Sustained stimulation of platelet thrombin receptor is associated with tyrosine dephosphorylation of a novel p67 peptide in a manner regulated by extracellular calcium.
Signaling pathways elicited by protease-activated receptor-1 (PAR-1) agonists, thrombin receptor-activating peptide (TRAP) and thrombin, are markedly different. Here we show that TRAP-induced disaggregation of platelets is a function of extracellular calcium. Chelation of calcium with EGTA after the onset of aggregation precluded subsequent destabilization of the aggregates in TRAP-stimulated platelets, whereas disaggregation was not observed in the platelets stimulated with thrombin. TRAP-induced disaggregation was independent of the activity of the calcium-dependent thiol protease, calpain. Inhibition of phosphoinositide 3-kinase activity provoked further destabilization of the platelet aggregates in the presence of calcium; however, EGTA attenuated this effect. Activation of protein kinase C (PKC) by phorbol ester prevented disaggregation of the TRAP-stimulated platelets independent of the extracellular calcium. Two proteins of relative mobilities 67 and 75 kD were found to be significantly dephosphorylated on tyrosine in calcium-pretreated platelets as compared to the EGTA-treated platelets following continued stimulation with either TRAP or thrombin for 15 min. Inhibition of phosphoinositide 3-kinase by two pharmacologically independent inhibitors also caused dephosphorylation of p67, which was completely abrogated by chelation of extracellular calcium. Platelet activation by phorbol ester was not associated with disaggregation, although dephosphorylation of p67 was induced under this condition. SHP-1, an abundant tyrosine phosphatase in platelets, co-migrated with the p67 protein and co-localized to the actin-based cytoskeleton of aggregated platelets; however, its identity with p67 was ruled out from immunoprecipitation studies.
Department of Biochemistry, Institute of Medical Sciences, Banaras Hindu University, Varanasi-221005, India.
Zubair A
ZA
Karim
Saikat
S
Mukhopadhyay
Amanchy S S
AS
Ramars
Debabrata
D
Dash
D001792:Blood Platelets / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism / Q000494:pharmacology*; D002154:Calpain / Q000378:metabolism; D003599:Cytoskeleton / Q000187:drug effects / Q000378:metabolism; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D010455:Peptides / Q000737:chemistry* / Q000378:metabolism*; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D019000:Phosphotyrosine / Q000378:metabolism*; D010974:Platelet Aggregation / Q000187:drug effects; D019039:Platelet Glycoprotein GPIIb-IIIa Complex / Q000378:metabolism; D011493:Protein Kinase C / Q000378:metabolism; D021381:Protein Transport; D053824:Protein Tyrosine Phosphatase, Non-Receptor Type 6; D017027:Protein Tyrosine Phosphatases / Q000037:antagonists & inhibitors / Q000378:metabolism; D018179:Receptors, Thrombin / Q000378:metabolism*
D047908:Intracellular Signaling Peptides and Proteins; D010455:Peptides; D019039:Platelet Glycoprotein GPIIb-IIIa Complex; D018179:Receptors, Thrombin; D019000:Phosphotyrosine; D019869:Phosphatidylinositol 3-Kinases; D011493:Protein Kinase C; C509249:PTPN6 protein, human; D053824:Protein Tyrosine Phosphatase, Non-Receptor Type 6; D017027:Protein Tyrosine Phosphatases; D002154:Calpain; D002118:Calcium