Glycoprotein IIb-IIIa-liposomes bind fibrinogen but do not undergo fibrinogen-mediated aggregation.
Although platelet cross-bridging is mediated primarily by the binding of fibrinogen to its activated membrane receptor, glycoprotein (GP) IIb-IIIa*, such an interaction may not be sufficient to support the aggregation process. As this question could potentially be answered by reconstituting GPIIb-IIIa* into a non-platelet environment such as liposomes, a protocol was developed for the generation of large lipid vesicles containing purified GPIIb-IIIa*. Flow cytometric techniques confirmed that the receptor was present in the lipid bilayer and were used to evaluate the characteristics of fibrinogen binding to the liposomes, which like fibrinogen-platelet interactions exhibited specificity, saturability, time dependence and calcium dependence. No fibrinogen-specific aggregation of GPIIb-IIIa* liposomes with stir or shear was observed, as determined by flow cytometric cell counting and microscopic examination of particles. In contrast, activated platelets rapidly bound Fg and rapidly formed large aggregates. The Fg associated with GPIIb-IIIa* in liposomes was 'normally' recognized by two fluorescently labelled antibodies: 4A5, which interacts with the Fg gamma chain C-terminus (residues 400-411) required for Fg-mediated cross-bridging of activated platelets in platelet aggregation (Shiba E, Lindon JN, Kushner L, Matsueda GR, Hawiger J, Kloczewiak M, Kudryk B, Salzman EW. Antibody-detectable changes in fibrinogen adsorption affecting platelet activation on polymer surfaces. Am J Physiol 1991; 260: C965-74), and anti-Fg-RIBS-I, which associates with an epitope on Fg (residues 373-385) expressed upon binding to GPIIb-IIIa. These data suggest that the Fg gamma-terminus is sterically accessible for particle cross-bridging and that an identical conformational change occurs for receptor-bound Fg on both liposomes and platelets. It thus appears that cellular elements aside from GPIIb-IIIa, such as cytoskeletal proteins proposed to be necessary for receptor 'anchoring', play a necessary role in flow-associated platelet aggregation.
Department of Physiology, McGill University, Montreal, Quebec, Canada.
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Sloan
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Brown
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Liu
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Frojmovic
D000085:Acetates / Q000494:pharmacology; D000911:Antibodies, Monoclonal; D001665:Binding Sites; D001792:Blood Platelets / Q000737:chemistry; D002118:Calcium / Q000494:pharmacology; D048429:Cell Size; D005340:Fibrinogen / Q000378:metabolism* / Q000494:pharmacology; D005434:Flow Cytometry; D006801:Humans; D007700:Kinetics; D008081:Liposomes / Q000187:drug effects / Q000378:metabolism*; D008863:Microspheres; D008956:Models, Chemical; D010316:Particle Size; D010974:Platelet Aggregation / Q000187:drug effects*; D010975:Platelet Aggregation Inhibitors / Q000494:pharmacology; D019039:Platelet Glycoprotein GPIIb-IIIa Complex / Q000032:analysis / Q000276:immunology / Q000378:metabolism*; D011233:Precipitin Tests; D011485:Protein Binding / Q000187:drug effects; D011956:Receptors, Cell Surface / Q000378:metabolism; D014443:Tyrosine / Q000031:analogs & derivatives / Q000494:pharmacology
D000085:Acetates; D000911:Antibodies, Monoclonal; D008081:Liposomes; D010975:Platelet Aggregation Inhibitors; D019039:Platelet Glycoprotein GPIIb-IIIa Complex; D011956:Receptors, Cell Surface; D014443:Tyrosine; D005340:Fibrinogen; C084062:lamifiban; D002118:Calcium