By serving as an activation dependent receptor for adhesive proteins, alphaIIbbeta3 mediates platelet aggregation, an essential event in thrombus formation. This function depends upon the capacity of alphaIIbbeta3 to transmit an """"""""inside-out"""""""" signal from its cytoplasmic domain to render its extracellular domain competent to bind fibrinogen. At the same time, platelets play a central role in the coagulation arm of the hemostatic/thrombotic response by providing a surface for prothrombin activation. A role of alphaIIbbeta3 in thrombin formation has been established; and a potential mechanism for this participation has been provided: prothrombin is a ligand for alphaIIbbeta3. The overall objective of this proposal is to develop a molecular understanding of ligand binding to alphaIIbbeta3 and the consequences of such interactions.
Three Aims will be explored with address specific aspects of the ligand binding function of the integrin. First, the capacity of alphaIIbbeta3 to bind prothrombin and accelerate thrombin formation will be explored. The hypothesis will be tested that platelets circulate in blood pre-armed with prothrombin bound via the RGD sequence in its thrombin domain to unactivated alphaIIbbeta3. Bound prothrombin is more readily activated to thrombin by Factor Xa, and thrombin is released from the receptor to stimulate platelets and activate alphaIIbbeta3, which then functions as an adhesion receptor to mediate platelet aggregation. Second, the nature of the ligand binding pocket(s) in alphaIIbbeta3 will be analyzed. The hypothesis will be tested that two very similar cyclic ligand peptides, cRGD and cHarGD, are high affinity surrogates for the RGD and fibrinogen gamma-chain recognition peptides and can bind to distinct, but allosterically linked sites in the receptor. These sites will be localized and characterized, and the reactivity of therapeutic GPIIb-IIIa blockers with these two sites will be analyzed. Third, the mechanism by which the cytoplasmic tails of the alphaIIb and beta3 subunits control activation of the ligand binding function of the receptor will be assessed. Based on a NMR structure, a """"""""molecular on-off switch"""""""" model for integrin activation by the cytoplasmic tail of alphaIIb has been proposed. This model will be tested using synthetic peptide, molecular biology, molecular modeling and further NMR analyses. The existence of a similar molecular switch in the beta3 subunit also will be evaluated. Taken together, these studies will provide fundamental insights into the ligand binding function of alphaIIbbeta3 and, by extrapolation, to that of other integrins as well. At the same time, these studies may have direct bearing on the use and future development of the GPIIb-IIIa blockers as antithrombotic agents.
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