A novel mouse whole blood thrombin generation assay sensitive to FXI- and FIX-mediated amplification of coagulation

Thrombin generation (TG) assays serve as valuable tools to study the amplifying roles of intrinsic pathway factors in human coagulation and provide functional insight into the increased bleeding observed in individuals deficient in factors (F) XI, IX or VIII. Mice are used extensively in hemostasis research due to the availability of coagulation factor deficient mice. However, phenotypic differences between mouse and human TG have become apparent. Herein, we describe a novel, calibrated mouse whole blood (WB) TG assay used to assess the amplifying roles of intrinsic pathway factors in mouse coagulation. WB- and plasma-TG was triggered with either silica or tissue factor (TF) in samples from wild-type mice and mice deficient for FXII, FXI or FIX. As expected, silica-triggered WB-TG and platelet poor plasma (PPP)-TG were significantly reduced by deficiencies for FXII, FXI or FIX. FXII deficiency had no effect on WB-TG or PPP-TG when triggered with TF. However, FXI deficiency resulted in significantly reduced WB-TG triggered by low concentrations of TF but had no effect on TF-triggered PPP-TG. FIX deficiency profoundly reduced WB-TG when triggered by low or high concentrations of TF whereas TG in PPP or platelet rich plasma was only moderately reduced under these conditions. In conclusion, we have developed a novel mouse WB-TG assay with enhanced sensitivity to FXI- and FIX-dependent amplification of coagulation compared to an established plasma-TG assay. The enhanced sensitivity of WB-TG to FXI and FIX dependent amplification of coagulation suggests an important role of blood cells in this process.