Purification and characterization of human vascular plasminogen activator derived from blood vessel perfusates.

A vascular plasminogen activator obtained by perfusion of the vessels of the lower extremities of human cadavers has been purified to apparent homogeneity by sequential precipitation in ammonium sulfate, reverse ammonium sulfate gradient solubilization, hydrophobic chromatography on octyl-Sepharose, and gel filtration on Sephadex G-75 and G-150. The isolation procedures were carried out in the presence of 0.3 to 1.0 M NaCl and 0.1 M arginine, as these conditions improved the maintenance of functional activity at each isolation step. The purified vascular plasminogen activator had an activity of 10,000 to 40,000 CTA units/ mg of protein as assessed by plasminogen activation and had no direct fibrinolytic activity. The isoelectric point of the purified vascular plasminogen activator was in the range of 7.8 to 8.8 with a peak at 8.2. The apparent molecular weight of the purified vascular plasminogen activator was in the range of 70,000 to 75,000 by gel filtration and by sucrose density ultracentrifugation and was in the same range when the [3H]diisopropyl phosphorofluoridate-labeled protein was analyzed by sucrose density gradient centrifugation. On reduction and alkylation followed by sodium dodecyl sulfate-gel electrophoresis, the [3H]diisopropyl phosphorofluoridate-labeled vascular plasminogen activator exhibited the same molecular weight of 67.000 as the unreduced labeled protein. The fun%ional adtivity of the purified vascular plasminogen activator was unaffected by concentrations of the IgG fractions of antisera specific for urokinase or plasma prekallikrein which completely inhibited the fibrinolytic activity of urokinase and kallikrein, respectively. Thus the human vascular plasminogen activator is a distinct cationic single polypeptide chain serine esterase.