RESUMEN
The substrate recognition region of tissue factor contains two residues, Lys165 and Lys166, which are important for macromolecular substrate activation by the tissue factor:factor VIIa complex. Replacement of these two residues with alanine in a soluble version of human tissue factor resulted in a mutant, hTFAA, which can bind factor VIIa but forms an enzymatically inactive complex. We found that hTFAA inhibits the activity of guinea pig factor VIIa, allowing us to evaluate hTFAA's effects on thrombosis and hemostasis in a guinea pig model of recurrent arterial thrombosis. In addition to heparin, the effects of hTFAA were compared to active site inhibited factor IXa (F.IXai) and factor Xa (F.Xai). We found that hTFAA, F.IXai and F.Xai were potent antithrombotics and may possess a decreased risk of hemorrhage when compared to unfractionated heparin. When administered at a dose that inhibited thrombosis by about 90%, hTFAA neither affected cuticle bleeding nor the activated partial thromboplastin time, and had only a modest effect on the prothrombin time. At equi-efficacious doses, F.IXai, F.Xai and heparin prolonged bleeding times by 20% (p >0.5), 50% (p <0.05) and 100% (p <0.01), respectively. In summary, our study demonstrates that, unlike heparin, specific inhibitors of factors VIIa, IXa and Xa can produce antithrombotic effects without or with only minimally disturbing normal hemostasis. The results further suggest that factor VIIa and factor IXa are especially promising targets for antithrombotic drug development.
Asunto(s)
Tromboplastina/genética , Trombosis/prevención & control , Sustitución de Aminoácidos , Animales , Arteriopatías Oclusivas/tratamiento farmacológico , Arteriopatías Oclusivas/etiología , Arteriopatías Oclusivas/prevención & control , Tiempo de Sangría , Trombosis de las Arterias Carótidas/tratamiento farmacológico , Trombosis de las Arterias Carótidas/etiología , Trombosis de las Arterias Carótidas/prevención & control , Dominio Catalítico , Modelos Animales de Enfermedad , Factor IXa/antagonistas & inhibidores , Factor IXa/farmacología , Factor VIIa/efectos de los fármacos , Factor VIIa/metabolismo , Factor Xa/farmacología , Inhibidores del Factor Xa , Fibrinolíticos/administración & dosificación , Fibrinolíticos/farmacología , Cobayas , Heparina/administración & dosificación , Heparina/farmacología , Humanos , Solubilidad , Tromboplastina/administración & dosificación , Tromboplastina/farmacología , Trombosis/tratamiento farmacológico , Trombosis/etiologíaRESUMEN
10C12, a human antibody F(ab')2, which specifically binds to the Gla domain of factor IX, interfered with all known coagulation processes that involve factor IX/IXa. These include the function of the intrinsic Xase complex and the activation of zymogen factor IX by factor XIa and by the tissue factor:factor VIla complex. Furthermore, 10C12 potently inhibited activated partial thromboplastin clotting times (APTT) in plasma of guinea pig and rat, thus enabling in-vivo evaluation. In guinea pigs, a bolus administration of 10C12 (10 microg/kg) prevented cyclic flow variations in damaged carotid arteries without affecting coagulation or bleeding parameters. At a 100-fold higher dose, 10C12 had no effect on normal hemostasis as assessed by the cuticle bleeding time. At this dose, 10C12 was also efficacious in a rat arterial thrombosis model, substantially reducing clot weight and duration of vessel occlusion while prolonging ex-vivo APTT only 1.2-fold. The dose of heparin required to produce comparable antithrombotic effects prolonged the APTT by 12-fold and increased the tail bleeding time (TBT) by 8-fold. In contrast, 10C12 had no effect on TBT. However, rat tails showed a tendency for rebleeding which 10C12 exacerbated. In conclusion, the antithrombotic potency of the 10C12 antibody in two species provides evidence for an important role of F.IX, and its Gla domain in particular, during thrombogenesis under arterial flow conditions. The relative safety at effective doses of this fully human antibody suggests that it may have therapeutic value for treatment of thrombotic disorders.
Asunto(s)
Ácido 1-Carboxiglutámico/inmunología , Factor IX/inmunología , Factor IX/metabolismo , Fibrinolíticos/metabolismo , Fibrinolíticos/farmacología , Fragmentos Fab de Inmunoglobulinas/metabolismo , Fragmentos Fab de Inmunoglobulinas/farmacología , Animales , Sitios de Unión , Coagulación Sanguínea/efectos de los fármacos , Modelos Animales de Enfermedad , Factor IX/antagonistas & inhibidores , Cobayas , Hemostasis/efectos de los fármacos , Heparina/farmacología , Humanos , Masculino , Ratas , Ratas Sprague-Dawley , Trombosis/sangre , Trombosis/terapiaRESUMEN
Ro 44-3888 is a potent and selective antagonist of GP IIb/IIIa. Following I.V. administration to rhesus monkeys, the (mean +/- SD.) clearance, volume of distribution and terminal half-life of Ro 44-3888 were 4.4 +/- 1.8 ml/min/kg, 0.8 +/- 0.4 l/kg and 2.5 +/- 0.8 h respectively. Oral administration of Ro 48-3657 (1 mg/kg), a doubly protected prodrug form, produced peak concentrations of Ro 44-3888 (152 +/- 51 ng/ml), 4.2 +/- 2.2 h after dosing. Terminal half-life and estimated bioavailability were 5.1 +/- 1.6 h and 33 +/- 6% respectively. No effect on blood pressure, heart rate or platelet counts were seen. Adenosine diphosohate (ADP) induced platelet aggregation (PA) and cutaneous bleeding times (CBT) were determined prior to and after the last of 8 daily oral administrations of Ro 48-3657 (0.25 or 0.5 mg/kg) to eight rhesus monkeys. Peak and trough plasma concentrations were proportional to dose and steady state was achieved after the second administration. Inhibition of PA and prolongation of CBT were concentration dependent. The ex vivo IC50 (82 nM) for ADP-mediated PA correlated with a value (58 nM) determined in vitro. The CBT response curve was displaced to the right of the PA curve. CBT was prolonged to > or = 25 min when levels of Ro 44-3888 exceeded 190 nM and PA was > 90% inhibited. Therefore, in rhesus monkeys, Ro 48-3657 is reproducibly absorbed and converted to its active form, is well tolerated, and has a concentration-dependent effect on PA and CBT. These properties make Ro 48-3657 an attractive candidate for evaluation in patients at high risk for arterial thrombosis.
Asunto(s)
Amidinas/farmacología , Piperidinas/farmacología , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/antagonistas & inhibidores , Profármacos/farmacocinética , Administración Oral , Amidinas/efectos adversos , Amidinas/farmacocinética , Animales , Esquema de Medicación , Pruebas Hematológicas , Macaca mulatta , Oximas/farmacología , Piperidinas/efectos adversos , Piperidinas/farmacocinética , Profármacos/efectos adversos , Profármacos/farmacologíaRESUMEN
One approach to developing safer and more efficacious agents for the treatment of thrombotic disease involves the design and testing of inhibitors that block specific steps in the coagulation cascade. We describe here the development of a mutant of human tissue factor (TF) as a specific antagonist of the extrinsic pathway of blood coagulation and the testing of this mutant in a rabbit model of arterial thrombosis. Alanine substitutions of Lys residues 165 and 166 in human TF have been shown previously to diminish the cofactor function of TF in support of factor X (FX) activation catalyzed by factor VIIa (FVIIa). The K165A:K166A mutations have been incorporated into soluble TF (sTF; residues 1-219) to generate the molecule "hTFAA." hTFAA binds FVIIa with kinetics and affinity equivalent to wild-type sTF, but the hTFAA x FVIIa complex shows a 34-fold reduction in catalytic efficiency for FX activation relative to the activity measured for sTF x FVIIa. hTFAA inhibits the activation of FX catalyzed by the complex formed between FVIIa and relipidated TF(1-243). hTFAA prolongs prothrombin time (PT) determined with human plasma and relipidated TF(1-243) or membrane bound TF, and has no effect on activated partial thromboplastin time, but is 70-fold less potent as an inhibitor of PT with rabbit plasma. The rabbit homologue of this mutant ("rTFAA") was produced and shown to have greater potency with rabbit plasma. Both hTFAA and rTFAA display an antithrombotic effect in a rabbit model of arterial thrombosis with rTFAA giving full efficacy at a lower dose than hTFAA. Compared to heparin doses of equal antithrombotic potential, hTFAA and rTFAA cause less bleeding as judged by measurements of the cuticle bleeding time. These results indicate that TF x FVIIa is a good target for the development of new anticoagulant drugs for the treatment of thrombotic disease.
Asunto(s)
Anticoagulantes/farmacología , Fibrinolíticos/farmacología , Tiempo de Tromboplastina Parcial , Tiempo de Protrombina , Tromboplastina/farmacología , Animales , Encéfalo/metabolismo , Arterias Carótidas/efectos de los fármacos , Arterias Carótidas/patología , Línea Celular , Clonación Molecular , Coagulantes/farmacología , Cartilla de ADN , Escherichia coli , Factor VIIa/metabolismo , Heparina/farmacología , Humanos , Cinética , Músculo Liso Vascular/efectos de los fármacos , Músculo Liso Vascular/patología , Mutagénesis Sitio-Dirigida , Reacción en Cadena de la Polimerasa , Conejos , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/farmacología , Solubilidad , Tromboplastina/biosíntesis , Tromboplastina/aislamiento & purificaciónRESUMEN
The potent and selective GP IIb-IIIa antagonist lamifiban (1, Ro 44-9883) is currently in clinical development as an injectable antithrombotic agent for treating and preventing acute coronary syndromes. However, for secondary prevention of thrombotic occlusions, orally active inhibitors are needed. By means of a prodrug strategy, the modest oral absorption of 1 in mice was improved by a factor of 9. In addition, these studies demonstrated that an amidoxime group can serve as a prodrug functionality for an amidino group. Application of this principle to the structurally related amidino carboxylate 13 led to the amidoxime ester 18 which was absorbed approximately 20 times better, after oral administration to mice, than 13. Due to the modification of the amidino group as well as of the carboxylate group, 18 completely lost its ability to interact with purified platelet GP IIb-IIIa. After oral administration of 18 to rats, dogs, and rhesus monkeys, the bioavailability of the active derivative 13 was 26 +/- 5, 25 +/- 6, and 33 +/- 6%, respectively, and the elimination half-life was 4.1 +/- 1.7, 11.4 +/- 1.1, and 5.1 +/- 1.4 h, respectively. On the basis of these properties, the orally active 18 (Ro 48-3657), a double prodrug of the potent and selective non-peptide GP IIb-IIIa antagonist 13 (Ro 44-3888), was selected as clinical candidate for evaluation as a prophylactic agent in patients at high risk for arterial thrombosis.
Asunto(s)
Amidinas/farmacología , Oximas/farmacología , Piperidinas/farmacología , Inhibidores de Agregación Plaquetaria/farmacología , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/antagonistas & inhibidores , Profármacos/farmacología , Acetatos/farmacología , Administración Oral , Amidinas/química , Amidinas/farmacocinética , Animales , Anticoagulantes/administración & dosificación , Anticoagulantes/síntesis química , Anticoagulantes/farmacocinética , Anticoagulantes/farmacología , Perros , Macaca mulatta , Espectroscopía de Resonancia Magnética , Espectrometría de Masas , Ratones , Estructura Molecular , Oximas/química , Oximas/farmacocinética , Piperidinas/química , Piperidinas/farmacocinética , Agregación Plaquetaria/efectos de los fármacos , Inhibidores de Agregación Plaquetaria/química , Inhibidores de Agregación Plaquetaria/farmacocinética , Profármacos/administración & dosificación , Profármacos/síntesis química , Profármacos/farmacocinética , Ratas , Tirosina/análogos & derivados , Tirosina/farmacologíaRESUMEN
Clinical experience suggests that thrombolytic-induced bleeding is associated with systemic activation of the thrombolytic system. Using fibrin specific variants of tissue-type plasminogen activator (t-PA) and making use of the apparent fibrin specificity of streptokinase (SK) in the rabbit we tested the hypothesis that minimizing systemic plasmin production and fibrinogenolysis will decrease hemorrhages in models of peripheral bleeding and embolic stroke. t-PA consumed 51% of the available fibrinogen; caused cerebral bleeds and increased peripheral bleeding time. Fibrin-specific variants of t-PA depleted less than 20% of the fibrinogen and did not cause peripheral or cerebral bleeding. However, an equipotent dose of SK converted only 12% of the available fibrinogen but increased bleeding time and caused hemorrhagic conversion in 75% of embolic stroke model animals treated. The data suggest that bleeding associated with tissue-type plasminogen activators is linked to systemic plasmin generation and subsequent fibrinogenolysis. This hypothesis does not explain the mechanism(s) of SK-induced bleeding.
Asunto(s)
Hemorragia/sangre , Plasminógeno/metabolismo , Estreptoquinasa/uso terapéutico , Activador de Tejido Plasminógeno/sangre , Animales , Tiempo de Sangría , Fibrina/metabolismo , Hemorragia/prevención & control , Conejos , Estreptoquinasa/metabolismoRESUMEN
Multiple clinical trials have proven that thrombolytic therapy is an effective treatment for acute myocardial infarction. Spontaneous intracranial hemorrhage (ICH) occurs in a small percentage of patients as a result of the treatment. The etiology of the ICH is unknown and there is currently no established experimental model for this side effect. A model of ICH during thrombolytic therapy has been developed using spontaneously hypertensive rats (SHR). The SHR were made susceptible to ICH during thrombolytic therapy by bilateral ligation of the external jugular veins. This procedure produced asymptomatic hemorrhagic lesions in the brains of the animals in the hours preceding the administration of t-PA/heparin. The incidence of ICH following the administration of test substances was assessed by histological examination and by measuring the red blood cell count in a sample of cerebrospinal fluid taken from the atlanto-occipital space. t-PA administration produced a low frequency of ICH in this model. The incidence and severity of ICH were dramatically increased, and significant mortality at 24h was observed, by combining heparin were administered sequentially rather than simultaneously. Furthermore, ICHs were observed whether the t-PA dose was administered over 4 h, 1 h, or as a double bolus 30 min apart. The potentiation of ICH by heparin was dose dependent and proportional to the prolongation of the aPTT. Although the precise mechanism of ICH during thrombolytic therapy is unknown, many similarities exist between the observations made in this model and in the human clinical experience.
Asunto(s)
Hemorragia Cerebral , Modelos Animales de Enfermedad , Fibrinolíticos/administración & dosificación , Hipertensión/complicaciones , Estreptoquinasa/administración & dosificación , Activador de Tejido Plasminógeno/administración & dosificación , Animales , Antitrombina III/administración & dosificación , Hemorragia Cerebral/etiología , Interacciones Farmacológicas , Heparina/farmacología , Hirudinas/administración & dosificación , Humanos , Masculino , Ratas , Ratas Endogámicas SHRRESUMEN
BACKGROUND: The thrombolytic properties of a new variant of tissue plasminogen activator (TPA) (T103N, N117Q, KHRR 296-299 AAAA, or TNK-TPA) with longer plasma half-life, greater fibrin specificity, and increased resistance to inhibition by plasminogen activator inhibitor (PAI-1) were investigated in a rabbit thrombosed carotid artery model. METHODS AND RESULTS: After 60 minutes of arterial occlusion, TPA (1.5, 3.0, 6.0, or 9.0 mg/kg as a front-loaded IV infusion for 90 minutes; n = 22) or TNK-TPA (0.38, 0.75, or 1.5 mg/kg as IV bolus; n = 16) was administered. Blood flow through the artery was monitored for an additional 120 minutes. Bleeding was assessed by weighing the amount of blood absorbed in a gauze pad placed in a subcutaneous muscular incision. Recanalization rates and duration of recanalization were dose dependent. The doses that produced > 80% recanalization rates with the longest duration of recanalization were 9.0 mg/kg for TPA and 1.5 mg/kg for TNK-TPA. At these doses, time to reperfusion (mean +/- SEM) was significantly faster (11 +/- 2 versus 23 +/- 7 minutes) and duration of recanalization longer (77 +/- 9 versus 51 +/- 18 minutes) for TNK-TPA compared with TPA (P < .025). Weights of the residual thrombi of the TPA group were greater than those of the TNK-TPA group (P = .004). Concentrations of fibrinogen, plasminogen, and alpha 2-antiplasmin at 120 minutes were significantly higher for TNK-TPA-treated animals compared with TPA-treated animals (P < .001). ANOVA of the blood loss data determined that there were significant differences between thrombolytic agents but not between doses. After correction for saline controls, total blood loss for pooled doses of TPA and TNK-TPA was 82 +/- 6 mg and 40 +/- 4 mg, respectively (P < .01). CONCLUSIONS: From these data, we conclude that TNK-TPA, given as a bolus, produces faster and more complete recanalization of occluded arteries in a rabbit experimental model compared with TPA, without increasing systemic plasmin generation or peripheral bleeding. In addition, we observed that TNK-TPA, unlike TPA, did not potentiate collagen-induced aggregation of platelets obtained from human plasma. This lack of effect on platelet aggregation by TNK-TPA potentially could be associated with a decreased risk of reocclusion after successful thrombolysis.
Asunto(s)
Trombosis de las Arterias Carótidas/tratamiento farmacológico , Fibrinolíticos/uso terapéutico , Activador de Tejido Plasminógeno/uso terapéutico , Animales , Fibrinolíticos/efectos adversos , Fibrinolíticos/química , Hemorragia/inducido químicamente , Humanos , Kringles , Masculino , Datos de Secuencia Molecular , Agregación Plaquetaria/efectos de los fármacos , Conejos , Activador de Tejido Plasminógeno/efectos adversos , Activador de Tejido Plasminógeno/químicaRESUMEN
BACKGROUND AND PURPOSE: We compared the activity of a new long-half-life, fibrin-specific tissue-type plasminogen activator (TPA) variant with that of wild-type TPA in rabbit models of embolic stroke and peripheral bleeding. METHODS: In the embolic stroke model. TPA-induced clot lysis is followed by continuous monitoring of a radiolabeled clot lodged in the middle cerebral artery. Twenty-four hours after embolization and treatment with either thrombolytic agent or excipient, the brains are removed, fixed, and evaluated for cerebral hemorrhage. In a parallel template bleeding time experiment, the effects of equipotent doses of the two TPA molecules were measured. RESULTS: Infusion of wild-type TPA or bolus administration of the TPA variant resulted in dose-dependent clot lysis. The TPA variant was found to be an order of magnitude more potent than wild-type TPA on a milligram-per-kilogram basis. Unlike wild-type TPA, the variant caused less systemic activation of plasminogen (P < .05) and fewer hemorrhagic transformations in this model (P < .05). The TPA variant did not extend template bleeding times. CONCLUSIONS: These findings show that by combining increased fibrin specificity with decreased plasma clearance, it is possible to produce a thrombolytic agent that is more convenient and more potent than wild-tpe TPA. At the same time the significant reduction in hemorrhagic conversions may be attributable to the conservation of systemic plasminogen seen with this molecule.
Asunto(s)
Trastornos Cerebrovasculares/tratamiento farmacológico , Fibrina/metabolismo , Hemorragia/inducido químicamente , Embolia y Trombosis Intracraneal/tratamiento farmacológico , Activador de Tejido Plasminógeno/uso terapéutico , Animales , Coagulación Sanguínea/efectos de los fármacos , Hemorragia Cerebral/inducido químicamente , Hemorragia Cerebral/patología , Relación Dosis-Respuesta a Droga , Fibrinógeno/análisis , Fibrinólisis/efectos de los fármacos , Semivida , Embolia y Trombosis Intracraneal/patología , Masculino , Plasminógeno/análisis , Conejos , Terapia Trombolítica , Activador de Tejido Plasminógeno/administración & dosificación , Activador de Tejido Plasminógeno/efectos adversos , Activador de Tejido Plasminógeno/sangre , alfa 2-Antiplasmina/análisisRESUMEN
Current treatment with tissue plasminogen activator (tPA) requires an intravenous infusion (1.5-3 h) because the clearance of tPA from the circulation is rapid (t 1/2 approximately 6 min). We have developed a tPA variant, T103N,N117Q, KHRR(296-299)AAAA (TNK-tPA) that has substantially slower in vivo clearance (1.9 vs. 16.1 ml per min per kg for tPA in rabbits) and near-normal fibrin binding and plasma clot lysis activity (87% and 82% compared with wild-type tPA). TNK-tPA exhibits 80-fold higher resistance to plasminogen activator inhibitor 1 than tPA and 14-fold enhanced relative fibrin specificity. In vitro, TNK-tPA is 10-fold more effective at conserving fibrinogen in plasma compared to tPA. Arterial venous shunt models of fibrinolysis in rabbits indicate that TNK-tPA (by bolus) induces 50% lysis in one-third the time required by tPA (by infusion). TNK-tPA is 8- and 13-fold more potent in rabbits than tPA toward whole blood clots and platelet-enriched clots, respectively. TNK-tPA conserves fibrinogen and, because of its slower clearance and normal clot lysis activity, is effective as a thrombolytic agent when given as a bolus at a relatively low dose.
Asunto(s)
Fibrinolíticos/química , Activador de Tejido Plasminógeno/química , Animales , Fibrina/metabolismo , Fibrinógeno/metabolismo , Fibrinólisis , Fibrinolíticos/metabolismo , Fibrinolíticos/farmacocinética , Humanos , Cinética , Mutagénesis Sitio-Dirigida , Conejos , Relación Estructura-Actividad , Activador de Tejido Plasminógeno/antagonistas & inhibidores , Activador de Tejido Plasminógeno/genética , Activador de Tejido Plasminógeno/farmacocinéticaRESUMEN
Site directed mutagenesis was used to construct a t-PA variant that contains an additional glycosylation site in the first kringle domain (T103N) combined with a tetra-alanine substitution in the protease domain (KHRR 296-299 AAAA). This combination variant has a plasma clearance rate that is 4.5-fold slower in rats and 5.4-fold slower in rabbits than t-PA. It is also less than one tenth as active as t-PA towards plasminogen in the presence of fibrinogen, and has approximately twice the normal activity in the presence of fibrin. It shows substantial resistance to the fast acting inhibitor, plasminogen activator inhibitor-1 (PAI-1), requiring a 10-fold greater molar excess of PAI-1 to reduce its activity by 50%, compared to t-PA. This is the result of a reduction of nearly 100-fold in the second order rate constant for PAI-1 inactivation. These results show that it is possible to combine mutations in different domains of t-PA to construct a variant which is simultaneously slower clearing, less reactive towards plasminogen in the absence of a fibrin clot, and resistant to inactivation by PAI-1.
Asunto(s)
Inhibidor 1 de Activador Plasminogénico/farmacología , Activador de Tejido Plasminógeno/farmacocinética , Animales , Células CHO , Cricetinae , Fibrina/metabolismo , Tasa de Depuración Metabólica , Mutagénesis Sitio-Dirigida , Inhibidor 1 de Activador Plasminogénico/metabolismo , Estructura Terciaria de Proteína , Conejos , Ratas , Especificidad por Sustrato , Activador de Tejido Plasminógeno/antagonistas & inhibidores , Activador de Tejido Plasminógeno/metabolismoRESUMEN
In the accompanying paper, we reported that the properties of decreased plasma clearance rate, increased fibrin specificity, and resistance to inactivation by PAI-1 could be effectively combined in the t-PA variant T103N, KHRR 296-299 AAAA. In the current study we evaluated the in vivo efficacy of this variant as well as variants containing the individual mutations T103N and KHRR 296-299 AAAA. Plasma clearance and in vivo lysis of whole blood and platelet-rich clots were determined in a rabbit arterio-venous shunt model. The T103N containing variants were administered as an intravenous (i.v.) bolus. KHRR 296-299 AAAA and t-PA were infused i.v. over 90 min. The clearance rate of the KHRR 296-299 AAAA variant was similar to t-PA. However, the clearance of the T103N and T103N, KHRR 296-299 AAAA variants were 8 and 6-fold reduced, respectively. Potency of the variants relative to t-PA on whole blood clots ranged from 0.9 (T103N, KHRR 296-299 AAAA) to 1.7 (T103N). Relative potency on platelet-rich clots ranged from 2.4 (T103N) to 4.2 (T103N, KHRR 296-299 AAAA). Fibrinogen concentrations in rabbits 120 min after dosing with a 2.5 mg/kg bolus were: 24, 16, 82, and 77% of initial for t-PA; T103N; KHRR 296-299 AAAA; and T103N, KHRR 296-299 AAAA treatment groups, respectively. These results suggest that the T103N, KHRR 296-299 AAAA variant of t-PA, given as a bolus, could result in greater efficacy, particularly on refractory platelet-rich clots, without inducing the severe systemic lytic state produced by a bolus of a less fibrin specific variant.
Asunto(s)
Fibrinolíticos/uso terapéutico , Terapia Trombolítica , Activador de Tejido Plasminógeno/farmacología , Animales , Derivación Arteriovenosa Quirúrgica , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Inyecciones Intravenosas , Tasa de Depuración Metabólica , Agregación Plaquetaria , Conejos , Proteínas Recombinantes/farmacología , Activador de Tejido Plasminógeno/administración & dosificación , Activador de Tejido Plasminógeno/farmacocinéticaRESUMEN
The dose-dependent effects of tissue-type plasminogen activator (t-PA) on the kinetics of cerebral clot lysis in a rabbit model of middle cerebral artery embolic stroke were investigated. The clots were formed in vitro and tagged with 99Tc for gamma-scintigraphic imaging. After embolization, groups of animals were treated with t-PA. Dose-response curves for the t-PA were generated, and in addition, long and short dosing schedules were assessed. The optimal doses for frequency and rate of cerebral clot lysis in this model are approximately 6.3 mg/kg given over 2 hr or 3.3 mg/kg given over 30 min. These dosing regimens for t-PA were accompanied by approximately 50% consumption of plasma plasminogen, fibrinogen and alpha 2-antiplasmin. Doses of t-PA on either side of this optimum caused attenuation in both the frequency and rate of cerebral clot lysis. Treatment with t-PA under either dosing regimen did not augment the frequency of hemorrhagic transformation, but the size of the resultant hemorrhage in those animals where intracranial bleeding occurred was reduced by 3.3 mg/kg t-PA given over 30 min.
Asunto(s)
Embolia y Trombosis Intracraneal/tratamiento farmacológico , Terapia Trombolítica , Activador de Tejido Plasminógeno/uso terapéutico , Animales , Arterias Cerebrales , Hemorragia Cerebral/tratamiento farmacológico , Hemorragia Cerebral/etiología , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Ensayo de Inmunoadsorción Enzimática , Embolia y Trombosis Intracraneal/complicaciones , Masculino , Conejos , Azufre Coloidal Tecnecio Tc 99mRESUMEN
The tetra-alanine substitution variant KHRR 296-299 AAAA of tissue-type plasminogen activator (t-PA) was previously shown to have enhanced fibrin specificity and enhanced activity in the presence of fibrin compared with the wild-type form of the molecule. The structural requirements for these alterations in enzymatic activity were investigated by constructing several amino acid substitution variants at each of the positions from 296 to 299 and evaluating their activities under a variety of conditions. Effects on plasminogen activator activity were common among the point mutants at positions 296-299; nearly all had a phenotype similar to the KHRR 296-299 AAAA variant. The greatest effects on enzymatic function were found with multiple substitution variants, but some single charge reversals and proline substitutions had substantial effects. The enhanced fibrin specificity of KHRR 296-299 AAAA t-PA results in less fibrinogenolysis than seen with wild-type t-PA. Approximately four times greater concentration of KHRR 296-299 AAAA compared with wild-type t-PA was required to consume 50% of the fibrinogen in human plasma.
Asunto(s)
Alanina/química , Activador de Tejido Plasminógeno/metabolismo , Secuencia de Aminoácidos , Fibrina/metabolismo , Fibrinógeno/metabolismo , Fibrinólisis , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Oligopéptidos/metabolismo , Plasma , Plasminógeno/metabolismo , Ingeniería de Proteínas , Activador de Tejido Plasminógeno/químicaRESUMEN
Modification of the carbohydrate structures of recombinant tissue-type plasminogen activator (rt-PA) can increase or decrease its rate of clearance in rabbits. When rt-PA was treated with sodium periodate to oxidize carbohydrate residues, the rate of clearance was decreased from 9.6 +/- 1.9 ml min-1 kg-1 to 3.5 +/- 0.6 ml min-1 kg-1 (mean +/- SD, n = 5). A similar change in the clearance of rt-PA was introduced by the use of endo-beta-N-acetyl-glucosaminidase H (Endo-H), which selectively removes high mannose asparagine-linked oligosaccharides; the clearance of Endo-H-treated rt-PA was 5.0 +/- 0.5 ml min-1 kg-1. A mutant of rt-PA was produced with an amino acid substitution at position 117 (Asn replaced with Gln) to remove a potential glycosylation site that normally contains a high mannose structure. The clearance of this material was also decreased, similar to the periodate and Endo-H-treated rt-PA. Conversely, when rt-PA was produced in the CHO 15B cell line, which can produce only high mannose oligosaccharide structures on glycoproteins, the clearance was increased by a factor of 1.8. These results demonstrate that the removal of rt-PA from the blood depends significantly upon the nature of its oligosaccharide structures.
Asunto(s)
Carbohidratos , Activador de Tejido Plasminógeno/farmacocinética , Animales , Células Cultivadas , Cricetinae , Glutamina/análisis , Manosa/análisis , Mutación , Oligosacáridos/análisis , Ácido Peryódico/farmacología , Conejos , Proteínas Recombinantes/farmacocinéticaRESUMEN
In vitro artifacts due to proteolysis may occur in blood samples containing recombinant tissue-type plasminogen activator (rt-PA) due to continued activation of plasminogen to plasmin by rt-PA. The aim of this study was to identify a rapid inhibitor of rt-PA that would not interfere in assays designed to monitor thrombolytic events. When rt-PA was added at 5 micrograms/ml to whole blood and incubated at 25 degrees C, fibrinogen decreased 50 percent, plasminogen levels decreased 90 percent and alpha 2-antiplasmin decreased below detectable levels. If D-Phe-Pro-Arg-chloromethylketone (PPACK) or aprotinin were added before the addition of rt-PA there was no significant loss of fibrinogen. Only PPACK completely inhibited changes in fibrin degradation products, plasminogen and alpha 2-antiplasmin. PPACK was also found to inhibit the binding of rt-PA to plasma protease inhibitors in vitro. Rhesus monkeys were infused with rt-PA and blood samples were taken with either PPACK or aprotinin in the collection syringe. There was a significant increase in the recovery of immunoreactive rt-PA and consistent measures of fibrinogen, FDPs, plasminogen, and alpha 2-antiplasmin in the PPACK group as compared to the aprotinin group which indicates that PPACK will prevent the in vitro formation of artifacts due to the presence of active rt-PA.
Asunto(s)
Clorometilcetonas de Aminoácidos/farmacología , Recolección de Muestras de Sangre , Fibrinolíticos , Proteínas Recombinantes/uso terapéutico , Trombina/antagonistas & inhibidores , Activador de Tejido Plasminógeno/uso terapéutico , Clorometilcetonas de Aminoácidos/uso terapéutico , Aprotinina/farmacología , Activación Enzimática , Fibrinógeno/metabolismo , Fibrinolisina/metabolismo , Humanos , Cinética , Plasminógeno/metabolismoRESUMEN
The effect of iron deficiency on work capacity was studied in groups of rats that had received diets with iron contents ranging between 9 and 50 mg/kg diet from 3 to 6 wk of age. Maximal O2 consumption (VO2max) declined only 16% with a decrease in hemoglobin (Hb) from 14 to 8 g/dl and fell sharply only below a Hb of 7 g/dl. Duration until exhaustion in a treadmill exercise of submaximal intensity (endurance) showed no significant depression between a Hb of 14 and 10 g/dl. However, endurance declined abruptly by 73% between a Hb of 10 and 8 g/dl. The VO2max results are in accord with known compensatory mechanisms that help to maintain delivery of O2 to tissues until anemia becomes severe. The sharp fall in endurance with relatively mild iron deficiency suggests a lack of similarly effective compensations for decreased oxidative capacity of muscle.
Asunto(s)
Anemia Hipocrómica/fisiopatología , Esfuerzo Físico , Anemia Hipocrómica/metabolismo , Animales , Grupo Citocromo c/metabolismo , Dieta , Hemoglobinas/análisis , Hierro/administración & dosificación , Lactatos/sangre , Ácido Láctico , Masculino , Contracción Muscular , Músculos/metabolismo , Consumo de Oxígeno , Resistencia Física , Ratas , Ratas EndogámicasRESUMEN
The concentration of plasma erythropoietin was determined by radioimmunoassay during the progression of and subsequent recovery from iron-deficiency anemia in the rat. During the development of anemia, the plasma erythropoietin level rose as the hemoglobin (Hgb) concentration declined, reaching maximal levels when the Hgb was lowest. During the recovery from iron-deficiency anemia after institution of the control diet, the plasma erythropoietin concentration rapidly declined to baseline or below baseline levels even before the Hgb had completely returned to control values. This early fall in the erythropoietin level was associated with a sustained decrease in blood oxygen affinity (increase in P50). The rise in P50 was associated with an increase in the number of circulating reticulocytes in addition to and independently of an increase in the concentration of 2,3-diphosphoglycerate (DPG) in red cells. Therefore, reticulocytosis may play a part in the recovery from anemia, not only by replenishing the red cell pool but also by temporarily facilitating oxygen delivery to the tissues.
Asunto(s)
Anemia Hipocrómica/fisiopatología , Eritropoyetina/sangre , Anemia Hipocrómica/sangre , Animales , Dieta , Ácidos Difosfoglicéricos/análisis , Hemoglobinas/análisis , Masculino , Ratas , Ratas EndogámicasRESUMEN
Red blood cells (RBC) from iron-deficient rats were found to generate more malonyldialdehyde after in vitro incubation with H2O2 than RBC from control rats (p less than 0.001). The iron-deficient RBC, however, had a higher content of superoxide dismutase (SOD) than control RBC (p less than 0.02). This finding suggests an increased formation of SOD compensatory to an increased oxidant stress.
Asunto(s)
Anemia Hipocrómica/enzimología , Eritrocitos/enzimología , Superóxido Dismutasa/sangre , Anemia Hipocrómica/sangre , Animales , Recuento de Eritrocitos , Hemoglobinas/análisis , Hierro/sangre , Masculino , Ratas , Ratas Endogámicas , Reticulocitos/citologíaRESUMEN
Three weeks of dietary iron deficiency in weanling rats resulted in anemia (Hb, 3.9 vs. 14.2 g/dl in controls) and decreased oxidative capacities of skeletal muscle (as much as 90% below control values). Whole-animal maximal O2 consumption (VO2max), measured in a brief treadmill run of progressively increasing work load, was approximately 50% lower for iron-deficient rats than for controls, and maximal endurance capacity (time to exhaustion in a separate treadmill run at a constant, sub-Vo2max work load) was 90% lower for iron-deficient rats than for controls. Exchange transfusion, with packed erythrocytes or plasma, was used to adjust Hb to an intermediate concentration of approximately 9.5 g/dl in both iron-deficient and and control rats. This procedure corrected the Vo2max of iron-deficient rats to within 15% of control values, whereas endurance capacity showed no improvement. Our experimental dissociation of Vo2max and endurance capacity provides further evidence that Vo2max is not the sole determinant of endurance. We propose that defects in Vo2max during iron deficiency result primarily from diminished O2 delivery, whereas decreased endurance capacity reflects impaired muscle mitochondrial function.