Determination of kinetic constants governing the cleavage of rP2 and rMZ by prothrombinase assembled on the activated platelet membrane. Prothrombinase assays were performed as described in Figure 3. Reaction mixtures contained 2 × 10⁷/mL thrombin-activated platelets, equimolar concentrations of added plasma-derived factors Va and Xa (5nM), and various concentrations of rP2 (A) or rMZ (B). Subsequent to initiation of the reaction, aliquots were removed at timed intervals and analyzed by SDS-PAGE followed by densitometric analyses of protein bands visualized by phosphorimaging. The initial rates of cleavage of the variants were plotted as a function of the substrate concentration. Substrate consumption was less than 10% under all conditions. The data were fit to the Michaelis-Menten equation using GraphPad Prism software v5.0. The data shown, including the calculated K_m and V_max values, are from one of 2 platelet donors and are representative of experiments performed using platelets from both persons.