Agonist-induced regulation of myosin phosphatase activity in human platelets through activation of Rho-kinase.
Human platelets contained about 15 times lower amounts of Rho-kinase than Ca2+/calmodulin-dependent myosin light chain (MLC) kinase. Anti-myosin-binding subunit (MBS) antibody coimmunoprecipitated Rho-kinase of human platelets, and addition of GTPgammaS-RhoA stimulated phosphorylation of the 130-kD MBS of myosin phosphatase and consequently inactivated myosin phosphatase. Two kinds of selective Rho-kinase inhibitors, HA1077 and Y-27632, reduced both GTPgammaS-RhoA-dependent MBS phosphorylation and inactivation of the phosphatase activity. Activation of human platelets with thrombin, a stable thromboxane A2 analog STA2, epinephrine, and serotonin resulted in an increase in MBS phosphorylation, and the agonist-induced MBS phosphorylation was prevented by pretreatment with the respective receptor antagonist. HA1077 and Y-27632 inhibited MBS phosphorylation in platelets stimulated with these agonists. These compounds also blocked agonist-induced inactivation of myosin phosphatase in intact platelets. In addition, HA1077 and Y-27632 inhibited 20-kD MLC phosphorylation at Ser19 and ATP secretion of platelets stimulated with STA2, thrombin (0.05 U/mL), and simultaneous addition of serotonin and epinephrine, whereas these compounds did not affect MLC phosphorylation or ATP secretion when platelets were stimulated with more than 0.1 U/mL thrombin. Thus, activation of Rho-kinase and the resultant phosphorylation of MBS is likely to be the common pathway for platelet activation induced by various agonists. These results also suggest that Rho-kinase-mediated MLC phosphorylation contributes to a greater extent to the platelet secretion induced by relatively weak agonists.
Suzuki
Y
Y
2nd and the 1st Departments of Internal Medicine, Mie University School of Medicine, Tsu, Mie, Japan.
Yamamoto
M
M
Wada
H
H
Ito
M
M
Nakano
T
T
Sasaki
Y
Y
Narumiya
S
S
Shiku
H
H
Nishikawa
M
M
D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine / Q000031:analogs & derivatives / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000097:blood; D000577:Amides / Q000494:pharmacology; D001792:Blood Platelets / Q000201:enzymology*; D004533:Egtazic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D019204:GTP-Binding Proteins / Q000502:physiology; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D007700:Kinetics; D043263:Myosin-Light-Chain Phosphatase; D009218:Myosins / Q000097:blood / Q000276:immunology; D010749:Phosphoprotein Phosphatases / Q000097:blood*; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors / Q000097:blood*; D011725:Pyridines / Q000494:pharmacology; D054460:rho-Associated Kinases; D020742:rhoA GTP-Binding Protein
D000577:Amides; D004791:Enzyme Inhibitors; D047908:Intracellular Signaling Peptides and Proteins; D011725:Pyridines; C108830:Y 27632; C070379:1,2-bis(2-aminophenoxy)ethane N,N,N',N'-tetraacetic acid acetoxymethyl ester; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D004533:Egtazic Acid; D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; D000255:Adenosine Triphosphate; D017346:Protein Serine-Threonine Kinases; D054460:rho-Associated Kinases; D010749:Phosphoprotein Phosphatases; D043263:Myosin-Light-Chain Phosphatase; D019204:GTP-Binding Proteins; D009218:Myosins; D020742:rhoA GTP-Binding Protein; C049347:fasudil