Purification and characterization of a novel, oligomeric, plasminogen kringle 4 binding protein from human plasma: tetranectin

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Abstract
Purification of α2‐plasmin inhibitor (α2PI) from human plasma by affinity chromatography on plasminogen‐Sepharose resulted in copurification of a contaminating protein with Mr 17000 as judged by sodium dodecyl sulphate/polyacrylamide gel electrophoresis. This contaminating protein could not be removed from the purified α2‐PI preparation by several types of gel chromatography applied. The use of the kringle 1–3 part of plasminogen, K(1 + 2 + 3), bound to Sepharose for affinity chromatography, instead of plasminogen‐Sepharose, resulted in an α2PI preparation without this contaminant. The contaminating protein was found to interact specifically with the kringle 4 part of plasminogen (K4) and not with K(1 + 2 + 3) or miniplasminogen. The K4‐binding protein was purified by ammonium sulphate precipitation, affinity chromatography on K4‐Sepharose, ion‐exchange chromatography and gel filtration on AcA 34. The relative molecular mass of the protein (Mr 68 000) was estimated by gel filtration. This suggests a tetrameric protein composed of four subunits (Mr 17000), that are dissociated by 1% sodium dodecyl sulphate. Dissociation into subunits was also demonstrated by gel filtration in the presence of 6 M guanidine hydrochloride. A specific antibody was raised in rabbits against the purified protein and this antibody was shown not to react with any known fibrinolytic components. The pI of the K4‐binding protein was found to be 5.8. The first three N‐terminal amino acids were determined to be Glu‐Pro‐Pro. The concentration of the protein in plasma was estimated to be 0.20 ± 0.03 μM (15 ± 2 mg/l). The electrophoretic mobility of the K4‐binding protein was shown by crossed immunoelectrophoresis to be influenced by the presence of Ca2+, EDTA and heparin. The protein was found to enhance plasminogen activation catalyzed by tissue‐type plasminogen activator (t‐PA) in the presence of poly(d‐lysine). The protein appeared to be a novel plasma protein tentatively called ‘tetranectin’.