RESUMEN
We have discovered and characterized a novel coagulation factor Xa inhibitor from the salivary gland of the black fly, Simulium vittatum. Salivary glands were surgically dissected from the flies and a crude salivary gland extract was tested for inhibition of a number of coagulation assays. The gland extract inhibited both thrombin and factor Xa. To purify further the factor Xa inhibitor, a factor Xa affinity column was utilized. Final purification of the black fly factor Xa inhibitor was achieved by reverse-phase C8 microbore high pressure liquid chromatography. Inhibition of factor Xa was nearly stoichiometric by the purified inhibitor with no inhibitor of thrombin detected. SDS-polyacrylamide gel electrophoresis indicated the inhibitor had a molecular weight of 18,000 and sequence analysis of the inhibitor revealed a blocked amino terminus. These data indicate that the blood-sucking black fly has evolved a highly potent inhibitor of mammalian coagulation factor Xa to disrupt its host normal hemostatic clotting mechanisms.
Asunto(s)
Antitrombina III/aislamiento & purificación , Dípteros/análisis , Inhibidores del Factor Xa , Aminoácidos/análisis , Animales , Antitrombina III/química , Cromatografía de Afinidad , Cromatografía Líquida de Alta Presión , Peso Molecular , Glándulas Salivales/químicaRESUMEN
A recent report indicated that an arrow poison used by the native Indians of Rondonia, Brazil, to kill small animals was associated with profuse bleeding. The arrow poison was prepared from the bark of a tree, known locally as Tike-Uba. We have obtained bark and sap specimens from this tree and have characterized a potent anticoagulant activity in both the crude bark and sap samples as well as in more highly purified preparations. An aqueous extract of the bark significantly prolongs both prothrombin times and activated partial thromboplastin times in plasma based assays. Further fractionation of the bark extract and sap by molecular weight indicated that all of the anticoagulant activity could be isolated in a molecular weight fraction of equal to or greater than 30,000 daltons. The anticoagulant activity was also further purified by C-18 reverse phase chromatography. When highly purified preparations of the anticoagulant activity from the Tike-Uba tree were examined in specific blood coagulation enzyme assays utilizing chromogenic substrates, the highest inhibitory potency was found versus thrombin, followed by factor Xa. These studies establish the presence of a compound(s) in a Brazilian arrow poison, which potently disrupts mammalian blood clotting, and which may account for some of the observed toxicities associated with the arrows.
Asunto(s)
Anticoagulantes/aislamiento & purificación , Plantas Tóxicas/análisis , Aminoácidos/análisis , Brasil , Compuestos Cromogénicos/metabolismo , Factor Xa/inmunología , Humanos , Oligopéptidos/metabolismo , Trombina/inmunología , TromboplastinaRESUMEN
As a factor Xa inhibitor, antistasin is a potent anti-coagulant and anti-metastatic agent that is found in the salivary gland of the Mexican leech Haementaria officinalis. cDNA clones that encode antistasin have been isolated. Subsequent sequence analysis and comparison with the amino acid sequence of the mature protein indicates that antistasin is produced as a pre-protein containing a 17-amino acid signal peptide. Antistasin exists as at least two variants. By sequence analysis of multiple cDNA clones, we found two additional sites for amino acid substitutions, confirming variants that differ from each other by amino acid changes at a minimum of four residues. These sequence variations appear to be the result of allelic variation rather than gene duplication as deduced from DNA blot analyses. Sequence data suggest that antistasin may have evolved from a smaller ancestral gene by a duplication event giving rise to a two-fold structural homology between the N- and C-terminal halves of the molecule. Insect cells transfected with a recombinant baculovirus expressed antistasin which was biologically active and had an electrophoretic mobility identical to that of the native molecule.
Asunto(s)
Anticoagulantes , Antineoplásicos , Clonación Molecular , ADN/genética , Regulación de la Expresión Génica , Hormonas de Invertebrados/genética , Proteínas y Péptidos Salivales/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Southern Blotting , Medios de Cultivo , ADN/aislamiento & purificación , Factor Xa , Variación Genética , Immunoblotting , Sanguijuelas , Datos de Secuencia Molecular , Metástasis de la Neoplasia/tratamiento farmacológico , Biosíntesis de Proteínas , ARN/aislamiento & purificación , ARN Mensajero/genética , Serina Endopeptidasas/análisis , Inhibidores de Serina Proteinasa , Transcripción GenéticaRESUMEN
The antiplatelet compound ticlopidine exerts its potent inhibitory activity through an as yet undetermined mechanism(s). The goal of this study was to determine the effect, if any, of ticlopidine ex vivo on platelet calcium mobilization. Ticlopidine inhibited ADP-induced platelet aggregation by 50-80%. In the presence of 1 mM EGTA, ticlopidine inhibited ADP- and thrombin-stimulated increases in [Ca2+]i in fura-2 loaded platelets. We evaluated further the effect of ticlopidine on calcium mobilization by examining both agonist-stimulated formation of inositol trisphosphate in intact platelets and the ability of inositol trisphosphate to release 45Ca from intracellular sites in permeabilized cells. We show here that while ticlopidine significantly affected agonist-induced intracellular calcium mobilization in intact platelets, the drug was without effect on agonist-stimulated formation of inositol trisphosphate in intact platelets and on inositol trisphosphate-induced 45Ca release in saponin-permeabilized platelets. Our study demonstrates that ticlopidine exerts at least part of its effect via inhibition of intracellular calcium mobilization but that its site of action remains to be determined.
Asunto(s)
Plaquetas/efectos de los fármacos , Calcio/metabolismo , Inhibidores de Agregación Plaquetaria/farmacología , Ticlopidina/farmacología , Animales , Plaquetas/metabolismo , Radioisótopos de Calcio , Inositol/farmacología , Masculino , ConejosRESUMEN
Clinical observations have suggested that gallstones are increased in frequency in Mexican-Americans compared to other ethnic groups. Past autopsy surveys have demonstrated a low prevalence in blacks compared to whites. We retrospectively reviewed 1,018 charts to study the prevalence of cholelithiasis in ambulatory women of these three racial/ethnic backgrounds. Mexican-American women were found to have a prevalence of gallbladder disease approximately three times that of black women, with Anglo prevalences falling in an intermediate range. The prevalence of cholelithiasis was also positively associated with increasing age and diabetes, but not with serum cholesterol, use of oral contraceptives or conjugated estrogens, parity, hypertension, menstrual status, or smoking. An association with obesity was shown but could not be shown to be independent of other risk factors.