STRUCTURE AND FUNCTION OF BLOOD PLATELET GPIIB-IIIA

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Platelet aggregation and platelet adhesion to components of the extracellular matrix are control events in hemostasis and thrombosis and are regulated through the binding of a set of adhesive proteins including fibrinogen, fibronectin and von Willebrand factor all which contain Arg- Gly-Asp (RGD) sequences. GPIIb-IIIa is a component of a common receptor for these adhesive proteins and binds to peptides containing the RGD sequence. Moreover, GPIIb-IIIa is a member of the Integrin adhesion receptor superfamily and shares the RGD recognition function with certain other integrins. The various alpha and beta subunits are homologous proteins and possess several highly conserved sequences. The binding of adhesive proteins to these receptors is divalent cation dependent, the amino acid sequences of the putative divalent cation binding sites in the Integrin alpha subunits is highly conserved, and the binding RGD containing ligands to GPIIb-IIA causes the expression of divalent cation regulated antigenic sites on GPIIb and GPIIIa. This proposal will test the hypothesis that one or more of these putative calcium binding sites participate in RGD binding. In addition, we will test the hypothesis that the highly conserved region of GPIIIa to which bound RGD peptides may be chemically crosslinked also contributes to RGD binding. We will first analyze the sequence of these regions in GPIIb and GPIIIa cDNAs derived from a natural mutant in which the GPIIb-IIIa hetrodimer lacks RGD binding function. Secondly, we will asses the effects of recombinant GPIIb-IIIa in a transient expression system utilizing a novel cytometric assay, affinity chromatography, and chemical crosslinking. Third, in those regions whose deletion abolishes RGD binding,we will identify those individual amino acids essential for the RGD recognition by saturation mutagenesis. Finally, in addition to those specific regions we will identify those regions of GPIIb-IIIa whose structural integrity is essential for normal adhesive protein binding function through random nucleotide insertion mutations throughout both GPIIb and GPIIIa. These studies will provide fundamental information about those regions of GPIIb-IIIa which are essential for the platelet adhesion and aggregation.