Fibrinolytic and antithrombotic protease from Ganoderma lucidum

A putative metalloprotease was purified from mycelium of Ganoderma lucidum. The enzyme was purified by anion exchange, chromatofocusing, and gel filtration chromatography. The Mr was determined to be 52 000 by SDS-PAGE and 100 000 by gel filtration on a Sephadex G-150 column, indicating that it is a dimer. The enzyme was inhibited by EDTA, 1,10-phenanthroline, and phosphoamidon. The presence of Zn2+ was detected by ICP mass spectral analysis as 1.1 mol of Zn2+ per mol of protease. This protease hydrolyzed Aα and Bβ chains of human fibrinogen, but did not cleave thrombin, albumin, hemoglobin, and immunoglobulin under the same condition. It also showed an anticoagulant activity in human plasma. The enzyme delayed the activated partial thromboplastin time and thrombin time, but not the clotting induced by reptilase, indicating Ganoderma protease is specific to thrombin. Ganoderma protease behaved as a competitive inhibitor of thrombin-catalyzed fibrin formation, but no inhibition of thrombin was found with a small synthetic peptide. These observations indicate that Ganoderma protease could bind thrombin at an anion binding exosite distinct from the active site and cause the delay of clotting time induced by thrombin.