RESUMEN
AIM: Peroxisome proliferator activated receptors (PPARs) are nuclear receptors involved in glucose and lipid metabolism. Three isoforms of PPARs have been identified with different tissue distribution and biological functions. Although the pharmacology of each receptor is well studied, the physiological effect of simultaneous activation of PPARalpha, gamma and delta is only starting to emerge. We sought to determine the biological effects of a novel PPAR pan activator and elucidate the physiological mechanisms involved. METHODS: Ob/ob, diet-induced obese (DIO) or PPARalpha knockout mice were administered a novel agonist that activates all PPARs to various degrees to determine the effect on body weight, body composition, food intake and energy expenditure. In addition, serum parameters including glucose, insulin, triglycerides and ketone bodies as well as tissue acylcarnitine were evaluated. The effect of the novel agonist on liver and skeletal muscle histopathology was also studied. RESULTS: We report that simultaneous activation of all PPARs resulted in substantial weight loss in ob/ob and DIO mice. Consistent with known PPAR pharmacology, we observed that agonist treatment increased lipid oxidation, although appetite suppression was mainly responsible for the weight loss. Agonist-induced weight loss was completely absent in PPARalpha knockout mice suggesting that PPARalpha pharmacology was the major contributor to weight regulation in mice. CONCLUSIONS: Our work provides evidence that simultaneous activation of PPARalpha, gamma and delta decreases body weight by regulating appetite. These effects of the pan agonist were completely absent in PPARalpha knockout mice, suggesting that PPARalpha pharmacology was the major contributor to weight loss.
Asunto(s)
Depresores del Apetito/farmacología , Obesidad/tratamiento farmacológico , PPAR alfa/agonistas , PPAR delta/agonistas , PPAR gamma/agonistas , Pérdida de Peso/fisiología , Animales , Regulación del Apetito/fisiología , Metabolismo Energético/fisiología , Resistencia a la Insulina/fisiología , Peroxidación de Lípido/fisiología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Obesidad/fisiopatología , PPAR alfa/genética , PPAR gamma/genética , Rosiglitazona , Tiazoles/farmacología , Tiazolidinedionas/farmacologíaRESUMEN
OBJECTIVE: To assess the antithrombotic and profibrinolytic effects of tiplaxtinin (PAI-039), an orally bioavailable antagonist of PAI-1, in rat models of thrombosis. METHODS AND RESULTS: Carotid artery and vena cava vascular injury was produced by application of FeCl3 and blood flow was monitored using ultrasonic technology. To assess efficacy in a thrombosis prevention paradigm, PAI-039 was administered orally 90 min before injury (1-30 mg kg(-1)). To assess efficacy in a thrombosis treatment paradigm, vascular injury and stable thrombus formation were followed 4 h later by recovery and PAI-039 administration. PAI-039 prevented carotid artery occlusion in 20, 68 and 60% of animals pretreated with 0.3, 1.0 and 3.0 mg kg(-1), respectively. Time to occlusive thrombosis was increased from 18.2 +/- 4.6 min in controls to 32.5 +/- 8.7 (P = ns), 46.1 +/- 7.0 (P < 0.05), and 41.6 +/- 11.3 min (P < 0.05) in the respective PAI-039 treatment groups. In the vena cava protocol, PAI-039 pretreatment significantly reduced thrombus weight at PAI-039 doses of 3, 10 and 30 mg kg(-1). When PAI-039 was dosed in a treatment paradigm 4 h after stable arterial and venous thrombosis, a significant reduction in thrombus weight was observed 24 h later at PAI-039 doses of 3, 10 and 30 mg kg(-1). PAI-039 (10, 30 and 100 mg kg(-1)) had no effect on platelet aggregation in response to ADP or collagen and was not associated with increased bleeding or prolonged prothrombin time. In animals bearing no vascular injury, PAI-039 had no effect on circulating, low-levels of PAI-1 activity. In contrast, circulating PAI-1 activity increased 5-fold following the induction of vascular injury, which was completely neutralized by PAI-039. CONCLUSIONS: PAI-039 exerts antithrombotic efficacy in rat models of arterial and venous vascular injury without effecting platelet aggregation.
Asunto(s)
Ácidos Indolacéticos/farmacología , Inhibidor 1 de Activador Plasminogénico , Trombosis/prevención & control , Administración Oral , Animales , Disponibilidad Biológica , Modelos Animales de Enfermedad , Ácidos Indolacéticos/administración & dosificación , Ácidos Indolacéticos/farmacocinética , Masculino , Agregación Plaquetaria/efectos de los fármacos , Tiempo de Protrombina , Ratas , Ratas Sprague-DawleyRESUMEN
The tissue factor-factor (F)VIIa complex (TF/FVIIa) is responsible for the initiation of blood coagulation under both physiological and pathological conditions. Recombinant nematode anticoagulant protein c2 (rNAPc2) is a potent inhibitor of TF/FVIIa, mechanistically distinct from tissue factor pathway inhibitor. The first aim of this study was to elucidate the pharmacokinetics and pharmacodynamics of a single intravenous (i.v.) dose of rNAPc2. The second aim was to study its effect on endotoxin-induced coagulation and inflammation. Initially, rNAPc2 was administered to healthy volunteers in three different doses. There were no safety concerns and the pharmacokinetics were consistent with previous studies, in which rNAPc2 was administered subcutaneously. rNAPc2 elicited a dose-dependent reduction of the endogenous thrombin potential and a selective prolongation of prothrombin time. Subsequently, the effect on endotoxin-induced coagulation and inflammation was studied. The administration of rNAPc2 completely blocked the endotoxin-induced thrombin generation, as measured by plasma prothrombin fragment F1+2. The endotoxin-induced effect on fibrinolytic parameters such as plasmin-antiplasmin complexes and plasminogen activator inhibitor type 1 was not affected by rNAPc2. The administration of rNAPc2 attenuated the endotoxin-induced rise in interleukin (IL)-10, without affecting the rise in other cytokines. In conclusion, rNAPc2 is a potent inhibitor of TF/FVIIa, which was well tolerated and could safely be used intravenously in this Phase I study in healthy male volunteers. A single i.v. dose rNAPc2 completely blocked endotoxin-induced thrombin generation without affecting the fibrinolytic response. In addition, rNAPc2 attenuated the endotoxin-induced rise in IL-10, without affecting the rises in other cytokines.
Asunto(s)
Endotoxemia/tratamiento farmacológico , Factor VIIa/antagonistas & inhibidores , Proteínas del Helminto/farmacología , Interleucina-10/biosíntesis , Tromboplastina/antagonistas & inhibidores , Adolescente , Adulto , Animales , Anticoagulantes/administración & dosificación , Anticoagulantes/efectos adversos , Anticoagulantes/farmacocinética , Anticoagulantes/farmacología , Coagulación Sanguínea/efectos de los fármacos , Citocinas/biosíntesis , Endotoxemia/sangre , Endotoxemia/inmunología , Fibrinólisis/efectos de los fármacos , Proteínas del Helminto/administración & dosificación , Proteínas del Helminto/efectos adversos , Proteínas del Helminto/farmacocinética , Humanos , Masculino , Proteínas Recombinantes/administración & dosificación , Proteínas Recombinantes/efectos adversos , Proteínas Recombinantes/farmacocinética , Proteínas Recombinantes/farmacología , Seguridad , Trombina/biosíntesisRESUMEN
Originally isolated from a haematophagous hookworm, recombinant nematode anticoagulant protein c2 (rNAPc2) is an 85-amino acid protein with potent anticoagulant properties. Unlike conventional anticoagulants that attenuate blood coagulation via inhibition of thrombin or activated factor X (FXa) at the downstream portion of the cascade, rNAPc2 is a potent inhibitor of the activated factor VII/tissue factor complex (FVIIa/TF), the key physiological initiator of blood coagulation. Its mechanism of action requires prerequisite binding to circulating FXa or zymogen factor X (FX) to form a binary complex prior to its interaction and inhibition of membrane-bound FVIIa/TF. The binding of rNAPc2 to FX results in an elimination half-life of longer than 50 h following either subcutaneous or intravenous administration. Recombinant NAPc2, like other inhibitors of FVIIa/TF including tissue factor pathway inhibitor (TFPI) and active site-blocked FVIIa (ASIS, FFR-rFVIIa or FVIIai), may have a promising role in the prevention and treatment of venous and arterial thrombosis, as well as potential efficacy in the management of disseminated intravascular coagulopathies because of their potent and selective inhibition of FVIIa/TF.
Asunto(s)
Anticoagulantes/uso terapéutico , Factor VIIa/antagonistas & inhibidores , Proteínas del Helminto/uso terapéutico , Trombosis/prevención & control , Coagulación Sanguínea/efectos de los fármacos , Factor X/metabolismo , Factor Xa/metabolismo , Humanos , Proteínas Recombinantes/uso terapéutico , Tromboplastina/metabolismo , Trombosis de la Vena/prevención & controlRESUMEN
BACKGROUND: With the best prophylactics now available, venous thromboembolism after total knee replacement remains substantial (25% to 27%). Recombinant nematode anticoagulant protein c2 (rNAPc2) is a potent inhibitor of factor VIIa/tissue factor complex that has the potential to reduce this risk. The present study was performed to determine an efficacious and safe dose of rNAPc2 for prevention of venous thromboembolism after elective, unilateral total knee replacement. METHODS AND RESULTS: This open-label, sequential dose-ranging study was conducted in 11 centers in Canada, Europe, and the United States. Five regimens were tested. Injections were administered subcutaneously on the day of surgery (day 1) and days 3, 5, and optionally, day 7. Primary efficacy outcome was a composite of overall deep vein thrombosis based on mandatory unilateral venography (day 7+/-2) and confirmed symptomatic venous thromboembolism recorded
Asunto(s)
Anticoagulantes/administración & dosificación , Artroplastia de Reemplazo de Rodilla , Factor VIIa/antagonistas & inhibidores , Proteínas del Helminto/administración & dosificación , Complicaciones Posoperatorias/prevención & control , Trombosis de la Vena/prevención & control , Anciano , Artroplastia de Reemplazo de Rodilla/efectos adversos , Canadá , Relación Dosis-Respuesta a Droga , Europa (Continente) , Femenino , Proteínas del Helminto/efectos adversos , Hemorragia/inducido químicamente , Humanos , Inyecciones Subcutáneas , Modelos Logísticos , Masculino , Oportunidad Relativa , Medición de Riesgo , Tasa de Supervivencia , Tromboplastina/antagonistas & inhibidores , Estados Unidos , Trombosis de la Vena/etiologíaRESUMEN
BACKGROUND: In view of the central role of the tissue factor-factor VIIa pathway in the initiation of blood coagulation, novel therapeutic strategies aimed at inhibiting this catalytic complex are currently being evaluated. A limitation of this new class of anticoagulants may be the lack of an appropriate strategy to reverse the effect if a bleeding event occurs. The aim of this study was to investigate the in vivo potential of recombinant factor VIIa (rVIIa) to induce thrombin generation in healthy subjects pretreated with recombinant nematode anticoagulant protein c2, a specific inhibitor of the tissue factor-factor VIIa complex, in a double-blind randomized crossover study. METHODS AND RESULTS: Administration of nematode anticoagulant protein c2 (3.5 microgram/kg) caused a prolongation of the prothrombin time from 13.7+/-0.6 to 16.9+/-1.2 seconds. The subsequent injection of rVIIa (90 microgram/kg) resulted in an immediate and complete correction of the prothrombin time and a marked generation of thrombin, reflected by increased levels of prothrombin activation fragment F1+2 and thrombin-antithrombin complexes from 0.75+/-0.64 to 3.29+/-6.3 nmol/L and from 2.4+/-0.6 to 10.7+/-3.9 microgram/mL, respectively. Factor X and IX activation peptides showed a 3.5-fold and a 3.8-fold increase, respectively, after the administration of rVIIa in the presence of nematode anticoagulant protein c2. CONCLUSIONS: During treatment with an inhibitor of the tissue factor-factor VIIa complex, the infusion of rVIIa resulted in thrombin generation. Our results indicate that rVIIa may be a good candidate as an antidote for inhibitors of tissue factor.
Asunto(s)
Anticoagulantes/farmacología , Factor VIIa/farmacología , Proteínas del Helminto/farmacología , Trombina/efectos de los fármacos , Adulto , Animales , Estudios Cruzados , Método Doble Ciego , Factor IX/efectos de los fármacos , Factor IX/metabolismo , Factor VIIa/metabolismo , Factor X/efectos de los fármacos , Factor X/metabolismo , Proteínas del Helminto/sangre , Humanos , Masculino , Tiempo de Tromboplastina Parcial , Tiempo de Protrombina , Proteínas Recombinantes/farmacología , Trombina/metabolismoRESUMEN
Recombinant nematode anticoagulant protein c2 (rNAPc2) is a potent, factor Xa (fXa)-dependent small protein inhibitor of factor VIIa-tissue factor (fVIIa.TF), which binds to a site on fXa that is distinct from the catalytic center (exo-site). In the present study, the role of other fX derivatives in presenting rNAPc2 to fVIIa.TF is investigated. Catalytically active and active site blocked fXa, as well as a plasma-derived and an activation-resistant mutant of zymogen fX bound to rNAPc2 with comparable affinities (K(D) = 1-10 nm), and similarly supported the inhibition of fVIIa.TF (K(i)* = approximately 10 pm). The roles of phospholipid membrane composition in the inhibition of fVIIa.TF by rNAPc2 were investigated using TF that was either detergent-solubilized (TF(S)), or reconstituted into membranes, containing phosphatidylcholine (TF(PC)) or a mixture of phosphatidylcholine and phosphatidylserine (TF(PCPS)). In the absence of the fX derivative, inhibition of fVIIa.TF was similar for all three conditions (K(i) approximately 1 microm), whereas the addition of the fX derivative increased the respective inhibition by 35-, 150-, or 100,000-fold for TF(S), TF(PC), and TF(PCPS). The removal of the gamma-carboxyglutamic acid-containing domain from the fX derivative did not affect the binding to rNAPc2, but abolished the effect of factor Xa as a scaffold for the inhibition of fVIIa.TF by rNAPc2. The overall anticoagulant potency of rNAPc2, therefore, results from a coordinated recognition of an exo-site on fX/fXa and of the active site of fVIIa, both of which are properly positioned in the ternary fVIIa.TF.fX(a) complex assembled on an appropriate phospholipid surface.
Asunto(s)
Cisteína Endopeptidasas/química , Precursores Enzimáticos/metabolismo , Factor VIIa/metabolismo , Factor Xa/metabolismo , Proteínas del Helminto/metabolismo , Proteínas de Neoplasias , Proteínas Recombinantes/metabolismo , Animales , Anticoagulantes/farmacología , Sitios de Unión , Células CHO , Dominio Catalítico , Membrana Celular/metabolismo , Cricetinae , Relación Dosis-Respuesta a Droga , Electroforesis en Gel de Poliacrilamida , Precursores Enzimáticos/química , Factor Xa/química , Humanos , Hidrólisis , Cinética , Modelos Biológicos , Modelos Químicos , Mutación , Fosfolípidos/metabolismo , Unión Proteica , Estructura Terciaria de Proteína , Resonancia por Plasmón de Superficie , Garrapatas , Factores de TiempoRESUMEN
Blood coagulation in vivo is initiated by factor VII (FVII) binding to its cellular receptor tissue factor (TF). FVII is the only known ligand for TF, so it was expected that FVII-deficient embryos would have a similar phenotype to TF-deficient embryos, which have defective vitello-embryonic circulation and die around 9.5 days of gestation. Surprisingly, we find that FVII-deficient (FVII-/-) embryos developed normally. FVII-/- mice succumbed perinatally because of fatal haemorrhaging from normal blood vessels. At embryonic day 9.5, maternal-fetal transfer of FVII was undetectable and survival of embryos did not depend on TF-FVII-initiated fibrin formation. Thus, the TF-/- embryonic lethal and the FVII-/- survival-phenotypes suggest a role for TF during embryogenesis beyond fibrin formation.
Asunto(s)
Factor VII/fisiología , Hemorragia/etiología , Animales , Coagulación Sanguínea/fisiología , Técnicas de Cultivo , Desarrollo Embrionario y Fetal , Factor VII/genética , Femenino , Muerte Fetal , Fibrina/fisiología , Marcación de Gen , Hemorragia/mortalidad , Hemostasis/fisiología , Intercambio Materno-Fetal , Ratones , Ratones Endogámicos C57BL , Mutagénesis , Embarazo , Tromboplastina/metabolismoRESUMEN
We describe the antithrombotic effects of recombinant nematode anticoagulant peptide (rNAP5), a selective and direct factor Xa inhibitor, after a single s.c. administration in canine models of arterial and venous thrombosis. The systemic anticoagulant effects of rNAP5 were evaluated initially in conscious dogs after s.c. dosing (0.03, 0.1 and 0.3 mg/kg) that resulted in a dose-dependent increase in the activated clotting time and the activated partial thromboplastin time. The antithrombotic effects of rNAP5 were evaluated in anesthetized dogs where saline or rNAP5 (0.03, 0.1 and 0.3 mg/kg s.c.) was administered 1 hr before the left circumflex coronary artery was subjected to electrolytic injury. In the saline group (n = 10), the left circumflex artery occluded in 79 +/- 9 min, and 5 of 10 animals progressed to sudden death due to ventricular fibrillation. rNAP5 significantly prolonged the time to occlusion in the 0.03 mg/kg (163 +/- 62 min) and 0.1 mg/kg (327 +/- 62) treatment groups (n = 6). In the 0.3 mg/kg group (n = 5), all of the injured vessels remained patent for 8 hr. There was a dose-dependent reduction in the thrombus mass in the rNAP5-treated animals as compared with controls, as well as a lower mortality rate. rNAP5, in the doses of 0.03 and 0.1 mg/kg, did not alter the bleeding time, whereas 0.3 mg/kg produced a 5-fold increase. In a separate study, we evaluated the efficacy of rNAP5 (0.1 mg/kg) in the prevention of carotid artery and jugular vein thrombosis. In response to endothelial injury, the carotid artery and jugular vein in the saline group (n = 6) occluded in 142 +/- 16 and 100 +/- 11 min, respectively, compared with rNAP5, which maintained vessel patency in the carotid artery (6/6) and jugular vein (5/6) and significantly decreased the thrombus weights. The results demonstrate that rNAP5 has antithrombotic efficacy in canine models of arterial and venous thrombosis after a single s.c. administration.
Asunto(s)
Ancylostoma/química , Anticoagulantes/uso terapéutico , Inhibidores del Factor Xa , Fibrinolíticos/uso terapéutico , Proteínas del Helminto/uso terapéutico , Trombosis/prevención & control , Secuencia de Aminoácidos , Animales , Perros , Relación Dosis-Respuesta a Droga , Heparina de Bajo-Peso-Molecular/uso terapéutico , Inyecciones Subcutáneas , Datos de Secuencia Molecular , Proteínas Recombinantes/uso terapéuticoRESUMEN
The prothrombinase complex assembles through reversible interactions between the protease, factor Xa, the cofactor, factor Va, and acidic phospholipid membranes in the presence of calcium ions. Changes in macromolecular recognition by factor Xa which may result from its interaction with factor Va in the prothrombinase complex have been probed using a recombinant derivative of tick anticoagulant peptide where Arg3 has been replaced with Ala (R3A-TAP). In contrast to the wild type inhibitor, R3A-TAP was a weak competitive inhibitor of factor Xa (Ki = 794 nM). The inhibition of the prothrombinase complex by R3A-TAP was characterized by slow, tight-binding kinetics with an increased affinity of approximately 4000-fold (Ki* = 0.195 nM) relative to that of solution-phase factor Xa. Stopped-flow measurements using p-aminobenzamidine (PAB) demonstrated that the reaction between solution-phase factor Xa and R3A-TAP could be adequately described by a single reversible step with rate constants that were consistent with equilibrium binding measurements. The rate-limiting bimolecular combination of R3A-TAP and factor Xa was competitive with PAB binding of the protease. In contrast, the reaction of R3A-TAP with prothrombinase measured using PAB yielded biphasic stopped-flow traces, indicating a multistep pathway for the reaction of the inhibitor with the enzyme complex. The kinetic measurements were consistent with the initial formation of a ternary complex between R3A-TAP, prothrombinase, and PAB followed by two unimolecular steps which lead to PAB dissociation from the enzyme. In this case, prior occupation of the active site by PAB had no effect on the bimolecular reaction between R3A-TAP and prothrombinase. Thus, the interaction of factor Xa with factor Va on the membrane surface alters recognition of R3A-TAP by the protease, leading to changes in the thermodynamics as well as in the observed kinetic mechanism for the reaction. Therefore, a single amino acid substitution in TAP reveals large changes in macromolecular recognition by factor Xa as a consequence of its interaction with the cofactor within the prothrombinase complex.
Asunto(s)
Factor V/antagonistas & inhibidores , Factor Va/farmacología , Factor X/antagonistas & inhibidores , Factor Xa/metabolismo , Péptidos/farmacología , Animales , Proteínas de Artrópodos , Benzamidinas/farmacología , Sitios de Unión/genética , Bovinos , Factor V/metabolismo , Factor X/metabolismo , Péptidos y Proteínas de Señalización Intercelular , Cinética , Modelos Químicos , Mutagénesis Sitio-Dirigida/genética , Péptidos/genética , Péptidos/metabolismo , Fosfatidilcolinas/metabolismo , Unión Proteica , Conformación Proteica , Inhibidores de Serina Proteinasa/farmacología , Espectrometría de FluorescenciaRESUMEN
BACKGROUND: We examined the oral efficacy of a direct thrombin inhibitor, CVS-1123 [(CH3CH2CH2)(2)-CH-CO-Asp (OCH3)-Pro-Arg-CHO; MW, 575]. The object was to determine whether thrombin inhibition could reduce the incidence of occlusive coronary artery thrombosis in response to arterial wall injury. METHODS AND RESULTS: Arterial wall injury was induced in conscious dogs by a 150-muA anodal current applied to the intimal surface of the circumflex coronary artery 30 minutes after oral CVS-1123 (20 mg/kg every 8 hours for three doses; n = 11) or placebo containing diluent (n = 10). Dogs were monitored for 8 hours and at 24 hours. The coronary artery remained patent for 24 hours in 8 of 11 CVS-1123-treated dogs. All dogs (n = 10) in the placebo group developed a sustained, occlusive arterial thrombus. Two hours after the initial oral dose, the plasma CVS-1123 concentration was 13 +/- 1 microgram/mL, reaching a maximum of 15 +/- 1 micrograms/mL after the second dose and 4.4 +/- 0.5 micrograms/mL at 24 hours. Ex vivo platelet aggregation to gamma-thrombin was inhibited and activated partial thromboplastin time was increased after treatment with CVS-1123 (P < .05). CONCLUSIONS: The direct thrombin inhibitor CVS-1123 is effective after oral administration in reducing the incidence of primary thrombus formation in an experimental model of arterial wall injury. Thrombin-specific inhibitors, such as CVS-1123, may be alternative antithrombotic agents in clinical settings in which heparin-associated thrombosis is a complicating factor or when long-term anticoagulation is required.
Asunto(s)
Antitrombinas/administración & dosificación , Trombosis Coronaria/prevención & control , Oligopéptidos/administración & dosificación , Administración Oral , Amidas/metabolismo , Animales , Antitrombinas/farmacología , Arterias , Velocidad del Flujo Sanguíneo/efectos de los fármacos , Presión Sanguínea/efectos de los fármacos , Circulación Coronaria/efectos de los fármacos , Perros , Frecuencia Cardíaca/efectos de los fármacos , Masculino , Oligopéptidos/sangre , Oligopéptidos/farmacocinética , Concentración Osmolar , Tiempo de Tromboplastina Parcial , Agregación Plaquetaria/efectos de los fármacos , Trombina/antagonistas & inhibidoresRESUMEN
A potent and specific inhibitor of the human coagulation protease thrombin was identified in salivary gland extracts of the tsetse fly, Glossina morsitans morsitans, an important vector of African trypanosomiasis. This low molecular weight peptide (MW = 3,530 Da as determined by laser desorption mass spectrometry) was purified using a combination of size-exclusion chromatography and reverse-phase, high-performance liquid chromatography, respectively. Amino terminal sequencing of the purified protein reveals no homology to any previously identified serine protease inhibitor or naturally occurring anticoagulant. The tsetse thrombin inhibitor (TTI) is a stoichiometric inhibitor of thrombin, with an apparent equilibrium dissociation inhibitory constant (Ki*) [corrected] of 584 x 10(-15)M. In addition, it is also a potent inhibitor of thrombin-induced platelet aggregation. Like other hematophagous arthropods, tsetse flies appear to have evolved a novel protease inhibitor capable of antagonizing host hemostasis and facilitating blood feeding.
Asunto(s)
Antitrombina III/aislamiento & purificación , Saliva/química , Inhibidores de Serina Proteinasa/aislamiento & purificación , Moscas Tse-Tse , Secuencia de Aminoácidos , Animales , Antitrombina III/química , Antitrombina III/fisiología , Femenino , Humanos , Masculino , Datos de Secuencia Molecular , Peso Molecular , Tiempo de Tromboplastina Parcial , Agregación Plaquetaria , Inhibidores de Serina Proteinasa/química , Inhibidores de Serina Proteinasa/fisiología , Especificidad por SustratoRESUMEN
A new class of divalent thrombin inhibitors is described that contains an alpha-keto-amide transition-state mimetic linking an active site binding group and a group that binds to the fibrinogen-binding exosite. The X-ray crystallographic structure of the most potent member of this new class, CVS995, shows many features in common with other divalent thrombin inhibitors and clearly defines the transition-state-like binding of the alpha-keto-amide group. The structure of the active site part of the inhibitor shows a network of water molecules connecting both the side-chain and backbone atoms of thrombin and the inhibitor. Direct peptide analogues of the new transition-state-containing divalent thrombin inhibitors were compared using in vitro assays of thrombin inhibition. There was no direct correlation between the binding constants of the peptides and their alpha-keto-amide counterparts. The most potent alpha-keto-amide inhibitor, CVS995, with a Ki = 1 pM, did not correspond to the most potent divalent peptide and contained a single amino acid deletion in the exosite binding region with respect to the equivalent region of the natural thrombin inhibitor hirudin. The interaction energies of the active site, transition state, and exosite binding regions of these new divalent thrombin inhibitors are not additive.
Asunto(s)
Antitrombinas/química , Proteínas Sanguíneas , Hirudinas/análogos & derivados , Péptidos/química , Trombina/antagonistas & inhibidores , Amidas/química , Secuencia de Aminoácidos , Antitrombinas/síntesis química , Antitrombinas/farmacología , Sitios de Unión , Gráficos por Computador , Cristalografía por Rayos X , Hirudinas/química , Hirudinas/farmacología , Enlace de Hidrógeno , Modelos Moleculares , Datos de Secuencia Molecular , Péptidos/síntesis química , Péptidos/farmacología , Conformación Proteica , Relación Estructura-Actividad , Trombina/químicaRESUMEN
Hookworms are hematophagous nematodes that infect a wide range of mammalian hosts, including humans. There has been speculation for nearly a century as to the identity of the anticoagulant substances) used by these organisms to subvert host hemostasis. Using molecular cloning, we describe a family of potent small protein (75-84 amino acids) anticoagulants from the hookworm Ancylostoma caninum termed AcAP (A. caninum anticoagulant protein). Two recombinant AcAP members (AcAP5 and AcAP6) directly inhibited the catalytic activity of blood coagulation factor Xa (fXa), while a third form (AcAPc2) predominantly inhibited the catalytic activity of a complex composed of blood coagulation factor VIIa and tissue factor (fVIIa/TF). The inhibition of fVIIa/TF was by a unique mechanism that required the initial formation of a binary complex of the inhibitor with fXa at a site on the enzyme that is distinct from the catalytic center (exo-site). The sequence of AcAPc2 as well as the utilization of an exo-site on fXa distinguishes this inhibitor from the mammalian anticoagulant TFPI (tissue factor pathway inhibitor), which is functionally equivalent with respect to fXa-dependent inhibition of fIIa/TF. The relative sequence positions of the reactive site residues determined for AcAP5 with the homologous regions in AcAP6 and AcAPc2 as well as the pattern of 10 cysteine residues present in each of the inhibitors suggest that the AcAPs are distantly related to the family of small protein serine protease inhibitors found in the nonhematophagous nematode Ascaris lumbricoides var. suum.
Asunto(s)
Ancylostoma/enzimología , Factores de Coagulación Sanguínea/antagonistas & inhibidores , Coagulación Sanguínea , Proteínas del Helminto/genética , Inhibidores de Serina Proteinasa/genética , Secuencia de Aminoácidos , Ancylostoma/genética , Animales , Sitios de Unión , Clonación Molecular , ADN Complementario/genética , Datos de Secuencia Molecular , Alineación de Secuencia , Tromboplastina/metabolismoRESUMEN
The CD11/CD18 leukocyte integrins are necessary for tissue localization of neutrophils, an early requisite event in inflammation. We have analyzed the contribution of CD11a/CD18 and CD11b/CD18 to local neutrophil accumulation and tissue injury in the reverse passive Arthus reaction in the rat dermis. Experimental groups comprised animals that received an intravenous infusion of (1) recombinant neutrophil inhibitory factor (NIF), a hookworm-derived antagonist of CD11b/CD18; (2) monoclonal antibody to CD11a/CD18 (TA-3); (3) a combination of these agents; (4) a monoclonal antibody to CD18 (WT.3); or (5) saline. Administration of recombinant NIF or anti-CD11a/CD18 monoclonal antibody alone produced a slight reduction in neutrophil accumulation but did not affect edema formation. In contrast, a combination of these antagonists yielded a significant reduction in neutrophil accumulation and a modest reduction in edema, equivalent to levels observed with either anti-CD18 antibodies or animals that were rendered neutropenic. These results indicate that neutrophil infiltration in rat dermal tissue in the reverse passive Arthus reaction is dependent predominantly on the leukocyte integrins CD11a/CD18 and CD11b/CD18 and that either of these integrins is sufficient for neutrophil trafficking in this inflammatory setting.
Asunto(s)
Reacción de Arthus/fisiopatología , Antígenos CD11/fisiología , Antígenos CD18/fisiología , Dermatitis/fisiopatología , Proteínas de la Membrana , Animales , Anticuerpos Monoclonales/farmacología , Reacción de Arthus/tratamiento farmacológico , Reacción de Arthus/patología , Antígenos CD11/efectos de los fármacos , Antígenos CD11/inmunología , Antígenos CD18/efectos de los fármacos , Antígenos CD18/inmunología , Células CHO , Cricetinae , Dermatitis/tratamiento farmacológico , Dermatitis/patología , Edema/tratamiento farmacológico , Edema/inmunología , Edema/patología , Glicoproteínas/farmacología , Proteínas del Helminto/farmacología , Masculino , Neutrófilos/efectos de los fármacos , Neutrófilos/inmunología , Neutrófilos/patología , Conejos , Ratas , Proteínas Recombinantes/farmacologíaRESUMEN
BACKGROUND: In the pathogenesis of (recurrent) thrombosis, clot-associated thrombin appears to play an important role. Antithrombin III-independent thrombin inhibitors have been shown to neutralize clot-bound thrombin effectively. We compared the sustained antithrombotic effects and the effects on endogenous fibrinolysis of several of these agents with recombinant tick anticoagulant peptide (rTAP), a selective factor Xa inhibitor, and low-molecular-weight heparin (LMWH) in an experimental venous thrombosis model. METHODS AND RESULTS: Rabbits received either recombinant hirudin (rHir), Hirulog-1, CVS#995 (a novel direct inhibitor of thrombin), rTAP, LMWH, or saline. The effect on thrombus growth was assessed by measuring the accretion of 125I-labeled fibrinogen onto preformed nonradioactive thrombi, and the effect on endogenous fibrinolysis was assessed by measuring the decline in radioactivity of preformed 125I-labeled thrombi in rabbit jugular veins. All direct thrombin inhibitors induced a sustained antithrombotic effect compared with either LMWH and rTAP. In addition, CVS#995 also further decreased thrombus size after stopping its infusion, which was due to a significant enhancement of endogenous fibrinolysis. CONCLUSIONS: Direct thrombin inhibition by rHir, Hirulog-1, or CVS #995 induces a sustained antithrombotic effect compared with rTAP and LMWH, which is most likely due to inhibition of clot-bound thrombin. CVS#995 was shown to also enhance the extent of endogenous fibrinolysis to a greater degree compared with rHir and might therefore be an interesting new antithrombotic agent for the treatment of venous and arterial thrombosis.
Asunto(s)
Antitrombinas/uso terapéutico , Inhibidores del Factor Xa , Heparina de Bajo-Peso-Molecular/uso terapéutico , Terapia con Hirudina , Venas Yugulares/patología , Fragmentos de Péptidos/uso terapéutico , Trombina/antagonistas & inhibidores , Trombosis/tratamiento farmacológico , Animales , Antitrombinas/farmacocinética , Factor Xa/metabolismo , Heparina de Bajo-Peso-Molecular/farmacocinética , Hirudinas/farmacocinética , Venas Yugulares/metabolismo , Fragmentos de Péptidos/farmacocinética , Conejos , Proteínas Recombinantes/farmacocinética , Proteínas Recombinantes/uso terapéutico , Trombina/metabolismo , Trombosis/metabolismoRESUMEN
Tick anticoagulant peptide (TAP) is a potent and selective inhibitor of factor Xa. TAP has shown good antithrombotic efficacy in experimental animal models of disseminated intravascular coagulation and venous and arterial thrombogenesis. In the present study we evaluated the effect of recombinant TAP (rTAP) on acute thrombus formation in human nonanticoagulated blood triggered either by tissue factor (TF) or by collagen at arterial shear conditions. The main goal was to establish the role of factor Xa in thrombus formation by use of an optimal inhibitory concentration of rTAP. Blood was drawn directly from an antecubital vein by a pump over the respective thrombogenic surfaces, which were positioned in a parallel-plate perfusion chamber. rTAP was mixed homogeneously into the flowing blood by a heparin-coated device positioned proximal to the perfusion chamber. The passage of blood through this device caused minor activation of coagulation but little activation of platelets. Fibrinopeptide A and beta-thromboglobulin levels after 5 minutes of blood perfusion were, on average, 14 ng/mL and 45 IU/mL, respectively. rTAP at a plasma concentration of 0.90 mumol/L completely inhibited TF/factor VIIa-dependent thrombus formation at wall shear rates of 650 and 2600 s-1. These shear conditions are comparable to those in medium-sized arteries and in moderately stenosed small arteries, respectively. In contrast to the TF-coated surface, rTAP was less efficient in reducing collagen-induced thrombus formation. While a significant reduction of 53% was observed at 650 s-1, thrombus formation at 2600 s-1 was not affected by rTAP.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Colágeno/fisiología , Factor VIIa/fisiología , Inhibidores del Factor Xa , Péptidos/farmacología , Tromboplastina/fisiología , Trombosis/etiología , Proteínas de Artrópodos , Coagulación Sanguínea/efectos de los fármacos , Plaquetas/efectos de los fármacos , Factor Xa/fisiología , Fibrinopéptido A/análisis , Humanos , Técnicas In Vitro , Péptidos y Proteínas de Señalización Intercelular , Proteínas Recombinantes/farmacología , Trombosis/prevención & control , beta-Tromboglobulina/análisisRESUMEN
A low molecular mass monomeric protein termed Fh-KTM (Fasciola hepatica Kunitz-type molecule) was isolated from the trematode Fasciola hepatica. Fh-KTM is a single polypeptide of 58 amino acids and a Mr of 6751. The complete amino acid sequence of Fh-KTM was determined and revealed significant similarity to the Kunitz-type (BPTI) family of proteinase inhibitors. Several polymorphisms were observed suggesting that more than one Fh-KTM molecule may be expressed by this parasite. Modified proline residues were shown to occur at all four positions in this protein as 3-hydroxy derivatives. This is the first report of 3-hydroxyproline residues in a Kunitz-type molecule. Indirect immunofluorescence and immunogold labelling revealed that Fh-KTM is an abundant molecule within the parasite localised to the gut, the parenchymal tissue and the tegument of adult F. hepatica. Serine protease inhibition assays revealed that Fh-KTM exhibited little or no inhibition against chymotrypsin, kallikrein, urokinase or key serine proteases of the blood coagulation pathways. However, Fh-KTM was able to inhibit trypsin even though the P1 reactive amino acid of Fh-KTM was a leucine residue.
Asunto(s)
Fasciola hepatica/genética , Proteínas del Helminto/genética , Secuencia de Aminoácidos , Animales , Aprotinina/química , Aprotinina/genética , Aprotinina/aislamiento & purificación , Fasciola hepatica/química , Proteínas del Helminto/química , Proteínas del Helminto/aislamiento & purificación , Inmunohistoquímica , Datos de Secuencia Molecular , Peso Molecular , Homología de Secuencia de Aminoácido , Inhibidores de Serina Proteinasa/química , Inhibidores de Serina Proteinasa/genética , Inhibidores de Serina Proteinasa/aislamiento & purificación , Inhibidores de Tripsina/química , Inhibidores de Tripsina/genéticaRESUMEN
The low molecular weight alpha-keto amide inhibitor CVS-1347, benzyl-SO2-Met(O2)-Pro-Arg(CO)((CONH)CH2)-phenyl, is a slow, tight binding inhibitor of alpha-thrombin amidolytic activity having a Ki = 1.28 x 10(-10) M. A complex between human alpha-thrombin and a hydrolysis product of CVS-1347 has been determined and refined using crystallography. The crystals belong to monoclinic space group C2 with cell dimensions of a = 71.08, b = 72.05 and c = 72.98 A and beta = 100.8 degrees. The structure was solved using isomorphous replacement methods and refined with resolution limits of (8.00-1.76) A to an R-value of 0.162. The Pro-Arg core of the inhibitor binds in the S2 and S1 subsites respectively, as is usually observed for Pro-Arg thrombin inhibitors. The Met(O2) side chain does not make any close contacts with the enzyme but influences the conformation of Glu192; the N-terminal benzylsulfonyl group makes an aromatic-aromatic contact with Trp215 in the hydrophobic part of the active site. The alpha-keto carboxylic acid of the proteolyzed inhibitor binds with the carboxylate group in the oxyanion hole, demonstrating that this region can accommodate an anion in a protease-peptide complex. The alpha-keto carbonyl group interacts closely with the two most important residues in the active site: the carbon atom is within a covalent bond distance of the active site Ser195 O gamma and the carbonyl oxygen is hydrogen bonded to His57.(ABSTRACT TRUNCATED AT 250 WORDS)