Recombinant fibrinogen reveals the differential roles of α‐ and γ‐chain cross‐linking and molecular heterogeneity in fibrin clot strain‐stiffening

Essentials Fibrinogen circulates in human plasma as a complex mixture of heterogeneous molecular variants. We measured strain‐stiffening of recombinantly produced fibrinogen upon clotting. Factor XIII and molecular heterogeneity alter clot elasticity at the protofibril and fiber level. This highlights the hitherto unknown role of molecular composition in fibrin clot mechanics. Summary Background Fibrin plays a crucial role in haemostasis and wound healing by forming strain‐stiffening fibrous networks that reinforce blood clots. The molecular origin of fibrin's strain‐stiffening behavior remains poorly understood, primarily because plasma fibrinogen is a complex mixture of heterogeneous molecular variants and is often contaminated by plasma factors that affect clot properties. Objectives and methods To facilitate mechanistic dissection of fibrin nonlinear elasticity, we produced a homogeneous recombinant fibrinogen corresponding to the main variant in human plasma, termed rFib610. We characterized the structure of rFib610 clots using turbidimetry, microscopy and X‐ray scattering. We used rheology to measure the strain‐stiffening behavior of the clots and determined the fiber properties by modeling the clots as semi‐flexible polymer networks. Results We show that addition of FXIII to rFib610 clots causes a dose‐dependent stiffness increase at small deformations and renders the strain‐stiffening response reversible. We find that γ‐chain cross‐linking contributes to clot elasticity by changing the force–extension behavior of the protofibrils, whereas α‐chain cross‐linking stiffens the fibers, as a consequence of tighter coupling between the constituent protofibrils. Interestingly, rFib610 protofibrils have a 25% larger bending rigidity than plasma‐purified fibrin protofibrils and a delayed strain‐stiffening, indicating that molecular heterogeneity influences clot mechanics at the protofibril scale. Conclusions Fibrinogen molecular heterogeneity and FXIII affect the mechanical function of fibrin clots by altering the nonlinear viscoelastic properties at the protofibril and fiber scale. This work provides a starting point to investigate the role of molecular heterogeneity of plasma fibrinogen in fibrin clot mechanics and haemostasis.