Both Rho-kinase and the Ca2+/calmodulin-dependent myosin light chain (MLC) kinase increase the phosphorylation of MLC. We show that upon thrombin receptor stimulation by low-dose thrombin or the peptide ligand YFLLRNP, or upon thromboxane receptor activation by U46619, shape change and MLC phosphorylation in human platelets proceed through a pathway that does not involve an increase in cytosolic Ca2+. Under these conditions, Y-27632, a specific Rho-kinase inhibitor, prevented shape change and reduced the stimulation of MLC-phosphorylation. In contrast, Y-27632 barely affected shape change and MLC-phosphorylation by adenosine diphosphate (ADP), collagen-related peptide, and ionomycin that were associated with an increase in cytosolic Ca2+ and inhibited by BAPTA-AM/EGTA treatment. Furthermore, C3 exoenzyme, which inactivates Rho, inhibited preferentially the shape change induced by YFLLRNP compared with ADP and ionomycin. The results indicate that the Rho/Rho-kinase pathway is pivotal in mediating the MLC phosphorylation and platelet shape change by low concentrations of certain G protein–coupled platelet receptors, independent of an increase in cytosolic Ca2+. Our study defines 2 alternate pathways, Rho/Rho-kinase and Ca2+/calmodulin-regulated MLC-kinase, that lead independently of each other through stimulation of MLC-phosphorylation to the same physiological response in human platelets (ie, shape change).
Oxidised low density lipoprotein (LDL) plays an important role in the pathogenesis of atherosclerosis. Here we demonstrate that mildly oxidised (mox) LDL engages the GTPase Rho and its effector molecule p160 Rho-kinase to induce phosphorylation of myosin light chain and of moesin leading to platelet shape change. Pretreatment of platelets with the selective Rho inhibitor C3-transferase from Clostridium botulinum or with the Rho-kinase inhibitor Y-27632 blocked mox-LDL-induced myosin light chain phosphorylation, moesin phosphorylation and shape change. Mox-LDL did not induce an increase in cytosolic Ca 2 during shape change. We propose that Rho/Rho-kinase inhibition could be a strategy for prevention of the pathologic platelet activation during atherogenesis.z 2000 Federation of European Biochemical Societies.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.