Purification and characterization of an anticoagulant from the salivary glands of the ixodid tick Rhipicephalus appendiculatus.
An anticoagulant isolated from salivary gland extracts of the ixodid tick Rhipicephalus appendiculatus was purified by gel filtration on Sephadex G-100, ion exchange on DEAE-cellulose, aprotinin-Sepharose, and by high-pressure-liquid size-exclusion chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the anticoagulant activity was associated with a protein of an apparent Mr of 65 kDa. The purified molecule had a pI in the range of 8.0-8.5 on chromatofocusing and was stable over a wide pH range, but was heat labile and susceptible to inactivation by trypsin and reductive alkylation. The anticoagulant did not inhibit thrombin-initiated fibrin formation and had no detectable fibrino(geno)lytic or phospholipase-like activities. Although it inhibited factor Xa-induced clotting of bovine plasma, it did not affect the amidase activity of factor Xa toward a synthetic substrate, suggesting that the anticoagulant acts at a site distinct from the active site of factor Xa or on other components of the prothrombinase complex.
Limo
M K
MK
Department of Veterinary Microbiology and Immunology, University of California, Davis 95616.
Voigt
W P
WP
Tumbo-Oeri
A G
AG
Njogu
R M
RM
ole-MoiYoi
O K
OK
D000818:Animals; D001777:Blood Coagulation; D002851:Chromatography, High Pressure Liquid; D005165:Factor V / Q000037:antagonists & inhibitors*; D005170:Factor X / Q000037:antagonists & inhibitors*; D015951:Factor Xa; D065427:Factor Xa Inhibitors; D006863:Hydrogen-Ion Concentration; D007526:Isoelectric Point; D008970:Molecular Weight; D009842:Oligopeptides / Q000378:metabolism; D011506:Proteins / Q000302:isolation & purification* / Q000378:metabolism / Q000494:pharmacology; D012469:Salivary Glands / Q000737:chemistry; D013696:Temperature; D013987:Ticks / Q000032:analysis*
D065427:Factor Xa Inhibitors; D009842:Oligopeptides; D011506:Proteins; C022475:prothrombinase complex; C015608:benzoyl-Ile-Glu-Gly-Arg-p-nitroanilide; D005165:Factor V; D005170:Factor X; D015951:Factor Xa