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

Summary

The integrin α₂β₁ is a major cellular receptor for collagen. The α₂subunit 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 α₂-I-domain encoding region (aa residues D₁₄₅ to S₃₃₄) 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-α₂-I allows the expression of the I-domain, fused to the C-terminus of maltose binding protein (mal). The α₂-I-mal is purified from the bacterial extract by affinity chromatography on an amylose column.

The purified α₂-I-mal has been characterized by ELISA’s. The animal bound to immobilised collagen type I in a concentration dependent manner and could be blocked by the functional monoclonal anti-α₂β₁ antibody 6F1.

The interaction of α₂-I-mal with collagen furthermore is Mg²⁺- dependent since the binding was inhibited in the presence of 10 mM EDTA or 10 mM Ca²⁺ but sustained in the presence of 10 mM Mg²⁺.

Finally, α₂-I-mal itself was able to inhibit adhesion of washed platelets to collagen immobilised on a microtiterplate in a dose-dependent manner (α₂-I-mal IC₅₀:0.7 μ M) as well as platelet aggregation induced by collagen type I (α₂-I-mal IC₅₀: 0.7 μM).

With these results we could confirm that the α₂-I-domain represents the collagen-binding site of α₂β₁ 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 α₂-I-mal and hence of α₂β₁ in platelet-collagen interaction.

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