Evidence that fibrinogen γ′ directly interferes with protofibril growth: implications for fibrin structure and clot stiffness

Summary.  Background:  Fibrinogen contains an alternatively spliced γ‐chain (γ′), which mainly exists as a heterodimer with the common γA‐chain (γA/γ′). Fibrinogen γ′ has been reported to inhibit thrombin and modulate fibrin structure, but the underlying mechanisms are unknown. Objective:  We aimed to investigate the molecular mechanism underpinning the influence of γ′ on fibrin polymerization, structure and viscoelasticity. Methods:  γA/γA and γA/γ′ fibrinogens were separated using anion exchange chromatography. Cross‐linking was controlled with purified FXIIIa and a synthetic inhibitor. Fibrin polymerization was analyzed by turbidity and gel‐point time was measured using a coagulometer. We used atomic force microscopy (AFM) to image protofibril formation while final clot structure was assessed by confocal and scanning electron microscopy. Clot viscoelasticity was measured using a magnetic microrheometer. Results:  γA/γ′ fibrin formed shorter oligomers by AFM than γA/γA, which in addition gelled earlier. γA/γ′ clots displayed a non‐homogenous arrangement of thin fibers compared with the uniform arrangements of thick fibers for γA/γA clots. These differences in clot structure were not due to thrombin inhibition as demonstrated in clots made with reptilase. Non‐cross‐linked γA/γA fibrin was approximately 2.7 × stiffer than γA/γ′. Cross‐linking by FXIIIa increased the stiffness of both fibrin variants; however, the difference in stiffness increased to approximately 4.6 × (γA/γA vs. γA/γ′). Conclusions:  Fibrinogen γ′ is associated with the formation of mechanically weaker, non‐uniform clots composed of thin fibers. This is caused by direct disruption of protofibril formation by γ′.