Purification Of Vasoactive Peptides From Human Fibrinogen Degraded By Human Leucocyte Elastase

The presence of leucocytes around extravascular fibrin deposits suggests that the leucocyte elastases might be partly responsible for the extravascular degradation of fibrin. Our previous studies have shown that the degradation of fibrin(ogen) by plasmin leads to the release of 2 small peptides which markedly increase vascular permeability and induce oedema e.g. in the lungs. The results of this investigation show that small peptides released from fibrinogen after degradation by leucocytes elastases also increase vascular permeability.

Human fibrinogen (Kabi, Grade L) was made plasminogenfree by affinity chromatography on Lysine-Sepharose 4B prior to use. The human leucocyte elastases were isolated from extracts of lysosome-like granules of human leukaemic myeloid cells by a combination of gel filtration, affinity chromatography and preparative agarose gel electrophoresis. The fibrinogen (0.5 %) and the leucocyte elastases (in a molar ratio of 100:1) were incubated together for 48 h at +37°C and at pH 8.5. The mixture was then cooled to +4°C to stop the lysis and ultrafiltrated on a DIAFLO PM 10 membrane until the retentate was approximately 10 % of the starting volume. The peptides in the diffusate accounted for about 20 % of the starting material as estimated from absorbance measurements at 280 nm. The diffusate was concentrated by lyophilization and fractionated by chromatography on a column of Bio-Gel P-6. At least 8 fractions were obtained of which only two showed a significant activity in their ability to increase vascular permeability in rat skin. The active peptides in these two fractions were further purified to homogeneity by column zone electrophoresis at various pHs and their amino acid compositions established.