S 2238

Chromogenic substrate S-2238 was purchased from Chromogenix (Milan, Italy); bovine thrombin and argatroban were purchased from Sigma-Aldrich (St. Louis, MO, USA). The phosphate buffer saline (PBS) (pH 7.2-7.4, 0.01 M) and dimethyl sulfoxide (DMSO) were purchased from Solarbio (Beijing, China). Standard substance of galangin, kaempferol, astragaline, fisetin, quercetin, quercitrin, isoquercitrin, rutin, isorhamnetin, isorhamnetin-3-O-neohespeidoside, typhaneoside, myricetin, myricitrin, baicalein, baicalin, wogonin, wogonoside, hispidulin, scutellarein, genkwanin, hydroxygenkwanin, luteolin, luteoloside, 6-methoxyluteolin, dihydroquercetin, dihydromyricetin, naringenin, naringin, narirutin, hesperetin, hesperidin, neohesperidin, L-epicatechin, (-)-epigallocatechin, (-)-epicatechin gallate, proanthocyanidin B1 with 98% purity on the basis of HPLC analysis were obtained from Chengdu Herbpurify Co., Ltd. (Chengdu, China). Herbacetin and morin were obtained from Chengdu Must Bio-Technology Co., Ltd. (Chengdu, China). chrysin, apigenin, diosmetin and kaempferol-7-O-β-d-glucopyranoside were purchased from Chengdu Desite Bio-Technology Co., Ltd. (Chengdu, China).

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.

The heparins were subject of amidolytic activity assessments of thrombin (FIIa) or factor Xa (FXa) by measuring the hydrolysis of chromogenic substrates as previously described10 (link)11 (link). Incubations were performed in 96-well plates with 10 nM antithrombin, 2.0 nM FXa or thrombin (all from Hematologic Technologies; Essex Junction, USA) and 0 to 2 μg mL⁻¹ heparins (dry weight) in TS/PEG buffer (0.02 M Tris/HCl, 0.15 M NaCl and 1.0 mg mL⁻¹ polyethylene glycol 8,000, pH 7.4). FXa or FIIa were added lastly to trigger the reaction. After incubation for 60 s at 37 °C, residual FXa or FIIa activities were determined by the addition of 100 μM of chromogenic substrates S-2765 or S-2238, respectively (both from Chromogenix; Molndal, Sweden). Absorbance (405 nm) was recorded for 300 s in a Thermomax Microplate Reader (American Devices; Sunnyvile, California). Anti-FXa and anti-FIIa activities were obtained by parallel line assays against the 6^th International Heparin Standard (NIBSC) using SoftMax Pro5.4.1 software (American Devices; Sunnyvile, California) and validated methods recommended by the pharmacopeias. When the results obtained with HB-I were adjusted to IU mL⁻¹ in the assays, they generate coincident curves with the 6^th International Heparin Standard (NIBSC).

After incubation with AgNP or AgMP for 4 h, samples were incubated with 5 nM factor Xa and 10 nM factor Va in Tyrode buffer (134 mM NaCl, 10 mM HEPES, 5 mM glucose, 2.9 mM KCl, 1 mM MgCl₂, 12 mM NaHCO₃, 0.34 mM Na₂HPO₄, 0.3% BSA, and 2 mM CaCl₂ at pH 7.4) for 3 min at 37 °C. Thrombin formation was initiated by addition of 2 μM prothrombin. Exactly 3 min after the addition of prothrombin, an aliquot of the suspension was transferred to a tube containing stop buffer (50 mM Tris-HCl, 120 mM NaCl, and 2 mM EDTA at pH 7.9). Thrombin activity was determined using the chromogenic substrate S2238 (chromogenic substrate for thrombin; Chromogenix, Milano, Italy). We calculated the rate of thrombin formation from the change in absorbance at 405 nm using a calibration curve generated with active-site–titrated thrombin.

The modulation of FXa and thrombin activity was evaluated by colorimetric assay.
Briefly, CTX (0.5-13.5 µg/mL) or PBS (control) was incubated with 43 mU/mL of
human FXa or thrombin (Sigma-Aldrich, St. Louis, USA) for 10 minutes.
Afterwards, specific chromogenic substrate S-2222 or S-2238 (400 µM,
Chromogenix, Milan, Italy), were added and the hydrolysis of chromogenic
substrate by FXa or thrombin was measured at 405 nm using a Spectramax 190
microplate reader (Molecular Devices, San Jose, USA). The reaction was carried
out in a 96-well plate at 25ºC in PBS, pH 7.4. The enzymatic activity was
calculated based on the slope of the activity curve, obtained from sequential
readings at 405 nm, and considered the values generated by the incubation of the
factors and PBS (control) to be 100% activity.

Prothrombinase assays were performed essentially as described previously.12, 20, 34 Prothrombin activation was initiated by addition of 0‐600 nmol/L prothrombin to a mixture of 8pM FVa, 5 nmol/L FXa, in the presence of 0‐50 μmol/L phospholipids (DOPC/DOPS; 90:10) and 2 mmol/L CaCl₂ in 25 mmol/L Tris (pH 7.4), 150 mmol/L NaCl containing 0.5 mg/mL ovalbumin.34To quantify the remaining FVa activity after APC‐mediated inactivation, assays were performed using 500 nmol/L prothrombin. Due to the reduced cofactor function of FVa^Nara, 16 pmol/L was used.
Reactions were allowed to proceed for 2 minutes at 37°C and then stopped by dilution into TBS, 20 mmol/L EDTA, 1% PEG 6000. Prothrombin activation was quantified by cleavage of S‐2238 (Chromogenix) and comparison to a thrombin standard curve.21