The goal of this research proposal is to attain a greater understanding of human platelet function by studying a critical protein component of platelets, the calcium pump. Since calcium has been shown to be a trigger for both platelet aggregation and the release of stored contents, the activity of the calcium pump is essential for maintaining normal platelet function. The project consists of two parts: (1) purification and characterization of the structure and function of the calcium pump protein, and (2) characterization of the membranes containing the Ca++ pump protein. We have now purified the Ca++ ATPase using a new methodology involving solubilization with the nonionic detergent octylglucoside followed by molecular sieve chromatography and hydroxylapatite adsorption. We plan to characterize this preparation which contains only 2 polypeptides, one of which is an inactive proteolytic fragment of the active ATPase, with respect to (1) kinetics with ATP and CA++ to enable comparison with the activity in muscle membranes, (2) molecular weight determined in the analytical ultracentrifuge, (3) calmodulin and lipid requirement, (4) immunochemical relationship to themuscle calcium pump and (5) amino acid analysis and peptide mapping. We also plan to examine the membrane fraction from human platelets which contains the Ca++ ATPase in terms of (1) Ca++ transport activity, (2) protein components and comparison with the sarcoplasmic reticulum, (3) release of stored calcium and comparison with this activity in sarcoplasmic reticulum (i.e. external Ca++, sulhydryl reagents, and heavy metals cause release of Ca++ from sarcoplasmic reticulum which may be related to the physiological function in excitation/contraction coupling) and (4) localization of these membranes in the platelet using immunohistochemical staining and electron microscopy. We have already isolated membranes enriched in the Ca++ ATPase by sarcoplasmic reticulum in terms of Ca++ transport and release, then the analogy of platelet activation to muscle excitation/contraction coupling would be strengthened. This would greatly clarify the complex set of events leading to platelet activation. Furthermore, since platelet defects can lead to thrombosis, the final event in coronary and cerebral occlusion, study of the control of platelet calcium metabolism should result in a better understanding of platelet dysfunction and cardiovascular disease in general.
