Proteomic studies of normal and abnormal platelet function

描述(由申请人提供)： 对正常和病理血小板功能的许多有用的见解都是通过专注于单分子的研究得出的。然而，还有许多问题没有得到解答，这些问题不适合“一次一个分子”的方法。这些问题将受益于技术的应用，这些技术可以使用可以合理地从单个个体或少量近亲繁殖的小鼠身上获得的血量，以定量的方式识别复杂的蛋白质混合物。其中一些问题将在构成这项提案的研究中得到解决。第一个特定目标的目标是确定血小板脱落穹顶的成分--当血小板被激活时，从血小板表面蛋白水解性脱落的一组蛋白质。从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。脱落还可以改变基本细胞表面受体的功能，从而调节血栓的生长和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和黏附分子的丢失，无论是由于突变还是长期服用治疗性拮抗剂，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令人感兴趣，是因为它们可以提供对正常血小板激活事件的洞察，也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有潜在的相关性。第三个具体目标将解决血小板功能和存活率下降的分子基础，这种下降发生在输血前储存人血小板时。我们将检验这一假设，即这些变化在一定程度上是由于关键膜蛋白的脱落。第四个具体目标将检验这样一个假设，即在其他方法无法做到这一点的情况下，对血小板蛋白质组进行无偏见的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，但尚未确定分子诊断。每一项拟议的研究都依赖于临床血液学、输血医学、血小板激活和以PI、co-PI及其合作者为代表的蛋白质组技术的综合经验。这些研究是利用我们最近应用于鉴定血小板膜蛋白的方法设计的。这包括允许对单个分子进行量化的新方法，以便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.