Proteomic studies of normal and abnormal platelet function

描述（由申请人提供）： 通过针对单分子的研究，我们获得了对正常和病理血小板功能的许多有用见解。然而，还有许多未解答的问题不适合“一次一个分子”的方法。这些问题将受益于技术的应用，这些技术可以使用从单个个体或少量近交小鼠中合理获得的血液量，以定量方式识别复杂的蛋白质混合物。其中一些问题将在构成本提案的研究中得到解决。第一个具体目标的目标是定义血小板脱落物的组成部分 - 当血小板被激活时，从血小板表面通过蛋白水解脱落的一组蛋白质。血小板脱落的蛋白质将生物活性分子释放到周围的血浆中。脱落还可以改变重要细胞表面受体的功能，从而调节血栓生长以及血小板与其他类型细胞的相互作用。第二个目标是了解关键信号传导和粘附分子的丧失（无论是通过突变还是通过长期施用治疗拮抗剂）如何对其他蛋白质的表达产生意想不到的影响。这种代偿性变化之所以引起人们的兴趣，是因为它们可以为血小板活化的正常事件提供见解，并且因为它们与可能影响疾病易感性和抗血小板药物反应的个体差异具有潜在相关性。第三个具体目标将解决输血前储存人类血小板时血小板功能和存活率下降的分子基础。我们将检验以下假设：这些变化部分是由于关键膜蛋白的脱落造成的。第四个具体目标将检验这样一个假设：当其他方法无法做到这一点时，血小板蛋白质组的公正筛选可以帮助建立分子诊断。将对两组患者的血小板进行研究，这些患者具有血小板功能障碍的临床证据，但尚未建立分子诊断。每项拟议的研究都基于以 PI、co-PI 及其合作者为代表的临床血液学、输血医学、血小板活化和蛋白质组技术的综合经验。这些研究是使用我们最近应用于血小板膜蛋白鉴定的方法设计的。这包括允许对单个分子进行定量的新方法，从而可以测量表达水平的变化。

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.