Diversity in catalytic properties of single chain and two chain tissue-type plasminogen activator

Abstract

Like many other serine proteases, human tissue-type plasminogen activator (t-PA) is synthesised and released from cells in a single chain (sc-) form. This may later be converted proteolytically to a two chain (tc-) form, in which the chains are held together by a disulfide bridge. With most serine proteases, the sc-form is a genuine zymogen with an activity several orders of magnitude smaller than that of the tc-form. But in the case of t-PA, the relative activity of the two forms has been a controversial issue. We review here old and new investigations of the problem, emphasizing recent experiments with genetically engineered t-PA mutants that cannot be cleaved to the tc-form. Provided steps are taken to prevent plasmin-catalysed conversion of sc- to tc-t-PA during the assays, the results consistently point to a plasminogen activation activity of sc-t-PA that is at least one order of magnitude smaller than that of tc-t-PA. Fibrin stimulates the activity of sc-t-PA strongly and to a higher extent than tc-t-PA, resulting in equal activities of the two forms in the presence of fibrin. On the other hand, there is no doubt that sc-t-PA as such possesses a distinct activity even in the absence of fibrin. It reacts with synthetic substrates, with reactive site reagents and with its natural inhibitors. These properties have important implications as to the way t-PA may fulfil its physiological function in fibrinolysis: sc-t-PA is not likely to circulate as a reservoir of potential plasminogen activation activity. Rather, plasmin generation and subsequent fibrinolysis is likely to be initiated by release of sc-t-PA from the endothelium and to depend on the presence of polymerised fibrin as a cofactor. Other mechanisms of t-PA activation may be involved in non-fibrinolytic functions of the activator.