Aberrant Platelet Mechanisms in Inherited Human Platelet Function Disorders

描述(申请人提供)：尽管我们对血小板生理学的了解有了巨大的进步，但在绝大多数具有遗传性血小板功能缺陷的患者中，潜在的分子机制仍然未知。机制，如膜糖蛋白缺陷和储存池缺陷，通常被考虑，但在一小部分患者中发生。这些患者大多以出血性疾病为特征，聚集功能受损，分泌致密的颗粒影响血小板的激活，目前被归入一个定义较宽松的组，称为“血小板分泌/激活缺陷”。这些患者的分子机制尚不清楚。多项研究表明，这些患者的信号转导机制可能存在异常。我们已经记录了血小板磷脂酶C-β2、Gaq和蛋白激酶C-theta的特殊缺陷。需要更新的方法来确定这类患者的分子缺陷，这些患者构成了新信息的未开发储存库。我们现在已经使用Affymetrix微阵列应用了全基因组血小板表达谱，显示了在遗传性血小板减少症患者中肌球蛋白轻链9(MYL9，减少70倍)和其他基因的独特表达降低，血小板反应受损(包括肌球蛋白和Pleckstrin磷酸化以及GPIIb-111a激活)，以及转录因子CBFA2(核心结合因子A2)的杂合突变，从而提供了概念证据，即表达谱分析确实可以用于对血小板功能障碍患者进行新的见解。到目前为止，微阵列技术还没有应用于这类患者。在这个项目中，我们提出了一种综合的方法，包括a)对血小板机制的详细生化和功能研究，b)血小板表达谱，以及c)随后使用功能、突变、生化和免疫学方法验证异常机制，以描绘15例遗传性血小板功能障碍患者的分子机制。我们在所有涉及的方法方面都有丰富的经验。该项目是两个具有强大互补性专业知识的实验室之间的合作努力。它代表了基因组学和人类基因组计划的最新进展应用于一组经常遇到但特征极差的患者。我们的研究将提供关于血小板机制和潜在的抗血栓治疗新靶点的丰富信息。

DESCRIPTION (provided by applicant): Despite tremendous advances in our understanding of platelet physiology, the underlying molecular mechanisms remain unknown in the vast majority of patients with inherited defects in platelet function. Mechanisms, such as deficiencies in membrane glycoproteins and the storage pool deficiency, are commonly considered but occur in a small proportion of patients. Most of these patients are characterized by a bleeding disorder, and impaired aggregation and dense granule secretion on platelet activation, and are currently lumped in a loosely defined group called "platelet secretion/activation defects." The molecular mechanisms in these patients are unknown. Several studies suggest that these patients may have abnormalities in the signal transduction mechanisms. We have documented specific defects in platelet phospholipase C-beta2, Gaq and protein kinase C-theta. Newer approaches are required to define the molecular defects in such patients who constitute an untapped reservoir of new information. We have now applied genome-wide platelet expression profiling using the Affymetrix microarrays to demonstrate a unique decrease in expression of myosin light chain 9 (MYL9, 70 fold decrease) and other genes in a patient with an inherited thrombocytopenia, impaired platelet responses (including myosin and pleckstrin phosphorylation and GPIIb-llla activation), and a heterozygous mutation in transcription factor CBFA2 (Core-binding factor A2), thereby providing proof of concept that expression profiling can indeed be applied to obtain new insights in patients with platelet dysfunction. The microarray technology has hitherto not been applied to such patients. In this project, we propose an integrated approach encompassing, a) detailed biochemical and functional studies on platelet mechanisms, b) platelet expression profiling and c) subsequent validation of the aberrant mechanisms using functional, mutational, biochemical and immunological approaches, to delineate the molecular mechanisms in 15 patients with inherited platelet dysfunction. We have extensive experience in all of the involved approaches. This project is a collaborative effort between two laboratories with strong complementary expertise. It represents application of the recent advances in genomics and the Human Genome Project to a group of patients who are frequently encountered but extremely poorly characterized. Our studies will provide a wealth of information on platelet mechanisms and on potential new targets for antithrombotic therapy.