Fluorescent microspheres can affect in vitro fibrinolytic outcomes.

Hemostasis is the cessation of bleeding due to the formation of a blood clot. After the completion of wound healing, the blood clot is typically dissolved through the natural process of fibrinolysis, the enzymatic digestion by plasmin of the fibrin fibers that make up its structural scaffold. In vitro studies of fibrinolysis reveal mechanisms regulating these processes and often employ fluorescent microscopy to observe protein colocalization and fibrin digestion. In this study, we investigate the effects of labeling a fibrin network with 20 nm diameter fluorescent beads (fluorospheres) for the purpose of studying fibrinolysis. We observed fibers and 2-D fibrin networks labeled with fluorospheres during fibrinolysis. We found that the labeling of fibrin with fluorospheres can alter fibrinolytic mechanisms. In previous work, we showed that, during lysis, fibrin fibers are cleaved into two segments at a single location. Herein we demonstrate that fibrinolysis can be altered by the concentration of fluorospheres used to label the fibers, with high concentrations of fluorospheres leading to very minimal cleaving. Furthermore, fibers that are left uncleaved after the addition of plasmin often elongate, losing their inherent tension throughout the imaging process. Elongation was especially prominent among fibers that had bundled together due to other cleavage events and was dependent on the concentration of fluorophores used to label fibers. Of the fibers that do cleave, the site at which they cleave also shows a predictable trend dependent on fluorosphere concentration; low concentrations heavily favor cleavage locations at either end of fibrin fiber and high concentrations show no disparity between the fiber ends and other locations along the fiber. After the initial cleavage event bead concentration also affects further digestion, as higher bead concentrations exhibited a larger population of fibers that did not digest further. The results described in this paper indicate that fluorescent labeling strategies can impact fibrinolysis results.

Inherent fibrin fiber tension propels mechanisms of network clearance during fibrinolysis.

Proper wound healing necessitates both coagulation (the formation of a blood clot) and fibrinolysis (the dissolution of a blood clot). A thrombus resistant to clot dissolution can obstruct blood flow, leading to vascular pathologies. This study seeks to understand the mechanisms by which individual fibrin fibers, the main structural component of blood clots, are cleared from a local volume during fibrinolysis. We observed 2-D fibrin networks during lysis by plasmin, recording the clearance of each individual fiber. We found that, in addition to transverse cleavage of fibers, there were multiple other pathways by which clot dissolution occurred, including fiber bundling, buckling, and collapsing. These processes are all influenced by the concentration of plasmin utilized in lysis. The network fiber density influenced the kinetics and distribution of these pathways. Individual cleavage events often resulted in large morphological changes in network structure, suggesting that the inherent tension in fibers played a role in fiber clearance. Using images before and after a cleavage event to measure fiber lengths, we estimated that fibers are strained ~23% beyond their equilibrium length during polymerization. To understand the role of fiber tension in fibrinolysis we modeled network clearance under differing amounts of fiber polymerized strain (prestrain). The comparison of experimental and model data indicated that fibrin tension enables 35% more network clearance due to network rearrangements after individual cleavage events than would occur if fibers polymerized in a non-tensed state. Our results highlight many characteristics and mechanisms of fibrin breakdown, which have implications on future fibrin studies, our understanding of the fibrinolytic process, and the development of thrombolytic therapies. STATEMENT OF SIGNIFICANCE: Fibrin fibers serve as the main structural element of blood clots. They polymerize under tension and have remarkable extensibility and elasticity. After the cessation of wound healing, fibrin must be cleared from the vasculature by the enzyme plasmin in order to resume normal blood flow: a process called fibrinolysis. In this study we investigate the mechanisms that regulate the clearance of individual fibrin fibers during fibrinolysis. We show that the inherent tension in fibers enhances the action of plasmin because every fiber cleavage event results in a redistribution of the network tension. This network re-equilibration causes fibers to buckle, bundle, and even collapse, leading to a more rapid fiber clearance than plasmin alone could provide.