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


OBJECTIVE
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.
Bozulic
L D
LD
Department of Biochemistry and Molecular Biology, University of Louisville, School of Medicine, Louisville, KY 40292, USA.
Malik
M T
MT
Dean
W L
WL
D000595:Amino Acid Sequence; D001792:Blood Platelets / Q000166:cytology* / Q000201:enzymology; D000252:Calcium-Transporting ATPases / Q000737:chemistry / Q000378:metabolism*; D002462:Cell Membrane / Q000201:enzymology; D006801:Humans; D007700:Kinetics; D010766:Phosphorylation; D010974:Platelet Aggregation; D014443:Tyrosine / Q000378:metabolism*
D014443:Tyrosine; D000252:Calcium-Transporting ATPases