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.


OBJECTIVE
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 CaCl(2) 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.
Department of Biochemistry, Cardiovascular Research Institute Maastricht, Maastricht University, Maastricht, The Netherland.
S N
SN
Tchaikovski
B J M
BJ
VAN Vlijmen
J
J
Rosing
G
G
Tans
D020016:Activated Protein C Resistance / Q000235:genetics*; D000328:Adult; D000818:Animals; D001331:Automation; D001780:Blood Coagulation Tests / Q000295:instrumentation* / Q000379:methods*; D001792:Blood Platelets / Q000378:metabolism; D001798:Blood Proteins / Q000378:metabolism; D002138:Calibration; D005260:Female; D006801:Humans; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D011516:Prothrombin / Q000378:metabolism; D012680:Sensitivity and Specificity; D013917:Thrombin / Q000737:chemistry* / Q000378:metabolism
D001798:Blood Proteins; D011516:Prothrombin; D013917:Thrombin