PKC inhibition markedly enhances Ca2+ signaling and phosphatidylserine exposure downstream of protease-activated receptor-1 but not protease-activated receptor-4 in human platelets

M T Harper, A W Poole

Research output: Contribution to journalArticle (Academic Journal)

24 Citations (Scopus)

Abstract

BACKGROUND: Cytosolic calcium concentration is a critical regulator of platelet activation, and so platelet Ca(2+) signaling must be tightly controlled. Thrombin-induced Ca(2+) signaling is enhanced by inhibitors of protein kinase C (PKC), suggesting that PKC negatively regulates the Ca(2+) signal, although the mechanisms by which this occurs and its physiological relevance are still unclear. OBJECTIVES: To investigate the mechanisms by which PKC inhibitors enhance thrombin-induced Ca(2+) signaling, and to determine the importance of this pathway in platelet activation. METHODS: Cytosolic Ca(2+) signaling was monitored in fura-2-loaded human platelets. Phosphatidylserine (PS) exposure, a marker of platelet procoagulant activity, was measured by annexin V binding and flow cytometry. RESULTS: PKC inhibition by bisindolylmaleimide-I (BIM-I) enhanced α-thrombin-induced Ca(2+) signaling in a concentration-dependent manner. PAR1 signaling, activated by SFLLRN, was enhanced much more strongly than PAR4, activated by AYPGKF or γ-thrombin, which is a potent PAR4 agonist but a poor activator of PAR1. BIM-I had little effect on α-thrombin-induced signaling following treatment with the PAR1 antagonist, SCH-79797. BIM-I enhanced Ca(2+) release from intracellular stores and Ca(2+) entry, as assessed by Mn(2+) quench. However, the plasma membrane Ca(2+) ATPase inhibitor, 5(6)-carboxyeosin, did not prevent the effect of BIM-I. PKC inhibition strongly enhanced α-thrombin-induced PS exposure, which was reversed by blockade of PAR1. CONCLUSIONS: Together, these data show that when PAR1 is stimulated, PKC negatively regulates Ca(2+) release and Ca(2+) entry, which leads to reduced platelet PS exposure.
Translated title of the contributionPKC inhibition markedly enhances Ca2+ signaling and phosphatidylserine exposure downstream of protease-activated receptor-1 but not protease-activated receptor-4 in human platelets
Original languageEnglish
Pages (from-to)1559 - 1607
Number of pages8
JournalJournal of Thrombosis and Haemostasis
Volume9
Issue number8
DOIs
Publication statusPublished - Aug 2011

Bibliographical note

© 2011 International Society on Thrombosis and Haemostasis.

Keywords

  • Thrombin
  • Plasma Membrane Calcium-Transporting ATPases
  • Dose-Response Relationship, Drug
  • Humans
  • Annexin A5
  • Maleimides
  • Protein Kinase Inhibitors
  • Phosphatidylserines
  • Indoles
  • Blood Platelets
  • Peptide Fragments
  • Flow Cytometry
  • Protein Kinase C
  • Time Factors
  • Oligopeptides
  • Receptor, PAR-1
  • Calcium Signaling
  • Receptors, Thrombin

Fingerprint Dive into the research topics of 'PKC inhibition markedly enhances Ca2+ signaling and phosphatidylserine exposure downstream of protease-activated receptor-1 but not protease-activated receptor-4 in human platelets'. Together they form a unique fingerprint.

Cite this