Ca(2+)-dependent activation of the 33-kDa protein kinase transmits thrombin receptor signals in human platelets.
Thrombin stimulation induces a dramatic increase in the activity of the 33-kDa serine/threonine kinase (PK33) in human platelets (10). The Arg-Gly-Asp (RGD) peptide, an inhibitor of the thrombin-mediated aggregation of platelets, did not affect the PK33 activation induced by thrombin suggesting that the activation of this kinase occurs independently from platelet aggregation. To identify a potential role of Ca2+ and calmodulin in the regulation of PK33, the effect of several Ca2+/calmodulin inhibitors on the thrombin-induced activation of PK33 was assessed using denaturation/renaturation method. Pretreatment of platelets with EGTA decreased the maximum PK33 activity induced by thrombin. The chelation of both the extra- and the intracellular Ca2+ by EGTA and by acetoxymethyl ester of 5,5'-dimethyl-bis-(0-aminophenoxy) ethane-N,N,N',N'-tetraacetic acid (BAPTA-AM) decreased further the PK33 activation by thrombin. Preincubation of platelets with the anticalmodulin agent, N-(4-aminobutyl)-5-chloro-2-naphthalenesulfonamide (W13), inhibited markedly the activation of PK33 by thrombin, whereas the inactive structural analog N-(4-aminobutyl)-2-naphthalenesulfonamide (W12) and the myosin light chain kinase inhibitor 1-(5-chloronaphthalene-1-sulfonyl)-1H-hexahydro-1,4-diazepine (ML9) showed very weak inhibitory effects. Treatment of resting platelets with the calcium ionophore, A23187, activated PK33 in a dose-dependent manner; phorbol 12-myristate 13-acetate enhanced this effect. However, the two foregoing agents did not induce similar degree of PK33 activities as thrombin. These results indicate that the activation of PK33 is independent of the formation of the GPIIb/IIIa-fibrinogen complex and that it might be regulated by a Ca(2+)-dependent pathway.
Ido
M
M
Department of Molecular Pathobiology, Mie University School of Medicine, Japan.
Kato
S
S
Ogawa
H
H
Hayashi
K
K
Komada
Y
Y
Zhang
X L
XL
Sakurai
M
M
Suzuki
K
K
D001792:Blood Platelets / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism*; D006801:Humans; D015539:Platelet Activation; D011494:Protein Kinases / Q000378:metabolism*; D018179:Receptors, Thrombin / Q000819:agonists / Q000378:metabolism*; D015398:Signal Transduction; D013917:Thrombin / Q000494:pharmacology
D018179:Receptors, Thrombin; D011494:Protein Kinases; D013917:Thrombin; D002118:Calcium