Erythrocyte-derived microparticles supporting PTase and Xase complexes.

<p>A) To 50 µL reaction mix (EryMPs, FXa and CaCl₂), 50 µL FVa and 150 µL prothrombin were added and incubated at 37°C. The reaction was stopped after 2 minutes by dilution in EDTA-containing stop buffer. The thrombin formed was measured kinetically with chromogenic substrate (S-2238). Human thrombin of known concentration was used to obtain a calibration curve for the conversion of the substrate. Final concentrations: 0.5 µM prothrombin, 5 nM hFXa, 40 pM FVa, 3 mM CaCl₂ and 0–6×10⁶ eryMPs/mL. B) To 60 µL FVIIIa (370 mU/mL) and FIXa (8.9 nM) 40 µL eryMPs (FC: 0–16×10⁶/mL) and bovine FX (FC: 0.5 µM) were added and incubated for 3 minutes. The reaction was stopped by dilution 1/16 in ice-cold EDTA-containing buffer. Formed FXa, was measured kinetically by conversion of the chromogenic substrate S-2765 (Chromogenix). Known concentrations of FXa were used to obtain a calibration curve for the conversion of the substrate.