Summary
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.
Keywords
Fibrinolysis - Endothelial cells - Soluble fibrin polymer
