STRUCTURAL STUDIES OF PLATELET GPIIB/IIIA ACTIVATION

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Following agonist stimulation, soluble fibrinogen and/or Willebrand factor binds to the integrin alphaIIbbeta3, enabling platelets to undergo homotypic aggregation. Because the plasma concentration of fibrinogen and von Willebrand factor (vWF) are substantially in excess of those required to saturate alphaIIbbeta3, circulating platelets maintain alphaIIbbeta3 in an inactive state to prevent spontaneous platelet aggregation. The objectives of this project are to clarify the mechanisms of alphaIIbbeta3 activation and to characterize the changes that alphaIIbbeta3 undergoes that enable to be blind soluble ligands. The project consists of two Specific Aims that are an extension of ongoing work and represent an integrated cell biological, molecular, and biophysical approach to understanding the structural changes that accompany alphaIIbbeta3 activation. The studies focus on motifs located in alphaIIbased on data indicating that sequences in alphaIIb are responsible for the relative resistance of rat alphaIIbbeta3 to inhibition by RGD-containing peptides. To identify the sequences conferring RGD resistance, the consequences of a series of sequences exchanges between the human and rat proteins will be determined. The results will then be correlated with the secondary and tertiary structure of polypeptides corresponding to the amino terminus of alphaIIb as determined by circular dichroism spectroscopy, analytical ultracentrifugation, and x-ray crystallography. Specific Aim 2 addresses the sequence motifs located in the cytoplasmic and transmembrane segments of alphaIIb and beta3 that are involved in alphaIIbbeta3 activation. Sequence motifs in the cytoplasmic domains that mediate cytoskeletal constrain of alphaIIbbeta3 function will be identified using site-directed mutagenesis. To address the hypothesis that alphaIIbbeta3 activation results from specific interactions between the alphaIIb and beta3 cytoplasmic and/or transmembrane domains, the consequences of "disulfide and asparagine scanning" of the transmembrane and the juxta-membrane regions of alphaIIb and beta3 on alphaIIbbeta3 function will be tested and compared to the results of biophysical studies of the structure and oligomeric equilibria of peptides corresponding to the transmembrane helix and cytoplasmic domains of alphaIIb and beta3 in a membrane-like environment.