Sarah Bos

92 CHAPTER 6 stored at -80°C until use. In vitro addition of pro- and anticoagulants We added the following agents to plasma samples of each patient and control: • Pooled normal plasma (to mimic fresh frozen plasma [FFP] transfusion – obtained by combining plasma from > 200 healthy volunteers, a generous gift from Dr. J.C. Meijers, Academic Medical Center Amsterdam, The Netherlands) – final concentration 20% (v/v) • Cofact (a 4-factor prothrombin complex concentrate (PCC), Sanquin, Amsterdam, Netherlands) – final concentration 0.5 U/ml • Recombinant factor VIIa (Novo Nordisk, Bagsvaerd, Denmark) – final concentration 50 nM • The low molecular weight heparin (LMWH) Clexane (Sanofi-Aventis BV, Gouda, the Netherlands) – final concentration 0.2 U/ml • Unfractionated heparin (UFH, Leo Pharma, Denmark) – final concentration 0.1 U/ml • Dabigatran (Alsachim, Illkirch Graffenstaden, France) – final concentration 300 ng/ml • Rivaroxaban (Alsachim, Illkirch Graffenstaden, France) – final concentration 25 ng/ml The final concentrations of the anticoagulant drugs were based on initial experiments in which drugs were added in various concentrations to pooled normal plasma after which thrombin generation was performed as described in the next paragraph. Those drugs concentrations which gave appreciable (but not maximal) inhibition of thrombin generation in pooled normal plasma were selected so it would be possible to detect both increased and decreased drug effects in patients compared to controls. The final concentrations of the procoagulant drugs were chosen to mimic clinically relevant doses. All drugs were added in the same volume – thrombin generation tests are performed with 80 μ l of plasma per well and in all experiments 3 μ l of plasma was replaced by vehicle or drug. The exception was the pooled normal plasma addition, in which per well 16 μ l of plasma was replaced by 16 μ l of pooled normal plasma. All pro- and anticoagulants were added to samples obtained after induction of anesthesia, whereas only procoagulants were added to intraoperative, and only anticoagulants were added to postoperative samples. Thrombin generation The thrombin generation test was performed using platelet-poor plasma with Calibrated Automated Thrombography® in absence or presence of the above-