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Correlation between cytosolic Ca2+ concentration, protein phosphorylation and platelet secretion
L Dalla Via1, M Stimamiglio, M Scapin
1Department of Biological Chemistry, University of Padova, Italy.
Cell Calcium
|November 1, 1996
Summary
Platelet calcium levels regulate serotonin secretion, with higher calcium concentrations promoting release. Protein phosphorylation, particularly tyrosine phosphorylation, plays a complex role, inhibiting secretion when it occurs.
Area of Science:
- Biochemistry
- Cell Biology
- Hematology
Background:
- Platelets are crucial for hemostasis and thrombosis.
- Calcium ions ([Ca2+]c) are key regulators of platelet activation and secretion.
- Protein phosphorylation is involved in signal transduction pathways controlling platelet function.
Purpose of the Study:
- To investigate the role of cytosolic calcium concentration ([Ca2+]c) in regulating serotonin secretion from platelets.
- To elucidate the involvement of protein phosphorylation, including serine/threonine and tyrosine phosphorylation, in platelet secretion.
- To explore the relationship between specific protein kinases and platelet secretory activity.
Main Methods:
- Platelets were treated with ionomycin (a calcium ionophore) in low calcium media.
- Cytosolic calcium concentration ([Ca2+]c) was measured.
- Serotonin secretion was quantified.
- Protein phosphorylation (serine/threonine and tyrosine) was analyzed using Western blotting.
- The effect of protein kinase inhibitors (genistein) was assessed.
Main Results:
- Ionomycin induced a dose-dependent increase in [Ca2+]c, correlating with serotonin secretion.
- Serotonin secretion was maximal at [Ca2+]c > 2 microM and ceased within 1 minute.
- Rapid phosphorylation of pleckstrin and myosin light chain occurred, while 27 kDa protein phosphorylation was [Ca2+]c-dependent.
- Ionomycin increased tyrosine phosphorylation of 60 and 72 kDa proteins; subsequent CaCl2 addition caused dephosphorylation, notably of p60c-src.
- Genistein inhibited tyrosine phosphorylation and enhanced ionomycin-induced secretion.
Conclusions:
- Platelet secretion is dependent on cytosolic calcium levels.
- Protein kinase C-dependent pleckstrin phosphorylation is essential for secretion.
- Serine/threonine phosphorylation of 27/68 kDa proteins and tyrosine phosphorylation of p60c-src are associated with decreased secretory activity, suggesting a regulatory role.