Plasminogen is the inactive precursor of the serine protease plasmin, which has a primary role in dissolving blood clots (fibrinolysis). α2-antiplasmin is a serine protease inhibitor (SERPIN) that acts as a rapid inhibitor of plasmin. Conventionally, SERPINs inhibit their active protease substrates via irreversible complex formation (1). Interestingly, several independent studies suggested that plasminogen has an affinity for α2-antiplasmin in vivo and in vitro (2-5). While it is clear that α2-antiplasmin binds plasmin to stop fibrinolysis, the effect of α2-antiplasmin-plasminogen interaction on regulation of fibrinolysis remains controversial (2,5-8).
To determine the effect of α2-antiplasmin on plasminogen function, we need to have a better understanding of how the interaction occurs. Preliminary studies have been carried out in our laboratory using plasminogen isolated from human plasma and recombinant α2-antiplasmin from bacterial expression. A number of techniques have been employed, these include; 1. Pull-down assays using purified proteins. This method allowed for high-throughput screening for ideal binding conditions but lacked kinetic information. Therefore following this are binding kinetics assays using techniques like 2. Isothermal titration calorimetry, a low throughput assay but it confers an advantage for observing interaction of protein in solution and 3. BLItz (ForteBio), a high-throughput assay but it requires immobilisation of protein. By the means of binding studies, we are going to determine the optimal conditions for α2-antiplasmin-plasminogen binding and analyse the binding interaction using X-ray crystallography. High-resolution structure information will allow us to examine the nature of interaction and from there deduce how α2-antiplasmin is likely to affect plasminogen’s function at the clot interface.