Proteolysis of plasma‐derived factor V following its endocytosis by megakaryocytes forms the platelet‐derived factor V/Va pool

Summary Background Central to appropriate thrombin formation at sites of vascular injury is the concerted assembly of plasma‐ and/or platelet‐derived factor (F) V a and FX a on the activated platelet surface. While the plasma‐derived procofactor, FV, must be proteolytically activated by α‐thrombin to FVa to function in prothrombinase, the platelet molecule is released from α‐granules in a partially activated state, obviating the need for proteolytic activation. Objectives The current study was performed to test the hypothesis that subsequent to its endocytosis by megakaryocytes, plasma‐derived FV is proteolytically processed to form the platelet‐derived pool. Methods & Results Subsequent to FV endocytosis, a time‐dependent increase in FV proteolytic products was observed in megakaryocyte lysates by SDS ‐ PAGE followed by phosphorimaging or western blotting. This cleavage was specific and resulted in the formation of products similar in size to FV/ V a present in a platelet lysate as well as to the α‐thrombin‐activated FVa heavy chain and light chain, and their respective precursors. Other proteolytic products were unique to endocytosed FV. The product/precursor relationships of these fragments were defined using anti‐FV heavy and light chain antibodies with defined epitopes. Activity measurements indicated that megakaryocyte‐derived FV fragments exhibited substantial FVa cofactor activity that was comparable to platelet‐derived FV/ V a. Conclusions Taken together, these observations suggest that prior to its packaging in α‐granules endocytosed FV undergoes proteolysis by one or more specific megakaryocyte protease(s) to form the partially activated platelet‐derived pool.