Therapeutic doses of recombinant factor VIIa in hemophilia generates thrombin in platelet-dependent and -independent mechanisms.

BACKGROUND

In hemophilia bypass therapy, a platelet-dependent mechanism is believed to be primarily responsible for recombinant factor VIIa (rFVIIa)'s hemostatic effect. rFVIIa may also possibly interact with other cells through its binding to endothelial cell protein C receptor (EPCR) or cell surface phospholipids.

OBJECTIVES

We aim to investigate the relative contribution of platelet-dependent and platelet-independent mechanisms in rFVIIa-mediated thrombin generation in hemophilic conditions at the injury site.

METHODS

Platelets were depleted in acquired and genetic hemophilia mice using anti-platelet antibodies. The mice were subjected to the saphenous vein injury, and the hemostatic effect of pharmacological concentrations of rFVIIa was evaluated by measuring thrombin generation at the injury site.

RESULTS

Administration of anti-mouse CD42 antibodies to mice depleted platelets by more than 95%. As expected, hemophilia mice, compared with wild-type mice, generated only a small fraction of thrombin at the injury site. The depletion of platelets in hemophilia mice further reduced thrombin generation. However, when pharmacological doses of rFVIIa were administered to hemophilia mice, substantial amounts of thrombin were generated even in the platelet-depleted hemophilia mice. No differences in thrombin generation were detected among FVIII-/- , EPCR-deficient FVIII-/- , and EPCR-overexpressing FVIII-/- mice depleted of platelets or not. Evaluation of platelets by flow cytometry as well as immunoblot analysis showed no detectable expression of EPCR.

CONCLUSIONS

Our data suggest that pharmacological concentrations of rFVIIa generate thrombin in hemophilia in both platelet-dependent and platelet-independent mechanisms.