Sulfated Glycans Inhibit Coagulation Factors

Sulfated glycans were assayed for their serpin-mediated inhibitory activity against IIa and Xa using effective concentrations of 10 nM of AT (# HCATIII-0120, Haematologic Technologies) or 25 nM HCII (# HCII-0190, Haematologic Technologies), 2 nM of IIa (# HCT-0020, Haematologic Technologies) or factor Xa (# HCXA-0060, Haematologic Technologies), and 0 to 100 μg/ml of sulfated glycans in 100 μl of TS/PEG buffer (20 mM Tris–HCl, 0.15 M NaCl, and 1.0 mg/ml PEG 8000, pH 7.4) as reported earlier (77 ). Sulfated glycans (10 μl) at varying concentrations were dispensed into a 96-well microtiter plate, followed by the addition of 40 μl AT (25 nM) or HCII (25 nM). A 50 μl aliquot of IIa (4 nM) or Xa (4 nM) was added last to initiate the reaction. The plate was then immediately incubated at 37 °C for 1 min. This was followed by the addition of 25 μl of chromogenic substrate S-2238 (# S820324, Chromogenix) for IIa or CS–11 (32 (link)) (# 229011, Hyphen-BioMed) for factor Xa. Absorbances were then measured at 405 nm for 300 s at an interval of 15 s. Wells without sulfated glycans served as controls and IIa/Xa activity in the control was considered 100%. Residual activity in treated wells was calculated relative to that observed in control wells. Heparin (180 IU/mg) was used in all assays as a positive control.