RESUMEN
BACKGROUND: Antithrombin (AT) is a plasma serpin inhibitor that regulates the proteolytic activity of procoagulant proteases of the clotting cascade. In addition to its anticoagulant activity, AT also possesses potent anti-inflammatory properties. OBJECTIVES: The objective of this study was to investigate the anti-inflammatory activity of wild-type AT (AT-WT) and a reactive centre loop mutant of AT (AT-RCL) which is not capable of inhibiting thrombin. METHODS: The cardioprotective activities of AT-WT and AT-RCL were monitored in a mouse model of ischemia/reperfusion (I/R) injury in which the left anterior descending coronary artery was occluded and then released. RESULTS: We demonstrate that AT markedly reduces myocardial infarct size by a mechanism that is independent of its anticoagulant activity. Thus, AT-RCL attenuated myocardial infarct size to the same extent as AT-WT in this acute injury model. Further studies revealed that AT binds to vascular heparan sulfate proteoglycans via its heparin-binding domain to exert its protective activity as evidenced by the therapeutic AT-binding pentasaccharide (fondaparinux) abrogating the cardioprotective activity of AT and a heparin-site mutant of AT exhibiting no cardioprotective property. We further demonstrate that AT up-regulates the production of prostacyclin in myocardial tissues and inhibits expression of pro-inflammatory cytokines tumor necrosis factor (TNF)-α and interleukin (IL)-6 in vivo by attenuating ischemia/reperfusion-induced JNK and NF-κB signaling pathways. CONCLUSIONS: The present results suggest that both AT and the non-anticoagulant AT-RCL, through their anti-inflammatory signaling effects, elicit potent cardioprotective responses. Thus, AT may have therapeutic potential for treating cardiac I/R injury.
Asunto(s)
Antitrombinas/química , Isquemia Miocárdica/tratamiento farmacológico , Isquemia Miocárdica/patología , Daño por Reperfusión/tratamiento farmacológico , Daño por Reperfusión/patología , Animales , Antiinflamatorios/química , Cardiotónicos/química , Fondaparinux , Heparina/química , Inflamación , Interleucina-6/metabolismo , Isquemia/patología , Leucocitos/efectos de los fármacos , MAP Quinasa Quinasa 4/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Mutación , FN-kappa B/metabolismo , Polisacáridos/química , Transducción de Señal , Trombina/antagonistas & inhibidores , Troponina I/química , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
BACKGROUND: Modulation of energy substrate metabolism may constitute a novel therapeutic intervention against ischemia/reperfusion (I/R) injury. AMP-activated protein kinase (AMPK) has emerged as a key regulator of favorable metabolic signaling pathways in response to myocardial ischemia. Recently, we demonstrated that activated protein C (APC) is cardioprotective against ischemia/reperfusion (I/R) injury by augmenting AMPK signaling. OBJECTIVES: The objective of this study was to determine whether the APC modulation of substrate metabolism contributes to its cardioprotective effect against I/R injury. METHODS: An ex vivo working mouse heart perfusion system was used to characterize the effect of wild-type APC and its signaling-proficient mutant, APC-2Cys (which has dramatically reduced anticoagulant activity), on glucose transport in the ischemic heart. RESULTS: Both APC and APC-2Cys (0.2 µg g(-1)) augment the ischemic stress-induced translocation of the glucose transporter (GLUT4) to the myocardial cell membrane, leading to increased glucose uptake and glucose oxidation in the ischemic heart (P < 0.05 vs. vehicle). Both APC derivatives increased the autophagic flux in the heart following I/R. The activity of APC-2Cys in modulating these metabolic pathways was significantly higher than APC during I/R (P < 0.05). Intriguingly, APC-2Cys, but not wild-type APC, attenuated the I/R-initiated fatty acid oxidation by 80% (P < 0.01 vs. vehicle). CONCLUSIONS: APC exerts a cardioprotective effect against I/R injury by preferentially enhancing the oxidation of glucose over fatty acids as energy substrates in the ischemic heart. Given its significantly higher beneficial metabolic modulatory effect, APC-2Cys may be developed as a potential therapeutic drug for treating ischemic heart disease without risk of bleeding.
Asunto(s)
Miocardio/metabolismo , Proteína C/fisiología , Animales , HumanosRESUMEN
Heparin promotes the antithrombin (AT) inactivation of factors IXa (fIXa) and Xa (fXa) through a conformational activation of the serpin and also by a template mechanism in the presence of physiological levels of Ca2+. Recently, it was reported that heparin induces conformational changes in the active-sites of fIXa and fXa, raising the possibility that heparin also modulates the reactivity of these proteases with AT by this mechanism. To test this possibility, we prepared an AT mutant in which four critical heparin-binding residues of the serpin (Arg-45, Arg-46, Lys-114, and Arg-129) were replaced with non-basic residues. This mutant lost its affinity for heparin, but retained its normal reactivity with coagulation proteases. Thus, the high-affinity AT-binding pentasaccharide fragment of heparin had no cofactor effect on the reactivity of the AT mutant with coagulation proteases. Full-length heparin-concentration dependence of the AT inhibition of fIXa and fXa revealed that in contrast to a greater than 4-5 orders of magnitude accelerating effect for heparin on the AT inhibition of fIXa and fXa, heparin exhibits a negligible cofactor effect (<2-fold) on the mutant AT inhibition of these proteases. The same results were obtained for the mutant AT inhibition of thrombin and factor VIIa, however, heparin accelerated the mutant AT inhibition of factor XIa ~10-fold. We conclude that, with the exception of factor XIa, heparin-mediated conformational modulation of the active-sites of coagulation proteases makes a minor contribution to the regulation of these proteases by AT.
Asunto(s)
Antitrombina III/farmacología , Factor IX/antagonistas & inhibidores , Factor X/antagonistas & inhibidores , Heparina/farmacología , Sustitución de Aminoácidos , Antitrombina III/genética , Sitios de Unión , Línea Celular , Activación Enzimática/efectos de los fármacos , Humanos , Conformación Proteica/efectos de los fármacosRESUMEN
BACKGROUND: There are two O-linked and two N-linked glycosylation sites on the activation peptide of factor X (FX) involving residues Thr-17, Thr-29, Asn-39 and Asn-49. OBJECTIVES: The purpose of this study was to explore the contribution of carbohydrates of the FX activation peptide to zymogen recognition by physiological activators. METHODS: The putative glycosylation sites were substituted individually or in combinations with Ala and mutants were expressed in mammalian cells. The entire activation peptide up to the P3 residue was deleted in another construct. RESULTS: It was discovered that activation of zymogen mutants by both FVIIa and FIXa on negatively charged phospholipid vesicles has been improved 2-40-fold independent of a cofactor. These mutants were activated with slightly lower catalytic efficiency (k(cat)/K(m)) by FVIIa in the extrinsic Xase complex, though both K(m) and k(cat) constants for mutants were elevated. With the exception of approximately 3-fold improvement in the activation of N49A, the catalytic efficiency of FIXa toward mutants was decreased 2-5-fold in the intrinsic Xase complex. CONCLUSIONS: The carbohydrate chains of the FX activation peptide play an important role in restricting the specificity of zymogen recognition by both FVIIa and FIXa, thereby preventing the cofactor-independent activation of FX by these proteases. On the other hand, the carbohydrates contribute to the cofactor-dependent recognition of the zymogen by both extrinsic and intrinsic Xase complexes.
Asunto(s)
Factor X/química , Procesamiento Proteico-Postraduccional , Secuencia de Aminoácidos , Conformación de Carbohidratos , Células Cultivadas , Cisteína Endopeptidasas/metabolismo , Activación Enzimática , Precursores Enzimáticos/metabolismo , Epítopos/química , Epítopos/genética , Factor IXa/metabolismo , Factor VIIa/metabolismo , Factor X/genética , Factor X/metabolismo , Factor Xa/metabolismo , Glicosilación , Humanos , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Proteínas de Neoplasias/metabolismo , Fragmentos de Péptidos/química , Fragmentos de Péptidos/metabolismo , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Especificidad por SustratoRESUMEN
We recently demonstrated that the substitution of the autolysis loop (residues 143 to 154 in the chymotrypsin numbering system) of activated protein C (APC) with the corresponding loop of factor Xa (fXa) renders the APC mutant (APC/fX143-154) susceptible to inhibition by antithrombin (AT) in the presence of pentasaccharide. Our recent results further indicated, that in addition to an improvement in the reactivity of APC/fX143-154 with AT, both the amidolytic and anti-factor Va activities of the mutant APC have also been significantly increased. Since the autolysis loop of APC is five residues longer than the autolysis loop of fXa, it could not be ascertained whether this loop in the mutant APC specifically interacts with the activated conformation of AT or if a shorter autolysis loop is responsible for a global improvement in the catalytic activity of the mutant protease. To answer this question, we prepared another APC mutant in which the autolysis loop of the protease was replaced with the corresponding loop of trypsin (APC/Tryp143-154). Unlike an approximately 500-fold improvement in the reactivity of APC/fX143-154 with AT in the presence of pentasaccharide, the reactivity of APC/Tryp143-154 with the serpin was improved approximately 10-fold. These results suggest that both the length and structure of residues of the autolysis loop are critical for the specificity of the coagulation protease interaction with AT. Further factor Va inactivation studies with the APC mutants revealed a similar role for the autolysis loop of APC in the interaction with its natural substrate.
Asunto(s)
Antitrombinas/metabolismo , Autólisis/enzimología , Coagulación Sanguínea/genética , Mutación/genética , Péptido Hidrolasas/genética , Proteína C/genética , Secuencia de Aminoácidos , Activación Enzimática , Factor Va/genética , Factor Va/metabolismo , Factor Xa/genética , Factor Xa/metabolismo , Humanos , Datos de Secuencia Molecular , Péptido Hidrolasas/metabolismo , Proteína C/metabolismo , Alineación de Secuencia , Especificidad por Sustrato/genéticaRESUMEN
We recently demonstrated that the substitution of the autolysis loop (residues 143 to 154 in the chymotrypsin numbering system) of activated protein C (APC) with the corresponding loop of factor Xa (fXa) renders the APC mutant (APC/fX143-154) susceptible to inhibition by antithrombin (AT) in the presence of pentasaccharide. Our recent results further indicated, that in addition to an improvement in the reactivity of APC/fX143-154 with AT, both the amidolytic and anti-factor Va activities of the mutant APC have also been significantly increased. Since the autolysis loop of APC is five residues longer than the autolysis loop of fXa, it could not be ascertained whether this loop in the mutant APC specifically interacts with the activated conformation of AT or if a shorter autolysis loop is responsible for a global improvement in the catalytic activity of the mutant protease. To answer this question, we prepared another APC mutant in which the autolysis loop of the protease was replaced with the corresponding loop of trypsin (APC/Tryp143-154). Unlike an ~500-fold improvement in the reactivity of APC/fX143-154 with AT in the presence of pentasaccharide, the reactivity of APC/Tryp143-154 with the serpin was improved ~10-fold. These results suggest that both the length and structure of residues of the autolysis loop are critical for the specificity of the coagulation protease interaction with AT. Further factor Va inactivation studies with the APC mutants revealed a similar role for the autolysis loop of APC in the interaction with its natural substrate.