SkM and CM myosin-mediated prothrombinase activity results from contaminating phospholipids. Varying concentrations of rabbit SkM (A) or bovine CM (E) myosin were incubated with 5 nM fVa, 0.2 nM fXa, and 1 µM prothrombin in the presence of 5 mM CaCl₂, 150 mM NaCl, and 50 mM Tris, pH 7.85. Reactions were stopped after 5 min with EDTA, fIIa substrate S-2238 was added, and color development was measured in kinetic mode on a VersaMax microplate reader (Molecular Devices). Substrate development was converted to U/mL IIa using a standard curve. Myosin supported generation of thrombin in the absence of added phospholipid. SkM, 64 nM, (B-D) or 16 nM CM (F-H) were preincubated with lactadherin (B,F), annexin A5 (C,G) or phospholipase A2 (D,H) for 5 min before the addition of fVa, fXa, and prothrombin. Lactadherin and annexin A5 inhibited myosin-mediated thrombin activation at similar concentrations (half-maximal inhibition: 0.35 ± 0.07 and 0.33 ± 0.02 nM, respectively, for SkM; 3.0 ± 0.8 and 1.4 ± 0.3 nM, respectively, for CM). Phospholipase concentrations >32 mU/mL inhibited SkM- and CM-mediated thrombin activation >99% and >95%, respectively. Support of CM for Gla-domain–deleted fXa (GD-fXa, 0.2 nM) or fXa for 10 min was evaluated (I). GD-fXa (open squares) supported <1% of the activity supported by full fXa (open circles). To test inhibition in the presence of GD-fXa, CM (160 nM) was incubated with either lactadherin (J) or PLA2 (K) for 5 min before the addition of 5 nM fVa, 1 nM GD-fXa, and 1 µM prothrombin. The reaction was run for 10 min before quenching with EDTA. Lactadherin and PLA2 inhibited ∼95% of myosin-mediated activity. Data shows mean ± standard deviation (SD) for 4 (SkM) and 3 (CM) experiments, each performed in duplicate.