Generation and characterization of peptide-specific antibodies that inhibit von Willebrand factor binding to glycoprotein IIb-IIIa without interacting with other adhesive molecules. Selectivity is conferred by Pro1743 and other amino acid residues adjacent to the sequence Arg1744-Gly1745-Asp1746.

S Berliner,K Niiya,J R Roberts,R A Houghten,Z M Ruggeri

doi:10.1016/s0021-9258(18)68526-6

Abstract

We have generated antibodies against a synthetic peptide corresponding to the sequence of human von Willebrand factor (vWF) between residues Glu1737-Ser1750 which includes the Arg-Gly-Asp sequence common to several adhesive molecules. Two anti-peptide antibodies, one polyclonal, and one monoclonal reacted with native vWF and inhibited its binding to platelet glycoprotein (GP) IIb-IIIa, but showed negligible cross-reactivity with fibrinogen, fibronectin, and vitronectin, three other molecules that contain the sequence Arg-Gly-Asp and bind to platelets. The structural bases for the specificity of the two antibodies were evaluated by testing the ability of peptides homologous to the parent sequence, but with single amino acid substitutions, to neutralize the binding of the two antibodies to vWF. The substitution of Pro1743, the residue immediately adjacent to the Arg-Gly-Asp sequence on the amino-terminal side, with Phe resulted in a peptide that failed to interact with either antibody. Thus, Pro1743 is important for maintaining a peptide conformation recognized by two antibodies specific for the GP IIb-IIIa-binding domain of vWF. Other residues important for optimal peptide reactivity with the polyclonal antibody were Ser1742, Arg1744, and Gly1745, whereas Gly1741, Gly1745, and Asp1746, but not Arg1744, were important for reactivity with the monoclonal antibody. The epitopes of both antibodies, therefore, included at least 2 of the residues in the sequence Arg-Gly-Asp considered the common cell-binding site of adhesive molecules that interact with GP IIb-IIIa. Nevertheless, both antibodies reacted only with vWF. These studies demonstrate that peptide-specific antibodies, unlike the promiscuous GP IIb-IIIa receptor, can recognize distinctive structural characteristics of the cell-binding domain of adhesive molecules imposed by residues adjacent to the sequence Arg-Gly-Asp.

Highlights

From the Divisionof Experimental Hemostasis, Department of Basic and Clinical Research, Research Institute of ScrippCs linic, La JoUa, California 92037
Thesubstitution of Pro1743,the residue immediately adjacent to the Arg-Gly-Asp sediscontinuation of vascular integrity, contribute to the arrest of bleeding [1].In pathological conditions, platelet thrombi may lead to theocclusion of vessels with consequent ischemic damage to organs [2].aclear definition of the molecular bases of platelet function is essential for understanding the mechanisms of normal hemostasis as well as the development of arterial thrombosis
The present report demonstratesthat antibodies generated against a synthetic peptide representing the sequence of the vWF subunit between residues G l ~ ' ~ ~ ~ - S erre'a~ct~w' 'ith the the other mixtures, the antibody was incubated with a modified synthetic peptide having a single substitution of phenylalanine ( F ) for one of the amino acids in the sequence, as indicated in the figure with the single letter abbreviation for the substituted residue

Summary

Introduction

Two anti-peptide antibodies, one polyclonal, and one monoclonal reacted with native vWF and inhibited its binding to platelet glycoprotein (GP) IIb-IIIa, but showed negligible cross-reactivity with fibrinogen, fibronectin, and vitronectin, three other molecules that contain the sequence Arg-Gly-Asp and bind to platelets. The present results provide information relevant for understanding the structural bases of the specificity of these antibodies and support the concept that the binding domains of different adhesive molecules containing the sequence Arg-Gly-Asp may have unique conformations.

Results

Conclusion