Proteomic studies normal and abnormal platelet function

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DESCRIPTION (provided by applicant): Many useful insights into normal and pathologic platelet function have come through studies that focused on single molecules. There are, however, many unanswered questions that do not lend themselves to a "one molecule at a time" approach. These questions would benefit from the application of technologies that can identify complex mixtures of proteins in a quantitative manner using amounts of blood that can reasonably be obtained from single individuals or small numbers of inbred mice. Some of those questions will be addressed in the studies that comprise this proposal. The goal of the first specific aim is to define the components of the platelet sheddome - the set of proteins that are proteolytically shed from the platelet surface when platelets are activated. Protein shedding from platelets releases bioactive molecules into the surrounding plasma. Shedding can also alter the function of essential cell surface receptors, thereby modulating thrombus growth and the interaction of platelets with other types of cells. The goal of the second aim is to understand how the loss of critical signaling and adhesion molecules, either by mutation or by the long term administration of therapeutic antagonists, can have unanticipated effects on the expression of other proteins. Such compensatory changes are of interest because of the insights that they can provide into the normal events of platelet activation, and because of their potential relevance to differences among individuals that may affect susceptibility to disease and responses to anti-platelet agents. The third specific aim will address the molecular basis for the decrease in platelet function and survival that occurs when human platelets are stored prior to transfusion. We will test the hypothesis that these changes are due in part to the shedding of critical membrane proteins. The fourth specific aim will test the hypothesis that an unbiased screen of the platelet proteome can help to establish a molecular diagnosis when other methods have failed to do so. Studies will be performed on platelets from two cohorts of patients with clinical evidence for platelet dysfunction, but no established molecular diagnosis. Each of the proposed studies rests on the combined experience in clinical hematology, transfusion medicine, platelet activation and proteomic technologies represented by the PI, co-PI and their collaborators. The studies are designed using methods that we have recently applied to the identification of platelet membrane proteins. This includes novel methods that permit quantitation of individual molecules so that changes in levels of expression can be measured.