The Integrin α2β1 (GPIa/IIa)-I-Domain Inhibits Platelet-Collagen Interaction

The integrin α 2 β 1 is a major cellular receptor for collagen. The α 2 subunit contains an ± 200 amino acids inserted domain (I-domain) in the N-terminal region. A certain degree of homology exists between the I-domains found in integrins, collagen and the A-domains of vWF. The α 2 -I-domain encoding region (aa residues D 145 to S 334 ) was obtained by RT-PCR from mRNA of non stimulated human PBL's. The primers were designed to introduce the necessary restriction sites for cloning of the DNA fragment in frame downstream of the malE gene, as well as a stop codon after the last triplet. The resulting construct pMAL-c2-α 2 -I allows the expression of the I-domain, fused to the C-terminus of maltose binding protein (mal). The α 2 -I-mal is purified from the bacterial extract by affinity chromatography on an amylose column. The purified α 2 -I-mal has been characterized by ELISA's. The α 2 -I-mal bound to immobilised collagen type I in a concentration dependent manner and could be blocked by the functional monoclonal anti-α 2 β 1 antibody 6F1. The interaction of α 2 -I-mal with collagen furthermore is Mg 2+ -dependent since the binding was inhibited in the presence of 10 mM EDTA or 10 mM Ca 2+ but sustained in the presence of 10 mM Mg 2+ . Finally, α 2 -I-mal itself was able to inhibit adhesion of washed platelets to collagen immobilised on a microtiterplate in a dose-dependent manner (α 2 -I-mal IC 50 : 0.7 μM) as well as platelet aggregation induced by collagen type I (α 2 -I-mal IC 50 : 0.7 μM). With these results we could confirm that the α 2 -I-domain represents the collagen-binding site of α 2 β 1 and we furthermore could indicate that this domain is able to prevent platelet adhesion to collagen and collagen-induced platelet aggregation, pointing to the primordial role of α 2 -I-mal and hence of α 2 β 1 in platelet-collagen interaction.