Principal Investigator: SANFORD SHATTIL
Abstract: During hemostasis, the platelet membrane becomes a conduit for the flow of information into and out of the cell. The long-term goal of this project is to test whether platelet adhesion, aggregation and procoagulant activity are regulated by specific interactions of plasma membrane constituents with intracellular regulatory molecules on the one hand and extracellular ligands on the other. The four specific aims will focus primarily on integrin GP IIb-IIIa, a receptor for RGD- containing adhesive proteins. First, the intracellular processes that regulate binding of fibrinogen to GP IIb-IIIa will be examined. To identify the signaling pathways involved, antibodies specific for and peptides derived from potential mediators, including GTP-binding proteins and protein kinases, will be introduced into permeabilized platelets, and their effects on fibrinogen receptor function measured by flow cytometry. A series of chemical cross-linking and immuno- precipitation strategies will also be employed to identify specific regulatory molecules that interact directly with the cytoplasmic tails of GP IIb or IIIa. Second, the process of inward signaling across GP IIb-IIIa will be characterized. To initiate fibrinogen binding without agonists, platelets will either be incubated with antibodies that directly induce ligand binding or allowed to adhere to immobilized fibrinogen. Then changes in cytoplasmic free Ca++, protein tyrosine and serine/threonine phosphorylation, F-actin assembly, and granule secretion will be monitored. In parallel studies, fluorescence resonance energy transfer techniques will be used to examine whether clustering of receptors is required for these fibrinogen-dependent platelet responses. Third, an antigen-antibody interaction will be studies as a model for integrin-fibrinogen interactions. PAC1 is an antibody specific for activated GP IIb-IIIa and its antibody-combining site mimics GP IIb-IIIa recognition sites in fibrinogen. To establish the molecular basis for this mimicry, recombinant Fab and Fv fragments of PAC1 will be expressed in mammalian and insect cell expression systems. Site-directed mutations will be introduced in the hyper- variable regions of PAC1 and their effects on antibody affinity and specificity determined. Fourth, a flow cytometric assay will be used to study clinical abnormalities of platelet membrane activation in whole blood. Platelets from individuals with reduced platelet aggregation responses will be studied to identify defects either in the structure or activation of GP IIb-IIIa or in signaling reactions distal to GP IIb-IIIa. An assay will also be developed to quantitate occupancy of GP IIb-IIIa by potential therapeutic agents, such as peptides (disintegrins), peptidomimetics and antibody fragments. Finally, we will test the hypothesis that the procoagulant activity of platelets contributes to thrombosis in two acquired disorders that affect the platelet membrane, paroxysmal nocturnal hemoglobinuria and the anti-phospholipid syndrome. Accordingly, platelets from bleeding time wounds in these patients will be studied for over-expression of membrane binding sites for factors Va and VIIIa. Overall, these studies should lead to a more detailed understanding of the platelet membrane events required for normal hemostasis and the role of membrane activation in hemorrhagic and thrombotic disorders.
Funding Period: 1993-04-01 - 1994-03-31
more information: NIH RePORT