Activation with plasmin of two-chain urokinase-type plasminogen activator derived from single-chain urokinase-type plasminogen activator by treatment with thrombin

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Abstract
Thrombin converts single‐chain urokinase‐type plasminogen activator (scu‐PA) to an inactive two‐chain derivative (thrombin‐derived tcu‐PA) by hydrolysis of the Arg‐156–Phe‐157 peptide bond. In the present study, we show that inactive thrombin‐derived tcu‐PA (specific activity 1000 IU/mg) can be converted with plasmin to active two‐chain urokinase‐type plasminogen activator (specific activity 43000 IU/mg) by hydrolysis of the Lys‐158–Ile‐159 peptide bond. This conversion follows Michaelis‐Menten kinetics with a Michaelis constant Km of 37 μM and a catalytic rate constant k2 of 0.013 s−1. The catalytic efficiency (k2/Km) for the activation of thrombin‐derived tcu‐PA by plasmin is about 500‐fold lower than that for the conversion of intact scu‐PA to tcu‐PA. tcu‐PA, generated by plasmin treatment of thrombin‐derived tcu‐PA, has similar properties to tcu‐PA obtained by digestion of intact scu‐PA with plasmin (plasmin‐derived tcu‐PA); its plasminogen activating potential and fibrinolytic activity in an in vitro plasma clot lysis system appear to be unaltered. These observations confirm that the structure of the NH2‐terminal region of the B chain of u‐PA is an important determinant for its enzymatic activity, whereas that of the COOH‐terminal region of the A chain is not.