Cleavage of prothrombin variants by prothrombinase assembled on activated platelets or PC/PS vesicles. Prothrombinase assays were performed, as described in the legend to Figure 3, using either the prothrombin variant rP2 (A-B), which may only be cleaved at Arg271 to generate fragment 1.2 (F1.2) and prethrombin-2 (P2), or rMZ (C-D), which may only be cleaved at Arg320 to generate meizothrombin consisting of F1.2.A and B chains. Reactions were performed using either: (A,C) 1 × 10⁸/mL thrombin-activated platelets, 5nM factor Xa, and 5nM factor Va; or (B,D) 20μM PC/PS vesicles, 5nM factor Xa, and 0.5nM factor Va. Aliquots were removed at timed intervals and analyzed by SDS-PAGE followed by phosphorimaging. The locations of rP2, rMZ, and their cleavage products are indicated at the right of each panel. Time points are indicated at the top, and molecular weight markers are indicated on the left.