Funding is requested to support studies on the structure and function of the membrane glycoprotein (GP) Ib-IX-V complex, a platelet receptor that binds von Willebrand factor (vWF) and alpha-thrombin. This proposal consists of four specific aims. 1) Analysis of interactions induced by exogenous modulators under static conditions indicates that the residues involved in vWF binding are located in the 45 kDa amino terminal domain of GP Ibalpha. To obtain information related to events in flowing blood, fluorescent beads will be coated with recombinant 45 kDa fragment as a mimic of the receptor on platelets. The interaction with vWF will be studied under flow, and selected mutations will be expressed to define amino acid residues relevant to function. 2) Platelets have unique properties that cannot be mimicked by other cells, notably the capacity to undergo homotypic aggregation forming thrombi in rapidly flowing blood. Human GP Ibalpha will be expressed in mouse platelets deficient in the endogenous homologue, thus obtaining membrane GP Ib-IX-V amenable to mutagenesis in the GP Ibalpha subunit. The information database on the human receptor will be used to introduce selected structural modifications in GP Ibalpha and define the functional consequences on vWF-dependent platelet adhesion and aggregation in flowing blood. Moreover, specific mutations in the intracytoplasmic domains of the protein will be evaluated with respect to the consequences on the interaction with vWF and the generation of outside-in signals important for platelet activation. 3) Activation of human platelets by alpha- thrombin involves the protease-activated receptor (PAR) 1 with the possible contribution of the homologous PAR-4. The significance of alpha-thrombin binding to GP Ibalpha is still debated. We will obtain mouse platelets expressing the human alpha-subunit in the GP Ib-IX-V complex, and generate mutants lacking alpha-thrombin but not vWF binding. We will use this approach to modulate alpha-thrombin binding to GP Ibalpha and define the consequences on platelet activation and function. 4) The beta-subunit of GP Ib is important for assembly of the GP Ib- IX-V complex. Moreover, biochemical studies suggest the GP Ibbeta may participate in platelet signaling, thus contributing directly to the adhesive properties of GP Ib-IX-V. A mouse model lacking active GP Ibbeta will be generated to address the in vivo relevance of its putative signaling function. The results obtained with the proposed studies will enhance our understanding of biological processes intimately involved with hemostasis and arterial thrombosis, likely benefiting patients through better prevention, diagnosis and treatment of atherosclerotic cardiovascular disease.
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