Skeletal muscle myosin promotes thrombin generation in purified prothrombinase reaction mixtures. (A) The effect of varying concentrations of myosin or prothrombin on the initial rate of prothrombin activation by factor Xa/factor Va. (B) The effects of myosin (●) or PCPS vesicles (Ο) on the initial rate of prothrombin activation by factor Xa/factor Va. (C) The effects on myosin’s enhancement of purified prothrombinase activity for 2 allosteric inhibitors of myosin (8 μM MyoVin-1 and 100 μM trifluoperazine) and for anti–myosin antibodies (abs) (0.9 mg/mL total protein), and the effect of pull-down of myosin using anti–myosin monoclonal antibody beads. (D) The effects of myosin on the initial rate of prothrombin activation by factor Xa/factor Va (●) and by DG-factor Xa/factor Va (Ο). Varying concentrations of myosin or phospholipid vesicles (PCPS, 80%/20% wt/wt) were incubated with factor Va (5 nM, final) and factor Xa (0.2 nM final) in TBS containing 0.5% BSA (TBSA) plus 5 mM CaCl₂ at room temperature. Thrombin generation was initiated by the addition of prothrombin (concentration as indicated for panel A, 1.5 μM final for panel B, and 0.75 μM final for panels C and D) in TBSA containing 5 mM CaCl₂. The reaction was quenched by adding EDTA (10 mM final) at 5 min (A) or 10 min (C,D). The reactions shown in panel B were quenched by EDTA (10 mM final) at 20, 40, and 60 seconds. Thrombin formation was quantified by the rate of substrate (Pefa TH) hydrolysis. For panel D, DG-factor Xa was used in place of factor Xa following the same protocol.