A Low Molecular Weight Fibrinolytic Enzyme from Polymorphonuclear Leukocytes (PMN)
Despite evidence implicating PMN in fibrinolysis, the enzymes involved are incompletely characterized.PMN were prepared from normal blood by dextran sedimentation and fibrinolytic activity assayed by l25I-fibrin solid phase assay (Blood 46:543, 1975). More than 80% of activity was associated with intact PMN, was stimulated by Na salicylate (+65%, 20 mg/100 ml) and inhibited by α1-anti-trypsin (α1AT, -48%, 2 × 10-6 M). Similar activity was found in a PMN membrane fraction prepared by homogenization, differential centrifugation and Sepharose 4B gel filtration, from which fraction it was released by freeze-thawing and/or IM KCl treatment. Soluble enzyme activity was inhibited by of α1AT (-80%, 10-6 M), PMSF (-98%, 10-3 M), FeCl3 and ZnCl3 (-100%, 10-2 M), vitamin E(-38%, 10-4 M) and trypan blue (-40%, 10-3 M), but not by EACA (10-2 M), tranexamic acid (10-2 M), TLCK (10-3 M) or TPCK (10-3 M). This activity had an alkaline proteinase pH-activity profile, was localized to a single cationic protein band on acid Polyacrylamide disc gel electrophoresis, with pl of 8.6-8.7 by isoelectric focussing, and eluted between lysozyme and myoglobin on Bio-Gel P-10. On Bio-Gel A 0.5m and P-10, 125I-fibrin degradation products eluted after myoglobin. These findings indicate the presence in PMN of a low molecular weight, membrane-associated fibrinolytic enzyme of alkaline proteinase and serine active site type.