Sustained stimulation of platelet thrombin receptor is associated with tyrosine dephosphorylation of a novel p67 peptide in a manner regulated by extracellular calcium

Zubair A. Karim, Saikat Mukhopadhyay, Amanchy S S Ramars, Debabrata Dash

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Abstract

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.

Original languageEnglish (US)
Pages (from-to)147-157
Number of pages11
JournalBiochimica et Biophysica Acta - Molecular Cell Research
Volume1693
Issue number2
DOIs
StatePublished - Aug 23 2004

Fingerprint

thrombin receptor peptide SFLLRNP
Thrombin Receptors
Tyrosine
Blood Platelets
Calcium
Peptides
Egtazic Acid
Thrombin
1-Phosphatidylinositol 4-Kinase
Phorbol Esters
Non-Receptor Type 6 Protein Tyrosine Phosphatase
PAR-1 Receptor
Calpain
Platelet Activation
Actin Cytoskeleton
Immunoprecipitation
Sulfhydryl Compounds
Protein Kinase C
Proteins
Peptide Hydrolases

Keywords

  • Platelet
  • Protease-activated receptor-1
  • Protein tyrosine kinase
  • Protein tyrosine phosphatase
  • Thrombin receptor-activating peptide

ASJC Scopus subject areas

  • Cell Biology
  • Molecular Biology
  • Biophysics

Cite this

@article{86f3d11f32f54d12bf03bc12924d488d,
title = "Sustained stimulation of platelet thrombin receptor is associated with tyrosine dephosphorylation of a novel p67 peptide in a manner regulated by extracellular calcium",
abstract = "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.",
keywords = "Platelet, Protease-activated receptor-1, Protein tyrosine kinase, Protein tyrosine phosphatase, Thrombin receptor-activating peptide",
author = "Karim, {Zubair A.} and Saikat Mukhopadhyay and Ramars, {Amanchy S S} and Debabrata Dash",
year = "2004",
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T1 - Sustained stimulation of platelet thrombin receptor is associated with tyrosine dephosphorylation of a novel p67 peptide in a manner regulated by extracellular calcium

AU - Karim, Zubair A.

AU - Mukhopadhyay, Saikat

AU - Ramars, Amanchy S S

AU - Dash, Debabrata

PY - 2004/8/23

Y1 - 2004/8/23

N2 - 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.

AB - 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.

KW - Platelet

KW - Protease-activated receptor-1

KW - Protein tyrosine kinase

KW - Protein tyrosine phosphatase

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JO - Biochimica et Biophysica Acta - Molecular Cell Research

JF - Biochimica et Biophysica Acta - Molecular Cell Research

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