Myosin is thrombogenic in ex vivo studies of fresh human blood under flow. The thrombogenicity of myosin was studied either (A) when myosin (or control vehicle) was added to whole blood that then flowed over collagen-coated surfaces or (B) when whole blood without myosin addition flowed over myosin-coated surfaces. Data represent observations at 4 minutes after flow initiation at 300 s⁻¹ wall shear rate. (A) Either skeletal muscle myosin (50 µg/ml) or control buffer vehicle was added to recalcified blood before its perfusion over a collagen-coated surface. Three-dimensional reconstruction of platelet aggregates and fibrin on a collagen-coated coverslip was generated from confocal z-sections serially collected after blood perfusion. Recalcified flowing human blood contained mepacrine (green) to visualize platelets and Alexa Fluor 546–labeled anti–fibrin antibody (red) to visualize fibrin. Yellow represents superposition of corresponding images at 4 minutes. White bars shown in the top panels indicate 20 μm. The amounts of fibrin and platelet deposition are shown in the bar graphs (average values from 2 experiments). (B) Recalcified human blood was perfused over immobilized myosin at 300 s⁻¹ shear rate. Three-dimensional reconstruction of platelet aggregates and fibrin on myosin or BSA coated coverslip was generated as described for panel A. Images of deposited platelets and fibrin formation after maintaining blood perfusion for 4 minutes are shown. The volumes of fibrin and platelet deposition are shown in the bar graphs (average values from 3 experiments). Human blood perfused over immobilized BSA at 300 s⁻¹ shear rate for 4 minutes did not form detectable platelet aggregates or fibrin (data not shown).