The integrative physiology of the plasma membrane calcium ATPase in platelets

Plasma Membrane Calcium-ATPases (PMCA’s) extrude calcium from a variety of cell types and have recently been shown to regulate signalling events and function in the cardiovascular system. PMCA (isoform 4) is expressed in platelets, however its functional role remains undefined. The aims of this project were to investigate the roles of PMCA in regulating calcium homeostasis and human platelet function during the various stages of platelet activation by evaluating the effects of a pharmacological inhibitor of PMCA, carboxyeosin, upon a range of in vitro and in vivo assays. Additional experiments were performed to determine the functional consequences of PMCA4 ablation using platelets from PMCA4 knock-out mice and the roles of PMCA were contrasted with other calcium transporters. Finally, the mechanism of action of PMCA in platelets was investigated by integrating molecular studies with the functional assays. The findings presented in this thesis suggest that PMCA plays an important role in regulating calcium 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 measured as adhesion and clot retraction respectively. PMCA also regulates platelet function in vivo. Experiments in PMCA4 knock-out mice implicated the PMCA4 isoform in the regulation of platelet function. Comparison of PMCA, SERCA (sarcoendoplasmic reticulum ATPase) and NCX (Na+/Ca2+-exchanger) pumps indicated that all three calcium transporters play pivotal but contrasting roles in platelet function and calcium homeostasis. In conclusion, PMCA is a key regulator of calcium homeostasis and differentially regulates the various stages of platelet activation. PMCA regulates intracellular signalling events in platelets although the mechanisms remain to be fully determined. PMCA is therefore a key modulator of haemostasis and thrombosis and may be a potential target in the treatment of conditions such as arterial thrombosis and stroke.EThOS - Electronic Theses Online ServiceGBUnited Kingdo