THE INTEGRIN ALPHA(2)BETA(1) (GPIA IIA)-I-DOMAIN INHIBITS PLATELET-COLLAGEN INTERACTION/

The integrin alpha(2) beta(1) is a major cellular receptor for collage n. The alpha(2) subunit contains an +/- 200 amino acids inserted domai n (I-domain) in the N-terminal region. A certain degree of homology ex ists between the I-domains found in integrins, collagen and the A-doma ins of vWF. The alpha(2)-I-domain encoding region (aa residues D-145 t o S-334) was obtained by RT-PCR from mRNA of non stimulated human PBL' s. The primers were designed to introduce the necessary restriction si tes for cloning of the DNA fragment in frame downstream of the malE ge ne, as well as a stop codon after the last tripler. The resulting cons truct pMAL-c2-alpha(2)-I allows the expression of the I-domain, fused to the C-terminus of maltose binding protein (mal). The alpha(2)-I-mal is purified from the bacterial extract by affinity chromatography on an amylose column. The purified alpha(2)-I-mal has been characterized by ELISA's. The alpha(2)-I-mal bound to immobilised collagen type I in a concentration dependent manner and could be blocked by the function al monoclonal anti-alpha(2) beta(1) antibody 6F1. The interaction of a lpha(2)-I-mal with collagen furthermore is Mg2+-dependent since the bi nding was inhibited in the presence of 10 mM EDTA or 10 mM Ca2+ but su stained in the presence of 10 mM Mg2+ Finally, alpha(2)-I-mal itself w as able to inhibit adhesion of washed platelets to collagen immobilise d on a microtiterplate in a dose-dependent manner (alpha(2)-I-mal IC50 : 0.7 mu M) as well as platelet aggregation induced by collagen type I (alpha(2)-I-mal IC50: 0.7 mu M). With these results we could confirm that the alpha(2)-I-domain represents the collagen-binding site of alp ha(2) beta(1) and we furthermore could indicate that this domain is ab le to prevent platelet adhesion to collagen and collagen-induced plate let aggregation, pointing to the primordial role of alpha(2)-I-mal and hence of alpha(2),beta(1) in platelet-collagen interaction.