Binding studies of the enzyme (factor IXa) with the cofactor (factor VIIIa) in the assembly of factor-X activating complex on the activated platelet surface.
Activated platelet membranes expose binding sites for the enzyme factor (F)IXa, the substrate (FX) and the cofactor (FVIIIa) that colocalize to assemble the FX-activating complex and promote optimal rates of FX activation. To determine the stoichiometry and affinity of binding to activated platelets, coordinate, equilibrium binding studies with enzyme (125I-FIXa) and cofactor (131I-FVIII or 131I-FVIIIa) were carried out in the presence of saturating concentrations of substrate (FX). Results of these studies indicate that in the presence of FX (1.5 micro m), the enzyme (active-site-inhibited Glu-Gly-Arg-FIXa, EGR-FIXa) and procofactor (FVIII) bind to an equal number (approximately 700 sites/platelet) of receptors whereas the active cofactor (FVIIIa) binds an additional approximately 500 high-affinity FVIIIa binding sites per platelet (Kd approximately 0.8 nm). With excess zymogen (FIX) to block shared FIX/FIXa-binding sites, the stoichiometry of 125I-FIXa and 131I-FVIIIa binding was 1:4. These FIXa/FVIIIa binding studies together with previously reported evidence of the coordinate binding of FVIIIa and FX to equivalent numbers of binding sites on activated platelets provide strong evidence to support the conclusion that FVIIIa comprises the receptor that presents FX to FIXa for efficient catalysis on the activated platelet membrane.
Ahmad
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SS
The Sol Sherry Thrombosis Research Center and Department of Biochemistry, Temple University School of Medicine, Philadelphia, PA 19140, USA.
London
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FS
Walsh
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PN
D001345:Autoradiography; D001665:Binding Sites; D001792:Blood Platelets / Q000378:metabolism / Q000648:ultrastructure; D002462:Cell Membrane / Q000737:chemistry / Q000378:metabolism; D003546:Cysteine Endopeptidases; D015949:Factor IXa / Q000378:metabolism*; D015944:Factor VIIIa / Q000378:metabolism*; D005170:Factor X / Q000378:metabolism*; D006801:Humans; D007457:Iodine Radioisotopes; D007700:Kinetics; D009363:Neoplasm Proteins; D015539:Platelet Activation; D011485:Protein Binding
D007457:Iodine Radioisotopes; D009363:Neoplasm Proteins; D015944:Factor VIIIa; D005170:Factor X; D015949:Factor IXa; D003546:Cysteine Endopeptidases; C030910:cancer procoagulant