Solid-phase binding of vitronectin to β3 integrins

α_Vβ₃ integrin (EMD Millipore) or αIIbβ3 (Molecular Innovations) was applied to microtiter wells (250 ng per well) overnight, then blocked with BSA. PDI–CCCC, PDI–AAAA, ERp57-CCCC or BSA were added with 0.3 mM DTT to 100 μl of recalcified plasma. The reaction proceeded for 10 min at 37 °C and terminated with 20 mM NEM. Plasma was diluted 1:100, added to the coated plates for 1 h, and bound vitronectin quantitated with rabbit anti-vitronectin antibody detected with anti-rabbit IgG conjugated to HRP.