Assessment of inhibitors with chromogenic substrates.

Blood plasma contains a number of proteins capable of inhibiting the activation of plasminogen and the action of plasmin. Except for urokinase, the nature and action of plasminogen inhibitors are not yet fully understood. Inhibitors of plasmin, on the other hand, are fairly well defined and their clinical significance recognised. Although there are a number of plasmin inhibitors in plasma-for example, onl-antitrypsin, inter-crinhibitor, antithrombin III, and Cl-inactivatoronly two, 0Y2-antiplasmin and 0c2-macroglobulin, appear to be important in vivo. 0X2-Antiplasmin (see D Collen at page 24) is a specific inhibitor of plasmin which binds the enzyme instantly and irreversibly in a stoichiometric manner (1:1). Its molar concentration in plasma is about 65% of that of plasminogen. Thus on complete activation of plasminogen in plasma only two-thirds of the resulting plasmin is inactivated by oC2-antiplasmin. The remainder is taken care of primarily by 0X2macroglobulin. This inhibitor differs greatly from OY2-antiplasmin. It is present in plasma in a much higher concentration, has very broad specificity (binds most of the proteases), and has a progressive action. It is generally accepted that the o2-macroglobulinenzyme complex is formed in two stages-firstly, there is a proteolytic cleavage of M2-macroglobulin by the enzyme and, secondly, a conformational change of the modified inhibitor which traps the enzyme. Owing to steric hindrance, access of high molecular weight substrates to the enzyme is prevented and the complex is practically inactive towards natural protein substrates. However, it retains its activity for small synthetic substrates. Since the access of other inhibitors to the complex is also dependent upon their size this activity cannot be blocked effectively by such commonly used inhibitors of plasmin as soya bean trypsin inhibitor and aprotinin. Assay of both CX2-antiplasmin and c2-macroglobulin has been greatly facilitated by new types of synthetic peptide compounds called chromogenic substrates. Several such substrates, composed of three amino-acid peptides with a para-nitroaniline group attached to the C-terminal end, have been synthesised. Depending on their amino-acid composition, these substrates are highly sensitive to plasmin, thrombin, factor Xa, urokinase, kallikrein, and other proteases. A schematic reaction of plasmin with a substrate S-2251 (Kabi, Stockholm) designated for its assay is: