Sta r coagulometer

Citrated human plasma from two consenting healthy volunteers were pre-tested with normal coagulation parameters. Samples were pooled and mixed with 50mM HEPES and 140mM NaCl at pH 7.5 and SMIPP-C proteins or SMIPP-C buffer (negative control) to a final plasma concentration of 40% and incubated for 10min at 37°C in duplicates. The Thrombin Clotting Time (TCT) was determined in duplicates using the STA—Thrombin kit (Diagnostica Stago, France) and a Sta-R coagulometer (Diagnostica Stago, France). Statistical analysis was performed using a one-way ANOVA and Dunnett’s comparison (JMP v13.1, SAS Institute).

The clotting of blood involves multiple serine proteases so we performed two standard tests (activated partial thromboplastin time, APTT; and the prothrombin time, PT) to determine whether the two recombinant EgKIs had any effect on the intrinsic and the extrinsic pathways of coagulation. Fresh healthy human blood (30 ml) was collected into sodium citrate vacutainers and the plasma was separated. Then, 800 μl plasma was mixed with 50 μl of each EgKI protein (final concentrations of 200 pM, 200 nM and 2 μM for EgKI-2 and 200 pM, 200 nM and 10 μM for EgKI-1) and incubated in a 37°C water bath for 10 min. After adding CaCl₂ to the mixture, the time taken for clot formation was measured by a Sta-R coagulometer (Diagnostica stago, Asnières, France). TriniCLOT APTT HS (Trinity Biotech, Bray, Co Wicklow, Ireland) and Thromborel S (Siemens, Malvern, PA, USA) kits were used for the determination of the APTT and the PT, respectively. Aprotinin (Sigma Aldrich, St Louis, USA) and FVII negative plasma (Helena Laboratories, Texas, USA) were used as positive controls for APTT and PT, respectively.

Anti-Xa activity was assessed by quantifying residual factor Xa activity by cleavage of a chromogenic substrate (STA-Liquid Anti Xa, Diagnostica Stago, Asnieres, France), reference value <0.05 kIU/L. Activated partial thromboplastin time (aPTT) was measured with STA APTT reagent containing cephalin as a source of the phospholipids and silica as a particulate activator (reference range 30–42 seconds). Determination of thrombin time (TT) was done by standard commercial STA Thrombin 2 test (Diagnostica Stago, Asnieres, France), adding thrombin to patient plasma and measuring the clotting time (reference range 14–21 seconds). Antithrombin was measured by chromogenic substrate method (STA-STACHROM AT III, Diagnostica Stago, Asnieres, France) (reference range 0.8–1.2 kIU/L). All analyses were performed on STA-R coagulometer (Diagnostica Stago, Asnieres, France).