Studies on the regulation of tissue plasminogen activator activity by physiological templates

Abstract

Myocardial infarction (MI) and ischemic stroke (IS) are caused by blood clots blocking arteries and cutting off the blood supply to tissues in the heart or brain. Clots may be cleared using plasminogen activators (“clot busters”) that convert plasminogen into plasmin, which in turn digests fibrin. The major plasminogen activator in the circulation is tissue plasminogen activator (tPA) and a recombinant form Alteplase was developed for use as a thrombolytic drug. However, newer engineered thrombolytics based on tPA have often been disappointing in clinical trials for MI, and there has been little progress in treatment for IS. Some of this lack of success may be due to gaps in our understanding of the details of the action of these drugs during clot dissolution as no rational models exists to understand the quantitative relationship between fibrin binding and plasminogen activation and hence fibrin lysis. Development of such models was the goal of this project. Fibrin binding is central in the regulation of tPA activity and binding is principally via 2 tPA domains, finger (F) and kringle 2 (K2), which were the focus of attention in the design of domain variants of tPA, including fusions with jelly fish fluorescent proteins. Protease activity was investigated initially with amidolytic substrates and then with plasminogen as substrate in the presence of templates: heparin, fibrinogen and fibrin, which co-localise tPA and substrate, plasminogen, to enhance plasmin generation. Kinetics of association and dissociation and equilibrium binding studies were investigated using surface plasmon resonance technology and ELISA-style plate binding assays. The results of these experiments showed that both the strength and pattern of binding of domain variants depends on the template. Models have been developed combining intrinsic enzyme kinetic and binding data to predict plasminogen activation rates in the presence of different templates. These studies are useful for optimising the design of thrombolytic drugs to treat MI and IS.