The Development of Novel Hemostatic Bypassing Molecules.

Abstract SCI-20

Blood coagulation factors VIII and IX are part of the intrinsic pathway and play a critical role in maintaining normal hemostasis by activating factor X. Deficiency of either of these proteins caused by mutations in the genes encoding FVIII or FIX leads to hemophilia A or B (HA and HB), respectively. In each disease, there is impairment of the intrinsic pathway with inadequate thrombin (IIa) generation and defective hemostasis. Unfortunately, ∼20-30% of patients with FVIII deficiency and ∼3-5% of patients with FIX deficiency develop inhibitory antibodies to infused factor replacement products. This has prompted the research community to develop so-called “bypass strategies” which use other coagulation factors to provide hemostasis in these patients. In principle, infusion of FXa should bypass deficiencies in the intrinsic pathway; however it is generally thought that it has limited utility since the infused FXa could cause excessive activation of coagulation and/or more importantly, FXa is rapidly inactivated by plasma inhibitors resulting in a very short half-life (<2 min). We recently characterized variants of FXa (FXa-I16L and FXa-V17a) which have “zymogen-like” properties that could circumvent these associated problems (JBC 2008; 283: 18627). For example we have found that i) these proteins have an incompletely formed active site, making them resistant to plasma protease inhibitors; ii) in the absence of FVa, the FXa variants are, in general, refractory to active site functions and thus do not activate FVII, FV, FVIII, and prothrombin very well; and iii) the variants are thermodynamically rescued by FVa; thus at the site of injury on the activated platelet surface, where FVa is present, prothrombinase rapidly forms generating a burst of thrombin. We have begun to exploit these unique properties and evaluate whether these FXa variants could be effective and safe in bypassing the hemophilic phenotype both in vitro and in vivo. Clotting and IIa generation assays in human HA, HB and inhibitor plasma revealed that FXa-I16L could completely restore IIa generation in a FVa-dependent fashion. Furthermore, the zymogen-like conformation protects FXa-I16L in human plasma as it has a prolonged half-life (∼2 hr) versus wt-FXa (<2 min). In vivo studies using HB mice revealed that administration of FXa-I16L via tail vein almost completely corrected the prolonged aPTT. The aPTT was shortened for more than 2 hours and returned close to the starting value after 24 hr. In these experiments, infusion of the protein was well tolerated as platelet levels were unaffected over the course of the experiment with little or no change in the values for TAT, D-dimer, and fibrinogen. Next we tested whether the improvement of the clotting times was associated with in vivo hemostatic performance. Using three separate injury models (tail clip assay, FeCl2 carotid artery injury model, and cremaster muscle laser injury model) infusion FXa-I16L provided effective hemostasis. This was in contrast to the infusion of wild-type FXa which was ineffective. Taken together our data show that FXa-I16L is highly effective in correcting the hemostatic defect in human hemophilic plasma. Furthermore it improves the hemophilic phenotype in HB mice following a series of hemostatic challenges and can restore thrombus formation upon injury at both micro and macrocirculation levels. Thus zymogen-like variants of FXa have properties that indicate their ability to serve as superior therapeutic procoagulants for bypassing deficiencies upstream of the common pathway.