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PAR‐1‐dependent pp60 src activation is dependent on protein kinase C and increased [Ca 2 + ] i : evidence that pp60 src does not regulate PAR‐1‐dependent Ca 2+ entry in human platelets
Author(s) -
HARPER M. T.,
SAGE S. O.
Publication year - 2006
Publication title -
journal of thrombosis and haemostasis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.947
H-Index - 178
eISSN - 1538-7836
pISSN - 1538-7933
DOI - 10.1111/j.1538-7836.2006.02207.x
Subject(s) - protein kinase c , microbiology and biotechnology , chemistry , phospholipase c , tyrosine kinase , kinase , biophysics , biology , signal transduction
Summary. Background: The role of the tyrosine kinase pp60 src in PAR‐1‐dependent Ca 2+ entry was investigated in human platelets. pp60 src plays a role in thapsigargin (TG)‐evoked store‐operated Ca 2+ entry (SOCE), which is thought to be a major component of thrombin‐evoked Ca 2+ entry. Methods: pp60 src tyr 416 phosphorylation was used to assess pp60 src activation. Fura‐2‐loaded platelets were used to monitor intracellular Ca 2+ concentration ([Ca 2+ ] i ). Results: Activation of PAR‐1 with the specific agonist SFLLRN increased pp60 src activation within 10 s. This required phospholipase C (PLC) activity, Ca 2+ release and a rise in intracellular Ca 2+ . PP2, an inhibitor of Src‐family tyrosine kinases, inhibited SFLLRN‐evoked Ca 2+ entry, but also inhibited Ca 2+ release and the extrusion of Ca 2+ by the plasma membrane Ca 2+ ATPase. Actin polymerization and conventional protein kinase C (cPKC) activity were required for TG‐ and SFLLRN‐evoked pp60 src activation. Although Gö6976, an inhibitor of cPKCs, inhibited TG‐evoked SOCE, it had little effect on SFLLRN‐ or thrombin‐evoked Ca 2+ entry. Conclusions: These data indicate that stimulation of PAR‐1 leads to activation of pp60 src in human platelets, through PLC and cPKC activation, Ca 2+ release and actin polymerization. However, as PKC and actin polymerization are not needed for SFLLRN‐evoked Ca 2+ entry, we suggest that pp60 src is also not required. The apparent inhibition of SFLLRN‐evoked Ca 2+ entry by PP2 is likely to be secondary to reduced Ca 2+ release. These data argue against a contribution of this SOCE pathway to PAR‐1‐dependent Ca 2+ entry.