(E-161) Biochemical and structural regulation of the enzymatic degradation of blood clots

Introduction:: Blood clots perform a critical biological function in cessation of bleeding following injury. However, their function is transient and after performing their physiological function must be resolved through a process known as fibrinolysis. Upon blood vessel injury, activated platelets release clotting factors that convert fibrinogen, a blood plasma protein, into a polymeric fibrin network. Blood cells, such as red blood cells and platelets are encased in the fibrin network, leading to the obstruction of blood flow in the vessel. Fibrinolysis, the breakdown of the clot, can occur in two ways: 1) exogeneous drug delivery of lytic enzymes (external fibrinolysis) or 2) endogenous/innate presence of lytic enzymes in the blood stream (internal fibrinolysis). Tissue plasminogen activator (tPA) converts free plasminogen into active plasmin, which binds to the fibrin network, breaking it down into fibrin degradation products and releasing the entrapped cells. Extensive studies have been done to understand the molecular dynamics of fibrin and tPA interactions during external fibrinolysis. It has been found that external fibrinolysis occurs as a lysis front in which the tPA molecules bind to the periphery of the network and propagate towards the core of the clot. However, it is poorly understood how changes in the fibrin structure and molecular interactions influence the biochemical regulation and behavior of internal fibrinolysis. Improved understanding of internal lysis can inform about the critical balance of resolution in preventing life threatening complications to aid in the development of better thrombolytics.


Materials and Methods:: Methods: We used a combination of experiments and mathematical modeling to study fibrin structure and molecular interactions that restrict internal fibrinolysis. For experiments, we altered the fibrin structure by varying the fibrinogen concentration. We used turbidimetric experiments to assess the biochemical regulation of fibrinolysis with varied fibrinogen concentration and a fixed concentration of tPA (Figure 1A). Next, to study the role of molecular dynamics, we preserved the ratio of tPA to fibrin molecules by increasing the tPA concentration for higher fibrinogen concentrations and less tPA for less fibrinogen (Figure 1B). A 3D stochastic multiscale computational model was used to probe the individual influence of changes in network density, pore size, and fiber diameter. We then performed confocal microscopy studies to characterize the microstructure of fibrinolysis in human blood plasma with varying network structure with and without tPA. Confocal microscopy timelapse images of clot formation and degradation were taken to monitor structure changes over time.


Results, Conclusions, and Discussions:: Results: Increasing fibrinogen concentration with a fixed concentration of tPA slows fibrin degradation (p< 0.0001, Figure 1C) and significantly delays time to lysis (p< 0.0001), resulting in a direct correlation between fibrinogen concentration and lysis times (p< 0.05, Figure 1D). Contrastingly, when a fixed ratio of tPA is dosed, there is no correlation between degradation rate or delay time and fibrinogen concentration (ns, Figure 1D); degradation rate is significantly slowed for lower concentrations of fibrinogen (p< 0.0001) and hastened for higher fibrinogen concentrations (ns). Modeling simulations suggested the mechanism of fibrinolytic resistance with a fixed concentration of tPA is driven by the densification of the network with smaller pore sizes leading for higher fibrinogen concentrations (Figure 2). Pore expansion occurs heterogeneously for lower fibrinogen concentrations but uniformly for higher fibrinogen concentrations (Figure 3A), measured as the variance of pore sizes of the clot (Figure 3B).



Conclusions: Analysis of simulations and experiments indicate that internal fibrinolysis is driven by pore expansion of the fibrin network. We show that this effect is strongly influenced by the ratio of protein:enzyme when compared to absolute enzyme concentration.



Discussion: Our findings suggest that fibrin structure, which is altered in pathological conditions, is an important contributor for the development of enzymatic treatments with increased efficacy in treating bleeding and/or thrombosis. It is essential to consider the molecular interactions when studying internal fibrinolysis.


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