Isolation and characterization of an anticoagulant from the salivary glands of the tick, Ornithodoros savignyi (Acari: Argasidae)

An inhibitor of activated coagulation factor X (fXa) was isolated from salivary gland extracts prepared from Ornithodoros savignyi using a two-step procedure, involving reversed-phase high-performance liquid chromatography (RP-HPLC) and diethylaminoethyl (DEAE) ion-exchange chromatography. From its behaviour during DEAF chromatography it could be deduced that it possesses an acidic pi (similar to 4.6). Capillary zone electrophoresis (CZE) of the purified inhibitor showed it to be homogeneous. The molecular mass was determined as 12 kDa using capillary gel electrophoresis (CGE) and as 7183.4 using laser desorption mass spectrometry (LDMS). The N-terminal amino acid sequence (residues 1-12) was determined and found to share a 66% identity with tick anticoagulant peptide (TAP). The O. savignyi peptide is a slow, tight-binding inhibitor of fXa (K-i=0.83+/-0.10 nM). The interaction of the first-inhibitor was found to be competitive and dependent on ionic strength. Preliminary investigations show that the inhibitor may be specific for fXa.