Signal Transduction Defects in Human Platelets

描述（由申请人提供）：在大多数遗传性血小板功能障碍患者中，潜在的分子机制尚不清楚。我们研究的总体长期目标是通过对血小板功能遗传缺陷患者的研究，获得对正常血小板机制和相关蛋白质的新见解。迄今为止，我们的研究首次描述了参与信号转导的两种主要血小板蛋白磷脂酶C-β 2（PLC-β 2）和GTP结合蛋白Galphaq中迄今未被认识到的缺陷。最近在我们的血小板PLC-β 2缺陷和G-α-q缺陷患者中的研究表明，蛋白水平降低与正常编码序列相关，但与相应基因的血小板mRNA水平降低相关，并且缺陷存在于血小板中而不是中性粒细胞中，表明造血谱系特异性缺陷。我们的假设是，患者在各自基因的转录调控或mRNA稳定性方面存在缺陷。到目前为止，很少有人知道有关的调控和启动子元件控制PLC-β 2和G-α-q在正常血小板的表达。为了阐明我们患者的机制，我们将进行详细的研究，包括转录起始和mRNA稳定性，确定启动子序列，研究3'和5'非翻译区（因为它们对mRNA稳定性的潜在影响），并研究同源核蛋白（转录因子）与DNA调控元件的结合（特异性目的1和2）。此外，我们还将对血小板进行全基因组表达谱分析，以确定mRNA降低的特异性，并确定它是孤立的还是特定连接途径的一部分。这些研究将确定患者正常基因和异常基因的调控机制。在具体目标3中，我们将进行详细的研究，以描述我们所示的患者的潜在机制，这些患者具有受损的受体介导的聚集、普列克底物蛋白（蛋白激酶C的底物）的磷酸化和GPIIb-IIIa的信号转导依赖性激活。在这个病人中，我们最近发现了转录因子CBFA 2（核心结合因子A2）的突变，该突变调节造血中发挥作用的几个基因。我们的其他研究已经证明存在PLC-β 2的两种可选剪接变体，命名为PLC-β 2a和PLC-β 2b，其在COOH末端序列中相差15个氨基酸残基（对应于氨基酸864-878）。我们的假设是PLC-β 2的两种剪接变体在它们被G-α-q激活的能力和它们的颗粒缔合方面不同。我们将研究两个PLC-β 2剪接变体在瞬时转染细胞中的这些方面的功能。总的来说，我们的研究将为参与血小板信号传导的两种关键血小板蛋白（G-α-q和PLC-β 2）的调节和血小板信号传导机制提供新的重要见解。

DESCRIPTION (provided by applicant): In most patients with inherited disorders of platelet function, the underlying molecular mechanisms are unknown. The overall longstanding goals of our studies have been to obtain new insights into normal platelet mechanisms and the involved proteins through the study of patients with inherited defects in platelet function. Our studies to date have lead to the first descriptions of hitherto unrecognized deficiencies in two major platelet proteins involved in signal transduction, phospholipase C-beta2 (PLC-beta2) and the GTP-binding protein Galphaq. Recent studies in our patients with the platelet PLC-beta2 deficiency and G-alpha-q deficiency show that the decreased protein levels are associated with a normal coding sequence but with decreased platelet mRNA levels of the respective gene, and that the defect is present in platelets but not neutrophils, suggesting a hematopoietic-lineage specific defect. Our hypothesis is that the patients have a defect in the transcriptional regulation of the respective gene or in mRNA stability. As of now, little is known regarding the regulatory and promoter elements governing the expression of PLC-beta2 and G-alpha-q in normal platelets. To elucidate the mechanisms in our patients, we will perform detailed studies including on transcription initiation and mRNA stability, define the promoter sequence, study the 3' and 5' untranslated regions (because of their potential impact on mRNA stability), and study the binding of cognate nuclear proteins (transcription factors) to the DNA regulatory elements (Specific Aims 1 and 2). In addition, we will perform genome-wide expression profiling of platelets to define the specificity of the mRNA decreases, and to determine if it is isolated or part of a specific linked pathway. These studies will define the regulatory mechanisms for the normal gene and the abnormality in the patient. In Specific Aim 3 we will perform detailed studies to delineate the underlying mechanisms in a patient shown by us to have impaired receptor-mediated aggregation, phosphorylation of pleckstrin (a substrate of protein kinase C), and signal transduction-dependent activation of GPIlb-IIIa. In this patient we have recently identified a mutation in the transcription factor CBFA2 (core binding factor A2), which regulates several genes that play a role in hematopoiesis. Our other studies have demonstrated the presence of two alternatively splice variants of PLC-beta2, designated as PLC-beta2a and PLC-beta2b, which differ by 15 amino acid residues (corresponding to amino acids 864-878) in the COOH terminal sequence. Our hypothesis is that the two splice variants of PLC-beta2 differ in their ability to be activated by G-alpha-q and in their particulate association. We will study the function of the two PLC-beta2 splice variants with respect to these aspects in transiently transfected cells. Overall, our studies will provide new important insights into the regulation of two key platelet proteins (G-alpha-q and PLC-beta2) involved in platelet signaling and into the signaling mechanisms in platelets.