Dichotomous regulation of myosin phosphorylation and shape change by Rho-kinase and calcium in intact human platelets.
Both Rho-kinase and the Ca(2+)/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 Ca(2+). 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 Ca(2+) 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 Ca(2+). Our study defines 2 alternate pathways, Rho/Rho-kinase and Ca(2+)/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).
Bauer
M
M
Institut für Prophylaxe und Epidemiologie der Kreislaufkrankheiten, Klinikum Innenstadt, Universität München, München, Germany.
Retzer
M
M
Wilde
J I
JI
Maschberger
P
P
Essler
M
M
Aepfelbacher
M
M
Watson
S P
SP
Siess
W
W
D019796:15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid / Q000494:pharmacology; D000577:Amides / Q000494:pharmacology; D001792:Blood Platelets / Q000378:metabolism* / Q000648:ultrastructure; D002118:Calcium / Q000378:metabolism*; D048429:Cell Size / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D019204:GTP-Binding Proteins / Q000378:metabolism*; D006490:Hemostatics / Q000494:pharmacology; D006801:Humans; D008024:Ligands; D008565:Membrane Proteins / Q000378:metabolism*; D018994:Myosin Light Chains / Q000378:metabolism*; D010766:Phosphorylation; D011725:Pyridines / Q000494:pharmacology; D018179:Receptors, Thrombin / Q000819:agonists / Q000378:metabolism; D013917:Thrombin / Q000494:pharmacology; D014662:Vasoconstrictor Agents / Q000494:pharmacology; D020743:rhoB GTP-Binding Protein
D000577:Amides; D004791:Enzyme Inhibitors; D006490:Hemostatics; D008024:Ligands; D008565:Membrane Proteins; D018994:Myosin Light Chains; D011725:Pyridines; D018179:Receptors, Thrombin; D014662:Vasoconstrictor Agents; C108830:Y 27632; D019796:15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; D013917:Thrombin; D019204:GTP-Binding Proteins; D020743:rhoB GTP-Binding Protein; D002118:Calcium