IDENTIFICATION OF THE EPITOPE FOR A MONOCLONAL-ANTIBODY THAT BLOCKS PLATELET-AGGREGATION INDUCED BY TYPE-III COLLAGEN

A library of eight conformation-dependent monoclonal antibodies that r eact with distinct epitopes on native human type III collagen has been examined for the ability of these antibodies to inhibit platelet aggr egation induced by this collagen. Six of these antibodies had no effec ts; one, 1E7-D7/Col3, delayed the onset and slowed the rate of platele t aggregation, while another, 2G8-B1/Col3, completely inhibited aggreg ation. In order to identify the epitope recognized by this inhibitory antibody, a series of peptides that could fold to form triple-helical fragments was examined. Each peptide included six Gly-Xaa-Yaa triplets from the human type III collagen sequence, where Xaa and Yaa represen t the particular amino acids in the sequence, and a C-terminal (Gly-Pr o-Hyp)(4) sequence to enhance triple-helical stability. Using these pe ptides we have identified the epitope as a nine-amino-acid sequence, G LACAOGLR (where O is the one-letter code for Lt-hydroxyproline), start ing at position 520 in the human type III collagen helical domain. Thi s sequence is proximal to the site proposed for the interaction of typ e III collagen with alpha(2) beta(1)-integrin of platelets.