cGMP signaling inhibits platelet shape change through regulation of the RhoA-Rho Kinase-MLC phosphatase signaling pathway

Abstract

Essentials Platelet shape change requires cytoskeletal rearrangement via myosin-mediated actin contraction. We investigated whether nitric oxide (NO) affected thrombin-induced platelet shape change. NO inhibits shape change, RhoA/ROCK signalling and myosin light chain (MLC) phosphorylation. NO promotes MLC phosphatase activity, thus prevents MLC phosphorylation and shape change.

Summary: Background Platelet shape change, spreading and thrombus stability require activation of the actin cytoskeleton contractile machinery. The mechanisms controlling actin assembly to prevent unwanted platelet activation are unclear. Objectives We examined the effects of nitric oxide on the signaling pathways regulating platelet actin-myosin activation. Results S-nitrosoglutathione (GSNO) inhibited thrombin-induced platelet shape change and myosin phosphorylation of the myosin light chain (MLC). Because thrombin stimulates phospho-MLC through the RhoA/ ROCK dependent inhibition of MLC phosphatase (MLCP) we examined the effects of NO on this pathway. Thrombin caused the GTP loading and activation of RhoA, leading to the ROCK-mediated phosphorylation of MLCP on threonine 853 (thr⁸⁵³ ), which is known to inhibit phosphatase activity. Treatment of platelets with GSNO blocked ROCK-mediated increases in phosphoMLCP-thr⁸⁵³ induced by thrombin. This effect was mimicked by the direct activator of protein kinase G, 8-pCPT-PET-cGMP, and blocked by the inhibition of guanylyl cyclase, but not inhibitors of protein kinase A. Further exploration of the mechanism demonstrated that GSNO stimulated the association of RhoA with protein kinase G (PKG) and the inhibitory phosphorylation (serine188) of RhoA in a cGMP-dependent manner. Consistent with these observations, in vitro experiments revealed that recombinant PKG caused direct phosphorylation of RhoA. The inhibition of RhoA by GSNO prevented ROCK-mediated phosphorylation and inhibition of MLCP activity. Conclusions These data suggest novel crosstalk between the NO-cGMP-PKG and RhoA/ROCK signaling pathways to control platelet actin remodeling.

Keywords: RhoA GTP-biding protein; cyclic GMP; myosin light chain phosphatase; nitric oxide; platelets; protein kinase A.