Aggregates Dramatically Alter Fibrin Ultrastructure

Abstract

Among the many factors influencing fibrin formation and structure (concentration, temperature, composition, pH, etc.), it has been suggested that the polydispersity of fibrinogen may play an important role. We propose here a detailed investigation of the influence of this parameter on fibrin multiscale structure. Two commercial fibrinogen preparations were used, a monodisperse and a polydisperse one. First, the respective compositions of both fibrinogen preparations were thoroughly determined by measuring the fibrin-stabilizing factor; fibronectin; α, β, and γ intact chain contents; the γ/γ' chains ratio; the N-glycosylation; and the post-translational modifications. Slight variations between the composition of the two fibrinogen preparations were found that are much smaller than the compositional variations necessary to alter significantly fibrin multiscale structure as observed in the literature. Conversely, multiangle laser light scattering-coupled size exclusion chromatography and dynamic light scattering measurements showed that the polydisperse preparation contains significant amounts of aggregates, whereas the other preparation is essentially monodisperse. The multiscale structure of the fibrins produced from those two fibrinogen preparations was determined by using x-ray scattering, spectrophotometry, and confocal microscopy. Results show that fibers made from the aggregate-free fibrinogen present a crystalline longitudinal and lateral structure and form a mikado-like network. The network produced from the aggregates containing fibrinogen looks to be partly built around bright spots that are attributed to the aggregate. The multiscale structure of mixtures between the two preparations shows a smooth evolution, demonstrating that the quantity of aggregates is a major determining factor for fibrin multiscale structure. Indeed, the effect of a few percent in the mass of aggregates is larger than any other effect because of compositional differences under the same reaction conditions. Finally, we propose a mechanistic interpretation of our results, which points at a direct role of the aggregates during polymerization, which disrupts the ideal ordering of monomers inside fibrin protofibrils and fibers.

Figure 1

(A) Molecular size distribution of Clottafact and Fib1 determined by SEC. (B) Multiscale structure of the fibrin clot. To see this figure in color, go online.

Figure 2

(A) Scattered intensity of SAXS for fibrin clots (1 mg/mL). (B and C) Relative intensity of around the 22.5-nm periodicity peak for respective FibWoA and WibWA. Continuous lines are the total fit, whereas dashed lines are the individual peaks. The R2 values are the goodness-of-fit coefficients. (D) Final number of protofibrils and (E) final protein density inside fibrin fibers. Inset: fibers radii versus fibrinogen concentration. Error bars are standard deviations calculated from at least three individual experiments. To see this figure in color, go online.

Figure 3

(A) Qualitative geometry of the fibrin networks. Smooth manifolds were extracted from 3D confocal microscopy stacks at [Fg] = 3 mg/mL. White arrows show curved fibers, whereas red arrows show bright dots. Frames are 67.5 μm wide. The insets are ×2 enlargements of a portion of the original SME image (12 × 12 μm). (B) Number of protofibrils, protein density, and radius (inset) versus content in FibWA. Closed circles represent protofibrils number; open circles represent protein density. Inset: fibers average radius is shown. Error bars are standard deviations calculated from at least three individual experiments. To see this figure in color, go online.

Figure 4

Proposed fibrin formation mechanism, (A) shown in the presence of aggregates (FibWA) and (B) shown in the absence of aggregates (FibWoA). To see this figure in color, go online.