Isolation and Properties of Human Vascular Plasminogen Activator

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Abstract
Improved methods for the isolation of human cadaveric endothelial vascular plasminogen activator have been developed. The enzyme was isolated as the native complex with soluble fibrin by a combination of polyethylene glycol precipitation and hydroxyapatite chromatography. The purified complex was then finally dissociated by affinity chromatography on lysine agarose. The dissociated enzyme was relatively unstable when bioassayed on fibrin plates, however its activity against low molecular weight chromogenic substrates was more stable. The enzymic activity was totally inhibited by DFP, PMSF or DTT and was totally resistant to iodoacetamide or Trasylol, indicating that it was a serine protease but differing in certain respects from urokinase. The specific activity of the native enzyme is ≥ 50,000 CTA units/mg protein, it has a molecular weight of 56,600 and will reform biologically active complexes with soluble fibrin polymers prepared in vitro. Both SFP and various poly-lysine preparations were shown to stimulate the activation of plasminogen by VPA in a spectrophotometric assay based on the rate of plasmin generation assayed against the chromogenic substrate S-2251. Considerable loss of material from purified preparations occurs by adsorption, thus with the limited amount of material available from cadavers, a radioisotopic labelling method was sought using iodination or 3H-DFP labelling, however neither approach was satisfactory. Comparison of VPA activity in cadaveric eluates and in venous occlusion plasma or following infusion of vasopressin analogues showed that all three activities behaved identically in the above purification steps. The affinity of VPA for insoluble fibrin was much higher than that of urokinase, or a human melanoma activator from tissue culture.