An Important Role for Rac1 and RhoA In Regulating Platelet Microparticle Formation and Phosphatidylserine Exposure

Abstract 2023

Platelets activated by physiological agonists such as thrombin and collagen shed procoagulant microparticles (MPs) and externalize the procoagulant phospholipid phosphatidylserine (PS), both of which are critical to hemostasis and play an important role in inflammation. To date, the signaling mechanisms that regulate agonist-induced MP formation and PS exposure in platelets remain unclear. In this study, we demonstrate that the small GTPases Rac1 and RhoA play important roles in regulating the procoagulant activity of platelets. Rac1 null (-/-) mouse platelets or human platelets treated with the Rac1 inhibitor NSC23766 (NSC) displayed a significant defect in MP formation and PS exposure induced by various agonists. Furthermore, Rac1^-/- platelets and NSC-treated human platelets displayed a defect in procoagulant activity as demonstrated by a prolonged coagulation time following recalcification of citrated PRP. The stimulatory role of Rac1 in platelet MP formation and PS exposure is distinct from the known function of Rac1 in facilitating platelet granule secretion and secretion-dependent amplification of platelet aggregation, because supplementation of the granule content ADP rescued the defect in platelet aggregation caused by Rac1 inhibition, but failed to rescue the defect in MP formation caused by Rac1 inhibition. In contrast to Rac1, RhoA plays an inhibitory role in regulating platelet procoagulant activity, because treatment of platelets with the Rho inhibitor C3-toxin (C3) significantly enhanced agonist-induced MP formation, PS exposure, and procoagulant activity. The enhancing effect of C3 on platelet procoagulant activity is not caused by an overall enhancement of platelet activation because C3 significantly inhibited platelet secretion and aggregation. Thus, our data demonstrates that while Rac1 and RhoA both play important stimulatory roles in platelet granule secretion and aggregation, they play opposing roles in MP formation and PS exposure in platelets. Rac1 is important for stimulating platelet MP formation, PS exposure, and procoagulant activity, which is antagonized by RhoA.