Structural and functional characterization of the inhibition of urokinase by .alpha.2-macroglobulin

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Abstract
The interaction of .alpha.2-macroglobulin (.alpha.2-M) with the serine proteinase urokinase, an activator of plasminogen was investigated. Urokinase formed sodium dodecyl sulfate stable complexes with purified .alpha.2M and with .alpha.2M in plasma. These complexes could by visualized after polyacrylamide gel electrophoresis by protein blots using 125I-labeled anti-urokinase antibody or by fibrin autography, a measure of fibrinolytic activity. According to gel electrophoretic analyses under reducing conditions, urokinase cleaved .alpha.2M subunits and formed apparently covalent complexes with .alpha.2M. Urokinase cleaved only .apprx. 60% of the .alpha.2M subunits maximally at a mole ratio of 2:1 (urokinase: .alpha.2M). Binding of urokinase to .alpha.2M protected the urokinase active site from inhibition by antithrombin III-heparin and inhibited, to a significant extent, plasminogen activation by urokinase. Reaction of urokinase with .alpha.2M caused an increase in intrinsic protein fluorescence and, thus, induced the conformational change in .alpha.2M that is characteristic of its interactions with active proteinases. Results indicate that both in plasma and in a purified system the .alpha.2M-urokinase reaction is functionally significant.