OC 29.1 - Both α- and γ-chain crosslinks mediated by FXIIIa affect fibrin fibre resistance to rupture

Background: Mechanical strength of individual fibrin fibres is essential to prevent clot breakdown and subsequent embolism. As the last step of clot formation, activated coagulation factor XIII (FXIIIa) crosslinks the α- and γ-chains of fibrin, thus increasing clot stiffness. We recently showed that the absence of γ-chain crosslinks leads to decreased rupture stress and toughness of fibrin fibres in murine plasma-purified fibrin (FGG3X, γQ398N/Q399N/K406R), resulting in more frequent pulmonary embolism (PNAS, 2021).

Aims: To investigate how α-crosslinks alter the mechanical behaviour of fibrin fibres, we generated a novel murine model (FGA4X, αQ241N/Q243N/Q257N/Q518N) with impaired α-α crosslinking, and compared individual fibre biomechanical behaviour with that of wild type (WT) fibres.

Methods: Clots from FGA4X, FGG3X or WT murine plasma were made on a striated surface using 8-times diluted plasma with the addition of 0.5 U/ml thrombin and 10 mM CaCl2. Individual fibrin fibres were stretched using lateral force sensing atomic force microscopy (AFM) combined with fluorescence microscopy, and the resulting stress-strain behaviour of each fibre was analysed.

Results: Compared with WT fibrin fibres, FGA4X fibres ruptured at 1.8-fold lower stress (p < 0.001) and had 1.3-fold lower toughness (ns). Similarly, a 1.9-fold decrease in rupture stress (p < 0.0001) was found for FGG3X fibres with a somewhat larger, 1.9-fold decrease for toughness (p < 0.0005), when compared with their WT counterparts. Both WT and 4X fibres gained ~70% extra length after rupture compared with their original length, showing a degree of unrecoverable deformation. Such deformation indicates permanently broken bonds, which represent potential places for rupture within the fibre.

Conclusion(s): Lower rupture stress and fibre toughness indicate that deficiency in both α- and γ-chain crosslinking results in fibres that are more prone to rupture. These data indicate that α- and γ-chain crosslinking play complementary roles in generating key biomechanical properties of fibrin clots for the prevention of embolism.