIIa-specific inhibitor, significantly moderated the reaction triggered by APCC/TF, but hirudin, FIIa-specific inhibitor, little affected this activation. We further evaluated the velocity of APCC-induced thrombin generation in the presence or absence of FVIII. The increase rate of thrombin production triggered by APCC/TF in the presence of FVIII was greater than that by APCC/TF in the absence of FVIII by ∼1.6-fold, whilst the increase was little observed in the absence of TF. To clarify its enhancing effects, we performed APCC-catalyzed FVIII cleavage in SDS-PAGE and Western blot. APCC contains FVII (mainly active form) and FII, FIX and FX (mainly non-active forms). In general, FVIIa/TF, FIIa, and FXa rapidly proteolyze the HCh at Arg372 (and Arg740), and FVIIa/TF and FXa proteolyze at Arg336. FVIII proteolysis by FVIIa/TF is dependent on the presence of PL, but not FIIa. Interestingly, APCC/TF proteolyzed the HCh at Arg372 and Arg740, followed by at Arg336 in the presence of PL, whilst did not proteolyze in its absence. The presence of TF accelerated the proteolysis by ∼6.6-fold compared to the absence of TF. However, the addition of E-76 significantly delayed these cleavages. These findings supported that APCC as well as rFVIIa possessed a potential to activation/inactivation of FVIII in early-timed coagulation phase, and that FVIIa in APCC appeared to play a major role in APCC-catalyzed FVIII activation/inactivation. Furthermore, to confirm this enhancing effect in the presence of anti-FVIII inhibitors, we prepared the anti-FVIII inhibitor IgGs (3 of anti-A2, 4 of anti-C2 with type 1, and 2 of anti-C2 with type 2). The presence of anti-FVIII inhibitors did not significantly affect the APCC-catalyzed FVIII activation (by ∼3-fold), independently of their epitopes, in one-stage clotting assay. Of surprise, anti-C2 with type 1 significantly moderated APCC-catalyzed FVIIIa inactivation, and the peak level of FVIIIa retained over 30 min. In contrast, the other inhibitors little affected this inactivation, similar to FVIIa/TF-catalyzed FVIII reaction. In conclusion, we demonstrated the putative mechanism of enhancing hemostatic effects in the combination therapy using FVIII and APCC. In addition, only a small amount of APCC relative to the standard dosage (1–2 U/mL) for clinical use could activate FVIII even in the presence of anti-FVIII inhibitors, and this combination therapy would provide new therapeutic strategy in congenital hemophilia A with inhibitor and/or acquired hemophilia A. Disclosures: Yada: Baxter Hemophilia Scientific Research and Education Fund 2011: Research Funding. Nogami:Bayer Award 2009: Research Funding.
30
Hsu, Ting-Chang, Kathleen P. Pratt, and Arthur R. Thompson. "The factor VIII C1 domain contributes to platelet binding." Blood 111, no. 1 (January 1, 2008): 200–208. http://dx.doi.org/10.1182/blood-2007-01-068957.
Анотація:
Activated factor VIII (FVIIIa) forms a procoagulant complex with factor IXa on negatively charged membranes, including activated platelet surfaces. Membrane attachment involves the FVIII C2 domain; involvement of the adjacent C1 domain has not been established. Binding of recombinant FVIII C1C2 and C2 proteins to platelets was detected by flow cytometry using (1) anti-C2 monoclonal antibody ESH8 followed by a phycoerythrin-labeled secondary antibody; (2) biotinylated C1C2 detected by phycoerythrin-labeled streptavidin, and (3) C1C2 and C2 site-specifically labeled with fluorescein. Highest binding and lowest background were obtained using fluorescein-conjugated proteins. More than 90% of activated platelets bound C1C2, compared with approximately 50% for equimolar C2. Estimates using fluorescent microbeads indicated approximately 7000 C1C2-binding sites per platelet, approximately 1400 for C2, and approximately 3000 for fluorescein-labeled FVIIIa. Unlike C2 or FVIII(a), C1C2 bound to approximately 700 sites/platelet before activation. C1C2 binding to activated platelets appeared independent of von Willebrand factor and was competed effectively by FVIII(a), but only partially by excess C2. Fluorescein-labeled FVIIIa was competed much more effectively by C1C2 than C2 for binding to activated platelets. Two monoclonal antibodies that inhibit C2 binding to membranes competed platelet binding of C2 more effectively than C1C2. Thus, the C1 domain of FVIII contributes to platelet-binding affinity.
31
Grushin, Kirill, Jaimy Miller, Daniela Dalm, and Svetla Stoilova-McPhie. "Factor VIII organisation on nanodiscs with different lipid composition." Thrombosis and Haemostasis 113, no. 04 (July 2015): 741–49. http://dx.doi.org/10.1160/th14-09-0725.
Анотація:
SummaryNanodiscs (ND) are lipid bilayer membrane patches held by amphiphilic scaffolding proteins (MSP) of ~10 nm in diameter. Nanodiscs have been developed as lipid nanoplatforms for structural and functional studies of membrane and membrane associated proteins. Their size and monodispersity have rendered them unique for electron microscopy (EM) and single particle analysis studies of proteins and complexes either spanning or associated to the ND membrane. Binding of blood coagulation factors and complexes, such as the Factor VIII (FVIII) and the Factor VIIIa - Factor IXa (intrinsic tenase) complex to the negatively charged activated platelet membrane is required for normal haemostasis. In this study we present our work on optimising ND, specifically designed to bind FVIII at close to physiological conditions. The binding of FVIII to the negatively charged ND rich in phosphatidylserine (PS) was followed by electron microscopy at three different PS compositions and two different membrane scaffolding protein (MSP1D1) to lipid ratios. Our results show that the ND with highest PS content (80 %) and lowest MSP1D1 to lipid ratio (1:47) are the most suitable for structure determination of the membrane-bound FVIII by single particle EM. Our preliminary FVIII 3D reconstruction as bound to PS containing ND demonstrates the suitability of the optimised ND for structural studies by EM. Further assembly of the activated FVIII form (FVIIIa) and the whole FVIIIa-FIXa complex on ND, followed by EM and single particle reconstruction will help to identify the protein-protein and protein-membrane interfaces critical for the intrinsic tenase complex assembly and function.
32
Russell, Joshua, Yesim Dargaud, Randal J. Kaufman, Claude Negrier, and Steven W. Pipe. "Inactivation-Resistant Recombinant Factor VIII Exhibits Superior Thrombin Generation Capacity in Comparison to Wild-Type and B Domain-Deleted Factor VIII." Blood 108, no. 11 (November 16, 2006): 1604. http://dx.doi.org/10.1182/blood.v108.11.1604.1604.
Анотація:
Abstract Activated factor VIII (FVIIIa) functions as a cofactor in the intrinsic hemostatic pathway leading to thrombin generation. Recombinant FVIII (rFVIII) has proven effective in replacement therapy for patients with hemophilia A (FVIII deficiency). The activity of FVIIIa in plasma is limited by both spontaneous dissociation of the A2 subunit and by cleavage by activated protein C (APC). Inactivation resistant FVIII (IR8) has been bioengineered to be resistant to both mechanisms of inactivation. The specific activity of purified IR8, as determined by one-stage clotting (aPTT) and two-stage chromogenic assays, was significantly higher (~7 to 20-fold) than that of wild-type (WT)-FVIII and B domain deleted (BDD)-FVIII. The specific activity was calculated based on ELISA antigen results and complemented by Western blots using commercial anti-FVIII antibodies. Since bioengineered IR8 may have altered immunoreactivity with anti-FVIII antibodies, an alternative functional assay was investigated to better characterize its potency. We evaluated WT-FVIII, BDD-FVIII and IR8 via the Calibrated Automated Thrombogram (CAT), a global assay of hemostasis, in platelet-free plasma (PPP) from 6 severe hemophilia A patients (<1 IU/dl FVIII) without inhibitors. The CAT test was chosen because of its ability to offer more valuable insight into the potential clinical value of IR8 than traditional clotting and chromogenic assays. Blood samples were taken into Corn trypsin inhibitor (CTI) to block contact activation and ensure that thrombin generation was triggered exclusively by tissue factor (TF) via the extrinsic hemostatic pathway. In an effort to demonstrate the dose dependency of each concentrate on its thrombin generating capacity, all 3 proteins were added to PPP along with a low TF concentration (1 pM) at varying FVIII activities (0, 25, 50 & 100 IU/dl). At each protein concentration, IR8 showed a significantly higher endogenous thrombin potential (ETP, the area under the thrombin generation curve) and peak height of the thrombin burst compared to either WT-FVIII or BDD-FVIII. The mean ETP values (nM*min) at 100 IU/dl, were WT-FVIII 650 and BDD-FVIII 725 (Mann Whitney test, p=0.69) and IR8 1107 (Mann Whitney test, p=0.04) with a mean ETP for FVIII <1% of 315 used as a control. Similar results were obtained in the presence of 1 nM thrombomodulin, which was added to sensitize the system to the action of APC. Consistent with the increased specific activity of IR8, the mean ETP of IR8 at 25 IU/dl was comparable to that of WT-FVIII at a concentration 4 times greater (100 IU/dl). Furthermore, no significant difference was found between the lag times of IR8 versus WT-FVIII and BDD-FVIII indicating that the advantage of IR8 does not lie in its ability to activate the initiation phase of thrombin generation, but rather in its persistent cofactor activity during the propagation phase of coagulation. These results are encouraging because the development of a rFVIII with markedly increased potency would potentially allow for reduced protein requirements in replacement therapy, thereby reducing costs and possibly decreasing inhibitor antibody development and would improve the efficacy of hemophilia A gene therapy without necessitating large improvements in genetic transfer strategies.
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Foster, PA, CA Fulcher, RA Houghten, and TS Zimmerman. "Synthetic factor VIII peptides with amino acid sequences contained within the C2 domain of factor VIII inhibit factor VIII binding to phosphatidylserine." Blood 75, no. 10 (May 15, 1990): 1999–2004. http://dx.doi.org/10.1182/blood.v75.10.1999.bloodjournal75101999.
Анотація:
The effective activation of factor X by factor IXa requires the co- factor activity of activated factor VIII (FVIII). Factor Xa formation is also dependent on the presence of negatively charged phospholipid. A phospholipid binding domain of FVIII has been reported to be present on the FVIII light chain. Recent observations on a subset of human FVIII inhibitors have implicated the carboxyl-terminal C2 domain of FVIII as containing a possible phospholipid binding site. The purpose of this study was to investigate directly the role of the C2 domain in phospholipid binding. Twenty-six overlapping peptides, which span the entire C2 domain of FVIII, were synthesized. The ability of these peptides to inhibit the binding of purified human FVIII to immobilized phosphatidylserine was evaluated in an enzyme-linked immunosorbent assay. Three overlapping synthetic FVIII peptides, 2303–2317, 2305- 2332, and 2308–2322, inhibited FVIII binding to phosphatidylserine by greater than 90% when tested at a concentration of 100 mumols/L. A fourth partially overlapping peptide, 2318–2332, inhibited FVIII binding by 65%. These results suggest that the area described by these peptides, residues 2303 to 2332, may play an important role in the mediation of FVIII binding to phospholipid.
34
Healey, John F., Ernest Parker, and John (Pete) S. Lollar. "Comparative Decay Rates of Human, Rhesus Macaque, Cynomolgus, and Porcine Activated Factor VIII." Blood 114, no. 22 (November 20, 2009): 3164. http://dx.doi.org/10.1182/blood.v114.22.3164.3164.
Анотація:
Abstract Abstract 3164 Poster Board III-104 The proteolytic conversation by thrombin of factor VIII (fVIII) to fVIIIa produces a A1/A2/A3-C1-C2 heterotrimer that spontaneously dissociates into inactive A1/A3-C1-C2 and A2 species. Human mutations that increase the rate of A2 subunit dissociation produce hemophilia A, indicating that A2 subunit dissociation is physiologically relevant and is an important regulatory feature of the blood coagulation mechanism. The A2 subunit dissociation rate from human fVIIIa is significantly faster than the corresponding dissociation rates from porcine or murine fVIIIa. The fast decay rate of human fVIIIa raises the question whether the f8 gene is under positive selection for this trait. To determine whether fast A2 dissociation occurs elsewhere in the primate lineage, we cloned cDNAs encoding B-domain deleted (BDD) fVIII from rhesus macaque and cynomolgus monkey liver. The deduced BDD amino acid sequences of rhesus and cynomolgus fVIII were 97.9 % and 98% identical to human fVIII, respectively, and were 99.9% identical to each other. The expression of rhesus and cynomolgus fVIII from baby hamster kidney-derived cells was similar to human fVIII and ten-fold lower than porcine fVIII. BDD human, rhesus, cynomolgus, and porcine fVIII molecules were purified to homogeneity by tandem ion-exchange chromatography. Concentrations of the purified constructs were calculated using a molar extinction coefficient at 280 nm based on their predicted tyrosine, tryptophan and cysteine compositions. Human, rhesus, and cynomolgus fVIII displayed similar specific coagulant activities by one-stage coagulation assay (6800, 4500, and 5200 units per mg, respectively). The kinetics of decay of human, rhesus, cynomolgus and porcine fVIIIa were measured following rapid activation of 1 nM fVIII by thrombin using a chromogenic substrate assay of purified intrinsic fXase complex under conditions in which fVIIIa was limiting. Decay curves were fit using nonlinear least-squares regression to a first-order model (Fig. 1). Decay rate constants for rhesus and cynomolgus fVIIIa were similar (0.31 and 0.27 min-1, respectively) and were slightly, but significantly lower than human fVIIIa (0.40 min-1). In contrast, the decay rate constant for porcine fVIIIa, 0.17 min-1, was 2.3-fold lower than human fVIIIa, consistent with previous observations. These results suggest that fast A2 subunit dissociation rates evolved before evolution of the primate lineage. Disclosures No relevant conflicts of interest to declare.
35
Novakovic, Valerie A., David B. Cullinan, Hironao Wakabayashi, Philip J. Fay, James D. Baleja, and Gary E. Gilbert. "Membrane-binding properties of the Factor VIII C2 domain." Biochemical Journal 435, no. 1 (March 15, 2011): 187–96. http://dx.doi.org/10.1042/bj20101797.
Анотація:
Factor VIII functions as a cofactor for Factor IXa in a membrane-bound enzyme complex. Membrane binding accelerates the activity of the Factor VIIIa–Factor IXa complex approx. 100000-fold, and the major phospholipid-binding motif of Factor VIII is thought to be on the C2 domain. In the present study, we prepared an fVIII-C2 (Factor VIII C2 domain) construct from Escherichia coli, and confirmed its structural integrity through binding of three distinct monoclonal antibodies. Solution-phase assays, performed with flow cytometry and FRET (fluorescence resonance energy transfer), revealed that fVIII-C2 membrane affinity was approx. 40-fold lower than intact Factor VIII. In contrast with the similarly structured C2 domain of lactadherin, fVIII-C2 membrane binding was inhibited by physiological NaCl. fVIII-C2 binding was also not specific for phosphatidylserine over other negatively charged phospholipids, whereas a Factor VIII construct lacking the C2 domain retained phosphatidyl-L-serine specificity. fVIII-C2 slightly enhanced the cleavage of Factor X by Factor IXa, but did not compete with Factor VIII for membrane-binding sites or inhibit the Factor Xase complex. Our results indicate that the C2 domain in isolation does not recapitulate the characteristic membrane binding of Factor VIII, emphasizing that its role is co-operative with other domains of the intact Factor VIII molecule.
36
Foster, PA, CA Fulcher, RA Houghten, and TS Zimmerman. "Synthetic factor VIII peptides with amino acid sequences contained within the C2 domain of factor VIII inhibit factor VIII binding to phosphatidylserine." Blood 75, no. 10 (May 15, 1990): 1999–2004. http://dx.doi.org/10.1182/blood.v75.10.1999.1999.
Анотація:
Abstract The effective activation of factor X by factor IXa requires the co- factor activity of activated factor VIII (FVIII). Factor Xa formation is also dependent on the presence of negatively charged phospholipid. A phospholipid binding domain of FVIII has been reported to be present on the FVIII light chain. Recent observations on a subset of human FVIII inhibitors have implicated the carboxyl-terminal C2 domain of FVIII as containing a possible phospholipid binding site. The purpose of this study was to investigate directly the role of the C2 domain in phospholipid binding. Twenty-six overlapping peptides, which span the entire C2 domain of FVIII, were synthesized. The ability of these peptides to inhibit the binding of purified human FVIII to immobilized phosphatidylserine was evaluated in an enzyme-linked immunosorbent assay. Three overlapping synthetic FVIII peptides, 2303–2317, 2305- 2332, and 2308–2322, inhibited FVIII binding to phosphatidylserine by greater than 90% when tested at a concentration of 100 mumols/L. A fourth partially overlapping peptide, 2318–2332, inhibited FVIII binding by 65%. These results suggest that the area described by these peptides, residues 2303 to 2332, may play an important role in the mediation of FVIII binding to phospholipid.
37
Livnat, Tami, Amy L. Dunn, Shirley Azar-Avivi, Wallace Hunter Baldwin, Gili Kenet, and Shannon L. Meeks. "Epitope Specific Discrepancies Between Antibody Mediated Inhibition Of Recombinant Factor VIII and Plasma Derived Factor VIII Containing Von Willebrand Factor Affecting All Domains Of Factor VIII." Blood 122, no. 21 (November 15, 2013): 1089. http://dx.doi.org/10.1182/blood.v122.21.1089.1089.
Анотація:
Abstract Background Previous results from our laboratory demonstrate an epitope specific response to high doses of FVIII within anti-C2 antibodies that was independent of antibody titer. In addition, for a panel of monoclonal antibodies (MAbs) directed against all FVIII domains the kinetics of inhibition influenced response to combinations of FVIII and recombinant factor VII (rFVIIa). The influence of inhibitor kinetics on response to treatment has also been demonstrated in inhibitor patient plasmas. The Bethesda assay detects the inhibitory capacity of anti-factor VIII (FVIII) antibodies to neutralize FVIII after 2 hours of incubation with pooled normal plasma (PNP). In this assay patient antibody is added to PNP as compared to the clinical scenario where recombinant FVIII (rFVIII) or plasma derived FVIII containing von Willebrand Factor (pdFVIII/VWF) is infused into the patient where antibody is already present. In this case the infused product is immediately available to interact with both VWF and anti-FVIII antibodies as compared to the Bethesda assay where VWF is already bound to FVIII when the antibody is added. Methods In this study we investigated the inhibitory kinetics of a panel of 20 anti-FVIII MAbs (Table) with known epitope specificity and inhibitory activity in a standard Bethesda assay. Inhibitor plasma consisted of a single MAb added to FVIII deficient plasma at 5 µg/ml. rFVIII and pd-FVIII/VWF were added to each inhibitor plasma and samples were sequentially removed at intervals between 5 and 90 minutes. FVIII activity was measured by a one-stage aPTT based assay. Results Of the MAb panel, 2 anti-A2 MAbs, 1D4 and 4A4, and the anti-A3 MAb F147 had full neutralization of both rFVIII and pd-FVIII/VWF. All 3 of these MAbs exhibit high inhibitory titers in the Bethesda assay. The majority of the other MAbs had improved neutralization kinetics and thus higher residual FVIII activity with pd-FVIII/VWF when compared to rFVIII. The figure below shows the residual FVIII activity at 15 minutes following the addition of rFVIII or pdFVIII/VWF into the inhibitor plasmas. Similar patterns were seen at the other time points. Three MAbs from the panel, two anti-A2 (4C7 and B157) and one anti-C2 (2-117), had significant inhibition of FVIII activity when rFVIII was added to the inhibitor plasma. This was not demonstrated in the standard Bethesda assay or when pd-FVIII/VWF was added to the inhibitor plasma. This demonstrates that the order of binding of VWF and anti-FVIII antibodies may be clinically relevant for a subset of FVIII epitopes. Conclusion The Bethesda assay in isolation neither predicts inhibitory kinetics nor defines response to various FVIII sources. FVIII source dependent neutralization kinetics and epitope mapping may be applied as additional tools for tailoring therapy in patients with inhibitors. Disclosures: No relevant conflicts of interest to declare.
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Lannert, Kerry W., Hilary S. Gammill, Barbara A. Konkle, and Jill M. Johnsen. "The Ability of Von Willebrand Factor (VWF) to Bind Factor VIII (FVIII) Can Decrease during Pregnancy." Blood 124, no. 21 (December 6, 2014): 4244. http://dx.doi.org/10.1182/blood.v124.21.4244.4244.
Анотація:
Abstract Background: The coagulation protein von Willebrand Factor (VWF) and its circulating partner protein, Factor VIII, are known to be elevated in pregnancy. However, the nature of changes in VWF which occur during pregnancy are not well understood. We previously presented results from a study of 46 healthy pregnancies in which we measured VWF antigen (VWF:Ag) and Factor VIII (FVIII determined both by activity and antigen measurements). We found that VWF:Ag and FVIII increased during pregnancy, consistent with less FVIII relative to VWF. We have also observed that VWF multimer size can decrease during pregnancy. We hypothesized that pregnancy can induced molecular changes in VWF, and that such changes could impact the interaction of VWF with its ligands, specifically FVIII. Methods: We adapted a Type 2N von Willebrand Disease (VWD) assay to test the ability of plasma VWF to bind recombinant FVIII. In brief, serial dilutions of plasma VWF were captured using a polyclonal anti-VWF antibody (DAKO), endogenous FVIII was stripped using a CaCl2 wash, recombinant FVIII (Recombinate) was applied, and bound FVIII was detected using an anti-FVIII antibody (Cedarlane). In parallel, a VWF:Ag ELISA was performed using the same anti-VWF capture antibody. The VWF-FVIII binding ratio (VWF-FVIIIb) was then determined by dividing the quantity of rFVIII detected by the amount of VWF:Ag immobilized on the plate. The range, reproducibility, and variance of the assay was first determined in a healthy blood donor repository. We then tested VWF-FVIIIb in healthy pregnancies for which 3rd trimester and non-pregnant baseline samples were available. Additional time points during pregnancy were also studied, when available. VWF-FVIIIb results for each trimester and the non-pregnant baseline were analyzed by one-way ANOVA (All Pairwise Multiple Comparison Procedures, Holm-Sidak method). Results: Forty-six healthy pregnancies with non-pregnant baseline samples were studied. Samples tested were drawn in the first (n=6), second (n=38), and third (n=43) trimesters, at 38 weeks gestational age (n=13), and at a non-pregnant time point (baseline). In non-pregnant samples, the VWF-FVIIIb was close to 1.0 (0.988 +/- 0.084), similar to a normal blood donor repository. We identified a significant decrease in VWF-FVIIIb in the second trimester (0.931 +/- 0.82; p = 0.013), which became more pronounced in the third trimester (0.899 +/- 0.75; p < 0.001) and at 38 weeks gestational age (0.88 +/- 0.072; p < 0.001). The means, quartiles, and standard deviations for each pregnancy time point are shown in Figure 1. Conclusions: These data support the hypothesis that VWF can acquire a decreased ability to interact with FVIII during pregnancy. A decreased capacity to carry FVIII could contribute to the increased VWF:FVIII ratio we and others have observed during pregnancy, although other factors, such as increased VWF production relative to FVIII, could also play a role. This work provides further evidence for a new model of acquired VWF changes in the setting of pregnancy. Disclosures No relevant conflicts of interest to declare.
39
Gangadharan, Bagirath, H. Trent Spencer, Ernest T. Parker, and Christopher B. Doering. "High-Level Expression of Porcine Factor VIII from Murine Mesenchymal Stem Cells." Blood 104, no. 11 (November 16, 2004): 5281. http://dx.doi.org/10.1182/blood.v104.11.5281.5281.
Анотація:
Abstract Low-level expression of human factor VIII (fVIII) has limited the success of clinical trials for hemophilia A using both ex vivo and in vivo gene transfer methods. Ex vivo genetic modification provides increased control of gene transfer and permits thorough characterization of transgene copy number, chromosomal integration site(s), and expression levels prior to transplantation. A subpopulation of adherent bone marrow-derived cell types, termed mesenchymal stem cells (MSCs), comprise an attractive target cell type for ex vivo gene transfer due to their accessibility, ability to differentiate into multiple cell types, and long-term survival following transplantation. Recently we demonstrated, in vitro, that recombinant B-domain-deleted porcine fVIII (rp-fVIII) is expressed at levels 10 – 14-fold greater than recombinant B-domain-deleted human fVIII (rh-fVIII) due to an enhanced rate of secretion from baby hamster kidney-derived (BHK-M) cells. Additionally we found that only the A1 and activation peptide-A3 domain sequences of porcine fVIII are necessary to retain high-level expression of hybrid human/porcine fVIII constructs. Here we report the expression of rp-fVIII from murine MSCs isolated from exon-16 fVIII knockout mice following ex vivo retroviral transduction. MSCs were transduced with ecotropic envelope-pseudotyped murine stem cell virus containing a rp-fVIIII transgene at an multiplicity of infection of 2 – 5 functional viral particles/target MSC. Following this transduction regimen the cell population harbored an average of 1.8 proviral genomes/cell as determined by quantitative real-time PCR. During culture in growth medium supplemented with 20% fetal bovine serum (FBS) rp-fVIII-transduced MSCs demonstrated a steady-state level of 2,400 fVIII mRNA transcripts/cell and an apparent fVIII production rate of 174 units/106 cells/24 hr as determined by quantitative real-time RT-PCR and one-stage clotting assay, respectively. However, when cultured in serum-free medium the mRNA levels decreased to 950 transcripts/cell and apparent fVIII production was reduced to 14 units/106 cells/24 hr. The latter mRNA/fVIII expression ratio is in agreement with that previously reported from stably transduced BHK-M clonal cell lines. In contrast the former mRNA levels are lower than predicted from the observed fVIII activity levels. The activation quotient (ratio of apparent fVIII activity following thrombin pre-treatment to non-thrombin treated baseline fVIII activity) increased 17-fold when the cells were cultured in serum-free versus serum-containing medium. Additionally, SDS-PAGE analysis of immunoprecipitated fVIII protein from serum-containing and serum-free medium revealed the presence of activated fVIII (fVIIIa) in conditioned medium samples containing FBS. Highly purified rp-fVIII from transduced-MSCs cultured in serum-free medium displayed similar relative mobility to BHK-M produced rp-fVIII upon SDS-PAGE analysis. These data warn against the determination of fVIII activity in serum-containing medium using the one-stage coagulation assay due to the presence of activated fVIII with high specific activity. Based on these results it is reasonable to predict that hemophilia A could be cured using high-level expression fVIII constructs by transplantation of a feasible number (106 - 108) of cells containing a single copy of rp-fVIII or other high-level expression hybrid human/porcine fVIII transgenes.
40
Minami, Hiroaki, Keiji Nogami, Takehisa Kitazawa, Kunihiro Hattori, and Midori Shima. "FVIII Heavy Chain Enhances Tenase Activity Induced By FVIIIa Mimicking Bispesific Antibody, ACE910." Blood 124, no. 21 (December 6, 2014): 1481. http://dx.doi.org/10.1182/blood.v124.21.1481.1481.
Анотація:
Abstract Background: ACE910, asymmetric bispecific monoclonal antibodies to activated factor IX (IXa) and factor X, mimics the cofactor function of activated factor VIII (VIIIa) by modulating an optimal position on the tenase assembly. The estimated therapeutic range of ACE910 shows ~30% of thrombin generation in native tenase assembly, supporting that the structure on ACE910-mimicking tenase assembly is different from that on native tenase. Being close to physiological structure consisting from factor IXa, factor X, and factor VIIIa is important for potentiating the clotting function. We examined the effects of factor VIII subunits (light chain, heavy chain, A1 and A2, C2) on ACE910-tenase. Materials/Methods: The factor VIII light chain and heavy chain were isolated from EDTA-treated recombinant factor VIII following chromatography on SP- and Q- Sepharose columns. The A2 and A1 subunits were purified from thrombin-cleaved factor VIII heavy chain by Heparin-, SP- Sepharose columns. Purified factor Xa generation assays was examined with (i) factor VIII subunit (0-40 nM), ACE910 (10 µg/ml), phospholipid (PL) (40 µM), factor IXa (1 nM) and factor X (200 nM), (ii, iii) the A2 or heavy chain (40 nM), ACE910 (10 µg/ml), PL (40 µM), factor IXa and factor X (1 or 0-80 nM, and 0-300 or 200 nM, respectively). These mixtures were reacted for five minutes (i, ii) or one minute (iii). These assays were conducted at 37 °C. Results: (i) The factor Xa generation in ACE910-tenase complex in the absence of factor VIIIa was 10.1±2.2 nM. With the intact heavy chain and A2, amounts of factor Xa were increased dose-dependently, resulting in 1.3- and 1.2-fold increases, respectively. While, the light chain and A1 subunit failed to increase at all. (ii) Vmax for factor X in ACE910-tenase was 173.0±7.0 nM and Km was 31.2±3.9 nM. Vmax obtained with the heavy chain or A2 was 175.9±6.1 or 159.0±6.1 nM, whilst Km was 17.0±2.2 or 31.9±3.5 nM, respectively, indicating that the heavy chain enhanced the binding affinity for factor X in ACE910-tenase. (iii) Vmax for factor IXa in ACE910-tenase was 43.8±2.7 nM and Km was 36.9±4.8 nM. With the heavy chain or A2, Vmax was 46.8±3.0 or 45.0±3.1 nM, and Km was 36.4±3.0 or 32.1±4.9 nM, respectively, indicating that either the heavy chain or A2 did not enhance the catalytic activity and the binding affinity for factor IXa in ACE910-tenase. Conclusion: ACE910-tenase assembly seems to be close to physiological structure by the presence of intact heavy chain interacting with factor X. In addition, ACE910 may substitute the position such as the factor VIII(a) light chain associated with FIXa and FX on ACE910-tenase assembly defecting factor VIII. Disclosures Minami: Chugai Pharmaceutical Co., Ltd.: Research Funding. Nogami:Chugai Pharmaceutical Co., Ltd.: Membership on an entity's Board of Directors or advisory committees, Research Funding. Kitazawa:Chugai Pharmaceutical Co., Ltd.: Employment, Equity Ownership, Patents & Royalties. Hattori:Chugai Pharmaceutical Co., Ltd.: Employment, Equity Ownership, Patents & Royalties. Shima:Chugai Pharmaceutical Co., Ltd.: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.
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Novakovic, Valerie A., James D. Baleja, and Gary E. Gilbert. "The Factor VIIII C2 Domain Shows Stereospecificity for Phosphatidyl-L-Serine and Increases Factor IXa Activity." Blood 112, no. 11 (November 16, 2008): 3090. http://dx.doi.org/10.1182/blood.v112.11.3090.3090.
Анотація:
Abstract Background: Factor VIII is an essential cofactor in the blood coagulation cascade and shows greatly increased activity when bound to phospholipid membranes. While high proportions of various negatively-charged phospholipids support binding of factor VIII, only phosphatidyl-L-serine (Ptd-L-Ser) supports stereospecific affinity when present at physiologically relevant proportions. Factor VIII binds to phospholipid membranes primarily through its C2 domain, but binding is also mediated by the C1 domain (Meems et al., abstract ASH 2008). However, the membrane-binding properties of the isolated C2 domain and the relationship of the C2 domain to factor IXa and factor X have not been fully studied. Methods: The factor VIII C2 domain (fVIII-C2) and a mutant in which residue Val2223 was replaced by Cys (fVIII-C2C) were produced in E. coli and purified from cytosol by metal ion affinity chromatography followed by cation exchange chromatography. fVIII-C2C was labeled with fluorescein maleimide (fVIII-C2C-fluor). Binding studies were performed by flow cytometry using membranes supported by glass microspheres (lipospheres) and by fluorescence resonance energy transfer between fVIII-C2 and dansyl-labeled vesicles. The factor Xase assay was utilized to infer the capacity of fVIII-C2 to influence membranebinding and protein-protein interactions. Results: fVIII-C2C-fluor bound to liposphere membranes containing at least 10% Ptd-LSer. The KD for binding to lipospheres was 150 ± 40 nM. The KD for fVIII-C2 binding to sonicated vesicles of composition Ptd-L-Ser:PE-dansyl:PC 20:5:75 was 230 ± 30 nM indicating that fVIII-C2 and fVIII-C2C-fluor bind with similar affinities. Binding was measurable at pH 6.0 but was at least 10-fold lower affinity at pH 7.8. Change of pH from 6.0 to 7.8 was associated with a change in intrinsic fluorescence and was not associated with increased light scatter, suggesting conformational change rather than formation of dimers or aggregates. Sonicated vesicles with 15% Ptd-L-Ser competed with lipospheres for binding of fVIII-C2C-fluor with 3-fold higher affinity than vesicles with 15% Ptd-D-Ser. Phosphatidylinositol or phosphatidic acid-containing vesicles bound fVIII-C2C-fluor with at least 4-fold lower affinity than Ptd-L-Ser. fVIII-C2 did not compete with factor VIII-fluor for binding to lipospheres at pH 6 or 7.8. At pH 6.0 the factor Xase assay was inhibited by addition of fVIII-C2 with a plateau of 30% inhibition at 1.5 μM. In the absence of intact factor VIII, fVIII-C2 enhanced the activity of factor IXa by 2-fold. The enhanced activity correlated to a reduced KM but not an altered apparent affinity for phospholipid vesicles. Conclusions: These results indicate that fVIII-C2 binds membranes containing Ptd-L-Ser in a stereospecific manner with approximately 30-fold lower affinity than intact factor VIII. Membrane binding is pH-dependent, likely requiring a conformational change in fVIII-C2. Lack of competition with intact factor VIII implies that fVIII-C2 does not recognize the initial membrane contact site of the full protein. Partial inhibition of the factor Xase complex and enhancement of factor IXa activity in the absence of intact factor VIIIa implies that fVIII-C2 binds either factor IXa or factor X. We speculate that the conformational change enabling membrane binding is caused by an acidic microenvironment in intact factor VIII produced by proximity to the C1 domain and/or the phospholipid membrane.
42
Rolli, Veronique, Nathalie Enjolras, Cecile Ducasse, Marie-Helene Rodriguez, Thomas Weimer, Hans-Peter Hauser, Claude Negrier, and Jean-Luc Plantier. "Generation of Factor VIII Molecules Partially Resistant to Activated Protein C." Blood 104, no. 11 (November 16, 2004): 1729. http://dx.doi.org/10.1182/blood.v104.11.1729.1729.
Анотація:
Abstract Following a vascular injury, factor VIII (FVIII) is rapidly activated by thrombin cleavage at arginine (Arg) 372, 740 and 1689. Activated FVIII, an heterotrimer composed by the association of A1, A2 and A3-C1-C2 domains, is rapidly degraded to limit thrombosis risk. Two main phenomenons that account for the disappearence of FVIIIa consist of an intrinsic dissociation of the trimer due to the loss of A2 domain, and the cleavage of the molecule by activated protein C (APC). APC cleaves FVIIIa at Arg 336 and 562. Mutant FVIII molecules were already generated, with one or two arginines substituted, and a subsequent APC cleavage diminished. Since the thrombotic potential of high plasma levels of FVIII has been described, we aimed to generate new factor VIII molecules where the APC cleavage was only modulated. The ultimate goal being to increase in vivo the FVIIIa half-life. Among the two APC cleavage sites, the sequence around the site 562 was the most conserved between species. This region was therefore chosen to be modified with a prior verification that the amino-acids to be modified were not described in any hemophilic phenotype. Subsequently, the six following mutations were realized in a BDD-FVIII cDNA: Q561N, G563A, N564D, N564A, N564Q and I566M as well as the controls R336I, R562K and R336I+R562K. The constructs were transiently expressed in BHK cells to determine the specific activities of the corresponding molecules. The mutant specific activities, as determined by one- and two-stage clotting assays, ranged from 40 to 94 % of the wild-type except for G563A that was almost inactive. CHO clones expressing FVIII molecules were obtained. The mutants and control FVIII molecules were produced, partially purified on heparin column and further analyzed. The comparative specific activities in a two-stage clotting assay were the following (+/− SD; n=6): BDD-FVIII 100 %, Q561N 105 % +/− 45, G563A 6 % +/− 6, N564D 50 % +/− 23, N564A 45 % +/− 21, N564Q 80 % +/− 26, I566M 100 % +/− 41. The one-stage clotting assay gave identical results than the two-stage assay for each mutant. The mutants were then activated by thrombin (1:1) and the occurrence of FVIII activity was monitored. A 20- to 25-fold increase in FVIII activity was measured within 2 minutes following the addition of thrombin for all mutants. The mutants, except for the inactive G563A, were then analyzed for their resistance to APC by three different assays: an APC resistance kit (Coatest, Chromogenix), an in vitro assay that measured APC sensitivity of FVIIIa and an immunoblot assay that visualized the cleavage efficiency of the A2 fragment. These three assays confirmed the APC resistance of the previously published R336I, R562K and R336I+R562K, as compared with wild-type FVIII. They also revealed that the mutants N564D, N564A and I566M behaved similarly to the wild-type FVIII whereas Q561N and N564Q mutants were partially resistant to APC. The APC resistance ratio were the following 2.3 +/− 0.3 for BDD-FVIII, 2.1 +/− 0.4 for N564Q, 1.7 +/− 0,1 for Q561N, 1.6 +/− 0.3 for R562K and 1.3 +/− 0.3 for R336I+R562K. The N564Q and Q561N mutants exhibited a profile intermediate between wild type FVIII and R562K regarding of the loss of FVIIIa activity that was confirmed on the immunoblot profile. In conclusion we have generated new factor VIII molecules that retained their full procoagulant function while possessing a reduced sensitivity to APC cleavage.
43
Markovitz, Rebecca C., John F. Healey, W. Hunter Baldwin, Ernest T. Parker, Shannon L. Meeks, and Pete Lollar. "Decreasing the Humoral Response to Factor VIII By Targeted Deletion of Factor VIII - Specific B Cells." Blood 124, no. 21 (December 6, 2014): 238. http://dx.doi.org/10.1182/blood.v124.21.238.238.
Анотація:
Abstract The development of neutralizing anti-factor VIII (fVIII) antibodies (inhibitors) remains the most significant complication in the treatment of hemophilia A patients. Treatment of inhibitor patients consists of management of bleeding episodes using bypassing agents or porcine fVIII. Inhibitors can be eradicated by immune tolerance induction (ITI) using thrice-weekly administration of large doses of fVIII. However, ITI fails in approximately 30% of patients. Additionally, the median time to tolerance in successful cases is ~18 months, making ITI expensive and inconvenient. In the current study, we used a murine E16 hemophilia A model to test a novel approach to both prevent and eradicate fVIII inhibitors. We hypothesized that conjugation of fVIII to the toxin saporin, a Type I ribosome-inactivating protein, would target fVIII-specific cell surface immunoglobulin and selectively delete fVIII-specific naïve and memory B cells. Recombinant full-length fVIII was covalently linked to saporin using the heterobifunctional crosslinker N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP). To test for eradication of existing fVIII inhibitors by fVIII-saporin, an adoptive transfer protocol was developed to measure fVIII-specific memory B cells. Hemophilia A donor mice were immunized with 2 μg of full-length fVIII by intravenous injection every other week for 8 weeks, followed by a final dose of 4 μg at ten weeks. Four weeks later, the mice were randomized into three treatment groups to receive equimolar doses of saporin, fVIII, or fVIII-saporin. Seven days after treatment, the mice were sacrificed and 4 x 106 plasma cell CD138+-depleted splenocytes were adoptively transferred as a source of fVIII-specific memory B cells into naïve recipient hemophilia A mice. At 24 hours, recipient mice were given a single injection of 0.5, 1.0 or 2.0 μg of recombinant full-length fVIII by tail vein injection. Anti-fVIII IgG antibodies in recipient mice were measured by ELISA 2 and 5 weeks following the fVIII injection. In the absence of fVIII-specific memory B cells from donor mice, naïve hemophilia A mice did not produce detectable anti-fVIII antibodies. Recipient hemophilia A mice receiving splenocytes from fVIII donor and saporin donor mice displayed a dose-dependent increase in anti-fVIII antibodies. In contrast, the slope of the anti-fVIII titer versus dose of fVIII was significantly decreased in recipient mice receiving splenocytes from fVIII-saporin donor mice. To test for prevention of fVIII inhibitor formation by fVIII-saporin, naïve hemophilia A mice were divided into three treatment groups to receive a single dose of saporin, fVIII, or fVIII-saporin by tail vein injection. Seven days after treatment, the mice were immunized by tail vein injection with 2 μg of full-length fVIII every other week for 10 weeks. Anti-fVIII IgG antibodies were measured 1 week after the fourth and sixth injections of fVIII. Anti-fVIII antibody titers were significantly lower in the fVIII-saporin group compared to the fVIII group (1,900 vs. 21,400 (p=0.027, n=4, Mann-Whitney test, see figure) after the fourth injection. After 6 injections, the average anti-fVIII titer of the fVIII group was 23,000 compared to 4,000 in the fVIII-saporin group (p=0.057, n=4, Mann-Whitney test, see figure). In conclusion, our results suggest that infusion of fVIII-saporin results in the depletion of both fVIII-specific naïve B cells and memory B cells. FVIII-saporin potentially could be used in the treatment of congenital hemophilia A patients with inhibitors and patients with acquired hemophilia A. In addition, fVIII-saporin potentially could be used in previously untreated patients with hemophilia A to prevent inhibitor development. Similar therapeutic strategies could be extended to other antigen-specific immune disorders. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.
44
Mazurier, Claudine, Armelle Parquet-Gernez, and Maurice Goudemand. "Validation of a Procedure for Potency Assessing of a High Purity Factor VIII Concentrate -Comparison of Different Factor VIII Coagulant Assays and Effect of Prediluent." Thrombosis and Haemostasis 64, no. 02 (1990): 251–55. http://dx.doi.org/10.1055/s-0038-1647295.
Анотація:
SummaryThe assessment of factor VIII coagulant activity (FVTII: C) in recently available highly purified and concentrated FVTII therapeutic products calls for careful evaluation of assay methodologies. We assayed more than 130 batches of a concentrate with a specific activity of about 150 FVTII :C units/mg protein, using one-stage and two-stage clotting and chromogenic methods. There was good agreement between the potency estimates obtained with the different methods. We also compared the FVTII :C potencies obtained after predilution in buffer or FVIII-deficient plasma using either calibrated plasma or FVTII concentrate as references. With the one-stage assay we found a marked discrepancy between the potency values obtained with buffer and with FVTII-deficient plasma used as prediluents. In order to validate our “in vitro” data we performed 6 “in vivo” analyses in severe haemophilia A patients. On the basis of the overall data obtained we chose to label FVIII potency by using FVIII-deficient plasma as prediluent, reference plasma as standard and the chromogenic assay method.
45
Planque, Stephanie, Miguel A. Escobar, Hiroaki Taguchi, Sangeeta Karle, Yasuhiro Nishiyama, Elizabeth Donnachie, and Sudhir Paul. "Irreversible Blockade of Factor VIII Antibodies." Blood 104, no. 11 (November 16, 2004): 1728. http://dx.doi.org/10.1182/blood.v104.11.1728.1728.
Анотація:
Abstract High titer antibodies (Abs) to Factor VIII (FVIII) can render FVIII replacement therapy ineffective in severe hemophilia A patients. An antigen capable of specific and covalent binding is predicted to reverse the effects of pathogenic Abs potently compared to conventional noncovalently binding antigens, as dissociation of the former type of antigen from the immune complexes is precluded. Recently, we described the apparently universal presence of enzyme-like nucleophiles in the antigen binding sites of Abs (Planque et al, J Biol Chem, 2003). Here, we report a covalently reactive antigen analog (CRA) of FVIII that inactivates anti-FVIII Abs specifically. Recombinant FVIII was derivitized at Lys side chains with phosphonate diester groups, positioning these electrophiles within the antigenic epitopes expressed by FVIII. Comparable binding of FVIII-CRA and underivitized FVIII by a monoclonal Ab and polyclonal Abs from 5 hemophilia patients was evident, suggesting maintenance of the FVIII epitope structure necessary for noncovalent Ab recognition. FVIII-CRA formed immune complexes with polyclonal anti-FVIII Abs from hemophilia patients that were not dissociated by sodium dodecyl sulfate (2%), a detergent that disrupts noncovalent interactions. Removal of free FVIII-CRA from the reaction mixtures by affinity chromatography on Protein G did not restore the FVIII-binding activity of the Abs, confirming irreversible occupancy of the Ab binding sites. No binding of the Abs by an irrelevant peptidyl CRAs was observed, suggesting the role of noncovalent antigen binding in imparting specificity to the covalent reaction. Ab inhibition of the functional role of FVIII in coagulation was studied by the chromogenic assay. Treatment of the Abs with the FVIII-CRA followed by removal of free FVIII-CRA relieved the inhibitory effect of the Abs on FVIII mediated activation of FIXa/FX. An irrelevant peptidyl CRA did not relieve the Ab inhibitory effect. Compared to FVIII, the FVIII-CRA activated FIXa/FX poorly, suggesting that the integrity of the FIXa binding site had been disrupted. These observations encourage the development of electrophilic CRA analogs of FVIII for treatment of patients with inhibitory Abs to FVIII.
46
Fu, Richard, Meghan J. Lyle, Xuefeng Wang, and Carol H. Miao. "Factor VIII-specific CAR regulatory T cells modulate murine anti-factor VIII immune responses." Journal of Immunology 196, no. 1_Supplement (May 1, 2016): 126.13. http://dx.doi.org/10.4049/jimmunol.196.supp.126.13.
Анотація:
Abstract The immune response to factor VIII protein (FVIII; F8 in constructs) limits the effectiveness of treatments for hemophilia A (HemA) patients. Previously we demonstrated that regulatory T cells (Tregs) play a pivotal role in modulating anti-FVIII immune responses. For application of adoptive Treg therapy, we successfully expanded highly suppressive murine polyclonal Tregs antigen specifically in vitro. However, the FVIII-specific Tregs in the polyclonal population are still in very small numbers. Thus, we explored the strategy to generate FVIII-specific Tregs using the chimeric antigen receptor (CAR) approach. Lentiviral vector (LV) incorporating a high-binding anti-FVIII scFv linked to the CAR signaling domains and fused with a Foxp3 cDNA (F8CAR-Foxp3-LV) was prepared and used to transduce murine CD4+T cells. Flow cytometry analysis confirmed extracellular scFv and intracellular Foxp3 expression in transduced cells (F8CAR-Tregs). In vitro FVIII-specific suppressive assay showed that transduced cells had significantly higher suppressive activity than untransduced cells towards murine effector T cells. In addition, 1×106 transduced cells and untransduced cells were adoptively transferred into HemA mice and the treated mice were subsequently challenged with FVIII plasmid injected hydrodynamically. The anti-FVIII antibody titers are evaluated overtime. It is expected that F8CAR-Foxp3-LV transduced cells will prevent or decrease the production of anti-FVIII antibodies in HemA mice. We anticipate that compared with nonspecific or polyclonally expanded Tregs, FVIII-specific CAR Tregs will exert superior suppressive activity towards anti-FVIII immune responses without triggering systemic immune suppression.
47
Hausl, Christina, Rafi U. Ahmad, Maria Sasgary, Christopher B. Doering, Pete S. Lollar, Guenter Richter, Hans Peter Schwarz, Peter L. Turecek, and Birgit M. Reipert. "Inhibition of Factor VIII-Specific Memory B Cell Responses by Supra-Physiological Concentrations of Factor VIII." Blood 104, no. 11 (November 16, 2004): 38. http://dx.doi.org/10.1182/blood.v104.11.38.38.
Анотація:
Abstract Inhibitory antibodies against factor VIII (FVIII) are the major complication experienced by hemophilia A patients treated with FVIII products. The most effective therapy to eradicate these antibodies is elevated doses of FVIII over a prolonged period. Despite clinical practice in using such protocols, nothing is known about the immunological mechanisms that cause the down-modulation of FVIII-specific immune responses and the induction of long-lasting immune tolerance against FVIII. Understanding the underlying mechanisms, however, would facilitate designing new therapeutic strategies. The re-stimulation of FVIII-specific memory responses after each dose of FVIII is probably the most important event in the maintenance of FVIII inhibitors in patients. Therefore, the eradication of these memory responses should be an essential step in the down-modulation of inhibitory antibodies and the induction of immune tolerance. We used a murine model of hemophilia A to answer the question whether FVIII-specific memory responses are sensitive to increasing doses of FVIII. In particular, we were interested in the differential effects of FVIII on memory-B-cell and memory-T-cell responses. For the analysis of FVIII-specific memory responses, we re-stimulated FVIII-specific memory B- and T-cells obtained from spleens of hemophilic mice treated with four doses of human FVIII or eight doses of murine FVIII as described (Sasgary et al.: Thromb Haemost2002; 87:266–72; Hausl et al.: Blood2004; 104:115–22). Our results show dose-dependent effects of FVIII on the re-stimulation of FVIII-specific memory B cells in vitro. Physiological concentrations of FVIII below 100 ng/ml re-stimulate memory B cells and induce their differentiation into anti-FVIII antibody-secreting plasma cells. Supra-physiological concentrations above 100 ng/ml, however, inhibit memory-B-cell re-stimulation. The inhibition of memory-B-cell re-stimulation is irreversible and seems to be due to an induction of apoptosis that is at least partly mediated by Fas-dependent mechanisms. Furthermore, the inhibition appears to be initiated by triggering the B-cell receptor (BCR) without the requirement of an excessive cross-linking of the BCR. The activation of FVIII-specific T cells is not affected by increasing doses of FVIII. We conclude that the induction of apoptosis in FVIII-specific memory B cells might be the first step in the induction of immune tolerance in hemophilia A patients with FVIII inhibitors who receive high doses of FVIII. The eradication of memory B cells would prevent their differentiation into antibody-secreting plasma cells and, moreover, might lead to a deficiency of effective antigen-presenting cells required for the re-stimulation of FVIII-specific memory T cells. The induction of regulatory T cells rather than effector T cells could be the consequence of this deficiency.
48
Mertens, Koen, David Gestel, Gunny van Stempvoort, Vincent Limburg, Ed E. Moret, and Alexander B. Meijer. "Somatic Mutations in Factor VIII Inhibitors Determine Their Factor VIII Binding and Inhibitory Properties." Blood 110, no. 11 (November 16, 2007): 1762. http://dx.doi.org/10.1182/blood.v110.11.1762.1762.
Анотація:
Abstract The formation of inhibitors against Factor VIII (FVIII) is one of the major adverse effects of FVIII substitution therapy in hemophilia A patients. Previously we have cloned a panel of antibodies from a phage display library from the VH gene repertoire of a single hemophilia A patient (Van den Brink et al, Blood 2002). One single-chain variable fragment (scFv), designated KM33, was found to display remarkably high affinity FVIII binding and FVIII nhibition. The epitope of this scFV was located in the FVIII light chain, in particular within the C1 domain. Using computational epitope prediction methods we identified three putative antibody binding C1 loops. By mutagenesis we found one of these (K2092-S2094) critical for KM33 binding. This region is the counterpart of one of the lipid-binding loops in the FVIII C2 domain. This suggest that KM33 inhibits FVIII activity by interfering in FVIII with membrane binding. Sequence analysis revealed that the scFV fragment KM33 was derived from the germ-line sequence VH 3–30 with a number of substitutions. As somatic mutations have the potential of contributing to affinity maturation and antigen neutralization, we addressed the contribution of individual mutations to the inhibitory properties of scFV KM33. Somatic mutations were reversed to germ-line residues, and purified KM33 variants were analyzed by Surface Plasmon Resonance (SPR) and by solid-phase binding and inhibition assays. Somatic mutations in positions H56 (S to N) and H57 (N to D) within the Complementarity Determining Region H2 (CDR H2) proved neutral with regard to affinity for FVIII. In contrast, the somatic mutations near the CDR H1 (H23 A to V, H24 A to D, and H27 F to L) proved of major impact. The equilibrium binding constant (Kd) of mutant VH-D24A (4.4 nM) and the triple mutant VH-(V23A-D24A-L27F) (10 nM) were 2–3 orders of magnitude higher than that of the wild-type KM33 (Kd = 0.05 nM). The SPR sensorgrams revealed that the difference in affinity resulted mainly from higher dissocation rates for the mutants. Molecular modeling and molecular dynamics suggested that Asp in position H24 plays a key role in that it forms a hydrogen bond network that stabilizes the CDR H1 in the mature KM33 antibody. The stabilization of CDR H1 in KM33 proved essential for its inhibitory properties and its detection as an anti-FVIII antibody. First, the KM33 D24A variant failed to display any inhibitory activity in the classical Bethesda assay, irrespective whether residual FVIII activity was assessed by one-stage assay or by chromogenic assay. Second, this variant tested negative in ELISA based assays for FVIII-binding, but non-inhibitory antibodies, presumably due to its relatively high dissociation rate from immobilized FVIII. This finding may have major implications for the detection of FVIII inhibitors. First, anti-FVIII antibodies may exist that are directed against functional epitopes on the FVIII molecule that escape detection in functional Bethesda assays. The same may hold for binding assays that are designed to detect typical non-inhibitory antibodies. While patients having such antibodies seem virtually inhibitor-free, a single somatic mutation within a pre-existing non-inhibitory antibody may result in the a strong FVIII inhibitory response. This may be particularly relevant in linking FVIII inhibitor development in individual patients to exposure to specific FVIII products.
49
Tang, Liang, Clark Pan, Harpartap Atwal, Jennifer Nixon, Thomas Barnett, John E. Murphy, Baisong Mei, and Michael Fournel. "PEGylation of Factor VIII Reduces the Inhibitory Effects of Human Antibody Inhibitors on the Factor VIII Molecule." Blood 108, no. 11 (November 16, 2006): 4017. http://dx.doi.org/10.1182/blood.v108.11.4017.4017.
Анотація:
Abstract Replacement therapy with plasma-derived or recombinant Factor VIII (FVIII) has successfully reduced mortality and morbidity and improved the quality of life for patients with hemophilia A. However, up to 30% of patients develop antibody inhibitors to the FVIII molecule. Inhibitors reduce the efficacy of FVIII, increase the cost of treatment, and greatly increase the risk for life-threatening bleeding events in these patients. In the hopes of developing a more efficacious FVIII therapeutic for hemophilia A patients with inhibitors, we initiated studies on the PEGylation of B-region deleted FVIII (FVIII-BDD) with different sizes of polyethylene glycol (PEG) and investigated the inhibitory effects of anti-FVIII inhibitors on the activity of PEGylated FVIII-BDDs. Applying a site-specific mutagenesis technique to the FVIII-BDD molecule, an amino-acid residue at the inhibitor binding site was changed to cysteine. Mutated FVIII-BDD was purified and PEG molecules of different sizes were added at the mutation sites through a chemical reaction with the maleimide group on the activated PEG. PEGylated FVIII-BDD molecules were further purified and tested for FVIII activity using the chromogenic assay. To investigate the effects of antibody inhibitors on the PEGylated FVIII-BDD, studies were carried out utilizing inhibitory plasmas collected from 8 hemophilia A patients with confirmed inhibitors and different monoclonal antibodies as controls. Of the 8 patient plasmas tested, 43 kD PEGylated FVIII-BDD was more resistant to antibody inhibitors in 4 patient plasma samples than unmodified FVIII-BDD. In one sample, pre-incubation of FVIII-BDD with inhibitor patient plasma (1:15 dilution) reduced FVIII activity to 5% of the originally activity added, according to the chromogenic assay. By contrast, approximately 20% of original activity was retained for monoPEGylated FVIII-BDD and >40% activity was retained for diPEGylated FVIII-BDD. Overall, the results suggest that PEGylated FVIII-BDDs may retain more FVIII activity in the presence of some FVIII antibody inhibitors compared to FVIII-BDD. It is important to note that there was a positive correlation between the size of PEG added to FVIII-BDD and the amount of FVIII activity retained. This study indicates that the addition of PEG to FVIII molecules through site-specific PEGylation has the potential to increase the resistance of FVIII to the effects of some antibody inhibitors in patients with hemophilia A.
50
Nogami, Keiji, Midori Shima, Tomoko Matsumoto, Katsumi Nishiya, Masahiro Takeyama, Kohei Tatsumi, Ichiro Tanaka, and Akira Yoshioka. "Mechanisms of Plasmin-Catalyzed Inactivation of the Factor VIII." Blood 106, no. 11 (November 16, 2005): 1017. http://dx.doi.org/10.1182/blood.v106.11.1017.1017.
Анотація:
Abstract Factor VIII (FVIII) functions as a cofactor for factor IXa in the intrinsic tenase complex. This tenase activity is down-regulated by activated protein C (APC) or factor Xa (FXa). Plasmin, the most potent fibrinolytic protease, inactivates FVIII as well as other clotting factors. However, the mechanism of FVIII inactivation by plasmin is poorly understood. FVIII activity reached to the peak value of ~2-fold increase at 3 min after the addition of plasmin in a one-stage clotting assay. Then, the activity was decreased rapidly and was undetectable within 30 min. This time-dependent reaction was not affected in the presence of von Willebrand factor and phospholipid. The activation of FVIII by plasmin was an ~50% level of that by FXa. The rate constant (min-1) of inactivation of FVIIIa by plasmin possessed ~11.3- and ~2.5-folds greater than those by FXa and APC in the presence of protein S, respectively. SDS-PAGE analysis showed that plasmin cleaved the 90~210-kDa heavy chain of FVIII to 50, 48,45, 40, and 38-kDa fragments via 90-kDa fragment. Using western blot and N-terminal sequence analyses, these fragments derived from the heavy chain were identified as A11-372, A1337-372-A2, A11-336, A2, and A137-336, respectively, by cleavages at Arg372, Arg740, Lys36 and Arg336 in the A1 domain. On the other hand, the 80-kDa light chain was cleaved to 67-kDa fragment via 70-kDa fragment by cleavages at Arg1721 and Arg1689, respectively, consistent with the pattern of cleavage by FXa. However, the cleavage at Arg336 by plasmin was much quicker than that at Arg372, contrast with that by FXa. Furthermore, this cleavage was faster than that by APC, consistent with rapid inactivation of FVIII. In addition, the cleavage at Arg336 of FVIIIa by plasmin was faster than that of isolated A1 or A1/A3-C1–C2 dimer, different with that by FXa. These results demonstrate the importance of cleavage at Arg336 for the mechanism of plasmin-catalyzed FVIII inactivation. Furthermore, this cleavage appears to be selectively modulated by the A2 domain that may interact with plasmin.