Cleavage of prothrombin by prothrombinase assembled on the activated platelet surface. Thrombin-activated platelets (1 × 10⁸/mL) were incubated with 1.4μM human prothrombin containing trace ¹²⁵I-prothrombin and 3μM DAPA. Prothrombinase assays were initiated by addition of 5nM factor Xa; aliquots were removed at timed intervals and analyzed by SDS-PAGE followed by phosphorimaging. (A) The assays were performed either relying on the platelet-released factor Va (left panel) or adding 5nM plasma-derived factor Va to saturate all factor Va binding sites on the platelet surface (right panel). (B) Prothrombin activation was similarly followed on the surface of either thrombin-activated platelets (left panel) or thrombin-activated platelets that had been washed 3 times after activation to remove any soluble materials released by the platelets (right panel). Under both conditions, plasma-derived factor Va (5nM) was added to saturate the activated platelet surface. All panels are labeled as described in Figure 2.