In vitro prothrombin studies

The prothrombin neutralizing anti-human monoclonal antibody {"type":"entrez-nucleotide","attrs":{"text":"AB730006","term_id":"429901813","term_text":"AB730006"}}AB730006 ba SP14-046 IgG2b was obtained from MedImmune (Cambridge, UK).

Blood samples intended for in vitro studies at AstraZeneca R&D, Gothenburg, Sweden were drawn from 5 healthy volunteers (3 males and 2 females) after informed consent and approval from the local Gothenburg ethical committee (ethical permit number: 033-10). The sample size was not determined from a formal power calculation for this particular study but was based on previous experience of the assay methods used. Whole blood was collected into polypropylene tubes from Sarstedt (Nümbrecht, Germany) by free flow from a 17-gauge Venflon needle from Becton Dickinson Infusion Therapy AB (Helsingborg, Sweden). The first 2 mL of the collected blood was discarded. Then nine volumes blood was mixed with one volume 0.11 M trisodium citrate. Of the 50 mL whole blood collected per donor 10 mL was used for preparation of platelet poor plasma by centrifugation of the citrated blood at 2000 x g in a swing-out rotor for 20 min at 20 °C prior to transfer of the plasma supernatant to a new sample tube.

Clot elasticity was measured by a standard coagulation test (EXTEM + STARTEM reagents, Tem Innovations GmbH, Munich, Germany) using ROTEM equipment (Tem Innovations GmbH). Citrated blood, as described above, was pre-incubated with prothrombin neutralizing anti-human antibody (MedImmune) 0.021, 0.041, 0.062 and 0.083 mg/mL to study the effect of a specific decrease in prothrombin. A serial dilution was also performed to study the effect of a simultaneous decrease in all coagulation and anticoagulation factors. This step-wise dilution was done with 13 mM sodium citrate buffer starting at 100% whole blood and then 70, 50, 40, 30, 20, 10 and 5% whole blood. Samples were kept at 37 °C for 10 min before starting ROTEM analysis. Sodium citrate was included in the dilution buffer to keep the citrate concentration constant during dilution of the citrated whole blood. Thus, the free calcium concentration following addition of calcium for recalcifying purposes as part of the standard assay was kept constant. EXTEM CT and EXTEM MCF were evaluated by adding 300 μL of the incubated blood samples into the ROTEM equipment together with 20 μl EXTEM reagent and 20 μL STARTEM reagent according to the manufacturer instructions. All samples were run for 60 min.

Citrated plasma was pre-incubated with prothrombin neutralizing anti-human antibody (MedImmune) 0.021, 0.041, 0.062, 0.083, 0.124 and 0.165 mg/mL or diluted with 20 mM sodium citrate starting at 100% and then 70, 50, 40, 30, 20, 10 down to 5% plasma and kept at 37 °C for 9 min before PT was measured by adding 25 μL plasma to the KC 10 A micro coagulometer (Amelung, Lemgo, Germany). Dilution with a sodium citrate buffer for ROTEM analysis was performed as described for the diluted whole blood samples although the concentration of citrate had to be increased to compensate for the lack of hematocrit in plasma. After a 1 min incubation period at 37 °C in the coagulometer, 50 μL of Thromborel^® S (Siemens Healthcare GmbH, Marburg, Germany) was added and the time to coagulation was measured. Maximum measurement time was 15 min.