OC 44.3 - A new mouse model of impaired fibrin α-chain crosslinking shows increased venous thrombembolism

Background: Factor XIII (FXIII) crosslinks fibrin and incorporates fibrinolysis inhibitors into the clot, increasing resistance to mechanical strain and proteolytic degradation. We recently demonstrated that in-vivo thromboembolisation is increased in a mouse strain with abolished fibrin α-chain crosslinking (PNAS, 2021). However, the role of α-chain crosslinking in clot formation and stability in- and ex-vivo is not yet known.

Aims: The aims of this study were to generate a new genetically modified murine model of impaired fibrin α-chain crosslinking and investigate its impact on venous thromboembolism.

Methods: Genetically modified FGA4X mice were generated by mutating the fibrin α-chain glutamine residues involved in crosslinking by FXIII (αQ241N/Q243N/Q257N/Q518N). Mice phenotype was characterised, and clot properties were analysed by ROTEM and clot contraction assay. Pulmonary embolism was investigated by Xtreme live optical imaging, following FeCl3 injury to the inferior vena cava.

Results: Compared to WT, FGA4X mice showed no differences in haematological parameters. Fibrinogen levels were reduced (1.0±0.1 vs 1.5±0.1 mg/ml). Fibrin α-α, but not γ-γ, crosslinking was significantly reduced (35.6±6.2 vs 83.7±4.0 %). ROTEM analysis of whole blood showed that clotting time was increased (37.0±4.0 vs 47.0±6.0 sec) and maximum clot firmness was reduced (15.8±2.9 vs 22.9±3.0 mm, FIBTEM). Clot contraction experiments showed that FGA4X mice generated larger (46.7±1.4 vs 30.8±1.5 μl) and heavier (44.3±0.7 vs 31.6±1.8 mg) clots than WT, containing more red blood cells (+21 %). Pulmonary embolism was significantly increased (~1.8-fold) in FGA4X mice compared to WT, at 0.5, 1, 2, 4, 24 hrs post-injury of the inferior vena cava.

Conclusion(s): Our data show that α-α crosslinking plays a significant role in clot stability during thrombosis, and protects against clot fragmentation and subsequent pulmonary embolism. In combination with our previous study, these data indicate that α- and γ-chain crosslinking play important complementary roles in protection against venous thromboembolism.