Abstract
Background: The plasma membrane calcium ATPase (PMCA) regulates localized signaling events in a variety of cell types, although its functional role in platelets remains undefined. Objectives: To investigate the role of PMCA in determining platelet intracellular calcium concentration ([Ca2+](i)) at rest and following agonist stimulation, and to define the corresponding effects upon different stages of platelet activation. Methods: [Ca2+](i) was continuously measured in Fura-2-loaded platelets and in vitro and in vivo functional analyses performed in the presence of the PMCA inhibitor carboxyeosin (CE). Results: Concentrations of CE that selectively inhibited Ca2+ extrusion through PMCA were established in human platelets. [Ca2+](i) was elevated by CE in resting platelets, although collagen-stimulated Ca2+ release was reduced. Impaired Ca2+ mobilization upon agonist stimulation was accompanied by reduced dense granule secretion and impaired platelet aggregation. Platelet aggregation responses were also reduced in PMCA4(-/-) mice and in an in vivo mouse model of platelet thromboembolism. Conversely, inhibition of PMCA promoted the early and later stages of platelet activation, observed as enhanced adhesion to fibrinogen, and accelerated clot retraction. Investigations into the signaling mechanisms underlying CE-mediated inhibition of platelet aggregation implicated cGMP-independent vasodilator-stimulated phosphoprotein phosphorylation. Conclusions: Disruption of PMCA activity perturbs platelet Ca2+ homeostasis and function in a time-dependent manner, demonstrating that PMCA differentially regulates Ca2+-dependent signaling events, and hence function, throughout the platelet activation process.