On The Conversion Of High Molecular Weight Urokinase To The Low Molecular Weight Form

The conversion of high molecular weight urokinase (HMW) to low molecular weight urokinase (LWM) by plasmin in vitro has been studied. The two molecular weight forms of urokinase were separated by SDS polyacrylamide gradient gel electrophoresis and active enzyme extracted from gel segments into isotonic saline after slicing the gel at 5 mm intervals. Extracts from gel segments were analyzed by the fibrin plate method, and electrophoretic separation of the two forms were shown to be complete by comparison with the migration of purified standards and by the absence of lytic zones between the peaks of activity. HMW was incubated with plasminogen and fibrinogen for various time intervals from 2.5 to 10 minutes, enzymatic activity inhibited with aprotinin, and the samples subjected to electrophoresis. Conversion from HMW to LMW was apparent in as little as 2.5 minutes and continued for the 10 minute duration of the experiments. Similar experiments starting with LMW showed no change in molecular weight. Incubation of HMW without plasminogen resulted in no conversion to LMW implying that this reaction was not autocatalytic. The same conversion may occur in vivo during therapeutic administration of urokinase when a “lytic state” is produced and plasmin activity is present. Possible conversion of HMW to LMW in vivo will need to be considered in evaluating the relative therapeutic efficacy of different urokinase preparations and in interpreting the results of clinical trials.