Abstract 1939: Genetic and Pharmacologic Targeting of Cdc42 GTPase Reveals its Critical Role in Regulation of Platelet Activation
A better understanding of the signaling mechanisms involved in platelet activation is essential for developing anti-platelet agents for prevention and management of atherothrombosis. Rac1 and Cdc42, members of the Rho family of small GTPases, play critical roles in reorganization of actin cytoskeleton in platelets. Rac1 GTPase has also been shown to be involved in regulation of platelet secretion and aggregation. In this study we investigated the role of Cdc42 in regulation of platelet function by investigating the effects of:
gene targeting of Cdc42 GTPase; or
inhibition of Cdc42 GTPase by a newly identified chemical inhibitor of Cdc42, CASIN (Cdc42 activity-specific inhibitor), on platelet activation.
Platelets from Cdc42−/− mice or human platelets pre-incubated with CASIN (10 μM) exhibited a complete lack of filopodia formation and spreading on collagen coated surfaces. Addition of CASIN, but not that of a pharmacologically inactive CASIN analog, to platelets blocked collagen induced activation of Cdc42 without detectably affecting Rac1 activity. Treatment of platelets with CASIN (1–10 μM) inhibited:
U46619-induced release of P-selectin and secretion of ATP from the α- and dense granules respectively;
aggregation induced by diverse agonists; and
collagen induced phosphorylation of Akt.
Furthermore, CASIN treatment inhibited:
collagen or phorbol 12, 13-dibutyrate induced aggregation in aspirinated platelets in the presence of apyrase; and
removal of CASIN from the platelet samples by repeated washings completely reversed inhibition of aggregation by collagen.
These data suggest that:
Cdc42 is uniquely involved in the regulation of platelet filopodia formation, spreading, secretion, and aggregation;
CASIN is capable of specifically and reversibly inhibiting Cdc42 activity in platelets; and
the Cdc42 targeting small molecule inhibitor, CASIN, may serve as a novel anti-platelet agent.
This research has received full or partial funding support from the American Heart Association, AHA Great Rivers Affiliate (Delaware, Kentucky, Ohio, Pennsylvania & West Virginia).