Regular Article Monitoring platelet dependent thrombin generation in mice Yesim Dargaud a,b, ⁎, Henri M.H. Spronk a , Peter Leenders a , H. Coenraad Hemker c , Hugo Ten Cate a a Laboratory for Clinical Thrombosis and Haemostasis, Department of Internal Medicine, and Cardiovascular Research Institute Maastricht, Maastricht University, Maastricht, The Netherlands b Unite d'Hemostase Clinique, Hopital Edouard Herriot, Universite Lyon 1, Lyon, France c Synapse bv, Cardiovascular Research Institute Maastricht, Maastricht, The Netherlands abstract article info Article history: Received 25 May 2010 Received in revised form 10 August 2010 Accepted 13 August 2010 Available online 16 September 2010 Keywords: Thrombin generation Mouse Platelet Corn trypsin inhibitor Activated protein C resistance Calibrated automated thrombin generation assay was adapted to measure thrombin generation in platelet rich plasma from mice. Vena cava phlebotomy appeared the best technique for blood sampling. The concentration-effect curves of tissue factor and platelet count have been determined. Corn trypsin inhibitor 2 μM inhibits contact activation effectively. Recombinant human thrombomodulin does not inhibit thrombin generation in mouse plasma but activated protein C (20 nM) does. Thrombin generation was dose dependently diminished by low molecular weight heparin and increased by high concentrations of exogenous factor VIII i.e. the assay can detect both hypo- and hypercoagulability. © 2010 Elsevier Ltd. All rights reserved. Introduction Laboratory mice have become an important and frequently used model for studying haemostasis. It has been previously shown that the routine laboratory assays used to assess human coagulation, e.g. prothrombin time, activated partial thromboplastin time, are also suitable to assess murine coagulation [1]. However, the correlation of these assays with the clinical risk of bleeding or thrombosis appears weak explaining the growing interest of physicians for global haemostasis assays such as thrombin genera- tion test (TGT) [2]. Recently, Tchaikovski et al. [3] developed a calibrated automated thrombography -based thrombin generation assay in mouse plasma enabling the detection of factor V Leiden, oral contraception and pregnancy-induced hypercoagulability in mouse plasma. In vivo thrombin generation is strongly dependent upon platelet procoagulant functions. Interactions between platelets and the clotting system escape our attention when clotting is studied in platelet poor plasma (PPP). Platelet rich plasma (PRP), however, is the optimal material to explore the haemostatic capacity of patients with inherited and acquired platelet disorders. Platelets are also intimately involved in several steps of thrombin generation induced by recombinant activated factor VII (rFVIIa) in haemophiliacs with inhibitors. We, therefore employed the Calibrated automated throm- bin generation assay (CAT) to measure thrombin generation in mouse PRP and optimized the assay conditions to detect hypo- and hyper- coagulability in mouse PRP. Materials and methods Animals and blood sampling Experiments have been performed in twenty four female mice of the C57BL/6 strain weighting approximately 25 grams and 8- 12 weeks of age. The mice had free access to water and diets. The animals were housed in usual cages in an environment with con- trolled temperature at 20 ± 2 °C. Mouse blood was collected from the inferior vena cava (IVC) under isoflurane 5% anesthesia, unless otherwise indicated. As previously described, the abdominal cavity was opened and 180 μL 3.2% sodium citrate was i.v. administrated in the IVC, using a 23 gauge needle and 1 mL plastic syringe, 15-20 seconds prior to blood drawing from the same vein into the syringe [4]. Platelet rich plasma (PRP) was prepared immediately after blood drawing by centrifugation at 200 × g during 3 minutes at room temperature. Platelet poor plasma (PPP) was obtained by double centrifugation: samples were centrifuged at 2500 × g during 15 min- utes at room temperature, subsequently plasma was centrifuged at 10,000 × g for 5 minutes to remove platelets and cell fragments. Mouse platelets were counted with a Coulter ACT diff analyzer (Beckman Coulter, Mijdrecht, The Netherlands). PRP was prepared immediately after blood drawing and was used within 2 hours. Thrombosis Research 126 (2010) 436–441 ⁎ Corresponding author. Unite d'Hemostase Clinique, Hopital Edouard Herriot, 5 place d'Arsonval, 69003 Lyon, France. Tel.: +33 4 72117370; fax: +33 4 72117312. E-mail address: ydargaud@univ-lyon1.fr (Y. Dargaud). 0049-3848/$ – see front matter © 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.thromres.2010.08.007 Contents lists available at ScienceDirect Thrombosis Research journal homepage: www.elsevier.com/locate/thromres