Abstract 11329: The Plasma Membrane Calcium ATPase Regulates Calcium Homeostasis, Function and Negative Signalling Events in Platelets
Plasma membrane calcium ATPases (PMCAs) extrude calcium from a variety of cell types and have recently been shown to regulate signalling events and function in the cardiovascular system. PMCAs are a major mechanism of calcium extrusion in platelets and we hypothesised that they are key regulators of calcium homeostasis and function in the platelet. We determined concentrations of carboxyeosin (CE) that selectively inhibited PMCA activity in platelets and demonstrated that 10–40 μM CE dose-dependently inhibited calcium extrusion through PMCA, elevated basal [Ca2+]i (by 20.5±6.6*, 30.1±6.3*, 40.7±5.4* and 52.4±9.1* nM respectively, n=5, P<0.05) and modified [Ca2+]i following activation with temporal specificity. CE also dose dependently inhibited collagen induced platelet aggregation in vitro (by 33.2±13.0*, 56.0±14.0*, 69.4±14.3* and 75.7±12.3* %, n=5, P<0.05, Fig A) and in vivo but enhanced both adhesion to fibrinogen and clot retraction. Similar functional observations were made in PMCA4 knock-out mice (Fig B) and with PMCA inhibitory caloxins (Fig C), confirming that the functional effects observed with CE were likely to be mediated through the PMCA4 isoform. The inhibitory effect of PMCA on platelet aggregation was not associated with pre-activation, measured as shape change or 5HT secretion, but increased VASP phosphorylation, associated with enhanced protein kinase activity, was observed using Western blotting and pharmacological approaches. In conclusion, PMCA regulates [Ca2+]i both basally and during platelet activation. PMCA positively regulates platelet aggregation through the modulation of negative signalling pathways but negatively regulates the earlier and later stages of activation, namely adhesion and clot retraction. Thus, PMCA is a key regulator of signalling events and differentially regulates the various stages of platelet activation, defining it as a potential therapeutic target in platelet-driven diseases.
- © 2010 by American Heart Association, Inc.