Labeling fibrin fibers with beads alter single fibrin fiber lysis, external clot lysis, and produce large fibrin aggregates upon lysis

Fluorescent beads are often used as a tool for visualizing fibrin fibers in single fiber mechanics studies and studies of single fiber lysis. Here we investigate the effect of beads on fibrin fiber lysis and extensibility to enhance understanding of this common research technique. We selected beads of the same diameter as those used in previous studies, as well as, beads of similar size to microparticles in the bloodstream. We used fluorescence microscopy, atomic force microscopy (AFM), and scanning electron microscopy (SEM) to quantify changes in fiber lysis, fiber extensibility, and clot structure in the presence and absence of beads. Fibrin clot structure and lysis were altered in the presence of beads. Fibrin clots formed with beads had a higher fiber density, smaller diameter fibers, and smaller pores. The rate of lysis for clots was reduced when beads were present. Lysis studies of bead-labeled individual fibers showed that beads, at concentrations similar to those reported for microparticles in the blood, cause a subset of fibers to resist lysis. In the absence of beads, all fibers lyse. These results demonstrate that beads alter fiber lysis through both a change in fibrin clot structure as well as changes to individual fiber lysis behavior. Additionally, the lysis of clots with beads produced large fibrin aggregates. This data encourages researchers to use careful consideration when labeling fibrin fibers with fluorescent beads and suggests that particles binding fibrin(ogen) in the bloodstream may be an underappreciated mechanism increasing the risk of thrombosis.