Antithrombin A total of 500 nM AT was mixed with 50 nM FXa or 50 nM thrombin at 37 °C in HBS/0.1% PEG/2 mM CaCl 2 (pH 7.4 or 7.0). At predetermined time intervals, an aliquot was removed and diluted 10fold (thrombin) or 20-fold (FXa) into HBS/0.1% PEG/ 20 mM EDTA (pH 7.4) containing 200 lM Spectrozyme TH or Spectrozyme Xa (Sekisui Diagnostics, Lexington, MA, USA). The absorbance at 405 nm was monitored immediately in a microplate reader (Molecular Devices, Sunnyvale, CA, USA). Rates of substrate hydrolysis at each time-point were converted to active enzyme concentrations and these data analyzed as a first-order process.
Tissue factor pathway inhibitor Characterization of the reaction between TFPI and FXa was performed as described previously [29] . FXa (1 nM final) was added to HBS/0.1% PEG/2 mM CaCl 2 (pH 7.4 or 7.0) at 37 °C. After the addition of TFPI (2 nM final), aliquots were removed at predetermined times and added to 200 lM Spectrozyme Xa. The reaction was diluted only 10% by the chromogenic substrate. The absorbance at 405 nm was monitored immediately as above. Rates of substrate hydrolysis at each time-point were converted to active enzyme concentrations and the resulting decay curves fitted with a double exponential to estimate the free FXa concentration present at equilibrium.
Tissue factor pathway inhibitor Characterization of the reaction between TFPI and FXa was performed as described previously [29] . FXa (1 nM final) was added to HBS/0.1% PEG/2 mM CaCl 2 (pH 7.4 or 7.0) at 37 °C. After the addition of TFPI (2 nM final), aliquots were removed at predetermined times and added to 200 lM Spectrozyme Xa. The reaction was diluted only 10% by the chromogenic substrate. The absorbance at 405 nm was monitored immediately as above. Rates of substrate hydrolysis at each time-point were converted to active enzyme concentrations and the resulting decay curves fitted with a double exponential to estimate the free FXa concentration present at equilibrium.