Specific Pharmacologic Targeting of Rho GTPases Rac1, Cdc42 and RhoA Reveals Their Differential and Critical Roles In Regulation of Platelet Activation

Abstract 2019

Rac1, Cdc42 and RhoA, members of the Rho family of small GTPases, play critical roles in reorganization of actin cytoskeleton and aggregation in platelets. Although they have been implicated in regulation of platelet activation, the unique and redundant roles of each of the Rho GTPase in various signaling cascades and the resulting functional outcomes have yet to be clearly defined. In this study we compared their roles in several aspects of platelet activation by utilizing three small molecule inhibitors, NSC23766, CASIN, and GO4, that specifically suppress endogenous Rac1, Cdc42, and RhoA activities, respectively. These novel pharmacological inhibitors are active in direct binding to their specific GTPase substrates, i.e. NSC23766 to Rac1, CASIN to Cdc42, and G04 to RhoA, and in interfering with the GTP loading exchange reactions of each Rho GTPase catalyzed by respective guanine nucleotide exchange factors at 5–50 uM concentration range. First, effector-domain pull down assays confirmed that treatment of platelets with NSC23766 (30 uM), CASIN (10 uM) or GO4 (30-50 uM) specifically blocked collagen induced Rac1-GTP, Cdc42-GTP, and RhoA-GTP formations, respectively. Incubation of platelets with NSC23766 (30 uM) or CASIN (10 uM) effectively inhibited collagen-induced phosphorylation of the Rac/Cdc42 effector, PAK1. Addition of GO4 (30 uM) to platelets prior to stimulation with thrombin blocked RhoA/ROCK mediated phosphorylation of myosin light chain (MLC). Second, incubation of aspirin treated platelets containing apyrase (3 U/ml) with CASIN (10 uM), but not NSC23766 (30 uM) or GO4 (30 uM), inhibited filopodia formation on immobilized fibrinogen or collagen-related peptide (CRP), a GPVI agonist. On the other hand, treatment of platelets with CASIN (10 uM) or GO4 (30 uM), but not with NSC23766 (30 uM), inhibited spreading of platelets on immobilized fibrinogen in the presence of aspirin and apyrase. Third, NSC23766 (3-30 uM), CASIN (3-10 uM), and GO4 (5-50 uM) all inhibited secretion from platelet granules and secretion-dependent aggregation induced by threshold concentration of ADP, collagen, CRP, or thrombin in a concentration-dependent manner. However, while CASIN (10 uM) or GO4 (30 uM) completely blocked collagen or CRP induced aggregation in aspirin treated platelets containing apyrase, NSC23766 (30 uM) showed no effect. Fourth, while pre-incubation of platelets with 5 uM CASIN or 10 uM G04 alone only partially (15%) inhibited CRP induced platelet aggregation in aspirin treated samples, CASIN at 10 uM or a combination of 5 uM CASIN and 5 uM G04 were able to inhibit platelet aggregation by 90%. Fifth, GO4 (30 uM) but not CASIN (10 uM) inhibited thrombin stimulated phosphorylation of p38-MAPK (137%) in aspirin treated platelets in the presence of apyrase. Addition of GO4 (30 uM) or CASIN (10 uM) to aspirin treated platelets containing apyrase inhibited CRP induced phosphorylation of ERK1/2 by 94% and 53% respectively, However, in the absence of aspirin and apyrase GO4 (30 uM), but not CASIN (10 uM), completely inhibited CRP induced phosphorylation of ERK1/2. Finally, although both GO4 (30 uM) and CASIN (10 uM) completely inhibited CRP induced phosphorylation of MLC in aspirin treated platelets containing apyrase, GO4 (30 uM) maximally (94%) while CASIN (10 uM) partially (36%) inhibited phosphorylation of MLC in the absence of aspirin and apyrase. Taken together, these data suggest that: (a) Cdc42 is involved in integrin alpha_IIbbeta₃ and GPVI mediated filopodia formation, RhoA is involved in regulation of integrin alpha_IIbbeta₃ induced platelet spreading, whereas Rac1 is critical in secondary mediators (ADP/TXA2) mediated lamellipodia formation; (b) Cdc42 and RhoA regulate platelet aggregation in parallel pathways, possibly by affecting the RhoA/ROCK-MAPK-dependent and -independent phosphorylation of MLC; and (c) the crosstalk among Cdc42, Rac1 and RhoA plays an important role in signaling cascades involved in platelet activation.