Evidence that αC region is origin of low modulus, high extensibility, and strain stiffening in fibrin fibers

Abstract

Fibrin fibers form the structural scaffold of blood clots and perform the mechanical task of stemming blood flow. Several decades of investigation of fibrin fiber networks using macroscopic techniques have revealed remarkable mechanical properties. More recently, the microscopic origins of fibrin's mechanics have been probed through direct measurements on single fibrin fibers and individual fibrinogen molecules. Using a nanomanipulation system, we investigated the mechanical properties of individual fibrin fibers. The fibers were stretched with the atomic force microscope, and stress-versus-strain data was collected for fibers formed with and without ligation by the activated transglutaminase factor XIII (FXIIIa). We observed that ligation with FXIIIa nearly doubled the stiffness of the fibers. The stress-versus-strain behavior indicates that fibrin fibers exhibit properties similar to other elastomeric biopolymers. We propose a mechanical model that fits our observed force extension data, is consistent with the results of the ligation data, and suggests that the large observed extensibility in fibrin fibers is mediated by the natively unfolded regions of the molecule. Although some models attribute fibrin's force-versus-extension behavior to unfolding of structured regions within the monomer, our analysis argues that these models are inconsistent with the measured extensibility and elastic modulus.

Figure 1

Setup for single fiber and network stretching experiments. Suspended fibrin fibers are labeled with fluorescent beads and then stretched with the AFM tip. Movies of the stretching are taken from below with epifluorescence imaging.

Figure 2

Stress-versus-strain plots of individual fibrin fibers. (A–D) AFM manipulation of a fibrin fiber suspended over micropatterned channel. The AFM tip (not visible) was brought in contact with a suspended fiber and stretched (B and C) to the point of failure (D). Scale bar = 10 μm. (E) Representative stress-versus-strain data of individual fibers with and without FXIII ligation. Both fibers show relatively linear behavior up to strain just above 1.0 followed by significant stiffening. (F) Tangent modulus illustrating the strain-dependent stiffness. These traces were found by numerically differentiating the traces in panel E. At high strains just before failure, the tangent moduli levels off and drops, indicating an end to stiffening. It is not clear this reflects the intrinsic properties of the fiber or is a result of slippage at constraint points.

Figure 3

Average tangent modulus at discrete strains for ligated (N = 14) and unligated (N = 14) fibers. At 0.25 strain, the ligated fibers have an elastic modulus of 2.1 ± 0.3 MPa whereas the unligated have a modulus of 1.1 ± 0.2 MPa (P < 0.003). The average modulus rises to 9.8 ± 1.2 MPa for ligated and 6.9 ± 1.3 MPa for unligated fibers (P < 0.05).

Figure 4

Western blot of fibrin showing α-, β-, and γ-bands without FXIIIa (A) and α-, β-, and γ-γ dimer after ligation with FXIIIa (B).

Figure 5

In situ fiber stiffness measurements. Fiber stiffness before (blue) and after (red) FXIII ligation.

Figure 6

Fibrin structure and corresponding mechanical model. (A) Crystal structure of fibrinogen. The α-, β-, and γ-polypeptides in blue, green, and red respectively, with a cartoon of the C-terminus region of the α-chain (in dashed blue). (B) Cartoon depiction of the fibrinogen. (C) (Upper model) Simplified fibrin fiber structure. Intraprotofibril FXIII-induced covalent interactions (red dashes). Protofibrils are connected through αC region interactions (αC regions in blue). We note that this picture is simplified for clarity. In a real fiber, each monomer has two αC regions extending. Lines (blue) spanning protofibrils represent αC/αC interactions. Though the figure suggests only pairwise αC interactions, it is known that αC regions typically form interactions with multiple other αC regions. (Dashed vertical lines) The 22.5-nm half-stagger periodicity within the fiber that is evidenced by banding in numerous TEM studies. (Lower model) Stretching of the fiber under stress. This cartoon depicts a model of one potential mechanism of extensibility. In this case, the αC domains, though linking protofibrils laterally, accommodate the tensile strain induced by applied force (green arrows). The stiff protofibrils, within this model, are represented by the stiff springs and accommodate little of the strain. (D) Simple mechanical model of the fibrin monomer. The linear spring (black) represents the stiff structured portion of the monomer (spring constant k). The random coil (green) represents the unstructured portions of the protein. These could be either those regions natively unfolded (αC domain) or any mechanically unfolded region of the protein such as the coiled-coil or portion of the D region. The WLC force-versus-extension relation is parameterized with persistence length, L_p, and contour length, L_c. (E) Simplified mechanical model of the fibrin fiber. There are M monomers in series and N single monomer chains in parallel. The model includes no lateral interactions because they have no relevance to uniform tensile stretching.

Figure 7

Force-versus-strain data (black circles) for unligated fiber (above) and ligated fiber below (5% of data points shown for clarity). (Red curve) Fit of Eq. 4.

Figure 8

Force per monomer as a function of fiber strain.