Platelet receptor occupancy with factor IXa promotes factor X activation.
To investigate the activated platelet surface as a locus for factor X activation, the functional consequences of factor IXa binding to platelets were studied. The concentration of factor IXa required for half-maximal rates of factor X activation in the presence of factor VIIIa and thrombin-activated platelets was 0.53 nM, which is close to the Kd (0.56 nM) for factor IXa binding to platelets under identical conditions, determined from equilibrium binding studies. In direct comparative experiments, there was a close correspondence between equilibrium binding of factor IXa to thrombin-activated platelets in the presence of factor VIIIa and kinetic determinations of factor X activation rates. Analysis by polyacrylamide gel electrophoresis revealed that 125I-labeled factor IXa bound to platelets was structurally intact and did not form covalent complexes with platelet proteins. Factor IXa active site-inhibited by 5-dimethylaminonaphthalene-1-sulfonyl glutamyl-glycylarginyl chloromethyl ketone was shown to be a competitive inhibitor of factor IXa binding in the absence (Ki = 2.3 nM) and presence (Ki = 0.43 nM) of factor VIIIa and factor X and of factor X activation (Ki = 0.4 nM) by factor IXa in the presence of factor VIIIa, indicating that the generation of factor Xa is not required for factor IXa binding and that factor IXa bound to activated platelets in the presence of factor VIIIa is closely coupled with rates of factor X activation. We conclude that factor IXa bound tightly to a platelet receptor in the presence of factor VIIIa is the enzyme active in factor X activation.
Ahmad
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SS
Department of Medicine, Temple University School of Medicine, Philadelphia, Pennsylvania 19140.
Rawala-Sheikh
R
R
Walsh
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PN
D001792:Blood Platelets / Q000502:physiology*; D004789:Enzyme Activation; D015949:Factor IXa / Q000378:metabolism*; D005170:Factor X / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008962:Models, Theoretical; D015539:Platelet Activation; D011956:Receptors, Cell Surface / Q000378:metabolism*; D013917:Thrombin / Q000502:physiology
D011956:Receptors, Cell Surface; C052276:factor IX-IXa receptor; D005170:Factor X; D015949:Factor IXa; D013917:Thrombin