Abstract/Summary

ABSTRACT Background: Active and passive biomechanical properties of platelets contribute substantially to thrombus formation. Actomyosin contractility drives clot contraction required for stabilizing the hemostatic plug. Impaired contractility results in bleeding but is difficult to detect using platelet function tests. Objectives: To determine how diminished myosin activity affects platelet functions including and beyond clot contraction. Methods: Using the myosin IIA-specific pharmacological inhibitor blebbistatin, we tuned myosin activity in platelets from healthy donors and systematically characterized platelet responses at various levels of inhibition using a range of complementary assays interrogating distinct platelet functions at each stage of thrombus formation. Results: Partial myosin IIA inhibition did not affect platelet von Willebrand Factor interactions under arterial shear nor spreading and cytoskeletal rearrangements on fibrinogen. However, it impacted stress fiber formation and the nanoarchitecture of cell-matrix adhesions, drastically reducing and limiting traction forces. Higher blebbistatin concentrations impaired platelet adhesion under flow, altered mechanosensing at lamellipodia edges, and eliminated traction forces without affecting spreading, α-granule secretion, or procoagulant platelet formation. Unexpectedly, myosin IIA inhibition reduced calcium influx, dense granule secretion and platelet aggregation downstream of glycoprotein (GP) VI and limited the redistribution of GPVI on the cell membrane, whereas aggregation induced by ADP or arachidonic acid were unaffected. Conclusions: Our findings highlight the importance of both active contractile and passive crosslinking roles of myosin IIA in the platelet cytoskeleton. They support the hypothesis that highly contractile platelets are needed for hemostasis and further suggest a supportive role for myosin IIA in GPVI signaling.