The platelet receptors for fibrinogen and von Willebrand factor play a central role in platelet physiology since adhesion to non-collagenous surfaces and aggregation induced by ristocetin or any of the agonists that are thought to act in vivo require the binding of one or the other of these proteins to their receptor(s) on the platelet surface. This proposal aims to: 1) improve our understanding of the biochemistry and physiology of these receptors by employing the murine monoclonal antibodies that the principle investigator made during the previous grant period that block the binding of these proteins to their receptor(s) and 2) prepare additional antibodies against other platelet receptors. Specific experiments will be directed at: 1) deciding the potential therapeutic usefullness of the antibodies in treating thrombotic disease by studies in animal models and pilot studies in humans, 2) applying the anti-GPIIb/IIIa antibody to carrier detection of thrombasthenia in a population (Iraqi-Jewish) with a high frequency of the gene, 3) discovering the precursors of GPIb, GPIIb and GPIIIa that are present in megakaryocytes, 4) characterizing the relationship between GPIb and the Mr 20,000 glycoprotein that copurifies with it, 5) evaluating whether glycocalicin circulates in plasma, 6) investigating whether GPIIb and GPIIIa exist as hetero-complexes in unactivated platelets, 7) defining whether the glycoproteins are transmembrane and whether they have free sulfhydryl groups, 8) determining the role of the different glycoproteins in binding von Willebrand factor when platelets are stimulated with various agonists, 9) assessing whether thrombospondin binds to a receptor on activated platelets separate from fibrinogen, 10) using fluorescence resonance energy transfer to determine whether GPIIb/IIIa and GPIb form homo- and/or hetero-clusters during platelet activation, and 11) employing a sensitive and specific assay for factor X activation to assess whether functionally significant factor VIII:C gains access to the platelet by being in complex with von Willebrand factor when the latter binds to platelets. These studies thus combine attempts to apply our present knowledge to clinical practice with the generation of new basic knowledge from which additional clinical applications may derive.
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