Platelet Receptor Recognition Site on Human Fibrinogen. Synthesis and Structure-Function Relationship of Peptides Corresponding to the Carboxy-Terminal Segment of the γ Chain

Marek Kloczewiak, Sheila Timmons, Thomas J. Lukas, Jacek Hawiger*

*Corresponding author for this work

Research output: Contribution to journalArticle

339 Scopus citations

Abstract

Binding of fibrinogen to human platelets depends on the interaction of the γ-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 γ chain were investigated in this study. Both pentadecapeptide (γ397-411) and dodecapeptide (γ400-411) inhibited binding of 125I-fibrinogen to ADP-treated platelets, with the concentration causing 50% inhibition (IC50) being 28 μM. In comparison, decapeptide (γ402-411) was almost 4 times less active (IC50 = 106 μM), thus suggesting that the two histidine residues (γ400-401) are required for a full inhibitory effect. A heptapeptide (>405-411) had a similar effect (IC50 =102 μM) whereas a pentapeptide (γ407-411) was even less inhibitory (IC50 =190 μM), indicating that the lack of lysine (γ406) further diminishes the reactivity of the platelet recognition site on the > chain of human fibrinogen. The heptapeptide (γ400-406) containing two 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-γ397-411 and the dimer cystinyl-(tyrosyl-γ397-411)2 obtained by the formation of a disulfide bond between two 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 (>400-411). A polyvalent conjugate of cysteinyl-tyrosyl-γ397-411 with human serum albumin was able to induce aggregation of ADP-stimulated platelets which was blocked by pentadecapeptide (>397-411) or dodecapeptide (γ400-411). Furthermore, monospecific antibody Fab fragment directed against the peptide, encompassing residues γ385-411, 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 γ chain of human fibrinogen. This segment binds to specific platelet receptors and is involved in the aggregation of platelets.

Original languageEnglish (US)
Pages (from-to)1767-1774
Number of pages8
JournalBiochemistry
Volume23
Issue number8
DOIs
StatePublished - Apr 1984

ASJC Scopus subject areas

  • Biochemistry

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