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J Thromb Haemost. 2007 May;5(5):1041-6.

Effects of plasma membrane Ca(2+) -ATPase tyrosine phosphorylation on human platelet function.

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1
Department of Biochemistry and Molecular Biology, University of Louisville, School of Medicine, Louisville, KY 40292, USA.

Abstract

BACKGROUND:

The plasma membrane Ca(2+)-ATPase (PMCA) plays an essential role in maintaining low intracellular Ca(2+) ([Ca(2+)](i)) in resting platelets. Earlier studies demonstrated that platelet activation by thrombin results in tyrosine phosphorylation of PMCA, which inhibits pump activity.

OBJECTIVES:

The objective was to determine the functional consequences of PMCA tyrosine phosphorylation.

METHODS:

A decapeptide including the tyrosine phosphorylation site of PMCA and a scrambled version were synthesized and introduced into human platelets using saponin. Fura-2 calcium monitoring and aggregometry were used to characterize the effects of inhibition of tyrosine phosphorylation.

RESULTS:

Western blot analysis of immunoprecipitates showed that introduction of the inhibitory peptide decreased tyrosine phosphorylation of PMCA by nearly 60% in saponin-permeabilized, thrombin-treated platelets as compared with the scrambled control peptide. Concomitant with inhibition of PMCA tyrosine phosphorylation was a significant decrease in [Ca(2+)](i) during thrombin-mediated platelet activation. The functional consequence of reduced PMCA tyrosine phosphorylation and decreased [Ca(2+)](i) was a significant delay in the onset of thrombin-mediated platelet aggregation.

CONCLUSIONS:

The results demonstrate that PMCA tyrosine phosphorylation regulates [Ca(2+)](i) during platelet activation, which affects downstream events in the activation process. Moreover, PMCA tyrosine phosphorylation and resultant inhibition of PMCA activity produces a positive feedback loop mechanism by enhancing the increase in [Ca(2+)](i) accompanying platelet activation.

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