Platelet receptor recognition site on human fibrinogen. Synthesis and structure-function relationship of peptides corresponding to the carboxy-terminal segment of the .gamma. chain

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Abstract
Binding of fibrinogen to human platelets depends on the interaction of the .gamma.-chain carboxy-terminal segment with specific receptors exposed by different agonists such as ADP, epinephrine and thrombin. The functions of a series of synthetic peptides encompassing the sequence of the 15 carboxy-terminal residues of the .gamma. chain were investigated in this study. Both pentadecapeptide (.gamma. 397-411) and dodecapeptide (.gamma.400-411) inhibited binding of 125I-fibrinogen to ADP-treated platelets, with the concentration causing 50% inhibition (IC50) being 28 .mu.M. Decapeptide (.gamma.402-411) was almost 4 times less active (IC50 = 106 .mu.M), thus suggesting that the 2 histidine residues (.gamma.400-401) are required for a full inhibitory effect. A heptapeptide (.gamma.405-411) had a similar effect (IC50 = 102 .mu.M) whereas a pentapeptide (.gamma.407-411) was even less inhibitory (IC50 = 190 .mu.M), indicating that the lack of lysine (.gamma.406) further diminishes the reactivity of the platelet recognition site on the .gamma. chain of human fibrinogen. The heptapeptide (.gamma.400-406) containing 2 histidine residues and derived from the dodecapeptide by proteolytic degradation with trypsin had very low inhibitory activity. The synthetic peptides inhibited fibrinogen-supported platelet aggregation in the same order of decreasing reactivity: pentadecapeptide = dodecapeptide > decapeptide = heptapeptide > pentapeptide. Modified synthetic pentadecapeptides bearing tyrosine or cysteinyltyrosine at the amino terminal were prepared to provide a means for radiolabeling and for formation of molecules of higher valency. Tyrosyl-.gamma.397-411 and the dimer cystinyl-(tyrosyl-.gamma.397-411)2 obtained by the formation of a disulfide bond between 2 single peptides had the same inhibitory activity toward the fibrinogen receptor on platelets. Radiolabeled tyrosyl-pentadecapeptide exhibited specific binding to human platelets which was inhibited by the dodecapeptide (.gamma.400-411). A polyvalent conjugate of cysteinyl-tyrosyl-.gamma.397-411 with human serum albumin was able to induce aggregating of ADP-stimulated platelets which was blocked by pentadecapeptide (.gamma.397-411) or dodecapeptide (.gamma.-400-411). Monospecific antibody Fab fragment directed against the peptide, encompassing residues .gamma.385-411, partially inhibited the platelet-aggregating function of the synthetic pentadecapeptide-albumin conjugate. Thus a polyvalent peptide conjugate functioned as a synthetic fibrinogen substitute in the platelet aggregation system. In conclusion, the continuous sequence of the 12 amino acid residues at the carboxy-terminal end constitutes the platelet recognition site for the .gamma. chain of human fibrinogen. This segment binds to specific platelet receptors and is involved in the aggregation of platelets.