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 microM), induced a dose-dependent (range 0.25-3 microM) 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 microM, whereas secretion was almost maximal at [Ca2+]c higher than 2 microM. 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 microM). 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 microM. Addition of 0.2 mM CaCl2 to platelets pretreated for 4 min with 0.5-1 microM ionomycin brought about a serotonin secretion remarkably lower than 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 p60c-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 p60c-src were found to be associated with a decrease in the secretory activity.
Dalla Via
L
L
Department of Biological Chemistry, University of Padova, Italy.
Stimamiglio
M
M
Scapin
M
M
Cesaro
L
L
Deana
R
R
D001792:Blood Platelets / Q000378:metabolism*; D001798:Blood Proteins / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism*; D002122:Calcium Chloride / Q000494:pharmacology; D003600:Cytosol / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D019833:Genistein; D006801:Humans; D015759:Ionomycin / Q000494:pharmacology; D007476:Ionophores / Q000494:pharmacology; D007529:Isoflavones / Q000494:pharmacology; D018994:Myosin Light Chains / Q000378:metabolism; D010750:Phosphoproteins; D010766:Phosphorylation; D011493:Protein Kinase C / Q000502:physiology; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors; D016392:Proto-Oncogene Proteins pp60(c-src) / Q000378:metabolism; D012701:Serotonin / Q000378:metabolism; D014443:Tyrosine / Q000378:metabolism
D001798:Blood Proteins; D004791:Enzyme Inhibitors; D007476:Ionophores; D007529:Isoflavones; D018994:Myosin Light Chains; D010750:Phosphoproteins; C039077:platelet protein P47; D012701:Serotonin; D014443:Tyrosine; D015759:Ionomycin; D019833:Genistein; D011505:Protein-Tyrosine Kinases; D016392:Proto-Oncogene Proteins pp60(c-src); D011493:Protein Kinase C; D002122:Calcium Chloride; D002118:Calcium