ISOLATION, SEQUENCE-ANALYSIS, AND CLONING OF HAEMADIN - AN ANTICOAGULANT PEPTIDE FROM THE INDIAN LEECH

A slow, tight-binding inhibitor of thrombin with an apparent molecular mass of about 5 kDa has been isolated from Haemadipsa sylvestris, an Indian leech of the family of Haemadipsidae. The inhibitory activity, called haemadin, is thrombin specific since it does not inhibit other proteases like trypsin, chymotrypsin, factor Xa, or plasmin. NH2-termi nal amino acid sequence analysis (residues 1-45) does not reveal any h omology to known serine protease inhibitors, including the thrombin-sp ecific inhibitor hirudin. The haemadin cDNA cloned by polymerase chain reaction techniques codes for a polypeptide of 57 amino acid residues preceded by 20 residues of a signal peptide sequence. A synthetic gen e coding for the mature haemadin was expressed in Escherichia coli. Re combinant haemadin displays a similar inhibition constant and specific activity as its natural counterpart. Although there is no obvious seq uence identity between haemadin and hirudin, both proteins seem to sha re common mechanisms for thrombin inhibition.