Hcatiii 0120

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.

HUVEC, GMVEC, LSEC, and fibroblast lysate/cell membrane fractions were collected without the use of the protease/phosphatase inhibitor cocktail. Thrombin activity was measured in 10 µl samples of undiluted lysate/cell membranes (7 µl lysis buffer/cm² cell surface area) and supernatant fractions (20 µl SF-media/cm² cell surface area) using the Abcam thrombin activity assay (ab234620). The cleavage of a specific thrombin substrate releases a p-nitroaniline chromophore that is detected at 405 nm. The change in mean 405 nm absorbance over time (measured every 30 min for 2 h) was recorded and cleavage rates were calculated. The minimum detectable value of human thrombin activity was stated by the manufacturer to be 0.031 AU/ml (0.0038 IU/ml). Samples below this threshold were denoted as negative. The manufacturer also reported that no significant cross-reactivity or interference has been observed with the thrombin-specific substrate. The specificity of the provided thrombin substrate was further tested by measuring the cleavage rate of 0.1 IU/ml thrombin after the addition of 1.78 U/ml AT (human antithrombin III, HCATIII-0120, Haematologic Technologies).