Inactivation-Resistant Recombinant Factor VIII Exhibits Superior Thrombin Generation Capacity in Comparison to Wild-Type and B Domain-Deleted Factor VIII.
Authors: Russell Joshua , Dargaud Yesim , Kaufman Randal J. , Negrier Claude , Pipe Steven W. 
Published: October 2019

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.