Structure and Function of Platelet Glycoprotein lb-IX-V Complex

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DESCRIPTION (provided by applicant): Platelet glycoprotein (GP) Ib-IX-V complex mediates initial platelet adhesion to the injured vessel wall and transduces signals to induce platelet activation and aggregation. Malfunction of this multi-subunit receptor complex leads to severe bleeding and can contribute to many cardiovascular diseases. Despite decades of intensive study, regulation of the complex function is not clear, nor is the process by which the platelet activating signal is mediated by the complex. We hypothesize that inter-subunit interactions are important to the functions and regulation of the complex. Since the 3-dimensional organization of the complex is unknown and inter-subunit interactions are largely unexplored, as a necessary prelude to test our hypothesis this project seeks to identify and characterize the inter-subunit interactions in the GP Ib-IX-V complex. In Specific Aim 1, we propose to test the hypothesis that the transmembrane domains play vital roles in inter-subunit associations and thus are important for expression and assembly of the GP Ib-IX-V complex. The studies are based on our observation that replacing the transmembrane domain of GP Ibalpha or lbbeta subunit with an unrelated sequence decreases significantly the complex expression level. Structural and functional roles of specific residues in the transmembrane domains, including polar residues and membrane-proximal disulfide forming cysteine residues, will be defined. The molecular basis for the subunit stoichiometry in the GP Ib-IXV complex will also be elucidated. In Specific Aim 2, we will define the interfacial regions and residues among the extracellular domains of the complex and explore their contribution to complex assembly and function. The studies are based on our observations that first, the extracellular domains of GP Ibbeta and IX subunits associate and stabilize each other, and second, glycocalicin, the soluble extracellular domain of GP Ibalpha, binds directly to the recombinant extracellular domain of GP IX. Overall, careful dissection of individual interactions in the isolated proteins, in the transfected mammalian cells and in the platelets will enable us to develop tools and reagents that specifically perturb the inter-subunit interaction of interest and will pave the way for studies of the likely structural changes in the GP Ib-IX-V complex in response to ligand binding or intracellular regulatory signals. This project will also provide a stepping stone for studies on the interaction of the GP Ib-IX-V complex with other receptors and may lead to novel therapeutic strategies to combat various complex-related hematological disorders and diseases. Relevance to public health: The glycoprotein Ib-IX-V complex in the platelet helps the blood to clot properly. Malfunction of this complex leads to severe bleeding and can contribute to many cardiovascular diseases. This project seeks to understand the structure of the Ib-IX-V complex and to learn how it functions