Summary
Bovine prothrombin was prepared by adsorption on barium sulfate. After elution, it
was passed through thick filter-cakes of Standard Super-Cel, which removed some venom
substrate (factor X). Almost all the remaining venom substrate was removed by repeated
passage through columns of DEAE-cellulose. Finally, the ratio of venom substrate to
prothrombin was considerably less than 1/1,000 that of plasma. The prothrombin was
also poor in factor V. It yielded very little thrombin upon incubation with Russell’s
viper venom, factor V, phospholipid and calcium chloride. However, inclusion of bovine
plasma at a final dilution of 1/10,000 caused the mixture to produce thrombin rapidly.
This system offers promise for the assay of venom substrate in plasma.
Thrombokinase derived from bovine plasma was able, at 0.000071 mg/ml, to substitute
for both the venom and its substrate in thrombin-producing systems. However, with
this small amount of thrombokinase, phospholipid was indispensable. The system was
sensitive to 0.00001 mg phospholipid/ml.
With 1,000 times as much thrombokinase, prothrombin was activated without addition
of accessory factors, in the presence of oxalate. Removal of venom substrate did not
affect this response of prothrombin. Nor did removal of venom substrate from the prothrombin
prevent its activation by crystallized trypsin in the presence of oxalate.
