Nonuniform Internal Structure of Fibrin Fibers: Protein Density and Bond Density Strongly Decrease with Increasing Diameter

Abstract

The major structural component of a blood clot is a meshwork of fibrin fibers. It has long been thought that the internal structure of fibrin fibers is homogeneous; that is, the protein density and the bond density between protofibrils are uniform and do not depend on fiber diameter. We performed experiments to investigate the internal structure of fibrin fibers. We formed fibrin fibers with fluorescently labeled fibrinogen and determined the light intensity of a fiber, I, as a function of fiber diameter, D. The intensity and, thus, the total number of fibrin molecules in a cross-section scaled as D1.4. This means that the protein density (fibrin per cross-sectional area), ρp, is not homogeneous but instead strongly decreases with fiber diameter as D−0.6. Thinner fibers are denser than thicker fibers. We also determined Young's modulus, Y, as a function of fiber diameter. Y decreased strongly with increasing D; Y scaled as D−1.5. This implies that the bond density, ρb, also scales as D−1.5. Thinner fibers are stiffer than thicker fibers. Our data suggest that fibrin fibers have a dense, well-connected core and a sparse, loosely connected periphery. In contrast, electrospun fibrinogen fibers, used as a control, have a homogeneous cross-section.

Molecular packing structure of fibrin fibers resolved by X-ray scattering and molecular modeling

“…The mass density ranged from ρ = 11.6 mg/ml for Np = 47 up to ρ = 248 mg/ml for Np = 368. This observation is qualitatively consistent with earlier reports of strong variations in fibrin fiber density with assembly conditions [39][40][41] . Note that for the Np = 2 networks we could only determine the mass-length ratio (and thus Np) of the fibers from light scattering and not the fiber radius (and thus ρ), because these thin fibers have radii of only 7.5-15 nm, smaller than the wavelength of light 9 .…”

“…This is more visible when we multiply the scattering intensities by q Df and adjust Df to make the resulting curves flat between q = 0.2 and 1 nm -1 (see Figure 5B). The bestfit values for Df vary between 1.3 and 1.7 ( Figure 6A), consistent with prior measurements based on SAXS 39 , fluorescence microscopy 49 , and single-fiber stretching 40,50 . As shown in Figure 5C, the Iq Df -curves nicely reveal the narrow Bragg peak at q = 0.29 nm -1 corresponding to the axial half-staggered packing order of fibrin, as well as a much broader peak centered around q = 0.47 nm -1 .…”

“…The peak position reveals a 13-nm repeat distance between protofibrils, close to values reported elsewhere 14,34,[36][37][38]42 . We found that the fractal dimension of the fibers is on the order of Df = 1.5, well within the range of other reported fractal dimensions for fibrin obtained by atomic force microscopy and SAXS [39][40]49 . However, we find that the fractal dimension is dependent on the fiber mass density.…”

“…Physically, this means that fibrin fibers would have a dense core and a loose periphery with sparsely arranged protofibrils. This model is supported by measurements of the fluorescence intensity 49 and stiffness 40 of fibrin fibers as a function of their diameter. The third model is essentially a hybrid of the other two models, because it considers the lateral packing as crystalline but with defects ( Figure 5A) 39 .…”

Fibrin Matrices as (Injectable) Biomaterials: Formation, Clinical Use, and Molecular Engineering

“…It is critical that fibrinopeptide A is cleaved before fibrinopeptide B for effective b:B interactions to occur; the latter have an affinity about six times lower than the a:A coupling ( K d = 140 µM vs K d = 25 µ m ), and are thought to play a role in fibril lateral aggregation (thicker fibers when a:A interactions are weakened by selective mutations), which becomes important after they reach a critical length of 600–800 nm . The lateral growth is largely irregular and the fibers become progressively less dense and softer the larger they grow, with density and stiffness both scaling with diameter D as D − 1.6 ; the general model therefore is of fibers containing a dense, highly interconnected core with a relatively sparse periphery where both the protein density and inter‐fibril bond density decrease as D −0.6 and D −1.5 , respectively . Competing with lateral aggregation, fibers may also undergo branching, typically in two forms: 1) bilateral branching involves the separation and divergence of two protofibrils due to incomplete lateral aggregation; 2) trimolecular junctions form from a terminating fibrin monomer with only 1 A:a bond growing its own independent strand …”