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.

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.

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.