Effect of bound coagulation factors’ surface density on FX activation. (A) The 25:75 and 5:95 PS/PC vesicles were incubated with FIXa (1 nM), FVIIIa (20 nM), and FX (400 nM) in buffer A with 2.5 mM CaCl₂ while keeping the PS concentration the same. The reaction was stopped after 4 minutes by the addition of EDTA to a final concentration of 10 mM. Generated FXa was determined from the rate of conversion of chromogenic substrate S-2765 (final concentration, 0.4 mM). The rate of substrate hydrolysis was monitored by absorbance at 405 nm. Mean ± SEM are shown for n = 3 independent experiments. (B) Scheme of membrane-dependent reactions of the tenase complex assembling on the phospholipid surface. FX and FIXa are assumed to bind to specific sites on the membrane and compete for them (reaction 1). FVIII is assumed to be tightly bound to the membrane with the possibility of deactivation during experiment. Their pairwise membrane interactions (reaction 2) lead to the formation of the enzyme-cofactor-substrate complex (reaction 3) and FX activation (reaction 4). (C) Comparison of the velocity of FXa formation in the computer simulation similar in condition to the experiment in (A). To achieve the drop in the amount of formed FXa observed in the experiments, the rate constants of the on-membrane factors interactions were reduced 10-fold. (D-G) Computational model of platelets with a “cap” with characteristics similar to real platelets. (D) Initial distribution of FVIIIa and binding sites for FX and FIXa. (E) Parameters of FVIIIa, and binding sites for FX and FIXa on the model platelet. (F) FXa concentration around platelets with a “cap” after 30 seconds of FX activation; the initial concentrations of FX and FIXa were 160 nM and 30 pM, respectively. (G) Time course of FXa production for a platelet with a “cap,” a platelet with uniform distribution of FVIIIa and binding sites, and with the assumption of membrane-dependent reactions slowing for low PS content. Molec, molecule; plt, platelet; SEM, standard error of the mean.