The effect of plasmin-mediated degradation on fibrinolysis and tissue plasminogen activator diffusion.

We modify a three-dimensional multiscale model of fibrinolysis to study the effect of plasmin-mediated degradation of fibrin on tissue plasminogen activator (tPA) diffusion and fibrinolysis. We propose that tPA is released from a fibrin fiber by simple kinetic unbinding, as well as by "forced unbinding," which occurs when plasmin degrades fibrin to which tPA is bound. We show that, if tPA is bound to a small-enough piece of fibrin that it can diffuse into the clot, then plasmin can increase the effective diffusion of tPA. If tPA is bound to larger fibrin degradation products (FDPs) that can only diffuse along the clot, then plasmin can decrease the effective diffusion of tPA. We find that lysis rates are fastest when tPA is bound to fibrin that can diffuse into the clot, and slowest when tPA is bound to FDPs that can only diffuse along the clot. Laboratory experiments confirm that FDPs can diffuse into a clot, and they support the model hypothesis that forced unbinding of tPA results in a mix of FDPs, such that tPA bound to FDPs can diffuse both into and along the clot. Regardless of how tPA is released from a fiber, a tPA mutant with a smaller dissociation constant results in slower lysis (because tPA binds strongly to fibrin), and a tPA mutant with a larger dissociation constant results in faster lysis.

Visualizing the degradation of fibrin fibers.

Polymeric fibrin provides the structural and mechanical stability of a blood clot. Fibrin fibers are rod-like and create a network mesh that holds blood cells. When a clot has performed its physiological function in wound healing and preventing excessive blood loss, it must be resolved by the enzymatic degradation of fibrin, otherwise known as fibrinolysis. If a blood clot forms when or where it is not needed, as occurs in ischemic strokes and myocardial infarctions, the blood clot (thrombus) can obstruct blood flow to downstream organs. Obstructive thrombi must be degraded or removed to prevent further complications. If a clot is not degraded on its own, lytic agents (i.e., tissue plasminogen activator, tPA) are given exogenously to induce fibrinolysis. Here, we fluorescently labeled both fibrin and tPA to visualize degradation at the edge of the clot. The fibers with bound tPA were looped or coiled while the fibers farther into the clot remain straight and stable displaying the diffusion of tPA and depth of lysis. This image provides (1) a new method to monitor fibrinolysis with a commercially available chamber with convenient inlets and (2) the visualization of tPA-bound fibrin and the behavior of fibers during degradation. Future work could utilize this technique to study tPA molecule and fibrin interactions, lysis front degradation, and fibrin fiber linearity to understand the mechanisms of intermolecular dynamics dependent on network structure. An enhanced insight into this process can aid in the development of optimized therapeutics to target stubborn clots.

Comprehensive Analysis of the Role of Fibrinogen and Thrombin in Clot Formation and Structure for Plasma and Purified Fibrinogen.

Altered properties of fibrin clots have been associated with bleeding and thrombotic disorders, including hemophilia or trauma and heart attack or stroke. Clotting factors, such as thrombin and tissue factor, or blood plasma proteins, such as fibrinogen, play critical roles in fibrin network polymerization. The concentrations and combinations of these proteins affect the structure and stability of clots, which can lead to downstream complications. The present work includes clots made from plasma and purified fibrinogen and shows how varying fibrinogen and activation factor concentrations affect the fibrin properties under both conditions. We used a combination of scanning electron microscopy, confocal microscopy, and turbidimetry to analyze clot/fiber structure and polymerization. We quantified the structural and polymerization features and found similar trends with increasing/decreasing fibrinogen and thrombin concentrations for both purified fibrinogen and plasma clots. Using our compiled results, we were able to generate multiple linear regressions that predict structural and polymerization features using various fibrinogen and clotting agent concentrations. This study provides an analysis of structural and polymerization features of clots made with purified fibrinogen or plasma at various fibrinogen and clotting agent concentrations. Our results could be utilized to aid in interpreting results, designing future experiments, or developing relevant mathematical models.

Internal fibrinolysis of fibrin clots is driven by pore expansion.

Blood clots, which are composed of blood cells and a stabilizing mesh of fibrin fibers, are critical in cessation of bleeding following injury. However, their action is transient and after performing their physiological function they must be resolved through a process known as fibrinolysis. Internal fibrinolysis is the degradation of fibrin by the endogenous or innate presence of lytic enzymes in the bloodstream; under healthy conditions, this process regulates hemostasis and prevents bleeding or clotting. Fibrin-bound tissue plasminogen activator (tPA) converts nearby plasminogen into active plasmin, which is bound to the fibrin network, breaking it down into fibrin degradation products and releasing the entrapped blood cells. It is poorly understood how changes in the fibrin structure and lytic protein ratios influence the biochemical regulation and behavior of internal fibrinolysis. We used turbidity kinetic tracking and microscopy paired with mathematical modeling to study fibrin structure and lytic protein ratios that restrict internal fibrinolysis. Analysis of simulations and experiments indicate that fibrinolysis is driven by pore expansion of the fibrin network. We show that this effect is strongly influenced by the ratio of fibrin:tPAwhen compared to absolute tPA concentration. Thus, it is essential to consider relative protein concentrations when studying internal fibrinolysis both experimentally and in the clinic. An improved understanding of effective internal lysis can aid in development of better therapeutics for the treatment of bleeding and thrombosis.

Neurovascular Relationships in AGEs-Based Models of Proliferative Diabetic Retinopathy.

Diabetic retinopathy affects more than 100 million people worldwide and is projected to increase by 50% within 20 years. Increased blood glucose leads to the formation of advanced glycation end products (AGEs), which cause cellular and molecular dysfunction across neurovascular systems. These molecules initiate the slow breakdown of the retinal vasculature and the inner blood retinal barrier (iBRB), resulting in ischemia and abnormal angiogenesis. This project examined the impact of AGEs in altering the morphology of healthy cells that comprise the iBRB, as well as the effects of AGEs on thrombi formation, in vitro. Our results illustrate that AGEs significantly alter cellular areas and increase the formation of blood clots via elevated levels of tissue factor. Likewise, AGEs upregulate the expression of cell receptors (RAGE) on both endothelial and glial cells, a hallmark biomarker of inflammation in diabetic cells. Examining the effects of AGEs stimulation on cellular functions that work to diminish iBRB integrity will greatly help to advance therapies that target vision loss in adults.