Development of a calibrated automated thrombography based thrombin generation test in mouse plasma

Background: Mouse models have become increasingly important in thrombosis research. However, only a limited number of assays are available for assessment of the coagulation system in mouse plasma. Objectives: To quantify tissue factor-initiated thrombin generation in murine platelet-rich and platelet-free plasma and to develop a test for measurement of resistance to activated protein C (APC) in mouse plasma. Methods: Thrombin generation was monitored with calibrated automated thrombography (CAT) using a low-affinity fluorogenic substrate for thrombin. Results: To overcome the higher activity of coagulation inhibitors in mouse plasma as compared with human plasma, the reaction temperature was lowered to 33 degrees C and the assay was carried out at a 2-fold higher final plasma dilution (1:3) than commonly used for CAT in human plasma. This increased the endogenous thrombin potential (ETP) 4- to 5-fold and enabled reliable measurement of thrombin generation in both platelet-free and platelet-rich mouse plasma. For the APC resistance measurement, the reaction conditions were further optimized with respect to tissue factor, phospholipid, APC and CaCl2 concentrations. The test was validated using plasma of mice with different genetic background with respect to the factor V Leiden mutation (FV Leiden). Mice homozygous for FV Leiden had higher APC sensitivity ratios (mean 5.46; 95% CI 4.88-6.03) than heterozygous FV Leiden mice (mean 4.21; 95% CI 3.53-4.89) and than wild-type mice (mean 2.71; 95%CI 2.15-3.27). Conclusions: We have established reaction conditions for measurement of thrombin generation and APC resistance in mouse plasma. This assay enables evaluation of the coagulation system and the function of the protein C system in mouse models.