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Characterization of a chimaeric plasminogen activator obtained by insertion of the second kringle structure of tissue-type plasminogen activator (amino acids 173 through 262) between residues Asp130 and Ser139 of urokinase-type plasminogen activator

par Lijnen, Henri Roger;Pierard, Laurent ;Reff, Mitchell;Gheysen, Dirk
Référence Thrombosis research, 52, 5, page (431-441)
Publication Publié, 1988-12

Article révisé par les pairs

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Résumé :

A chimaeric recombinant plasminogen activator (rscu-PA-∇K2) obtained by insertion of the second kringle (K2) of tissue-type plasminogen activator (t-PA) (amino acids 173-262) between residues Asp130 and Ser139 of single chain urokinase-type plasminogen activator (scu-PA) was purified from the conditioned medium of mouse myeloma cells transfected with the previously described plasmid pULB9137 (Piérard et al., J. Biol. Chem. 262, 11771-11778, 1987). Approximately 22 ug of purified protein was obtained per liter of conditioned medium with a yield of approximately 25 percent. On sodium dodecylsulfate gel electrophoresis under reducing conditions, rscu-PA-VK2 migrated with an apparent Mr of 65,000. Plasmin caused a time-and concentration-dependent conversion to an amidolytically active two chain derivative (rtcu-PA-VK2) with a specific activity of 45,000 IU/mg. Both rscu-PA-∇K2 and rtcu-PA-VK2 activated plasminogen directly with Km: 2.0 μM and k2= 0.00063 s-1 and Km= 100 μM and k2= 4.1 s-1 respectively, rscu-PA-VK2 did not bind exlusively to fibrin. It caused concentration-dependent lysis of 125I-fibrin-labeled plasma clots immersed in human plasma with a comparable specific activity and fibrin-specificity as rscu-PA. It is concluded that insertion in scu-PA of the second kringle of t-PA, which is believed to be involved in its fibrin affinity, does not significantly alter the enzymatic properties of scu-PA, but does not confer marked fibrin-affinity to the molecule. © 1988.