Abstract

The anti-bleeding therapy, recombinant factor VIIa (rFVIIa), is thought to bind to the platelet’s surface and increase thrombin generation in hemophilia. However, high plasma levels of rFVIIa are required, in part, due to the weak binding of rFVIIa to platelets. We hypothesized that the efficacy of the therapy could be improved by administering rFVIIa already bound to platelets. A recently described protocol involving pretreatment of platelets with paraformaldehyde permits platelets to be lyophilized while preserving many platelet functions. Such platelets could be used for binding rFVIIa ex vivo and then administered to hemophilic patients during a bleeding event. In this study, we have characterized the ability of reconstituted, lyophilized (RL) platelets to support thrombin generation under normal and hemophilic conditions and in the presence of rFVIIa. First, freshly-isolated (control) or RL platelets were incubated with factors IXa, VIII(a), X, V and II in the presence of 3 mM CaCl2 and assayed for thrombin generation. In these assays, both freshly-isolated and RL platelets supported thrombin generation (1.15x10−4 +/− 5.37x10−5 mOD/min2/platelet and 8.46x10−3 +/− 4.78x10−3 mOD/min2/platelet, respectively). In the absence of factor IX (hemophilia B), thrombin generation was significantly reduced on both freshly-isolated and RL platelets (4.19x10−6 +/− 4.50x10−6 mOD/min2/platelet and 8.25x10−4 +/− 1.13x10−6 mOD/min2/platelet, respectively). Interestingly, RL platelets supported 10 – 100-fold higher thrombin generation rates than fresh thrombin-activated platelets. Second, we examined the activity of rFVIIa on RL platelets in the absence of factors IX and VIII. RFVIIa increased thrombin generation on RL platelets in a rFVIIa-concentration dependent manner (between 1nM and 150nM), similar to that seen when using fresh platelets. An inhibitory anti-tissue factor (TF) antibody did not affect rFVIIa-mediated thrombin generation on RL platelets, indicating that the activity of rFVIIa on RL platelets is independent of TF. Finally, we examined the effect of different platelet agonists (thrombin, convulxin, and A23187) on fresh and RL platelets. When fresh platelets are stimulated with A23187 or co-stimulated with thrombin and convulxin, they become more procoagulant than platelets activated with thrombin alone. However, stimulation of RL platelets with A23187 or co-stimulation with thrombin and convulxin did not increase thrombin generation versus thrombin alone. RL platelets stimulated with thrombin, alone, had 3.1-fold higher activity than thrombin- and convulxin-costimulated fresh platelets, but 1.4-fold lower activity than A23187-stimulated fresh platelets. These data suggest that RL platelets are in a maximally active state prior to the addition of platelet agonists. We conclude that RL platelets are procoagulant and can support rFVIIa-mediated thrombin generation in the absence of factor IX. We hypothesize that co-administration of RL platelets with rFVIIa may increase the efficacy of rFVIIa treatment.

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