α-α Cross-links increase fibrin fiber elasticity and stiffness

Abstract

Fibrin fibers, which are ~100 nm in diameter, are the major structural component of a blood clot. The mechanical properties of single fibrin fibers determine the behavior of a blood clot and, thus, have a critical influence on heart attacks, strokes, and embolisms. Cross-linking is thought to fortify blood clots; though, the role of α-α cross-links in fibrin fiber assembly and their effect on the mechanical properties of single fibrin fibers are poorly understood. To address this knowledge gap, we used a combined fluorescence and atomic force microscope technique to determine the stiffness (modulus), extensibility, and elasticity of individual, uncross-linked, exclusively α-α cross-linked (γQ398N/Q399N/K406R fibrinogen variant), and completely cross-linked fibrin fibers. Exclusive α-α cross-linking results in 2.5× stiffer and 1.5× more elastic fibers, whereas full cross-linking results in 3.75× stiffer, 1.2× more elastic, but 1.2× less extensible fibers, as compared to uncross-linked fibers. On the basis of these results and data from the literature, we propose a model in which the α-C region plays a significant role in inter- and intralinking of fibrin molecules and protofibrils, endowing fibrin fibers with increased stiffness and elasticity.

Figure 1

Recombinant fibrinogen preparation and gel electrophoresis. SDS polyacrylamide gel electrophoresis of fibrin. Lane I is the molecular mass standard. Lanes II and III are uncross-linked γQ398N/Q399N/K406R fibrin; the α-, β- and γ-chains are visible. Lanes IV and V are cross-linked γQ398N/Q399N/K406R fibrin. The β- and γ-chains remain while the α-chain disappears due to cross-linking and appears at the top of the gel as a high molecular mass α multimer. Lanes VI and VII are cross-linked native (wild-type) fibrin. Both the α- and γ-chains disappear and a γ-dimer band appears at 94 kDa, as well as the α multimer band at the top of the gel. Lane VIII is uncross-linked native (wild-type) fibrin showing the α, β, and γ bands.

Figure 2

Fibrin fiber mechanical properties. (A–C) Optical images of γQ398N/Q399N/K406R fiber extensibility measurement. The AFM tip is indicated by a green asterisk; the fibrin fiber, attached to 7 μm wide ridges (white bands), extends across a 13 μm wide grove (dark band). (C) Fiber ruptures at a strain of 350%. (D) Top view schematic of the experimental setup. The initial, unstretched fiber, with length L_initial, is shown in gray; the stretched fiber, with length, L', is shown in black, and the AFM tip is shown as a green dot. The AFM tip travels a distance s, using the Pythagorean Theorem, the fiber is stretched to L' = (s² + L_initial²)^1/2. s is obtained from the nanoManipulator AFM data file, L_initial is determined from the optical image.

Figure 3

Fibrin fiber modulus. (A) Stress-strain plots. The modulus of individual fibers corresponds to the slope of the stress-strain graphs. (B) Modulus; NU – native, uncross-linked; NX – native, cross-linked; VU – variant, uncross-linked; VX – variant, cross-linked. (C) Representative force-extension curve.

Figure 4

Fibrin fiber extensibility and elasticity. (A) Extensibility; and (B) elasticity of uncross-linked and cross-linked fibers; NU – native, uncross-linked; NX – native, cross-linked; VU – variant, uncross-linked; VX – variant, cross-linked.

Figure 5

Fibrin fiber model. (A) Cartoon model of unstrained fiber. The main interactions for directed protofibril assembly are the short A:a interactions. The 60 nm long α-C connector can form intermolecular and interprotofibrillar interactions. Addition of FXIII covalently links the α-C domains to the α-C connectors; α multimers can be formed because there are several Glutamine donors on each α-C connector and several Lysine acceptors on each α-C domain. This results in a loose, polymer chain-like structure. Such a structure would result in elastic and extensible fibers. Longitudinal γ–γ cross-links are shown between abutting fibrin monomers, though transverse γ–γ cross-links have also been proposed (40). (B) Cartoon model of strained fiber (ε = 100%). The α-C connectors are stretched, and the α-helical coiled coils are converted to extended β-strands; the α-C connectors provide elasticity to revert back to the unstrained fiber. γ–γ cross-linking stiffens fibers by channeling more of the strain through the coiled coils of the fibrin monomer.