Studies on a human blood platelet protease with elastolytic activity

Abstract

The fractionation by ammonium sulfate precipitation and gel filtration on Sephadex G-150 of Triton X-100 extracts of human blood platelets is described, as well as the determination of the enzymatic (proteolytic and esterolytic) activities of the fractions obtained with 125I-labeled elastin, hemoglobin and synthetic substrates AcAla 3OMe, BzArgOEt and AcTyrOEt. Activity towards AcAla 3OMe was significantly higher in all fractions than that towards AcTyrOEt and BzArgOEt. The highest specific activity towards AcAla 3OMe and 125I-labeled elastin was obtained in the 2nd Sephadex peak of the 70% ammonium sulfate precipitate. The increase in specific activity towards AcAla 3OMe was of the order of 1200, with respect to the Triton extract. The 2nd Sephadex peak of the 70% ammonium sulfate precipitate gives three bands on acrylamide electrophoresis, the 2nd band containing all elastolytic activity. Preincubation with trypsin significantly increases the 125I-labeled elastin splitting activity of the Triton extract and of the 40% ammonium sulfate precipitate. No or only a very slight activation was observed in the 70% ammonium sulfate precipitate. These results suggest the presence of an inactive precursor in the 40% ammonium sulfate precipitate and of an activated protease in the 70% precipitate. The high specific activity of the purified fraction (2nd peak of 70% ammonium sulfate precipitate) with AcAla 3OMe and 125I-labeled elastin as substrate, as well as the absence of activity on other substrates (hemoglobin, BzArgOEt and AcTyrOEt) justified the denomination suggested of “platelet elastase” for the protease in this fraction.