Abstract
Platelet membrane GPIIbIIIa is a member of the family receptors named integrins that recognize RGD sequences in their ligands. GPIIbIIIa interacts with at least three different adhesive ligands: fibrinogen, fibronectin, and von Willebrand factor. These interactions are inhibited by RGD-containing peptides and by peptides corresponding to a sequence unique to fibrinogen in the COOH-terminal domain of its gamma chain (HLGGAKQAGDV). Two RGD sequences are present in fibrinogen A alpha chain: an RGDS sequence at A alpha 572-575, and an RGDF sequence at A alpha 95-98. Polyclonal antibodies raised against the RGDF sequence and the gamma COOH-terminal domain both reacted specifically with fibrinogen in solid phase enzyme-linked immunosorbent assays and immunoprecipitated the protein in solution. The Fab fragments prepared from these antibodies inhibited fibrinogen-platelet interaction and aggregation. These results demonstrate that these two sequences are both accessible within the fibrinogen molecule and are both implicated in ligand binding and cell-cell interaction. In addition, by further examining the interaction of the gamma chain peptide with platelets, it was found that RGDF and the gamma peptide produced a similar dose-dependent inhibition of the binding of the labeled gamma peptide to ADP-stimulated platelets. These results provide evidence that the RGDF sequence present at the A alpha 95-98 constitutes with the gamma 401-411 sequence two recognition sites interacting with the same site or with mutually exclusive sites on GPIIbIIIa.
Highlights
From the SDepartement de Recherche Fondamentale/Laboratoired’Hematologie, Inserm U217, CEN G 85X Grenoble 38041, France and the §Departmentof Immunology, Research Institute of Scripps Clinic, La Jolla, California 92037
The says, the anti-NIMEILRGDF antibody reacted with fibrinodecapeptide, NIMEILRGDF, which includes the NHz-termi- gen or with NIMEILRGDF coated onto microtiter wells and nal adjacent sequencesto RGDF in the A a chain of fibrinogen, failed to react with the y peptide LGGAKQAGDV (Fig. 2 A )
The platelet fibrinogen receptor, GPIIbIIIa, is an adhesion receptor with a relaxed recognition specificity. This membrane protein interacts with fibronectin and von Willebrand factor. All three of these adhesive proteins contain an RGDX sequence with X varying from a hydrophilic to a hydrophobic residue
Summary
Comparative Effects of RGDF and y Peptides onFibrinogen Binding and Platelet Aggregation-The efficienciesof RGDFcontaining peptides and y peptides in inhibiting fibrinogen binding to the same platelet preparation were compared. This confirmed that inhibitory activity of RGD-containing fibrinogen or with the y chain peptide andnot with the peptides is greatly influenced by the adjacent amino acids as NIMEILRGDF peptide (Fig. 2B). Teaued at 60%, while the decapeptide produced complete inhibition These data suggest that the anti-NIMEILRGDF antibody contains an antibody population that recognizes RGDF as well as one that recognizes other peptide regions. Neither RGDS nor the y chain peptide inhibited the antibody binding of the decapeptide.Binding of anti-y peptide antibody was inhibited by the y peptide LGGAKQAGDV but not by NIMEILRGDF (Fig. 3B) These results establish the specificity of each antibody and indicate that the NIMEILRGDF and LGGAKQAGDV sequences are both accessible on the TABLEI. Effects of Antibodieson Fibrinogen Binding and Platelet Aggregation-Fab fragments of the anti-apeptide and anti-y on platelets from different donors
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