RESUMEN
Protein S functions as an activated protein C (APC)-independent anticoagulant in the inhibition of intrinsic factor X activation, although the precise mechanisms remain to be fully investigated. In the present study, protein S diminished factor VIIIa/factor IXa-dependent factor X activation, independent of APC, in a functional Xa generation assay. The presence of protein S resulted in an c. 17-fold increase in K(m) for factor IXa with factor VIIIa in the factor Xase complex, but an c. twofold decrease in K(m) for factor X. Surface plasmon resonance-based assays showed that factor VIII, particularly the A2 and A3 domains, bound to immobilized protein S (K(d); c. 10 nmol/l). Competition binding assays using Glu-Gly-Arg-active-site modified factor IXa showed that factor IXa inhibited the reaction between protein S and both the A2 and A3 domains. Furthermore, Sodium dodecyl sulphate polyacrylamide gel electrophoresis revealed that the cleavage rate of factor VIIIa at Arg(336) by factor IXa was c. 1.8-fold lower in the presence of protein S than in its absence. These data indicate that protein S not only down-regulates factor VIIIa activity as a cofactor of APC, but also directly impairs the assembly of the factor Xase complex, independent of APC, in a competitive interaction between factor IXa and factor VIIIa.
Asunto(s)
Cisteína Endopeptidasas/metabolismo , Regulación hacia Abajo/efectos de los fármacos , Factor VIIIa/metabolismo , Proteínas de Neoplasias/metabolismo , Proteína S/farmacología , Unión Competitiva , Relación Dosis-Respuesta a Droga , Ensayo de Inmunoadsorción Enzimática/métodos , Factor IXa/metabolismo , Factor IXa/farmacología , Factor X/metabolismo , Factor X/farmacología , Humanos , Fosfolípidos/metabolismo , Proteína C/fisiología , Proteína S/metabolismo , Proteínas Recombinantes/metabolismo , Factor de von Willebrand/metabolismoRESUMEN
Factor VIII consists of a heavy chain (A1A2B domains) and light chain (A3C1C2 domains), whereas the contiguous A1A2 domains are separate subunits in the cofactor, factor VIIIa. The intrinsic instability of the cofactor results from weak affinity interactions of the A2 subunit within factor VIIIa. The charged residues Glu272, Asp519, Glu665, and Glu1984 appear buried at the interface of the A2 domain with either the A1 or A3 domain, and thus may impact protein stability. To determine the effects of these residues on procofactor/cofactor stability, these residues were individually replaced with either Ala or Val, and stable BHK cell lines expressing the B-domainless proteins were prepared. Specific activity and thrombin generation parameters for 7 of the 8 variants were more than 80% the wild-type value. Factor VIII activity at 52 degrees C to 60 degrees C and the decay of factor VIIIa activity after thrombin activation were monitored. Six of the 7 variants showing wild-type-like activity demonstrated enhanced stability, with the Glu1984Val variant showing a 2-fold increase in thermostability and an approximately 4- to 8-fold increase in stability of factor VIIIa. These results indicate that replacement of buried charged residues is an effective alternative to covalent modification in increasing factor VIII (VIIIa) stability.
Asunto(s)
Sustitución de Aminoácidos , Aminoácidos/metabolismo , Factor VIII/química , Factor VIII/metabolismo , Proteínas Mutantes/metabolismo , Sustitución de Aminoácidos/efectos de los fármacos , Animales , Bioensayo , Western Blotting , Línea Celular , Cricetinae , Electroforesis en Gel de Poliacrilamida , Factor IXa/farmacología , Humanos , Mutación Puntual/efectos de los fármacos , Estructura Terciaria de Proteína , Temperatura , Termodinámica , Trombina/metabolismoAsunto(s)
Factores de Coagulación Sanguínea/efectos adversos , Protrombina/efectos adversos , Tromboembolia/etiología , Animales , Coagulación Sanguínea/efectos de los fármacos , Factores de Coagulación Sanguínea/química , Factor IXa/administración & dosificación , Factor IXa/farmacología , Hemofilia B/tratamiento farmacológico , Técnicas In Vitro , Protrombina/administración & dosificación , ConejosRESUMEN
It has been well documented that there is an uncertainty over the true factor (F)VIII level in postinfusion samples due to assay discrepancies. The thrombin generation test (TGT) was used as a potentially more physiological approach to assess and compare FVIII concentrates. FVIII concentrates were added to artificial FVIII-deficient plasma. Thrombin generation was initiated by the addition of FIXa (14 nm), phospholipid and CaCl2. Thrombin was measured by subsampling into fibrinogen, and curves quantified as area under the curve (AUC) and time taken to half-maximum (t(1/2)max). Addition of one plasma-derived concentrate to as little as 0.005 IU mL-1 gave a normal AUC, but prolonged t(1/2)max. Increasing FVIII to 1 IU mL-1 had little effect on AUC, but did reduce the t(1/2)max to 64 s (normal 114 s). A range of plasma-derived and recombinant concentrates were tested at 1 IU mL-1; results were similar, except the B-domain deleted concentrate, which had the most rapid initial rate of thrombin generation (t(1/2)max 48 s, P < 0.05). Two hemophilic plasmas (< 0.01 IU mL-1) produced large amounts of thrombin (AUC 65% and 69%), although t(1/2)max was prolonged. Addition of a FVIII antibody abolished thrombin generation, indicating that these plasmas contained low levels of FVIII. Decreasing the FIXa concentration (0.2 nm) minimized thrombin generation in hemophilic plasma but not in normal plasma. These results indicate that FVIII < 0.01 IU mL-1 can generate significant quantities of thrombin depending upon the amount of FIXa present. The TGT could prove useful for patient monitoring in gene therapy and prophylaxis.
Asunto(s)
Pruebas de Coagulación Sanguínea/métodos , Factor VIII/análisis , Trombina/biosíntesis , Relación Dosis-Respuesta a Droga , Monitoreo de Drogas/métodos , Factor IXa/metabolismo , Factor IXa/farmacología , Factor VIII/farmacología , Factor VIII/uso terapéutico , Hemofilia A/sangre , Humanos , Modelos Biológicos , Reproducibilidad de los Resultados , Trombina/efectos de los fármacosRESUMEN
Factor IX (FIX) consists of an N-terminal gamma-carboxyglutamic acid (Gla) domain followed by two epidermal growth factor (EGF)-like domains, and the C-terminal serine protease domain. During physiologic coagulation, one of the activators of FIX is the FVIIa/tissue factor (TF) complex. In this reaction, the Gla and EGF1 domains of FIX are thought to interact with TF. The FIXa that is generated then combines with FVIIIa on the platelet surface to activate FX in the coagulation cascade. In this assembly, the protease domain and possibly the EGF2 domain of FIXa are thought to provide the primary specificity in binding to FVIIIa. Disruption of the interaction of FIX/FX with TF and of the FIXa:FVIIIa interface may provide a pharmacologic target as an alternative strategy for the development of antithrombotic agents.
Asunto(s)
Factor IX/química , Factor IX/farmacología , Factor IXa/química , Factor IXa/farmacología , Relación Estructura-Actividad , Coagulación Sanguínea/efectos de los fármacos , Humanos , Transducción de Señal/efectos de los fármacosRESUMEN
The coagulation cascade protease thrombin is a fibroblast mitogen, but the proliferative potential of other coagulation proteases is not known. In this study we show that factor Xa stimulated human fetal lung fibroblast DNA synthesis in a concentration-dependent manner from 1 nM onward with a fourfold increase at 200 nM. The mitogenic effect of factor Xa was confirmed using a colorimetric proliferation assay and direct cell counting. Factor Xa and thrombin had equivalent potencies, and their stimulatory effects followed a similar time course. Comparable results were also obtained with primary human adult fibroblasts derived from lung, kidney, heart, skin, and liver. Factor VIIa also stimulated fibroblast proliferation, but only at concentrations >10 nM, whereas factor IXa had no effect. To begin to address the mechanism by which factor Xa is acting, we show that human fibroblasts express effector-cell protease receptor-1 and that blocking antibodies to this receptor and the catalytic site of factor Xa inhibited its mitogenic effect. Furthermore, factor Xa upregulated platelet-derived growth factor-A (PDGF-A) mRNA expression, whereas PDGF-B could not be detected, and a blocking antibody to PDGF inhibited the mitogenic effect of factor Xa. We conclude that factor Xa acts as a fibroblast mitogen via binding to effector-cell protease receptor-1 and the autocrine release of PDGF.
Asunto(s)
Comunicación Autocrina/fisiología , Factor Xa/fisiología , Fibroblastos/fisiología , Mitógenos/fisiología , Factor de Crecimiento Derivado de Plaquetas/metabolismo , Receptores de Superficie Celular/metabolismo , Adulto , División Celular/efectos de los fármacos , Factor IXa/farmacología , Factor VIIa/farmacología , Factor Xa/metabolismo , Factor Xa/farmacología , Fibroblastos/citología , Humanos , Proteínas Inhibidoras de la Apoptosis , Masculino , Mitógenos/metabolismo , Péptido Hidrolasas/metabolismo , Factor de Crecimiento Derivado de Plaquetas/fisiología , Survivin , Trombina/farmacologíaRESUMEN
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
The effects of a fucoidan (C-II), which was purified from the brown seaweed Ecklonia kurome, on the generation of thrombin and factor Xa have been investigated by measuring the amidolytic activities by using the respective specific chromogenic substrates in both plasma and purified systems. C-II inhibited significantly the generation of thrombin in both the intrinsic and the extrinsic pathways, although the intrinsic inhibitory effect by C-II was more remarkable than the extrinsic one. On the other hand, C-II was a good inhibitor of the factor Xa generation in the intrinsic pathway, while it was a poor one in the extrinsic pathway. In the purified systems C-II also inhibited the formation of prothrombin-activating complex (i.e., prothrombinase), but not its activity. The concentration of C-II required for 50% inhibition of thrombin generation was about one-tenth to one-seventh of that of the activity of the generated thrombin in plasma. These results indicate that C-II has an inhibitory effect on the generation of thrombin by blocking the formation of prothrombinase and by preventing the generation of intrinsic factor Xa in addition to its antithrombin activity, and also that the generation-inhibitory effect is more remarkable than C-II's enhancement effect on the antithrombin activity by heparin cofactor II in plasma.
Asunto(s)
Inhibidores del Factor Xa , Factor Xa/biosíntesis , Polisacáridos/farmacología , Trombina/antagonistas & inhibidores , Trombina/biosíntesis , Animales , Anticoagulantes/farmacología , Dermatán Sulfato/farmacología , Endopeptidasas/farmacología , Factor IXa/farmacología , Factor X/metabolismo , Factor Xa/efectos de los fármacos , Fucosa/farmacología , Cofactor II de Heparina/farmacología , Humanos , Poliéster Pentosan Sulfúrico/farmacología , Phaeophyceae/química , Protrombina/metabolismo , Daboia , Algas Marinas , Ésteres del Ácido Sulfúrico/farmacología , Trombina/efectos de los fármacos , Tromboplastina/antagonistas & inhibidores , Tromboplastina/biosíntesis , Venenos de Víboras/farmacologíaAsunto(s)
Cisteína Endopeptidasas/farmacología , Lipoproteínas/genética , Músculo Liso Vascular/citología , Músculo Liso Vascular/metabolismo , Animales , Aorta/citología , Factores de Coagulación Sanguínea/farmacología , Movimiento Celular/efectos de los fármacos , Células Cultivadas , Medios de Cultivo , Factor IXa/farmacología , Factor VIIIa/farmacología , Factor 2 de Crecimiento de Fibroblastos/farmacología , Expresión Génica , Masculino , Proteínas de Neoplasias/farmacología , Factor de Crecimiento Derivado de Plaquetas/farmacología , Conejos , Trombina/farmacologíaRESUMEN
The complement protein C4b-binding protein plays an important role in the regulation of the protein C anticoagulant pathway. C4b-binding protein can bind to protein S, thereby inhibiting the cofactor activity of protein S for activated protein C. In this report, we describe a new role for C4b-binding protein in coagulation. We observed inhibition of the intrinsic factor X activating reaction by the complex of C4b-binding protein and protein S. At the plasma concentration of protein S, the factor X activation was inhibited for 50% and addition of C4b-binding protein led to a potentiation of the inhibition to almost 90%. Because C4b-binding protein alone had no effect on the activation of factor X, we hypothesized that binding of C4b-binding protein to protein S was a prerequisite for optimal inhibition of factor X activation. C4b-binding protein lacking the beta-chain, which is unable to bind to protein S, did not potentiate the inhibitory effect of protein S. In an earlier study, we observed that C4b-binding protein increased the binding affinity of protein S for factor VIII. Therefore, a possible interaction of C4b-binding protein with factor VIII was investigated. C4b-binding protein bound to factor VIII and to thrombin activated factor VIII in a saturable and specific way. Also, factor VIII in complex with von Willebrand factor was able to bind C4b-binding protein. The beta-chain of C4b-binding protein was not required for the interaction with factor VIII because C4b-binding protein lacking the beta-chain also bound to factor VIII. Monoclonal antibodies directed against the alpha-chain of C4b-binding protein inhibited the binding to factor VIII, whereas monoclonal antibodies directed against the beta-chain had no effect on the binding to factor VIII. This finding indicates that the binding site for factor VIII on C4b-binding protein is localized on the alpha-chains of C4b-binding protein. The potentiation by C4b-binding protein of the inhibition of the factor X activation by protein S was blocked by a monoclonal antibody directed against the alpha-chain of C4b-binding protein. This finding indicates that the potentiation of the inhibitory effect of protein S was mediated via an interaction of C4b-binding protein with factor VIII. C4b-binding protein did not bind to factor V and was not able to potentiate the inhibitory effect of protein S on prothrombinase activity.(ABSTRACT TRUNCATED AT 400 WORDS)
Asunto(s)
Proteínas Portadoras/farmacología , Proteínas Inactivadoras de Complemento , Factor X/metabolismo , Glicoproteínas , Proteína S/farmacología , Anticuerpos Monoclonales/farmacología , Sitios de Unión , Proteínas Portadoras/química , Proteínas Portadoras/metabolismo , Sinergismo Farmacológico , Factor IXa/farmacología , Factor VIII/metabolismo , Factor VIII/farmacología , Factor Xa/metabolismo , Humanos , Cinética , Proteína S/metabolismo , Factor de von Willebrand/metabolismoRESUMEN
Tissue factor is the major initiator of coagulation. Both factor IX and factor X are activated by the complex of factor VIIa and tissue factor (VIIa/TF). The goal of this study was to determine the specific roles of factors IXa and Xa in initiating coagulation. We used a model system of in vitro coagulation initiated by VIIa/TF and that included unactivated platelets and plasma concentrations of factors II, V, VIII, IX, and X, tissue factor pathway inhibitor, and antithrombin III. In some cases, factor IX and/or factor X were activated by tissue factor-bearing monocytes, but in some experiments, picomolar concentrations of preactivated factor IX or factor X were used to initiate the reactions. Timed samples were assayed for both platelet activation and thrombin activity. Factor Xa was 10 times more potent than factor IXa in initiating platelet activation, but factor IXa was much more effective in promoting thrombin generation than was factor Xa. In the presence of VIIa/TF, factor X was required for both platelet activation and thrombin generation, while factor IX was only required for thrombin generation. We conclude that VIIa/TF-activated factors IXa and Xa have distinct physiologic roles. The main role of factor Xa that is initially activated by VIIa/TF is to activate platelets by generating an initial, small amount of thrombin in the vicinity of platelets. Factor IXa, on the other hand, enhances thrombin generation by providing factor Xa on the platelet surface, leading to prothrombinase formation. Only tiny amounts of factors IX and X need to be activated by VIIa/TF to perform these distinct functions. Our experiments show that initiation of coagulation is highly dependent on activation of small amounts of factors IXa and Xa in proximity to platelet surfaces and that these factors play distinct roles in subsequent events, leading to an explosion of thrombin generation. Furthermore, the specific roles of factors IXa and Xa generated by VIIa/TF are not necessarily reflected by the kinetics of factor IXa and Xa generation.
Asunto(s)
Coagulación Sanguínea , Plaquetas/fisiología , Factor IXa/fisiología , Factor Xa/fisiología , Monocitos/fisiología , Activación Plaquetaria/fisiología , Tromboplastina/fisiología , Plaquetas/efectos de los fármacos , Western Blotting , Separación Celular , Células Cultivadas , Factor IXa/biosíntesis , Factor IXa/farmacología , Factor X/análisis , Factor X/biosíntesis , Factor Xa/farmacología , Humanos , Cinética , Monocitos/citología , Activación Plaquetaria/efectos de los fármacos , Trombina/biosíntesis , Tromboplastina/farmacología , Factores de TiempoRESUMEN
Rates of factor X activation on endothelial cells were compared with activation rates on other vascular cells, platelets, monocytes and negatively charged phospholipid vesicles. Factor VIIa-mediated factor X activation was observed on smooth muscle cells and fibroblasts in the absence of cell-perturbing agents, whereas endothelial cells required activation in order to allow extrinsic activation of factor X. On the other hand, unperturbed endothelial cells did promote intrinsic, factor VIII/IXa-dependent activation of factor X. The rate of factor X activation on these cells was about one-sixth of that on ionophore A23187-stimulated platelets. Also, smooth muscle cells and fibroblasts were able to activate factor X through the intrinsic pathway, although to a lesser extent than endothelial cells. Monocytes were ineffective in this respect. Prothrombin fragment 1, the prothrombin fragment containing the gamma-carboxyglutamic acid domain known to mediate binding of vitamin K-dependent coagulation factors to phospholipid surfaces, inhibited factor VIII/IXa-dependent factor X activation on endothelial cells (IC50 3.2 microM) to a lesser extent than on phospholipid vesicles (IC50 0.2 microM). Therefore, besides negatively charged phospholipids, other membrane constituents seem to be involved in endothelial cell mediated, intrinsic activation of factor X. Perturbation of endothelial cells with phorbol myristate acetate (PMA) or lipopolysaccharide (LPS) was without effect on intrinsic activation of factor X. This observation indicates that membrane constituents of endothelial cells involved in factor VIII/IXa-dependent activation of factor X are constitutively expressed.
Asunto(s)
Plaquetas/metabolismo , Vasos Sanguíneos/citología , Endotelio Vascular/citología , Factor X/metabolismo , Monocitos/metabolismo , Coagulación Sanguínea/fisiología , Células Cultivadas , Factor IXa/farmacología , Factor VIII/farmacología , Factor VIIa/farmacología , Factor X/efectos de los fármacos , Factor Xa/biosíntesis , Humanos , CinéticaRESUMEN
Factor VIIIa is inactivated by both factor IXa and activated protein C. The latter protease rapidly attacked a site at Arg562 (A2 subunit), whereas both proteases slowly cleaved factor VIIIa at Arg336 (A1 subunit). Cofactor inactivation catalyzed by activated protein C was 8-fold faster than that catalyzed by factor IXa. Simultaneous reaction of factor VIIIa with the two enzymes resulted in a rate of inactivation intermediate to that observed for the individual proteases. Under these conditions, the activated protein C-catalyzed cleavage at Arg562 was inhibited such that cofactor inactivation resulted primarily from cleavage at Arg336. Substitution of factor IXa modified in its active site with 6-(dimethylamino)-2-naphthalenesulfonyl-glutamylglycylarginyl++ + chloromethyl ketone (DEGR-IXa) for the native enzyme yielded a similar rate of activated protein C-catalyzed cleavage at the A1 site, whereas cleavage at the A2 site was virtually eliminated. However, the inclusion of protein S resulted in a marked increase in cleavage at the A2 site that correlated with an increased rate of cofactor inactivation. Active site-modified activated protein C inhibited the factor IXa-dependent enhancement of factor VIIIa reconstitution from isolated subunits. In addition, the factor VIIIa-dependent fluorescence enhancement of DEGR-activated protein C was inhibited by EGR-IXa. These results indicate that factor IXa can reduce the rate of activated protein C-catalyzed cofactor inactivation by selectively blocking cleavage at the A2 domainal site, an effect reversed by protein S. One mechanism consistent with the reciprocal inhibitory effects of the proteases is that activated protein C and factor IXa occupy overlapping sites on the cofactor. Thus, factor IXa may protect factor VIIIa by preventing activated protein C binding.
Asunto(s)
Compuestos de Dansilo , Factor IXa/farmacología , Factor VIIIa/metabolismo , Proteína C/metabolismo , Clorometilcetonas de Aminoácidos/farmacología , Secuencia de Aminoácidos , Arginina , Sitios de Unión , Factor VIIIa/aislamiento & purificación , Humanos , Cinética , Sustancias Macromoleculares , Proteína S/farmacología , Espectrometría de Fluorescencia , Especificidad por SustratoRESUMEN
The effect of hyperlipidaemia on endothelial cell haemostatic properties was examined using ex vivo studies on aortic segments obtained from fat-fed Chinchilla rabbits, mounted in a template device which exposed the luminal surface. Exposure of arterial endothelium to lipids resulted in marked enhancement of externally exposed anionic phospholipids, detected using either fluorescence microscopy with the probe merocyanine 540 or by binding of 125I-polymyxin B and 125I-Annexin V. Consistent with the known procoagulant properties of anionic phospholipid, following the lipid and cholesterol-rich diet intake, intact endothelial cells demonstrated enhanced binding of radioiodinated factors IX/IXa and Xa, and enhanced factor IXa/VIII-dependent factor X activation and factor Xa-factor Va-mediated prothrombin activation. Both factor Xa and thrombin formation were blocked, in large part, by polymyxin B, suggesting dependence of the reaction on anionic phospholipids. Consistent with these results, evidence of increased activation of the coagulation mechanism in vivo was observed in hyperlipidaemic animals, as assessed by a three-fold increase in levels of circulating antithrombin-protease complexes, compared with normolipidaemic controls.
Asunto(s)
Factores de Coagulación Sanguínea/metabolismo , Endotelio Vascular/metabolismo , Hipercolesterolemia/metabolismo , Animales , Aniones , Anexina A5/metabolismo , Antitrombina III/metabolismo , Aorta/metabolismo , Colesterol en la Dieta/farmacología , Endotelio Vascular/efectos de los fármacos , Factor IX/metabolismo , Factor IXa/metabolismo , Factor IXa/farmacología , Factor VIII/farmacología , Factor Va/farmacología , Factor X/metabolismo , Factor Xa/farmacología , Lípidos/farmacología , Masculino , Microscopía Fluorescente , Fosfolípidos/metabolismo , Polimixina B/metabolismo , Protrombina/metabolismo , ConejosRESUMEN
Although patients with thromboembolic disease frequently have family histories of thrombosis, well-defined defects such as inherited deficiencies of anticoagulant proteins are found only in a minority of cases. Based on the hypothesis that a poor anticoagulant response to activated protein C (APC) would predispose to thrombosis, a set of new coagulation assays was developed that measure the anticoagulant response in plasma to APC. A middle-aged man with a history of multiple thrombotic events was identified. The addition of APC to his plasma did not result in a normal anticoagulant response as measured by prolongation of clotting time in an activated partial thromboplastin time (APTT) assay. Four of the proband's relatives had medical histories of multiple thrombotic events, and they and several other family members responded poorly to APC in the APTT-based assay. Subnormal anticoagulant responses to APC were also found in factor IXa- and Xa-based assays. Several possible mechanisms for the observed phenomenon were ruled out, such as functional protein S deficiency, a protein C-inhibitory antibody, or a fast-acting protease inhibitor against APC. Moreover, restriction fragment-length polymorphism analysis excluded possible linkage of the underlying molecular defect to factor VIII and von Willebrand factor genes. We now describe a previously unrecognized mechanism for familial thromboembolic disease that is characterized by poor anticoagulant response to APC. This would appear to be explained best by a hypothesized inherited deficiency of a previously unrecognized cofactor to APC. As we have identified two additional, unrelated cases with thrombosis and inherited poor anticoagulant response to APC, this may constitute an important cause for familial thrombophilia.
Asunto(s)
Trastornos de la Coagulación Sanguínea/metabolismo , Coagulación Sanguínea/genética , Proteína C/metabolismo , Trombosis/metabolismo , Adulto , Coagulación Sanguínea/efectos de los fármacos , Trastornos de la Coagulación Sanguínea/genética , Pruebas de Coagulación Sanguínea/métodos , Factor IXa/farmacología , Femenino , Humanos , Masculino , Linaje , Proteína C/farmacología , Tromboplastina/farmacología , Trombosis/genéticaRESUMEN
To study the requirements for factor-IXa binding to platelets and factor-X activation, we examined the consequences of chemical modification (factor IXMOD) or enzymatic removal (factor IXDES) of gamma-carboxyglutamic acid (Gla) residues. In the presence of factor VIIIa and factor X, there were 344 (+/- 52) binding sites/platelet for factor IXaMOD (apparent dissociation constant [kdapp] = 4.5 +/- 0.9 nmol/L) and 275 (+/- 35) sites/platelet for factor IXaDES (kdapp = 5.0 +/- 0.8 nmol/L) compared with 580 (+/-65) sites/platelet for normal factor IXa (factor IXaN) (kdapp = 0.61 +/- 0.1 nmol/L) and 300 (+/-62) sites/platelet for factor IX (kdapp = 2.9 +/- 0.29 nmol/L). The concentrations of factor IXaN, factor IXaMOD and factor IXaDES required for half-maximal rates of factor-Xa formation were 0.67 nmol/L, 3.5 nmol/L, and 6.7 nmol/L. Whereas maximal velocities (Vmax) of factor Xa formation by factor IXaMOD (approximately 0.8 nmol/L.min-1) and factor IXaN (approximately 10.5 nmol/L.min-1), turnover numbers (kcat expressed as moles of factor Xa formed per minute per mole of factor IXa bound), and values of catalytic efficiency (kcat/Km) were normal, indicating that the decreased rates of factor X activation observed with factor IXaMOD and factor IXaDES are solely a consequence of the abnormal binding of these proteins to thrombin-activated platelets in the presence of factor VIIIa and factor X. Thus, factor IXa binding to platelets is mediated in part, but not exclusively, by high-affinity Ca2+ binding sites in the Gla domain of factor IX.
Asunto(s)
Ácido 1-Carboxiglutámico/sangre , Plaquetas/metabolismo , Factor IXa/química , Factor X/metabolismo , Ácido 1-Carboxiglutámico/química , Sitios de Unión , Calcio/metabolismo , Factor IXa/metabolismo , Factor IXa/farmacología , Humanos , Cinética , Activación Plaquetaria/efectos de los fármacos , Relación Estructura-Actividad , Trombina/farmacologíaRESUMEN
To assess the contribution of Factor IX/IXa, to intravascular thrombosis, a canine coronary thrombosis model was studied. Thrombus formation was initiated by applying current to a needle in the circumflex coronary artery. When 50% occlusion of the vessel developed, the current was stopped and animals received an intravenous bolus of either saline, bovine glutamyl-glycyl-arginyl-Factor IXa (IXai), a competitive inhibitor of Factor IXa assembly into the intrinsic Factor X activation complex, bovine Factor IX, or heparin. Animals receiving saline or Factor IX developed coronary occlusion due to a fibrin/platelet thrombus in 70 +/- 11 min. In contrast, infusion of IXai prevented thrombus formation completely (greater than 180 min) at doses of 460 and 300 micrograms/kg, and partially blocked thrombus formation at 150 micrograms/kg. IXai attenuated the accumulation of 125I-fibrinogen/fibrin at the site of the thrombus by approximately 67% (P less than 0.001) and resulted in approximately 26% decrease in serotonin release from platelets in coronary sinus (P less than 0.05). Hemostatic variables in animals receiving IXai, remained within normal limits. Animals given heparin in a concentration sufficient to prevent occlusive thrombosis had markedly increased bleeding, whereas heparin levels that maintained extravascular hemostasis did not prevent intracoronary thrombosis. This suggests that Factor IX/IXa can contribute to thrombus formation, and that inhibition of IXa participation in the clotting mechanism blocks intravascular thrombosis without impairing extravascular hemostasis.
Asunto(s)
Coagulación Sanguínea/efectos de los fármacos , Trombosis Coronaria/prevención & control , Factor IXa/farmacología , Animales , Trombosis Coronaria/fisiopatología , Modelos Animales de Enfermedad , Perros , Fibrinógeno/metabolismo , Heparina/farmacologíaRESUMEN
Activated protein C inactivates factor VIII by proteolytic cleavage of the heavy chain of factor VIII. Protein S and calcium ions are cofactors in this reaction. We have examined the effects of several potential modulators of this reaction, including phospholipids, von Willebrand factor, and factor IXa, all of which bind factor VIII. Our results indicate that neither resting nor stimulated platelets nor phospholipid vesicles protect factor VIII from inactivation by activated protein C in either the presence or the absence of protein S. However, the addition of von Willebrand factor decreases the inactivation of factor VIII by activated protein C by 20% to 30%, and factor IXa, which is known to protect factor VIII from inactivation by activated protein C, confers additional protection with von Willebrand factor. The active site of factor IXa is necessary for the protective effect, because native factor IX and active site-inhibited factor IXa do not protect factor VIII from inactivation. Thus there is an additive protective effect when von Willebrand factor and factor IXa are present with factor VIII, leading to a decrease in the inactivation by activated protein C. These factors may be particularly important in stabilizing factor VIII in the circulation and during the early stages of coagulation.