title: Proteolysis of plasma-derived factor V following its endocytosis by megakaryocytes forms the platelet-derived factor V/Va pool.
abstract: BACKGROUND
  Central to appropriate thrombin formation at sites of vascular injury is the concerted assembly of plasma- and/or platelet-derived factor (F) Va and FXa 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.
  
  
  OBJECTIVE
  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.
  
  
  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/Va 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/Va.
  
  
  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.
authors.affiliation: Department of Biochemistry, University of Vermont College of Medicine, Burlington, VT 05405, USA.
authors.forename: F
authors.initials: F
authors.lastname: Ayombil
authors.forename: S
authors.initials: S
authors.lastname: Abdalla
authors.forename: P B
authors.initials: PB
authors.lastname: Tracy
authors.forename: B A
authors.initials: BA
authors.lastname: Bouchard
mesh_terms: D018952:Antigens, CD34 / Q000378:metabolism; D001779:Blood Coagulation Factors / Q000378:metabolism*; D001792:Blood Platelets / Q000378:metabolism; D004705:Endocytosis; D000939:Epitopes / Q000378:metabolism; D005165:Factor V / Q000378:metabolism*; D015943:Factor Va / Q000378:metabolism*; D006801:Humans; D008533:Megakaryocytes / Q000166:cytology*; D015539:Platelet Activation; D059748:Proteolysis; D013917:Thrombin / Q000378:metabolism; D013925:Thromboplastin / Q000378:metabolism
chemical_list: D018952:Antigens, CD34; D001779:Blood Coagulation Factors; D000939:Epitopes; C037685:platelet factor V; D015943:Factor Va; D005165:Factor V; D013925:Thromboplastin; D013917:Thrombin
keywords: factor V; megakaryocyte; platelet; protease; prothrombinase