Contribution of nascent cohesive fiber-fiber interactions to the non-linear elasticity of fibrin networks under tensile load

Abstract

Fibrin is a viscoelastic proteinaceous polymer that determines the deformability and integrity of blood clots and fibrin-based biomaterials in response to biomechanical forces. Here, a previously unnoticed structural mechanism of fibrin clots' mechanical response to external tensile loads is tested using high-resolution confocal microscopy and recently developed three-dimensional computational model. This mechanism, underlying local strain-stiffening of individual fibers as well as global stiffening of the entire network, is based on previously neglected nascent cohesive pairwise interactions between individual fibers (crisscrossing) in fibrin networks formed under tensile load. Existence of fiber-fiber crisscrossings of reoriented fibers was confirmed using 3D imaging of experimentally obtained stretched fibrin clots. The computational model enabled us to study structural details and quantify mechanical effects of the fiber-fiber cohesive crisscrossing during stretching of fibrin gels at various spatial scales. The contribution of the fiber-fiber cohesive contacts to the elasticity of stretched fibrin networks was characterized by changes in individual fiber stiffness, the length, width, and alignment of fibers, as well as connectivity and density of the entire network. The results show that the nascent cohesive crisscrossing of fibers in stretched fibrin networks comprise an underappreciated important structural mechanism underlying the mechanical response of fibrin to (patho)physiological stresses that determine the course and outcomes of thrombotic and hemostatic disorders, such as heart attack and ischemic stroke. STATEMENT OF SIGNIFICANCE: Fibrin is a viscoelastic proteinaceous polymer that determines the deformability and integrity of blood clots and fibrin-based biomaterials in response to biomechanical forces. In this paper, a novel structural mechanism of fibrin clots' mechanical response to external tensile loads is tested using high-resolution confocal microscopy and newly developed computational model. This mechanism, underlying local strain-stiffening of individual fibers as well as global stiffening of the entire network, is based on previously neglected nascent cohesive pairwise interactions between individual fibers (crisscrossing) in fibrin networks formed under tensile load. Cohesive crisscrossing is an important structural mechanism that influences the mechanical response of blood clots and which can determine the outcomes of blood coagulation disorders, such as heart attacks and strokes.

Keywords: Blood clot; Cohesion; Computational model; Fibrin network; Viscoelasticity.

Figure 1.

Schematics of in silico fibrin clot stretching experiment and description of the individual fibrin fiber model. (A) Representative image of a three-dimensional fibrin network used in simulations. Black arrows show the direction of applied force. Zoomed section shows the detailed fibers. (B) Spatial discretization of a single fiber using 6 interior sub-nodes connected by WLC springs. i and j are the main nodes of the fiber. Zoomed section illustrates bending springs in a fiber using angular springs between nodes (including non-rotating both main and sub-nodes). (C) Representation of two fibers and a cohesive interaction between them.

Figure 2.

Visualization and quantitative structural analysis of non-deformed and stretched fibrin clots. (A) Reconstructed confocal microscopy-based 3D image of a fluorescently labeled non-deformed isotropic fibrin network. (B) Fibrin network stretched to a 20% strain in the y-direction. Fiber branch points are shown by yellow squares in (A) and fiber-fiber cohesion points are indicated by yellow circles in (B). Bent fibers are indicated by white arrows. (C,D) Quantification shows a node connectivity degree (C) and node density (D) in non-deformed fibrin clots and in the same clots stretched to a 20% strain (90 nodes analyzed in each fibrin clot prepared from 3 independent donors). The results in (C) and (D) are presented as M±SEM. A two-tailed Mann-Whitney U test, *P<0.05.

Figure 3.

Mechanical and structural changes of fibrin networks under uniaxial stretching. (A) Characterization of the mechanical and structural changes in the fibrin network in terms of the unidirectional tensile stress-strain response (M±SD, n=10). (B) Average fiber alignment in the central 50% region of the network. (C) Percent of the stretched fibrin clot cross-section area covered by fibers. The solid line shows the nonlinear model simulation results, circles are experimental data[5].

Figure 4.

Impact of fiber-fiber cohesion on the tensile stress-strain response and fiber strain distribution in stretched fibrin clots. (A) Tensile stress-strain responses (M±SD, n=5) for fibrin networks of cohesive and non-cohesive fibers of density 5 fiber/μm^3. (B) Corresponding strain distributions of individual fibers at the fibrin network strain.

Figure 5.

Strain-dependence of the node connectivity for fibrin networks with cohesive fibers. The fiber density ρ_f = 5 fibers/μm³. Dotted, dashed and solid lines in the columns correspond to the network connectivity degrees of 3, 4, and >4, respectively.

Figure 6.

Fibrin clot mechanical response and fiber strain probability distributions for different fiber densities. (A) Tensile stress-strain responses (M±SD, n=5) for fibrin networks of different fiber densities, ρ_f. The inset image shows the stress on a logarithmic scale. (B) Individual fiber strain distributions for the network strain Γ = 2.

Figure 7.

Impact of cohesive fiber-fiber interactions on fiber network alignment. Joint distributions for network alignment and network strain are shown for cohesive and non-cohesive networks at 300% strain for fiber density 1/μm³ (A, B) and 5/μm³ (C, D). Simulated networks are shown alongside the joint density distribution to illustrate the corresponding location of aligned fibers. The Y-axis represents the scaled position along the length of the fiber network, z* =z/z₀. Z₀ is the original clot length and z ranges over the current network length from bottom (z = 0) top (z = 3 * z₀) Greyscale at each point corresponds to the relative number of fibers oriented along the direction of the strain.