Structure and Function of the Platelet Integrin

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The objective of this project is to correlate the structure and function of the platelet membrane integrin alphaIIb/beta3, alphalIb/beta3 is a calcium-dependent heterodimer whose binding site for ligands such as fibrinogen and yon Willebrand factor is exposed by platelet stimulation. Because ligand binding to alphaIIb/Beta3 is responsible for platelet aggregation, regulation of the exposure of the tigand binding site on alphaIIb/Beta3 is a critical step in platelet function. Despite several decades of intensive study, the mechanism that enables alphaIIb/Beta3 to bind ligands is not known, nor have the tigand binding sites in alphaIIb/Beta3 been convincingly identified. The Specific Aims of this project addresses these questions. In Specific Aim 1, structural features of the alphallb and Beta3 transmembrane and cytoplasmic domains that regulate the alphaIIb/Beta3 activation state will be identified. The studies are based on our observations first, that proteins corresponding to the transmembrane and cytoplasmic domains of alphaIIb and Beta3, dispersed in lipid micelles, are present in monomer-dimer and monomer-trimer equilibria, respectively, and second, that the introduction of a polar asparagine at specific sites in the Beta3 transmembrane helix results in constitutive alphaIIb/Beta3 ligand binding activity. Proposed experiments will measure the igand binding activity of specific transmembrane and cytoplasmic domain mutants using transfected CHO cells and lymphocytes and ptatelets from transgenic mice. In Specific Aim 2, sequence motifs in the extracellular domains of alphaIIb and Beta3 that are responsible for ligand binding and for heterodimer formation will be studied, again using transfected CHO cells and transgenic mice. The proposed experiments will take advantage of the recently reported crystal structure of the extracellular domain of alpha upsilon Beta3 to identify specific alphaIIb/Bata3 regions and residues of interest. In Specific Aim 3, we will use laser tweezers to measure the function of alphaIIb/Beta3, other platelet receptors, and other integrins. Laser tweezers are an optical system that use laser light to trap and manipulate dielectric particles such as small beads or cells. Using this system, we have reproducibly and accurately measured the strength of fibrinogen and osteopontin binding to alphaIIb/Beta3 and alpha upsilon Beta3, respectively, both on agonist-stimulated living human and murine platelets, as well as on transfected tissue culture cells. In this Aim, we will extend these measurements to the function of alphaIIb/Beta3 mutants, to the interaction of von Willebrand factor with both alphaIIb/Beta3 and GPIb-IX-V, and to the interaction of Beta2 integrins with ligands such as ICAM- 1.