Interaction of Recombinant Factor VIIa with Rehydrated, Lyophilized Platelets.

The experiments presented here were undertaken to determine if factor VIIa (rFVIIa, the Novo Nordisk product NovoSeven^™) will directly bind to rehydrated, lyophilized (RL) platelets (Stasix^™ platelets, Entegrion, Inc. trade) for the formation of a catalytic surface with an enhanced ability to generate thrombin. The relationship of rFVIIa to the RL platelet surface was examined by measuring equilibrium and non-equilibrium binding of the coagulation factor to the cells, by studying the subcellular localization of the coagulation factor on RL platelets, and by following the effects of the surface modification on the kinetics of thrombin generation. The association of rFVIIa with RL platelets occurred with an on rate of 3.6x10³ sec⁻¹moles⁻¹. Saturation occurred in minutes and was calcium dependent. Disassociation (in plasma or citrated saline) was slow, with over half of the coagulation factor remaining bound after two hours (with slow and fast rate constants of 5.0x10⁻⁵ and 4.1x10⁻⁴ sec⁻⁵ respectively). These results define a binding site with an apparent equilibrium constants of 110 nM. Equilibrium binding of rFVIIa to RL platelets was analyzed with flow cytometry and Western analysis. The rFVIIa was bound to RL platelets in a dose-dependent manner when incubated at concentrations of 0.3 to 10.0 uM rFVIIa and 3x10⁴ to 10⁶ RL platelets/ul in citrated saline. When high concentrations of rFVIIa were bound to RL platelets densities of over one million molecules of rFVIIa per RL platelet was obtained. Fluorescent microscopy analysis revealed that the rFVIIa was localized to the surface membrane and that some rFVIIa localized internally to the outer surface of the surface connected open canalicular system and/or sites of internal trafficking. Flow cytometric analysis with annexin V demonstrated that considerable quantities of phosphatidylserine were present on the external surface of the RL platelet membrane for potential facilitation of rFVIIa binding. The effect of RL platelet surface modification by rFVIIa on thrombin generation was investigated by following the hydrolysis of the thrombin-specific fluorogenic substate D-phe-pro-arg-ANSNHin plasma. rFVIIa and RL platelets accelerated thrombin generation in this system with rFVIIa being approximately twice as effective (per molecule of the recombinant protein) when added to the assay system pre-bound to RL platelets as compared to being initially free in the plasma. Similar results were obtained when free and RL platelet bound rFVIIa were tested in factor IX-deficient plasma. These experiments show that rFVIIa retains activity when super-saturated on the RL platelet membrane. The results of the studies presented here suggest that RL platelets can be used to concentrate rFVIIa at sites of vascular injury.