Purification and characterization of a novel anti-coagulant from the leech Hirudinaria manillensis

Protease inhibitors have been reported rarely from the leech Hirudinaria manillensis.In this study,we purified a novel protease inhibitor(bdellin-HM-2)with anticoagulant properties from H.manillensis.With a molecular weight of 1.4x104,bdellin-HM-2 was also characterized with three intra?molecular disulfide bridges at the N-terminus and multiple HHXDD and HXDD motifs at theC-terminus.cDNAcloning revealed that the putative nucleotide-encoding protein of bdellin-HM-2 contained 132 ami no acids and was encoded by a 399 bp open reading frame(ORF).Sequence alignment showed that bdellin-HM-2 shared similarity with the“non-classical"Kazal-type serine protease inhibitors,but had no inhibitory effect on trypsin,elastase,chymotrypsin,kallikrein,factor Xlla(FXIIa),factor Xia(FXIa),factor Xa(FXa),thrombin,or plasmin.Bdellin-HM-2 showed anticoagulant effects by proIonging the activated partial thromboplastin time(aPTT),indicating a role in enabling H.manillensis to obtain a blood meal from its host.Our results suggest that bdellin-HM丒2 may play a crucial role in blood-sucking in this leech species and may be a pote ntial can didate for the development of clinical anti-thrombotic drugs.

Protease inhibitors have been reported rarely from the leech Hirudinaria manillensis. In this study, we purified a novel protease inhibitor(bdellin-HM-2) with anticoagulant properties from H. manillensis. With a molecular weight of 1.4 x10^4, bdellin-HM-2 was also characterized with three intra-molecular disulfide bridges at the N-terminus and multiple HHXDD and HXDD motifs at the C-terminus. c DNA cloning revealed that the putative nucleotide-encoding protein of bdellin-HM-2 contained 132 amino acids and was encoded by a 399 bp open reading frame(ORF).Sequence alignment showed that bdellin-HM-2 shared similarity with the"non-classical"Kazal-type serine protease inhibitors, but had no inhibitory effect on trypsin, elastase, chymotrypsin, kallikrein, factor XIIa(FXIIa), factor XIa(FXIa), factor Xa(FXa), thrombin,or plasmin. Bdellin-HM-2 showed anticoagulant effects by prolonging the activated partial thromboplastin time(a PTT), indicating a role in enabling H. manillensis to obtain a blood meal from its host. Our results suggest that bdellin-HM-2 may play a crucial role in blood-sucking in this leech species and may be a potential candidate for the development of clinical anti-thrombotic drugs.