Coordinate binding studies of the substrate (factor X) with the cofactor (factor vIII) in the assembly of the factor X activating complex on the activated platelet surfacet

The assembly of the factor X activating complex on the platelet surface requires the occupancy of three receptors: (1) enzyme factor IXa, (2) cofactor factor VIII(a), and (3) substrate factor X. To further evaluate this three-receptor model, simultaneous binding isotherms of I-125-factor X and I-131-factor VIII(a) to activated platelets were determined as a function of time and also as a function of the concentrations of both ligands in the presence of active site-inhibited factor IXa (45 nM) and 5 mM CaCl2. In the presence of active site-inhibited factor IXa and factor VIIIa there are two independent factor X binding sites: (1) low affinity, high capacity (similar to9000 sites/platelet; K-d similar to380 nM) and (2) low capacity, high affinity (1700 sites/platelet; K-d similar to30 nM). A single specific and selective factor X binding site was expressed (1200 sites/platelet; K-d similar to9 nM) when the shared factor X/factor II site was blocked by excess factor II (4 muM). In the presence of active site-inhibited factor IXa (4 nM) and factor II (4 muM), factor X binds to 3-fold more platelet sites than procofactor VIII with relatively low affinity (K-d similar to250 nM). The activation of procofactor VIII to factor VIIIa increases the affinity of binding to platelets of both factor VIIIa (-4-fold to K-d similar to0.8-1.5 nM) and factor X (similar to25-50-fold to K-d similar to5-9 nM). In the presence of excess zymogen factor IX, which blocks the shared factor IX/factor IXa binding site, the substrate, factor X, and the active cofactor, factor VIIIa, form a 1: 1 stoichiometric complex. These coordinate binding studies support the conclusion that factor X initially binds to a high-capacity, low-affinity platelet binding site shared with prothrombin, which then presents factor X to a specific high-affinity site consisting of factor VIIIa bound to a high-affinity, low-capacity receptor on activated platelets.