Signal Transduction Defects in Human Platelets

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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.