Platelets play a major role in hemostasis and congenital defects in platelet function are associated with bleeding manifestations. The goal of this project is to define the biochemical mechanisms underlying the platelet dysfunction in patients with congenital platelet function defects and thereby enhance our understanding of the normal platelet activation mechanisms which is vital to the development of newer therapeutic strategies for both bleeding and thrombotic disorders. The vast majority of patients with congenital platelet defects are generally characterized by impaired aggregation responses and dense granule secretion on activation; most of them have normal dense granule stores. These patients are lumped into a loosely defined group called """"""""platelet secretion defects"""""""" or """"""""activation defects."""""""" In them the underlying biochemical and molecular mechanisms are totally unknown. The hypothesis to be tested is that these patients have defects in the signal transduction mechanisms. This project focuses on specific patients in whom the applicant has demonstrated hitherto undescribed deficiencies in two major proteins involved in signal transduction mechanisms, namely, a) phospholipase C (Aim 1), and b) GTP-binding protein Gaq (Aim 2). These proteins play fundamental roles in cellular signaling mechanisms in a wide array of cells. Detailed studies in two patients suggest an unique abnormality in PLC activation with decreased PLC-b2 expression.
In Aim 1 Dr. Rao will characterize the molecular defect in PLC-b2 by cloning, sequencing and expressing PLC-b2 CDNA. The mutant PLC-b2 will be studied.
In Aim 2, the applicant will characterize the molecular defect in a patient identified with impaired G-protein-function associated with abnormal Ca2+ mobilization, arachidonate release and a hitherto undescribed selective deficiency in platelet Gaq subunit.
In Aim 3, Dr. Rao will study neutrophils and monocytes from these patients to determine if they share the defect in signal transduction mechanisms and define the impact on leucocyte function. This project represents application of state-of-the-art techniques to define the molecular mechanisms of platelet dysfunction in a group of patients who are very poorly characterized at present and contribute an untapped sour of new information. These studies will provide new information on the role of two major proteins, PLC-b2 and Gaq, in platelet signal transduction mechanisms.
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