Glycoprotein IIb-IIIa-liposomes bind fibrinogen but do not undergo fibrinogen-mediated aggregation

Although platelet cross-bridging is mediated primarily by the binding of fi brinogen to its activated membrane receptor, glycoprotein (GP) IIb-IIIa*, s uch an interaction may not be sufficient to support the aggregation process . As this question could potentially be answered by reconstituting GPIIb-II Ia* into a non-platelet environment such as liposomes, a protocol was devel oped for the generation of large lipid vesicles containing purified GPIIb-I IIa*. Flow cytometric techniques confirmed that the receptor was present in the lipid bilayer and were used to evaluate the characteristics of fibrino gen binding to the liposomes, which like fibrinogen-platelet interactions e xhibited specificity, saturability, time dependence and calcium dependence. No fibrinogen-specific aggregation of GPIIb-IIIa* liposomes with stir or s hear was observed, as determined by flow cytometric cell counting and micro scopic examination of particles. In contrast, activated platelets rapidly b ound Fg and rapidly formed large aggregates. The Fg associated with GPIIb-I IIa* in liposomes aas 'normally' recognized by tao fluorescently labelled a ntibodies: 4A5, which interacts with the Fg gamma chain C-terminus (residue s 400-411) required for Fg-mediated cross-bridging of activated platelets i n platelet aggregation (Shiba E, Lindon JN, Kushner L, Matsueda GR, Hawiger J, Kloczewiak M, Kudryk B, Salzman EW. Antibody-detectable changes in fibr inogen adsorption affecting platelet activation on polymer surfaces. Am J P hysiol 1991; 260: C965-74), and anti-Fg-RIBS-I, which associates with an ep itope on Fg (residues 373-385) expressed upon binding to GPIIb-IIIa. These data suggest that the Fg gamma-terminus is sterically accessible for partic le crossbridging and that an identical conformational change occurs for rec eptor-bound Fg on both liposomes and platelets. It thus appears that cellul ar elements aside from GPIIb-IIIa, such as cytoskeletal proteins proposed t o be necessary for receptor 'anchoring', play a necessary role in flow-asso ciated platelet aggregation.