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.

Microscale structural changes of individual fibrin fibers during fibrinolysis.

Fibrinolysis is the enzymatic digestion of fibrin, the primary structural component in blood clots. Mechanisms of fibrin fiber digestion during lysis have long been debated and obtaining detailed structural knowledge of these processes is important for developing effective clinical approaches to treat ischemic stroke and pulmonary embolism. Using dynamic fluorescence microscopy, we studied the time-resolved digestion of individual fibrin fibers by the fibrinolytic enzyme plasmin. We found that plasmin molecules digest fibers along their entire lengths, but that the rates of digestion are non-uniform, resulting in cleavage at a single location along the fiber. Using mathematical modeling we estimated the rate of plasmin arrival at the fiber surface and the number of digestion sites on a fiber. We also investigated correlations between local fiber digestion rates, cleavage sites, and fiber properties such as initial thickness. Finally, we uncovered a previously unknown tension-dependent mechanism that pulls fibers apart during digestion. Taken together these results promote a paradigm shift in understanding mechanisms of fibrinolysis and underscore the need to consider fibrin tension when assessing fibrinolytic approaches. STATEMENT OF SIGNIFICANCE: We developed a method for interrogating lysis of individual fibrin fibers, enabling the time-resolved observation of individual fiber digestion for the first time. Our results resolve longstanding disagreements about fibrinolytic processes and reveal previously unknown mechanisms that also play a role. Also, we developed the first microscale mathematical model of plasmin-fibrin interaction, which predicts the number of plasmin molecules on each fiber and can serve as a framework for investigating novel therapeutics.