α2–plasmin Inhibitor And α2-Macroglobulin-Plasmin Complexes In Plasma. Quantitation By An Enzyme-Linked Immunosorbent Assay

An enzyme-linked immunosorbent assay has been developed for the quantitation of α2-plasmin inhibitor-plasmin and α2-macroglobulin-plasmin complexes. In this method, the inhibitor-plasmin complex is bound to a surface by an inhibitor specific antibody, then the plasmin bound to the inhibitor is quantified by a second antibody labeled with alkaline phosphatase. Inhibitor-enzyme complexes were generated in plasma by the addition of plasmin or of urokinase (UK). The concentration of plasmin added was well below the plasma concentration of α2-plasmin inhibitor or of α2-macroglobulin so that neither inhibitor would be fully saturated with enzyme. Under these conditions, increasing amounts of plasmin generated an increase in both α2-plasmin inhibitor-plasmin and α2-macroglobulin- plasmin complexes. Varying amounts of plasmin were incubated with each of the purified inhibitors and the complexes that formed were quantitated by immunoassay. These studies made it possible to quantitate the distribution of plasmin between the two inhibitors in plasma. In plasmin-treated plasma, 10% or less of the plasmin bound to both inhibitors was in complex with α2-macroglobulin. In contrast, between 19-51% of the plasmin generated in UK-activated plasma was bound to α2-macroglobulin. Thus major changes in the distribution of plasmin were observed depending upon whether plasmin was added to plasma or whether plasminogen was activated endogenously. 23 normal individuals had low levels of α2-plasmin inhibitor-plasmin complexes (4.1±3.5 fmol/ml) whereas six patients with laboratory evidence for DIC demonstrated a 16 to 35-fold increase in the concentration of these complexes. These data indicate that a useful new probe for the study of the fibrinolytic enzyme system has been developed.