RESUMEN

Essentials Heparin-binding site (HBS) variants of antithrombin (AT) are associated with thrombosis risk. HSB variants have, in general, normal progressive inhibitory activity but reduced heparin affinity. Thrombosis in HSB carriers has been primarily attributed to the loss of heparin cofactor activity. Results here demonstrate that HSB variants of AT also lack anti-inflammatory signaling functions. SUMMARY: Background Several heparin-binding site (HBS) variants of antithrombin (AT) have been identified that predispose carriers to a higher incidence of thrombosis. Thrombosis in carriers of HBS variants has been primarily attributed to a loss in their heparin-dependent anticoagulant function. Objective The objective of this study was to determine whether HSB mutations affect the anti-inflammatory functions of variants. Methods Two HBS variants of AT (AT-I7N and AT-L99F), which are known to be associated with a higher incidence of thrombosis, were expressed in mammalian cells and purified to homogeneity. These variants were characterized by kinetic assays followed by analysis of their activities in established cellular and/or in vivo inflammatory models. The possible effects of mutations on AT structure were also evaluated by molecular modeling. Results The results indicated that, whereas progressive inhibitory activities of variants were minimally affected, their heparin affinity and inhibitory activity in the presence of heparin were markedly decreased. Unlike wild-type AT, neither AT variant was capable of inhibiting activation of nuclear factor-κB or downregulation of expression of cell adhesion molecules in response to lipopolysaccharide (LPS). Similarly, neither variant elicited barrier protective activity in response to LPS. Structural analysis suggested that the L99F substitution locally destabilizes AT structure. Conclusions It is concluded that the L99F mutation of AT is associated with destabilization of the serpin structure, and that the loss of anti-inflammatory signaling function of the HBS variants may also contribute to enhanced thrombosis in carriers of HBS mutations.

Asunto(s)

Antitrombina III/metabolismo , Heparina/metabolismo , Animales , Antitrombina III/química , Antitrombina III/genética , Sitios de Unión , Modelos Animales de Enfermedad , Células Endoteliales/metabolismo , Células HEK293 , Humanos , Inflamación/sangre , Inflamación/genética , Inflamación/prevención & control , Cinética , Ratones , Mutación , Unión Proteica , Conformación Proteica , Transducción de Señal , Relación Estructura-Actividad , Trombosis/sangre , Trombosis/genética

RESUMEN

Essentials Polyphosphate (polyP) activates mTOR but its role in Wnt/ß-catenin signaling is not known. PolyP-mediated cyclin D1 expression (ß-catenin target gene) was monitored in endothelial cells. PolyP and boiled platelet-releasates induced the expression of cyclin D1 by similar mechanisms. PolyP establishes crosstalk between mTOR and Wnt/ß-catenin signaling in endothelial cells. SUMMARY: Background Inorganic polyphosphate (polyP) elicits intracellular signaling responses in endothelial cells through activation of mTOR complexes 1 and 2. Glycogen synthase kinase 3 (GSK-3) is known to be a negative regulator of mTOR and Wnt/ß-catenin signaling pathways. Objective The objective of this study was to investigate the effect of polyP on the expression, degradation and subcellular localization of the Wnt/ß-catenin target gene, cyclin D1, in endothelial cells. Methods Regulation of cyclin D1 expression, phosphorylation and subcellular localization by polyP or platelet releasates was monitored in the absence and presence of pharmacological inhibitors and/or siRNA for specific molecules of the upstream mTOR/Wnt/ß-catenin signaling network by established methods. Results Both synthetic polyP and boiled-platelet releasates induced the phosphorylation-dependent inactivation of GSK-3, thereby increasing the expression and nuclear localization, but inhibiting the degradation of cyclin D1. Inhibitors of mTORC1 (PI3K, AKT, PLC, PKC), rapamycin and siRNA for raptor (mTORC1-specific component) and ß-catenin, all inhibited polyP-mediated regulation of cyclin D1 expression, phosphorylation and subcellular localization in endothelial cells. The signaling effect of polyP was effectively inhibited by the recombinant extracellular domain of the receptor for advanced glycation end products (RAGE) and/or by the RAGE siRNA. Specific pharmacological inhibitors and siRNA knockdown of ERK1/2 and NF-κB pathways indicated that polyP-mediated cyclin D1 expression and nuclear localization are IKKɑ and ERK1/2 dependent, whereas its inhibitory effect on phosphorylation-dependent degradation of cyclin D1 is IKKß-dependent. Conclusion We conclude that a RAGE-dependent polyP-mediated crosstalk between mTOR and the GSK-3/Wnt/ß-catenin signaling network can modulate important physiological processes in endothelial cells.

Asunto(s)

Ciclina D1/metabolismo , Glucógeno Sintasa Quinasa 3/metabolismo , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Polifosfatos/química , Serina-Treonina Quinasas TOR/metabolismo , Vía de Señalización Wnt , Células Endoteliales/metabolismo , Productos Finales de Glicación Avanzada/metabolismo , Humanos , Fosforilación , ARN Interferente Pequeño/metabolismo , Transducción de Señal

RESUMEN

BACKGROUND: Inorganic polyphosphate (polyP) elicits pro-inflammatory signaling responses in endothelial cells through interaction with two receptors, RAGE and P2Y1 . It is known that polyP activates mTOR signaling in breast cancer cells. OBJECTIVES: The objective of this study is to understand the mechanism of the polyP-mediated signaling pathway in endothelial cells and to determine whether polyP exerts its pro-inflammatory effect through activation of mTOR. METHODS: mTOR activation by polyP or platelet releasates in cellular and animal models was monitored in the absence and presence of pharmacological inhibitors and/or siRNA knockdown of specific signaling molecules. RESULTS: PolyP effectively induced phosphorylation of mTOR complex 1 (mTORC1) substrate, p70S6K, in endothelial cells by an AKT-dependent but ERK-independent mechanism. The siRNA knockdown of both RAGE and P2Y1 or specific inhibitors of the PI3K/PLC/PKC/Ca(2+) signaling axis inhibited polyP-mediated p70S6K phosphorylation. Moreover, either rapamycin or siRNA knockdown of raptor (mTORC1-specific component) abrogated polyP-mediated phosphorylation of p70S6K. By contrast, the siRNA knockdown of rictor (mTOR complex 2-specific component) but not raptor eliminated the barrier-disruptive effect of polyP. Specific NF-κB inhibitors abrogated polyP-mediated phosphorylation of p70S6K and rapamycin suppressed polyP-induced activation of NF-κB. Finally, specific inhibitors of the mTOR signaling network eliminated polyP-mediated vascular leakage and leukocyte recruitment in animal models. CONCLUSIONS: PolyP, through interaction with RAGE and P2Y1 , activates both the mTORC1 and mTORC2 signaling network. Both the pro-inflammatory and mTOR signaling functions of polyP are linked.

Asunto(s)

Endotelio Vascular/metabolismo , Inflamación/inducido químicamente , Compuestos Inorgánicos/farmacología , Complejos Multiproteicos/metabolismo , Polifosfatos/farmacología , Serina-Treonina Quinasas TOR/metabolismo , Animales , Endotelio Vascular/citología , Diana Mecanicista del Complejo 1 de la Rapamicina , Diana Mecanicista del Complejo 2 de la Rapamicina , Ratones , FN-kappa B/metabolismo

RESUMEN

Antithrombin (AT) is a heparin-binding serpin in plasma which regulates the proteolytic activity of procoagulant proteases of the clotting cascade. In addition to being an anticoagulant, AT also exhibits antiinflammatory activities when it binds to cell surface heparan sulfate proteoglycans (HSPGs) on the endothelium via its basic residues of D-helix to elicit intracellular signalling responses. By contrast to AT, α1-proteinase inhibitor (α1-PI) is a non-heparin-binding serpin that exhibits very slow reactivity with coagulation proteases and possesses no HSPG-dependent antiinflammatory properties. To determine whether the antiinflammatory signaling specificity of AT can be transferred to α1-PI, we replaced the D-helix of human α1-PI with the corresponding sequence of human AT and expressed the chimeric serpin α1-PI/D-helix) in a bacterial expression system. High molecular weight heparin bound to α1-PI/D-helix and accelerated the inhibition of thrombin by the serpin mutant by a template mechanism reminiscent of the cofactor effect of heparin on inhibition of thrombin by AT. Like AT, α1-PI/D-helix exhibited antiinflammatory properties in both cellular and animal models. Thus, α1-PI/D-helix inhibited the barrier-disruptive effect of proinflammatory cytokines and inhibited the activation of nuclear factor-κB transcription factor in lipopolysaccharide-stimulated endothelial cells by a concentration-dependent manner. Furthermore, the chimeric serpin reduced lipopolysaccharide-mediated lethality, elicited a vascular protective effect and inhibited infiltration of activated leukocytes to the peritoneal cavity of mice in an HMGB1-mediated inflammatory model. These results suggest that grafting the D-helix of AT to α1-PI confers antiinflammatory properties on the serpin and that the chimeric serpin may have therapeutic utility for treating inflammatory disorders.

Asunto(s)

Antitrombinas/fisiología , Células Endoteliales/fisiología , Inflamación/inmunología , Estructura Secundaria de Proteína , alfa 1-Antitripsina/metabolismo , Animales , Coagulación Sanguínea/genética , Movimiento Celular/genética , Células Cultivadas , Heparina/análogos & derivados , Heparina/metabolismo , Humanos , Inflamación/terapia , Ratones , Modelos Animales , Mutación/genética , FN-kappa B/metabolismo , Ingeniería de Proteínas , Estructura Secundaria de Proteína/genética , Proteoglicanos/metabolismo , Transducción de Señal/genética , alfa 1-Antitripsina/genética

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/metabolismo

RESUMEN

Protein C is a vitamin K-dependent serine protease zymogen in plasma which upon activation to activated protein C (APC) by thrombin down-regulates the clotting cascade by limited proteolysis of the procoagulant cofactors Va and VIIIa. In addition to its anticoagulant activity, APC also exhibits potent cytoprotective and anti-inflammatory activities. While the anticoagulant activity of APC is enhanced by the cofactor function of protein S on membrane phospholipids, the cytoprotective intracellular signalling activity of APC requires complex formation with endothelial protein C receptor (EPCR) expressed on the vascular endothelium. Two natural variants of APC [Arg-147 to Trp substitution (R147W) and Lys-150 deletion (K150del)] have been identified in the Chinese population as hotspot mutants occurring with high frequencies of 27.8% and 13.9%, respectively, among 36 protein C-deficient subjects. The affected individuals exhibit variable thrombotic tendencies. To understand the underlying cause of the thrombotic phenotype in these patients, we expressed these two protein C variants in mammalian cells and characterised their anticoagulant and anti-inflammatory properties using established in vitro and cellular assays. Our results suggest that both R147W and K150del variants have normal amidolytic and proteolytic activities in the absence of cofactors. However, the R147W mutant exhibits ~3 times lower affinity for binding to EPCR and the K150del variant has ~2-3-fold impaired anticoagulant activity in the presence of protein S. These results provide some insight into the possible pathogenic mechanism of protein C deficiency in Chinese patients carrying these mutations.

Asunto(s)

Coagulación Sanguínea , Deficiencia de Proteína C/sangre , Proteína C/metabolismo , Trombosis de la Vena/sangre , Antígenos CD/metabolismo , Pueblo Asiatico/genética , Coagulación Sanguínea/genética , China/epidemiología , Células Endoteliales/metabolismo , Receptor de Proteína C Endotelial , Frecuencia de los Genes , Predisposición Genética a la Enfermedad , Células HEK293 , Humanos , Cinética , Mutación , Fenotipo , Unión Proteica , Proteína C/genética , Deficiencia de Proteína C/etnología , Deficiencia de Proteína C/genética , Receptores de Superficie Celular/metabolismo , Transducción de Señal , Transfección , Trombosis de la Vena/etnología , Trombosis de la Vena/genética

RESUMEN

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 , Humanos

RESUMEN

BACKGROUND: Recent results have indicated that polyphosphate, released by activated platelets, can function as a procoagulant to modulate the proteolytic activity of serine proteases of the blood clotting cascade. OBJECTIVE: To determine whether polyphosphate is involved in inducing signal transduction in cellular and animal models. METHODS: The effect of polyphosphate on human umbilical vein endothelial cells was examined by monitoring cell permeability, apoptosis and activation of NF-κB after treating cells with different concentrations of polyphosphate. Moreover, the expression of cell surface adhesion molecules (VCAM-1, ICAM-1 and E-selectin) and the adhesion of THP-1 cells to polyphosphate-treated cells were monitored using established methods. In the in vivo model, the pro-inflammatory effect of polyphosphate was assessed by monitoring vascular permeability and migration of leukocytes to the peritoneal cavity of mice injected with polyphosphate. RESULTS: Polyphosphate, comprised of 45, 65 and 70 phosphate units, enhanced the barrier permeability and apoptosis in cultured endothelial cells and up-regulated the expression of cell adhesion molecules, thereby mediating the adhesion of THP-1 cells to polyphosphate-treated endothelial cells. These effects of polyphosphate were mediated through the activation of NF-κB and could not be recapitulated by another anionic polymer, heparin. Polyphosphate also increased the extravasation of the bovine serum albumin (BSA)-bound Evans blue dye and the migration of leukocytes to the mouse peritoneal cavity, which was prevented when activated protein C (APC) was intravenously (i.v.) injected 2 h before the challenge. CONCLUSION: Polyphosphate, in addition to up-regulation of coagulation, can elicit potent pro-inflammatory responses through the activation of NF-κB, possibly contributing to the pro-inflammatory effect of activated platelets.

Asunto(s)

Células Endoteliales de la Vena Umbilical Humana/metabolismo , Mediadores de Inflamación/metabolismo , Inflamación/metabolismo , Polifosfatos/metabolismo , Proteína C/metabolismo , Animales , Apoptosis , Permeabilidad Capilar , Adhesión Celular/efectos de los fármacos , Permeabilidad de la Membrana Celular , Células Cultivadas , Quimiotaxis de Leucocito , Técnicas de Cocultivo , Relación Dosis-Respuesta a Droga , Selectina E/metabolismo , Femenino , Heparina/farmacología , Células Endoteliales de la Vena Umbilical Humana/efectos de los fármacos , Células Endoteliales de la Vena Umbilical Humana/inmunología , Células Endoteliales de la Vena Umbilical Humana/patología , Humanos , Inflamación/inmunología , Inflamación/patología , Inflamación/prevención & control , Inyecciones Intraperitoneales , Inyecciones Intravenosas , Molécula 1 de Adhesión Intercelular/metabolismo , Ratones , Ratones Endogámicos ICR , FN-kappa B/metabolismo , Polifosfatos/administración & dosificación , Proteína C/administración & dosificación , Regulación hacia Arriba , Molécula 1 de Adhesión Celular Vascular/metabolismo

RESUMEN

BACKGROUND: Activated protein C (APC) is a vitamin K-dependent plasma serine protease that down-regulates clotting and inflammatory pathways. It is known that APC exerts a cardioprotective effect by decreasing apoptosis of cardiomyocytes and inhibiting expression of inflammatory mediators after myocardial ischemia. OBJECTIVES: The objective of this study was to understand the mechanism of the APC-mediated cardioprotection against ischemic injury. METHODS: Cardioprotective activities of wild-type APC and two derivatives, having either dramatically reduced anticoagulant activity or lacking signaling activity, were monitored in an acute ischemia/reperfusion injury model in which the left anterior descending coronary artery (LAD) was occluded. RESULTS: APC reduced the myocardial infarct size by a mechanism that was largely independent of its anticoagulant activity. Thus, the non-anticoagulant APC-2Cys mutant, but not the non-signaling APC-E170A mutant, attenuated myocardial infarct size by EPCR and PAR-1-dependent mechanisms. Further studies revealed that APC acts directly on cardiomyocytes to stimulate the AMP-activated protein kinase (AMPK) signaling pathway. The activation of AMPK by APC ameliorated the post-ischemic cardiac dysfunction in isolated perfused mouse hearts. Moreover, both APC and APC-2Cys inhibited production of TNFα and IL-6 in vivo by attenuating the ischemia/reperfusion-induced JNK and NF-κB signaling pathways. CONCLUSIONS: APC exerts a cardioprotective function in ischemic/reperfusion injury through modulation of AMPK, NF-κB and JNK signaling pathways.

Asunto(s)

Proteínas Quinasas Activadas por AMP/metabolismo , Cardiotónicos/farmacología , Daño por Reperfusión Miocárdica/prevención & control , Miocitos Cardíacos/patología , Proteína C/uso terapéutico , Transducción de Señal , Animales , Apoptosis , Células Cultivadas , Corazón , Técnicas In Vitro , Sistema de Señalización de MAP Quinasas , Ratones , Daño por Reperfusión Miocárdica/tratamiento farmacológico , Miocitos Cardíacos/efectos de los fármacos , FN-kappa B/metabolismo , Proteína C/farmacología

RESUMEN

Protein C is a vitamin K-dependent anticoagulant serine protease zymogen in plasma which upon activation by the thrombin-thrombomodulin complex down-regulates the coagulation cascade by degrading cofactors Va and VIIIa by limited proteolysis. In addition to its anticoagulant function, activated protein C (APC) also binds to endothelial protein C receptor (EPCR) in lipid-rafts/caveolar compartments to activate protease- activated receptor 1 (PAR-1) thereby eliciting antiinflammatory and cytoprotective signaling responses in endothelial cells. These properties have led to FDA approval of recombinant APC as a therapeutic drug for severe sepsis. The mechanism by which APC selects its substrates in the anticoagulant and antiinflammatory pathways is not well understood. Recent structural and mutagenesis data have indicated that basic residues of three exposed surface loops known as 39-loop (Lys-37, Lys-38, and Lys-39), 60-loop (Lys-62, Lys- 63, and Arg-67), and 70-80-loop (Arg-74, Arg-75, and Lys-78) (chymotrypsin numbering) constitute an anion binding exosite in APC that interacts with the procoagulant cofactors Va and VIIIa in the anticoagulant pathway. Furthermore, two negatively charged residues on the opposite side of the active-site of APC on a helical structure have been demonstrated to determine the specificity of the PAR-1 recognition in the cytoprotective pathway. This article will review the mechanism by which APC exerts its proteolytic function in two physiologically inter-related pathways and how the structure- function insights into determinants of the specificity of APC interaction with its substrates in two pathways can be utilized to tinker with the structure of the molecule to obtain APC derivatives with potentially improved therapeutic profiles.

Asunto(s)

Antiinflamatorios/metabolismo , Anticoagulantes/metabolismo , Proteína C/metabolismo , Antiinflamatorios/química , Antiinflamatorios/uso terapéutico , Anticoagulantes/química , Anticoagulantes/uso terapéutico , Antígenos CD/metabolismo , Receptor de Proteína C Endotelial , Humanos , Proteína C/química , Proteína C/uso terapéutico , Receptor PAR-1/metabolismo , Receptores de Superficie Celular/metabolismo , Sepsis/terapia , Transducción de Señal , Especificidad por Sustrato , Trombomodulina/metabolismo

RESUMEN

SUMMARY BACKGROUND: Activated protein C (APC) in complex with endothelial protein C receptor (EPCR) can reverse the barrier-disruptive and cytotoxic effects of proinflammatory cytokines by cleaving protease-activated receptor 1 (PAR-1). Recently, it was reported that the PAR-1-dependent vascular barrier-protective effect of APC is mediated through transactivation of the angiopoietin (Ang)-Tie2 signaling pathway. The antagonist of this pathway, Ang2, is stored in Weibel-Palade bodies within endothelial cells. OBJECTIVES: To determine whether the occupancy of EPCR by its ligand can switch the PAR-1-dependent signaling specificity of thrombin through the Ang-Tie2 axis. METHODS: We activated endothelial cells with thrombin before and after treating them with the catalytically inactive Ser195-->Ala substitution mutant of protein C. The expression levels of Ang1, Ang2 and Tie2 in response to thrombin were measured by both an enzyme-linked immunosorbent assay and a cell permeability assay in the absence and presence of small interfering RNA and a blocking antibody to Tie2. RESULTS: Thrombin upregulated the expression of both Ang1 and Tie2 but downregulated the expression of Ang2 when EPCR was occupied by its ligand. The Ang1-Tie2-dependent protective effect of thrombin was initiated through protein C inhibiting the rapid mobilization of Ang2 from Weibel-Palade bodies. Interestingly, the protein C mutant also inhibited the thrombin mobilization of P-selectin. CONCLUSIONS: These results suggest a physiologic role for the low concentration of thrombin in maintaining the integrity of the EPCR-containing vasculature through the PAR-1-dependent inhibition of Ang2 and P-selectin release from Weibel-Palade bodies.

Asunto(s)

Angiopoyetina 2/fisiología , Selectina-P/fisiología , Receptor TIE-2/fisiología , Trombina/fisiología , Regulación hacia Arriba/fisiología , Cuerpos de Weibel-Palade/fisiología , Línea Celular Transformada , Ensayo de Inmunoadsorción Enzimática , Humanos , Fosforilación

RESUMEN

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 fármacos

RESUMEN

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 Sustrato

RESUMEN

SUMMARY BACKGROUND: Recent studies have indicated that antithrombin (AT) possesses both anti-inflammatory and antiangiogenic properties. OBJECTIVES: The purpose of this study was to investigate the mechanism of the intracellular signaling activities of AT using wild-type and mutant serpins that have reduced anticoagulant activities due to mutations in either the reactive center loop (RCL) or the heparin-binding site. METHODS: Direct cellular effects of the AT derivatives were compared in the LPS-stimulated endothelial cells by employing permeability and neutrophil adhesion assays in the absence and presence of pertussis toxin (PTX) and siRNAs for either syndecan-4 or sphingosine 1-phosphate receptor 1 (S1P(1)). Furthermore, the roles of prostacyclin and nuclear factor (NF)-kappaB in modulating these effects were investigated. RESULTS: Both wild-type and the RCL mutant, AT/Proth-2, exhibited similar potent barrier protective activities and inhibited the adhesion of neutrophils to endothelial cells via inhibition of the NF-kappaB pathway. Indomethacin abrogated both activities. The heparin-binding site mutants, AT-K114E and AT-K125E, did not exhibit any protective activity in either one of these assays, but a potent pro-apoptotic activity was observed for the AT-K114E in endothelial cells. Both PTX and siRNA for syndecan-4 inhibited the protective effect of AT, but the siRNA for S1P(1) was inconsequential. CONCLUSIONS: The interaction of AT with syndecan-4 is required for its prostacyclin-dependent protective effect through a PTX-sensitive and non-S1P(1)-related G(i)-protein coupled receptor. The RCL mutant, AT/Proth-2, with a markedly reduced anticoagulant but normal protective signaling properties, may potentially be developed as a safer anti-inflammatory drug without increasing the risk of bleeding.

Asunto(s)

Antitrombinas/metabolismo , Mutagénesis , Transducción de Señal , Apoptosis/efectos de los fármacos , Adhesión Celular/efectos de los fármacos , Humanos , Lipopolisacáridos/farmacología , Modelos Moleculares , Neutrófilos/metabolismo , Toxina del Pertussis/farmacología , ARN Interferente Pequeño , Receptores de Lisoesfingolípidos/genética , Sindecano-4/genética

RESUMEN

BACKGROUND: The endothelial protein C receptor (EPCR)-dependent cleavage of protease activated receptor 1 (PAR-1) by either activated protein C (APC) or thrombin in lipid rafts initiates protective signaling responses in endothelial cells. OBJECTIVES: To investigate the mechanism by which APC and thrombin interact with and cleave PAR-1 in lipid rafts. METHODS: We constructed two types of PAR-1 cleavage reporter constructs in which a secreted alkaline phosphatase (ALP) was fused to the extracellular domain of PAR-1. The first construct has a transmembrane domain capable of uniformly anchoring the fusion protein to the membrane surface, while the second construct has the recognition sequence for targeting the fusion protein to lipid rafts/caveolae in transfected cells. RESULTS: Both APC and the Gla-domainless (GD)-APC cleaved the PAR-1 exodomain with similar efficiency in HUVECs transfected with the first construct. Unlike APC, GD-APC did not cleave PAR-1 in cells transfected with the second construct; however, prior treatment of cells with S195A mutants of either protein C or thrombin led to the GD-APC cleavage of PAR-1 with a comparable or higher catalytic efficiency. The same results were obtained if the cellular signaling properties of APC and GD-APC were monitored in the TNF-alpha-induced endothelial cell apoptosis and permeability assays. CONCLUSIONS: The lipid raft localization renders the scissile bond of the PAR-1 exodomain unavailable for interaction with coagulation proteases. The binding of either the Gla-domain of protein C to EPCR or exosite-1 of thrombin to the C-terminal hirudin-like sequence of PAR-1 changes the membrane localization and/or the conformation of the PAR-1 exodomain to facilitate its recognition and subsequent cleavage by these proteases.

Asunto(s)

Células Endoteliales/metabolismo , Microdominios de Membrana/química , Receptor PAR-1/metabolismo , Trombina/metabolismo , Fosfatasa Alcalina/metabolismo , Apoptosis , Factores de Coagulación Sanguínea/metabolismo , Humanos , Lípidos/química , Modelos Biológicos , Permeabilidad , Proteína C/metabolismo , Estructura Terciaria de Proteína , Receptores de Superficie Celular/metabolismo , Transducción de Señal , Trombina/química , Transfección

RESUMEN

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/genética

RESUMEN

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.

Asunto(s)

Humanos , 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 , Datos de Secuencia Molecular , Péptido Hidrolasas/metabolismo , Proteína C/metabolismo , Alineación de Secuencia , Especificidad por Sustrato/genética

RESUMEN

The molecular basis of the substrate and inhibitor specificity of factor Xa, the serine proteinase of the prothrombinase complex, was investigated by constructing two mutants of human antithrombin (HAT) in which the reactive site loop of the serpin from the P4-P4' site was replaced with the corresponding residues of the two factor Xa cleavage sites in prothrombin (HAT/Proth-1 and HAT/Proth-2). These mutants together with prethrombin-2, the smallest zymogen form of thrombin containing only the second factor Xa cleavage site, were expressed in mammalian cells, purified to homogeneity and characterized in kinetic reactions with factor Xa in both the absence and presence of cofactors; factor Va, high affinity heparin and pentasaccharide fragment of heparin. HAT/Proth-1 inactivated factor Xa approximately 3-4-fold better than HAT/Proth-2 in either the absence or presence of heparin cofactors. In the absence of a cofactor, factor Xa reacted with the HAT/Proth-2 and prethrombin-2 with similar second-order rate constants (approximately 2-3x10(2) M(-1)s(-1)). Pentasaccharide catalyzed the inactivation rate of factor Xa by the HAT mutants 300-500-fold. A similar 10(4)-10(5)-fold enhancement in the reactivity of factor Xa with prethrombin-2 and the HAT mutants was observed in the presence of the cofactors Va and heparin, respectively. Factor Va did not influence the reactivity of factor Xa with either one of the HAT mutants. These results suggest that (1) in the absence of a cofactor, the P4-P4' residues of HAT and prethrombin-2 primarily determine the specificity reactions with factor Xa, (2) factor Va binding to factor Xa is not associated with allosteric changes in the catalytic pocket of enzyme that would involve interactions with the P4-P4' binding sites, and (3) similar to allosteric activation of HAT by heparin, a role for factor Va in the prothrombinase complex may involve rearrangement of the residues surrounding the scissile bond of the substrate to facilitate its optimal docking into the catalytic pocket of factor Xa.

Asunto(s)

Antitrombinas/metabolismo , Factor Xa/metabolismo , Antitrombinas/biosíntesis , Antitrombinas/genética , Precursores Enzimáticos/metabolismo , Factor V/metabolismo , Factor Va/farmacología , Inhibidores del Factor Xa , Vectores Genéticos , Heparina/farmacología , Humanos , Cinética , Mutación , Fragmentos de Péptidos/farmacología , Protrombina/biosíntesis , Protrombina/química , Protrombina/metabolismo , Proteínas Recombinantes/aislamiento & purificación , Alineación de Secuencia

RESUMEN

Heparin is a commonly used anticoagulant drug. It functions primarily by accelerating the antithrombin inhibition of coagulation proteinases, among which factor Xa and thrombin are believed to be the most important targets. There are conflicting results as to whether anticoagulant heparins can catalyze the antithrombin inhibition of factor Xa in the prothrombinase complex (factor Va, negatively charged membrane surfaces, and calcium ion), which is the physiologically relevant form of the proteinase responsible for the activation of prothrombin to thrombin during the blood coagulation process. In this study, a novel assay system was developed to compare the catalytic effect of different molecular-weight heparins in the antithrombin inhibition of factor Xa, either in free form or assembled into the prothrombinase complex during the process of prothrombin activation. This assay takes advantage of the unique property of a recombinant mutant antithrombin, which, similar to the wild-type antithrombin, rapidly inhibits factor Xa, but not thrombin, in the presence of heparin. A direct prothrombinase inhibition assay, monitoring thrombin generation under near physiological concentrations of prothrombin and antithrombin in the presence of therapeutic doses of low- and high-molecular-weight heparins, indicates that factor Xa in the prothrombinase complex is protected from inhibition by antithrombin more than 1000 times, independent of the molecular size of heparin.

Asunto(s)

Antitrombina III/farmacología , Factor V/efectos de los fármacos , Inhibidores del Factor Xa , Factor Xa/efectos de los fármacos , Heparina/farmacología , Protrombina/farmacología , Secuencia de Aminoácidos , Antitrombina III/genética , Catálisis , Factor Xa/metabolismo , Humanos , Sustancias Macromoleculares , Peso Molecular , Proteínas Recombinantes de Fusión/farmacología , Especificidad por Sustrato , Trombina/antagonistas & inhibidores

RESUMEN

Activated protein C (APC) is a natural anticoagulant in plasma that down-regulates the coagulation cascade by degrading factors Va and VIIIa. In addition to its anticoagulant function, APC is also known to possess a profibrinolytic property. This property of APC has been attributed to its ability to neutralize PAI-1, thereby increasing the concentration of tissue plasminogen activator in plasma leading to up-regulation of the fibrinolytic cascade. This hypothesis, however, has not been well established, since the concentration of PAI-1 in plasma is low, and its reactivity with APC is very slow in a purified system. Here we demonstrate that vitronectin enhances the reactivity of PAI-1 with APC approximately 300-fold making PAI-1 the most efficient inhibitor of APC thus far reported (k(2) = 1.8 x 10(5) m(-)1 s(-)1). We further show that PAI-1 inhibition of the Glu(192) --> Gln mutant of APC is enhanced approximately 40-fold, independent of vitronectin, suggesting that vitronectin partially overcomes the inhibitory interaction of PAI-1 with Glu(192). Additionally, we show that PAI-1 inhibition of the Lys(37)-Lys(38)-Lys(39) --> Pro-Gln-Glu mutant of APC is severely impaired, suggesting that, similar to tissue plasminogen activator, the basic 39-loop of APC plays a critical role in the reaction. Together, these results suggest that vitronectin functions as a cofactor to promote the profibrinolytic activity of APC.

Asunto(s)

Fibrinólisis , Inhibidor 1 de Activador Plasminogénico/farmacología , Proteína C/metabolismo , Vitronectina/metabolismo , Sustitución de Aminoácidos , Activación Enzimática , Humanos , Cinética , Proteína C/antagonistas & inhibidores , Proteínas Recombinantes/farmacología