Collagen type 1 coated coverslips

HUVECs (Lonza, Catalogue # C2519A) were grown on collagen type I–coated coverslips (BD Biosciences) in EGM-2 media (Lonza). Serum-starved washed cells were activated with human α-thrombin (1 U ml⁻¹) and 1 mM MnCl₂ for 10 min at 37 °C. After washing, thrombin was inhibited with hirudin (5 U ml⁻¹). Cells were treated with GRGESP (5 μM), GRGDSP (5 μM) or an anti-α_Vβ₃ monoclonal antibody (clone LM609 (10 μg ml⁻¹; EMD Millipore) for 30 min. PDI-CCCC (15 μM) or PDI-AAAA (15 μM) was added to recalcified plasma (100 μl) containing 5 μM GPRP and 0.3 mM DTT. The reaction was incubated at 37 °C for 5 min, diluted 1:10 with EGM-2 medium containing 1 mM MnCl₂ then added to the activated HUVECs. After 30 min, the cells were washed, fixed with 4% paraformaldehyde then blocked with 10% goat serum. Cells were stained for DAPI and vitronectin using rabbit anti human vitronectin and goat-anti rabbit labelled with Alexa 488, and imaged by fluorescence microscopy.