Aggregates dramatically alter fibrin ultrastructure

Among the many factors influencing fibrin formation and structure (concentration, temperature, composition, pH,…), 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 FXIII, fibronectin, α,β and γ intact-chains contents, theγ/γ’ chains ratio, the N-glycosylation and the post-translational modifications. Slight variations between the composition of the two fibrinogen preparations were found which are much smaller than the compositional variations necessary to alter significantly fibrin multiscale structure as observed in the literature. Conversely, MALLS coupled SEC and DLS measurements showed that the polydisperse preparation contains significant amounts of aggregates while 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 from the monodisperse fibrinogen present a crystalline longitudinal and lateral structure and form a needle-like network. The internal structure of fibers produced from the polydisperse fibrinogen looks amorphous with star-like branching nodes. 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 mass of aggregates is larger than any other effect due to 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 disrupt the ideal ordering of monomers inside fibrin protofibrils and fibers.