Discrepancies exist for some of the modified coagulation factors when assayed with different one-stage clotting and chromogenic substrate assay reagents. The aim of this study was to evaluate the performance of a recombinant factor VIII Fc fusion protein (rFVIIIFc), currently in clinical development for the treatment of severe haemophilia A, in a variety of one-stage clotting and chromogenic substrate assays in clinical haemostasis laboratories. Haemophilic plasma samples spiked with rFVIIIFc or Advate® at 0.05, 0.20 or 0.80 IU mL−1 were tested by 30 laboratories using their routine procedures and plasma standards. Data were evaluated for intra- and inter-laboratory variation, accuracy and possible rFVIIIFc-specific assay discrepancies. For the one-stage assay, mean recovery was 95% to 100% of expected for both Advate® and rFVIIIFc at 0.8 IU mL−1. Intra-laboratory percent coefficient of variance (CV) ranged from 6.3% to 7.8% for Advate®, and 6.0% to 10.3% for rFVIIIFc. Inter-laboratory CV ranged from 10% for Advate® and 16% for rFVIIIFc at 0.8 IU mL−1, to over 30% at 0.05 IU mL−1 for both products. For the chromogenic substrate assay, the average FVIII recovery was 107% ± 5% and 124% ± 8% of label potency across the three concentrations of Advate® and rFVIIIFc, respectively. Plasma rFVIIIFc levels can be monitored by either the one-stage or the chromogenic substrate assay routinely performed in clinical laboratories without the need for a product-specific rFVIIIFc laboratory standard. Accuracy by the one-stage assay was comparable to that of Advate®, while marginally higher results may be observed for rFVIIIFc when using the chromogenic assay.

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

The role of the factor IXa heparin-binding exosite in coagulation was assessed with mutations that enhance (R170A) or reduce (R233A) stability of the proteasefactor VIIIa A2 domain interaction. After tissue factor (TF) addition to reconstituted factor IX-deficient plasma, factor IX R170A supported a 2-fold increase in velocity index (slope) and peak thrombin concentration, whereas factor IX R233A had a 4-to 10-fold reduction relative to factor IX wild-type. In the absence of TF, 5 to 100 pM of factor IXa increased thrombin generation to approach TF-stimulated thrombin generation at 100% factor IX. Factor IXa R170A demonstrated a 2-to 3-fold increase in peak thrombin concentration and 5-fold increase in velocity index, whereas the response for factor IXa R233A was blunted and delayed relative to wild-type protease. In hemophilia B mice, factor IX replacement reduced the average time to hemostasis after saphenous vein incision, and the time to occlusion after FeCl 3 -induced saphenous vein injury. At 5% factor IX, the times to occlusion for factor IX wild-type, R170A, and R233A were 15.7 minutes, 9.1 minutes (P < . IntroductionThrombin is the penultimate product of the coagulation cascade, generated in an explosive burst on stimulation of plasma with limiting concentrations of tissue factor. 1,2 The measurement of plasma thrombin generation has merits as a global test of coagulation, and enhanced levels of thrombin generation have been associated with increased risk of recurrent venous thrombosis. 3 Thus, the rate and magnitude of thrombin generation may be predictive of the coagulation phenotype of patients. 4,5 In vitro and ex vivo modeling of the coagulation cascade indicates that factor X activation by the intrinsic tenase complex (factor IXa-factor VIIIa) is the rate-limiting step for thrombin generation. 1,2,6 Intrinsic tenase activity is unstable because of the diffusional loss of the noncovalently bound factor VIIIa A2 domain. 7,8 The instability of this enzyme complex is presumed to be an important regulator of the coagulation response.The mechanism(s) for activation of factor IXa within the intrinsic tenase complex are poorly understood. Factor VIII circulates as a heterodimer of A1-A2-B and A3-C1-C2 peptides with domain structure and metal-binding functions similar to ceruloplasmin. 9 Factor VIII undergoes proteolytic activation by thrombin, resulting in an unstable, metal-dependent A1/A2/A3-C1-C2 heterotrimer with cofactor activity. 10,11 Factor VIII or factor VIIIa light chain (A3-C1-C2 domains) bind to factor IXa on the phospholipid surface with an affinity that approaches intact factor VIIIa but lack cofactor activity. 12,13 The isolated factor VIIIa A1 domain also lacks cofactor activity. In contrast, the factor VIIIa A2 domain directly modulates the catalytic activity of factor IXa, which is further enhanced by the A1 domain, markedly increasing the k cat for factor X activation. 14,15 Although the isolated A2 domain binds with low affinity to factor IXa, it contributes significantly to protease-c...

IntroductionDepolymerized holothurian glycosaminoglycan (DHG) is a low molecular weight (average MW 12 500) fucosylated chondroitin sulfate isolated from the sea cucumber Stichopus japonicus and prepared by partial oxidative depolymerization with hydrogen peroxide. 1,2 DHG demonstrates antithrombotic efficacy in models of murine thrombin-induced pulmonary thromboembolism, thrombin-induced venous thrombosis in the rat, and canine dialysis during renal failure. 3-6 DHG does not bind antithrombin with high affinity, and exhibits antithrombin-independent antithrombotic efficacy in vivo. 3,7 Compared with equitherapeutic doses of unfractionated or low-molecular-weight heparins (LMWHs), DHG demonstrates significantly reduced effects on tail transection and template bleeding assays in rat and dog models. 4,5,8 Thus, DHG has potential as an antithrombotic agent with reduced bleeding risk relative to heparin. In vitro testing has suggested that DHG accelerates thrombin inhibition by heparin cofactor II (HCII), inhibits factor-VIII activation by thrombin, and inhibits factor X activation by the intrinsic tenase complex. 9-11 Herein, we investigate the relevant mechanism(s) for the antithrombotic effect of DHG in human plasma.In vitro and ex vivo modeling of the coagulation cascade indicates that factor X activation by the intrinsic tenase complex (factor IXa-factor VIIIa) is the rate-limiting step for thrombin generation. [12][13][14][15] The heparin-binding exosite on factor IXa is the interactive site for the factor VIIIa A2 domain, contributing to stabilization of cofactor activity and allosteric activation of the protease within the enzyme complex. [16][17][18] The physiologic importance of this exosite is demonstrated by its critical role in the regulation of thrombin generation in human plasma and saphenous vein thrombosis in the mouse. 19 In an experimental system with purified components, the factor IXa heparin-binding exosite is the molecular target for antithrombin-independent inhibition of the intrinsic tenase complex by both LMWH and DHG. 11,17 Since in vitro data demonstrates that DHG inhibits the intrinsic tenase complex by interacting with heparin-binding exosite of factor IXa, and this exosite is a critical regulator of plasma thrombin generation and murine venous thrombosis, we hypothesized that DHG regulates thrombin generation via interaction with the factor IXa heparin-binding exosite. The effect of DHG on plasma thrombin generation was evaluated by fluorogenic substrate cleavage and Western blot analysis in HCII-or mockimmunodepleted plasma, factor VIII-or IX-deficient human plasma, and factor IX-deficient plasma reconstituted with recombinant factor IX(a) possessing selected mutations in the heparinbinding exosite. The results demonstrate that DHG inhibits plasma thrombin generation by targeting the heparin-binding exosite of factor IXa. Inhibition of plasma thrombin generation by DHG was independent of effects on factor VIII or IX activation or acceleration of thrombin inhibition by HCII. These data...