Recombinant human αiibβ3

Recombinant human αIIbβ3 and GPVI monomer were obtained from R&D Systems (Abingdon, Oxford, UK). Recombinant extracellular domain of GPVI (GPVIex, comprising D1D2 (amino acids 1–214; 42 kDa) fused with the Fc domain of human IgG (GPVI‐Fc2, 150 kDa) was prepared as previously described.24HB 96‐well plates (Nunc, Langenselbold, Germany) were coated with recombinant GPVI monomer or dimer (10 μg/ml in Phosphate‐Buffered Saline [PBS]) for 1 h at 24°C. All further incubations were performed at room temperature for 1 h unless otherwise stated. The wells were washed three times with adhesion buffer (1 mg/ml BSA in PBS containing 0.1% [v/v] Tween‐20) between each incubation step. The wells were then blocked with 50 mg/ml BSA in TBS prior to the addition of MMP‐13 at a concentration of 83 nmol L⁻¹ (unless otherwise stated) for 1 h at 24°C in adhesion buffer. Rabbit anti‐MMP‐13, raised against MMP‐13 hinge region (Abcam, Cambridge, UK), and goat anti‐rabbit HRP (Dako, Stockport, UK) were added at a dilution of 1:2000 in adhesion buffer prior to the addition of a TMB substrate system (Sigma, UK) and the plates read at 450 nm.