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 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.

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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.

A critical function of the immune system is the search and elimination of foreign pathogens by macrophages, neutrophils and other cells. These cells eliminate large particles (>0.5mm) by consuming them through a process called phagocytosis. There are several receptor-ligand pairs and associated signaling pathways that initiate phagocytosis. The best understood is the FcY receptor (FcYR)-immunoglobulin G (IgG) pair. Recognition by FcYR of an IgG coated particle triggers the cell's cytoskeleton to form a pseudopod that reaches around and engulfs the particle. Evaluating the mechanics of macrophage target engulfment requires careful monitoring of forces and high quality florescence imaging of the membrane and cytoskeleton. We combined an atomic force microscope (AFM) with a versatile optics system, a first of its kind, to monitor piconewton scale forces while imaging the phagocytosis engulfment from the side using Pathway Rotated Imaging for Sideways Microscopy with vertical light sheet (PRISM-LS). The macrophage produces a dynamic response a few nanonewtons during its envelopment of an IgG covered bead attached to an AFM cantilever. We observe the macrophages pull on the bead with downward forces in the range of 300pN-8nN. The macrophage exerts these downward forces before the pseudopod envelops past the midpoint of the bead. These downward forces can last from 5 seconds to 2 minutes. The phagocytic cup formation is associated with punctate brightening of actin underneath the bead. In addition, the versatile optics system can be used to record twochannel, three dimensional images of cells while engaging with the AFM. Bessel beam light sheet imaging with controlled force data will inform mechanical models of phagocytosis which will improve understanding of this important immunological process and inform mammalian disease progression. 2059-PosFibrin Density and Tension Regulates Fibrinolytic Susceptibility Fibrin fibers provide structural and mechanical integrity to blood clots until their dissolution during fibrinolysis. This work seeks to deconvolve the respective fibrinolytic influences of enzyme kinetics and enzyme perfusion rates into clots from clot structure and fiber properties. Here we consider the lysis of fibers within small, 2-D networks, which have polymerized between ridges. We tracked changes in fiber morphology within a 24-hour period during lysis. We find that fibers follow several possible degradation pathways during the lytic process including transverse cleavage, fiber bundling, structural elongation due to network rearrangements, tension loss, and network collapse. Transverse cleavage of individual fibers results in network rearrangements due to the redistribution of the tension in the yet-to-be-cleaved fibers. Network rearrangements and remnants of cleaved fibers often lead to fibers bundling into thicker fibers which are more resistant to fibrinolysis. When the final strand holding the network in place is transversely cleaved, the remaining fibers recoil and collapse onto the ridge. These effec...