Fibrin Plate Studies of Plasminogen Activator and Inhibitor from Pig Leucocytes

Abstract

A neutral proteinase isolated from pig leucocytes by DEAE-chromatography, gel filtration on Sephadex G-100 and CM-cellulose chromatography was tested on Astrup’s fibrin plates prepared with and without plasminogen. In this way its activity of plasminogen activator was determined. The inhibitory effect of EACA, AMCHA, PAMBA and Trasylol on the activator was tested with the same method. EACA in concentration 10−2 M and 20 KIE/ml of Trasylol 100% inhibited the activator. 10−2 M AMCHA inhibited the activator by 30% in contrast to PAMBA where 70% inhibition was obtained already in concentration of 10−3 M.With the aid of CM-cellulose chromatography and gel filtration on Sephadex G-100 two inhibitors were isolated from leucocyte cytoplasmic fraction (post granular supernatant). The inhibitory effect of the main inhibitor was determined on fibrin plates with granular extract as test enzyme, having neutral proteinase activity. The extent of inhibition of granule extract depended on the concentration of inhibitor added. Complete inhibition was achieved in the ratio 2 volumes of granule extract: 1 volume of inhibitor, protein content of the former being 800–1200 γ N2/ml and the latter 360 γ N2/ml.