Purification and Characterization of Human Antiplasmin, the Fast‐Acting Plasmin Inhibitor in Plasma

Abstract

A simple and rapid purification procedure for antiplasmin is described using affinity chromatography on plasminogen‐Sepharose, ion‐exchange chromatography on DEAE‐Sephadex and chromatography on Concanavalin‐A‐Sepharose. The final product was shown to be homogeneous using several polyacrylamide gel electrophoretic systems. It is a single‐chain glycoprotein with a molecular weight of about 70000 as determined both by dodecyl sulphate/polyacrylamide gel electrophoresis and by sedimentation equilibrium analysis. The sedimentation constant was estimated to be 3.45 S and the carbohydrate content about 14%. Sequence analysis showed the NH2‐terminal amino acid sequence to be: Asn‐Gln‐Glu‐Gly‐. Immunochemically this protein is different from all the known protease inhibitors in plasma. It was also shown that antiplasmin is a very stable protein if the pH is kept above 6. Antiplasmin rapidly forms a very stable complex with plasmin. Reduction of the complex shows that it is the B‐chain of plasmin which is involved. The purified partially reduced and S‐carboxy‐methylated B‐chain of plasmin can also progressively form a complex with antiplasmin. If the reaction between plasmin and antiplasmin is studied in an excess of plasmin a peptide bond seems to be clipped somewhere in the complex between the plasmin B‐chain and antiplasmin leading to release of a 14000‐Mr fragment (after reduction). This is, however, an event which occurs after complex formation. Copyright © 1977, Wiley Blackwell. All rights reserved