Phosphorylation-dependent and -independent pathways of platelet aggregation.
We have used the non-specific inhibitor of protein kinases, staurosporine, to investigate the role of protein phosphorylation during aggregation, the mobilization of intracellular Ca2+ (Ca2+)i and intracellular pH (pHi) in thrombin-stimulated platelets. The concentration of staurosporine chosen for these studies, 1 microM, was previously reported to inhibit protein phosphorylation completely but to have no effect on the activation of phospholipase C in thrombin-stimulated human platelets [Watson, McNally, Shipman & Godfrey (1988) Biochem. J. 249, 345-350]. Aggregation induced by phorbol dibutyrate is slow (several minutes) and is inhibited completely by staurosporine. In contrast, aggregation induced by thrombin, platelet-activating factor or ionophore A23187 is rapid (occurs within 60 s), and is slowed, but not inhibited, in the presence of staurosporine. On the other hand, staurosporine causes a small potentiation of the peak [Ca2+]i signal induced by thrombin and a marked increase in the half-life of decay of this signal, but has no effect on pHi. Under conditions designed to prevent an increase in [Ca2+]i (presence of Ni2+ to prevent Ca2+ entry, and depletion of the intracellular Ca2+ stores), aggregation induced by thrombin resembles that by phorbol dibutyrate and is now inhibited completely by staurosporine. Taken together, these results provide evidence for two signalling pathways for aggregation, a relatively rapid phosphorylation-independent route mediated by Ca2+ and a slower, phosphorylation-dependent, pathway mediated by protein kinase C. Since staurosporine slows aggregation induced by thrombin, it appears that under normal conditions these pathways interact synergistically.
Watson
S P
SP
Department of Pharmacology, University of Oxford, U.K.
Hambleton
S
S
D000470:Alkaloids / Q000494:pharmacology; D000584:Amiloride / Q000031:analogs & derivatives / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D006801:Humans; D010766:Phosphorylation; D010974:Platelet Aggregation / Q000187:drug effects*; D011493:Protein Kinase C / Q000037:antagonists & inhibitors; D019311:Staurosporine; D013917:Thrombin / Q000494:pharmacology
D000470:Alkaloids; D000584:Amiloride; D011493:Protein Kinase C; D013917:Thrombin; D019311:Staurosporine; D002118:Calcium; C039614:ethylisopropylamiloride