Functional characterization of human platelet-released factor V and its activation by factor Xa and thrombin.
The functional characterization of human platelet-released factor V and its activation by factor Xa and thrombin was studied by functional assessment of cofactor activity and Western blotting analyses of platelet releasates, obtained by stimulating washed suspensions of platelets with various agonists, including collagen, collagen with ADP, and the calcium ionophore A23187. Platelet factor V was released as a partially proteolyzed molecule that was bound to platelet microparticles, irrespective of the agonist used. Radiolabeled plasma factor V was not cleaved for up to 30 min following release when added to platelets prior to stimulation, suggesting that platelet factor V was stored in a partially proteolyzed form. Released platelet factor V possessed significant cofactor activity that was increased only 2-3-fold by either factor Xa or thrombin. The factor V subunits that expressed cofactor activity were isolated and found to consist of peptides of Mr = 220,000 and 150,000. Incubation of released platelet factor V with factor Xa or thrombin yielded the same cleavage pattern, in which two peptides of Mr = 105,000 and 74,000 appeared to be electrophoretically indistinguishable from thrombin-activated plasma factor V. Under the conditions of these studies, factor Xa activated platelet-released factor V 50-100 times more effectively than thrombin. This observation may be due in part to the existence of platelet factor V in a partially proteolyzed state, or its association with platelet microparticles following platelet stimulation. These data collectively suggest that platelet-released factor V may be the foremost initiator of prothrombinase complex assembly and function during the early stages of coagulation with additional cofactor activation accomplished by factor Xa.
Monković
D D
DD
Department of Biochemistry, University of Vermont, Burlington 05405.
Tracy
P B
PB
D000244:Adenosine Diphosphate / Q000494:pharmacology; D000328:Adult; D001792:Blood Platelets / Q000187:drug effects / Q000201:enzymology*; D000001:Calcimycin / Q000494:pharmacology; D003094:Collagen / Q000494:pharmacology; D004789:Enzyme Activation; D005165:Factor V / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D015951:Factor Xa / Q000378:metabolism*; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D046911:Macromolecular Substances; D008970:Molecular Weight; D013917:Thrombin / Q000378:metabolism*
D046911:Macromolecular Substances; D000001:Calcimycin; D000244:Adenosine Diphosphate; D005165:Factor V; D003094:Collagen; D013917:Thrombin; D015951:Factor Xa