Purpose of this grant application is to perform studies aimed at understanding the structure, structure-function relationships, and molecular pathology of the platelet membrane receptor, glycoprotein (GP) Ib-IX complex, GP Ib-IX is essential for normal platelet adhesion to and activation on thrombogenic surfaces. It exerts its function by interacting with the adhesive molecule, von Willebrand factor (vWF). and with the platelet agonist, thrombin. Deficiency or defective function of GP Ib-IX complex leads to the congenital bleeding disorder known as Bernard-Soulier syndrome. The interaction of platelets with vWF through the GP Ib-IX receptor has also been shown to be relevant for the development of pathological platelet thrombi responsible for vascular occlusion. Therefore, studies on the structure and function of GP Ib-IX are relevant for understanding the pathogenesis of the thrombotic complications of atherosclerosis, as well as for unraveling the physiological mechanisms of platelet function. We propose to use an integrated approach based on the use of proteolytic fragments, synthetic peptides, monoclonal antibodies, and expressed mutant molecules to identify the amino acid residues responsible for vWF and thrombin binding to GP Ib-IX. The amino terminal domain of the alpha-chain of GP Ib (the 45 kDa domain),previously shown to contain the vWF- and thrombin-binding sites, will be subjected to limited proteolysis to generate smaller fragments retaining binding function. Synthetic peptides will be constructed to represent the sequence of functionally relevant epitopes of GP Ib-Alpha. The identification of amino acid residues related to the binding function of GP Ib-IX will represent the basis for selective mutagenesis experiments that will be used to obtain final confirmation of the chemical structure of functional domains. Moreover, the molecular pathology of different forms of Bernard-Soulier syndrome, including a newly described variant, will be explored to establish the bases of the genetic abnormalities responsible for this diseases, and also to obtain information on the functionally relevant structures of the GP Ib-IX complex from the study of naturally occurring mutants with defective binding activity. These studies will provide concepts and reagents to probe the functional relevance of the GP Ib-IX complex in normal hemostasis and pathological thrombosis.
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