Correlation between cytosolic Ca2+ concentration, protein phosphorylation and platelet secretion

Addition of the calcium-ionophore ionomycin to acetylsalicylate-treated platelets suspended in a low Ca2+ concentration-containing medium (about 0.1 mu M), induced a dose-dependent (range 0.253 CIM) and transient increase in the cytosolic Ca2+ concentration ([Ca2+](c)). Less than 10% of the maximal releasable amount of serotonin was secreted at [Ca2+](c) lower than 1 mu M, whereas secretion was almost maximal at [Ca2+](c) higher than 2 mu M. In all cases the secretion stopped after about 1 min even if the [Ca2+](c) was kept constant by repeated small additions of CaCl2 (25-40 mu M). A rapid phosphorylation of pleckstrin (47 kDa) and myosin light chain (20 kDa) was found in all cases, whereas a weak phosphorylation of a 27 kDa protein occurred at [Ca2+](c) lower than 1.5. Addition of 0.2 mM CaCl2 to platelets pretreated for 4 min with 0.5-1 mu M ionomycin brought about a serotonin secretion remarkably lower than that obtained by the simultaneous addition of CaCl2 and ionophore. Platelets suspended in a low calcium-containing medium and exposed to ionomycin showed a major increase in tyrosine phosphorylation of 60 and 72 kDa proteins and a slight increment in tyrosine phosphorylation of 115 and 130 kDa proteins. Subsequent addition of 0.2 mM CaCl2 induced a widespread phosphotyrosine dephosphorylation, particularly evident in the 60 kDa protein identified as p60(c-src) kinase. The protein kinase inhibitor genistein caused, together with a marked prevention of the protein tyrosine phosphorylation, a remarkable increase in the ionomycin-elicited secretory activity of platelets. All together these results indicate that protein kinase C-dependent pleckstrin phosphorylation is a prerequisite of platelet secretion, but that the latter process is apparently regulated by a network of phosphoproteins, in particular the serine/threonine phosphorylation of 27 and 68 kDa proteins and the tyrosine phosphorylation of the p60(s-src) were found to be associated with a decrease in the secretory activity.