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

Abstract

SUMMARY Background Central to appropriate thrombin formation at sites of vascular injury is the concerted assembly of plasma- and/or platelet-derived factor Va and factor Xa on the activated platelet surface. While the plasma-derived procofactor, factor V, must be proteolytically activated by α-thrombin to factor Va 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 factor V is proteolytically processed to form the platelet-derived pool. Methods & Results Subsequent to factor V endocytosis, a time-dependent increase in factor V 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 factor V/Va present in a platelet lysate as well as to the α-thrombin-activated factor Va heavy chain and light chain, and their respective precursors. Other proteolytic products were unique to endocytosed factor V. The product/precursor relationships of these fragments were defined using anti-factor V heavy and light chain antibodies with defined epitopes. Activity measurements indicated that megakaryocyte-derived factor V fragments exhibited substantial factor Va cofactor activity that was comparable to platelet-derived factor V/Va. Conclusions Taken together, these observations suggest that prior to its packaging in α-granules endocytosed factor V undergoes proteolysis by one or more specific megakaryocyte protease(s) to form the partially activated platelet-derived pool.

Platelet‐Derived Factor V Is a Critical Mediator of Arterial Thrombosis

“…The work of Paula Tracy and Ken Mann 9 has shown that human platelet FV released from α‐granules is in a partially activated state, contributing to localized assembly of the prothrombinase complex. Proteolysis takes place in megakaryocytes before packaging within platelet α‐granules . In summary, these observations elucidated factors potentially contributing to the prothombotic properties of platelet FV and suggested that platelet FV could significantly influence thrombosis.…”

A mathematical model of flow-mediated coagulation identifies factor V as a modifier of thrombin generation in hemophilia A:

“…However, it is unknown what the true correlation is between the two FV pools. Platelet FV is distinct from plasma FV due to modifications in megakaryocytes (39) and is more procoagulant than plasma FV (36,40). In our model both FV pools have the same biochemical characteristics.…”

Thrombin generation in a patient with an acquired high-titre factor V inhibitor

“…In contrast to mice, human platelets/ megakaryocytes do not express the protein, but vesiclestored factor V is taken up and partially activated by bone marrow megakaryocytes [3,4]. Activated factor V (FVa) functions as a procoagulant cofactor for FXa in the prothrombinase complex that converts prothrombin to thrombin, whereas noncleaved factor V has anticoagulant activities by accelerating inactivation of FVIIIa by activated protein C [5].…”