Regulation of internal and external fibrinolysis in health and disease

Cardiovascular diseases, such as heart attacks and stroke, are leading causes of morbidity and mortality worldwide. These diseases are linked through excessive blood clotting (thrombosis) that restricts blood flow to downstream organs. Diseases such as cancer, diabetes, and COVID-19 can lead to predisposition to excessive blood clotting. Blood clots are structurally and mechanically stable due to the formation of fibrin fibers, which are a network that encase the cellular components. It is essential that the blood clot is enzymatically degraded to restore blood flow, which can be done through a process known as fibrinolysis or the breakdown of the fibrin network. Fibrinolysis can occur in two ways: 1) the physiological presence of lytic enzymes such as tissue plasminogen activator (tPA), termed internal fibrinolysis, or 2) the exogeneous delivery of tPA to fully formed clots as would be seen in the clinic after heart attack or stroke, termed external fibrinolysis. The formation of the fibrin network is influenced by the blood composition and the resultant structure distinctively modulates the susceptibility to degradation. While some is known about this topic, this dissertation addresses alterations in fibrin structure formed under (patho)physiological conditions to identify improved treatments to degrade blood clots. Here, we utilize a combination of turbidimetric assays to understand the bulk view of clot formation and fibrinolysis as well as microscopy techniques to relate the kinetics to the fibrin structure. These wet-lab experiments are complemented by mathematical modeling to isolate parameters in a way that cannot be done experimentally. We found fibrin network pore size is the driving force restricting or enhancing degradation, compared to fiber diameter; dense fibrin networks can impair both internal and external fibrinolysis. Therefore, there is a need to develop therapeutics that are able to penetrate dense fibrin networks to accelerate impaired external fibrinolysis. Moreover, we found that the elevation of fibrinolytic inhibitor (plasminogen activator inhibitor 1) in pancreatic cancer patient plasma and the activation of fibrinolytic inhibitors (thrombin activatable fibrinolytic inhibitors) due to anticoagulants provides a unique target to prevent hypercoagulability under these conditions. These inhibitors played a more significant role in slowing fibrinolysis compared to network structure in these conditions. In combination, these results highlight the contributions of both fibrin network structure and fibrinolytic inhibitors in regulating fibrinolysis depending on the conditions that led to clot formation. Understanding the relationship between the process of clot formation, resulting clot structure, and dynamics of degradation aids in the development of improved therapeutics to degrade stubborn clots and restore blood flow to downstream organs.