Spectrozyme xa

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.

The BL and BI heparins were supplemented in pooled normal human plasma and tested in a concentration range of 0 to 10 mg/mL. The clot-based activated partial thromboplastin time (aPTT) was performed on the ACL 300 Plus kinetic coagulation analyzer (Instrumentation Laboratory, Lexington, Massachusetts) using the Triniclot S aPTT reagent (Diagnostica Stago, Parsippany, New Jersey). The amidolytic anti-IIa and anti-Xa assays were run as the following. For BI1-6 samples, BIOPHEN Heparin Anti-IIa (or Anti-Xa) kit (ANIARA Diagnostica, West Chester, Ohio) was used in accordance with the manufacturer's instructions. The absorbance was measured at 405 nm using Spectra Max M5 (Molecular Devices, Sunnyvale, California). For all the other samples, bovine Xa and human thrombin were purchased from Enzyme Research Laboratories (South Bend, Indiana), and chromogenic substrates, Spectrozyme Xa and TH, were obtained from Sekisui Diagnostics (Stamford, Connecticut). The assay was run by ACL ELITE (Instrumentation Laboratory). As a standard for all of the samples, USP Heparin Sodium for Assays RS (The United States Pharmacopeial Convention, Inc, Maryland) was used (Lot #: F0I187). This USP RS has an assigned potency of 2144 USP U/ampule. The potency of each of the heparins was calculated based on the calibration curve prepared with the USP RS.