Thrombotrak solo coagulometer

Serially diluted, sulphated carbohydrate samples (25 μl) were incubated with normal human citrated plasma (50 μl; NHSBT) and Pathromtin SL reagent (50 μl; Siemens) for 2 min at 37 C prior to the addition of calcium chloride (25 μl; 50 mM). The time taken for clot formations to occur (an upper maximal of 2 mins was observed) were recorded using a Thrombotrak Solo coagulometer (Axis-Shield) as per the manufacturer’s instructions. Water and sodium porcine mucosal heparin (203 IU/mg; VWR) were assayed as controls. The EC₅₀ values of all semi-synthetic, sulphated carbohydrates were determined using a sigmoidal dose response curved fitted post normalisation (with a 100% upper maximal at 2 mins; 0% lower maximal represented by the time required for the water control to clot normal human citrated plasma) with GraphPad Prism 6 software and compared to those obtained for the heparin control.

Glycosaminoglycan samples (or control), pooled, normal human plasma (citrated; Technoclone, Vienna, Austria) and Pathromtin SL reagent (Siemens, Munich, Germany) were incubated for 120 s (37 °C) prior to the addition of 50 mM CaCl₂ (VWR, Lutterworth, UK; Vtot = 175 uL, 1:2:2:2 v/v). Clot formation times were determined using a Thrombotrak Solo coagulometer (Axis-Shield, Dundee, UK). An upper maximal of 120 s, representing 100% inhibition of clotting was adopted. Water (0% clot inhibition, representing a normal aPTT clotting time, of around 37–40 s) and porcine heparin (193 IU.mg⁻¹; Celsus, Cincinnati, OH, USA) were used as controls. EC₅₀ values were calculated by the fitting of a sigmoidal dose response curve (GraphPad Prism 7, San Diego, CA, USA).

Serially diluted GAG samples (25 μL) were incubated with pooled, normal human citrated plasma (50 μL; Technoclone, Surrey, UK) and Pathromtin SL reagent (50 μL; Siemens, Erlangen, Germany) for 2 mins at 37 °C prior to the addition of calcium chloride (25 μL, 50 mM; Alfa Aesar, Heysham, UK). The time taken for clot formations to occur (an upper maximal of 2 mins was imposed, represented as 100% inhibition of clotting) was recorded using a Thrombotrak Solo coagulometer (Axis-Shield). HPLC-grade H₂O (0% inhibition of clotting, representing a normal aPTT clotting time, of ≈ 37–40 seconds) and porcine mucosal heparin (193 IU mg⁻¹; Celsus, Cincinnati, OH, USA) were screened as controls. The EC₅₀ values of all test and control samples were determined using a sigmoidal dose response curve fitted with Prism 7 (GraphPad Software, San Diego, CA, USA).