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BACKGROUND AND GOALS: Active inflammatory bowel diseases (IBD) represent an independent risk factor for venous thromboembolism. The authors investigated the hemostatic profile of IBD patients before and after induction treatment with infliximab, vedolizumab, and methylprednisolone. STUDY: This prospective study included 62 patients with active IBD starting infliximab, vedolizumab, and/or methylprednisolone, and 22 healthy controls (HC). Plasma was collected before (w0) and after induction therapy (w14). Using a clot lysis assay, amplitude (marker for clot intensity), time to peak (Tmax; marker for clot formation rate), area under the curve (AUC; global marker for coagulation/fibrinolysis), and 50% clot lysis time (50%CLT; marker for fibrinolytic capacity) were determined. Plasminogen activator inhibitor-1 (PAI-1) and fibronectin were measured by ELISA. Clinical remission was evaluated at w14. RESULTS: At baseline, AUC, amplitude, and 50%CLT were significantly higher in IBD patients as compared with HC. In 34 remitters, AUC [165 (103-229)% vs. 97 (78-147)%, P=0.001], amplitude [119 (99-163)% vs. 95 (82-117)%, P=0.002], and 50%CLT [122 (94-146)% vs. 100 (87-129)%, P=0.001] decreased significantly and even normalized to the HC level. Vedolizumab trough concentration correlated inversely to fibronectin concentration (r, -0.732; P=0.002). The increase in Tmax for infliximab-treated remitters was significantly different from the decrease in Tmax for vedolizumab-treated remitters (P=0.028). The 50%CLT increased (P=0.038) when remitters were concomitantly treated with methylprednisolone. CONCLUSIONS: Control of inflammation using infliximab most strongly reduced those parameters that are associated with a higher risk of venous thromboembolism.
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Enfermedades Inflamatorias del Intestino , Trombosis , Fibrinólisis , Humanos , Enfermedades Inflamatorias del Intestino/tratamiento farmacológico , Infliximab/efectos adversos , Estudios ProspectivosRESUMEN
The clinical response in ankylosing spondylitis (AS) patients treated with biologic agents can be influenced by pharmacokinetic variability among and within these patients. Therapeutic drug monitoring is seen as a valuable tool to improve patient care. The aim of this study was to generate a panel of mAbs toward etanercept (ETN) and to determine ETN and anti-ETN concentrations in AS patients. mAbs against ETN (MA-ETN) were generated using hybridoma technology. For quantification of ETN concentrations, a mAb-based TNF-coated ELISA and a mAb/mAb-based sandwich-type ELISA were developed. For evaluation of the anti-ETN Ab response, a bridging ELISA, as well as a functional cell-based assay, were constructed. Disease activity of the AS patients was measured with the AS Disease Activity Score (ASDAS). Active disease was defined as ASDAS ≥ 2.1. A total of 59 of 76 generated mAbs were ETN specific and were characterized further. Fifty-one mAbs revealed inhibitory properties in a cell-based assay. Analysis of serum concentrations of 21 ETN-treated AS patients with the TNF/MA-ETN68C5-HRP ELISA and the MA-ETN63C8/MA-ETN61C1-HRP ELISA revealed a good Pearson's r (+0.974) but a poor intraclass correlation coefficient (+0.528) as the result of underestimation of the values in the former ELISA. At 24 wk, ETN concentrations were similar in patients with ASDAS < 2.1 and ≥ 2.1. Anti-ETN Abs were not detected in any of the patient samples tested. In conclusion, highly sensitive mAb-based immunoassays were developed for quantification of ETN and anti-ETN concentrations. The impact of these methods needs to be evaluated further in clinical practice.
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Anticuerpos Bloqueadores/metabolismo , Anticuerpos Monoclonales/metabolismo , Etanercept/uso terapéutico , Espondilitis Anquilosante/terapia , Ensayo de Inmunoadsorción Enzimática , Etanercept/inmunología , Etanercept/metabolismo , Humanos , Hibridomas , Monitoreo Fisiológico/métodos , Variaciones Dependientes del Observador , Índice de Severidad de la Enfermedad , Espondilitis Anquilosante/inmunología , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Circulating thrombin-activatable fibrinolysis inhibitor (TAFI) and plasminogen activator inhibitor-1 (PAI-1) are causal factors for thrombolytic failure. Therefore, we evaluated an antibody-engineered bispecific inhibitor against TAFI and PAI-1 (heterodimer diabody, Db-TCK26D6x33H1F7) in several mouse models of thrombosis and stroke. Prophylactic administration of the diabody (0.8 mg/kg) in a thromboplastin-induced model of thromboembolism led to decreased lung fibrin deposition. In a model of cerebral ischemia and reperfusion, diabody administration (0.8 mg/kg, 1 hour postocclusion) led to a mitigated cerebral injury with a 2.3-fold reduced lesion and improved functional outcomes. In a mouse model of thrombin-induced middle cerebral artery occlusion, the efficacy of the diabody was compared to the standard thrombolytic treatment with recombinant tissue-type plasminogen activator (tPA). Early administration of diabody (0.8 mg/kg) caused a twofold decrease in brain lesion size, whereas that of tPA (10 mg/kg) had a much smaller effect. Delayed administration of diabody or tPA had no effect on lesion size, whereas the combined administration of diabody with tPA caused a 1.7-fold decrease in lesion size. In contrast to tPA, the diabody did not increase accumulative bleeding. In conclusion, administration of a bispecific inhibitor against TAFI and PAI-1 results in a prominent profibrinolytic effect in mice without increased bleeding.
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Anticuerpos Biespecíficos/uso terapéutico , Fibrinolíticos/uso terapéutico , Histona Acetiltransferasas/inmunología , Serpina E2/inmunología , Accidente Cerebrovascular/terapia , Factores Asociados con la Proteína de Unión a TATA/inmunología , Terapias en Investigación/métodos , Factor de Transcripción TFIID/inmunología , Tromboembolia Venosa/terapia , Animales , Anticuerpos Biespecíficos/química , Anticuerpos Biespecíficos/metabolismo , Modelos Animales de Enfermedad , Femenino , Inmunoterapia , Masculino , Ratones , Ratones Endogámicos C57BL , Multimerización de Proteína , Accidente Cerebrovascular/patología , Tromboembolia Venosa/patologíaRESUMEN
BACKGROUND AND PURPOSE: Cerebral ischemia and reperfusion is associated with activation of the coagulation cascade and fibrin deposition in cerebral microvessels. Both thrombin-activatable fibrinolysis inhibitor (TAFI) and plasminogen activator inhibitor-1 (PAI-1) attenuate fibrinolysis and are therefore attractive targets for the treatment of ischemic stroke. METHODS: TAFI and PAI-1 were inhibited by monoclonal antibodies in a mouse model of transient middle cerebral artery occlusion. Twenty-four hours after stroke, mice were neurologically scored, cerebral thrombotic burden was assessed, and brain infarct sizes were calculated. RESULTS: Inhibition of TAFI or PAI-1 significantly decreased cerebral infarct sizes by 50% 24 hours after stroke. This reduction in cerebral damage was associated with a significant decrease in fibrin(ogen) deposition in the ischemic brain. Concurrently, functional recovery of the animals was improved. Interestingly, combined targeting of TAFI and PAI-1 using low, and by themselves inactive, doses of antibodies improved cerebral blood flow and reduced cerebral fibrin(ogen) deposition and infarct sizes by 50%. When dual treatment was delayed to 1 hour after the start of reperfusion, it still reduced brain injury; however, this was not statistically significant. CONCLUSIONS: Targeting of PAI-1 and TAFI is protective in an ischemic stroke model by attenuating fibrin(ogen) deposition, thereby improving reperfusion. Combined inhibition has a co-operative effect that could become useful in ischemic stroke therapy.
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Anticuerpos Monoclonales/uso terapéutico , Isquemia Encefálica/tratamiento farmacológico , Encéfalo/efectos de los fármacos , Carboxipeptidasa B2/inmunología , Inhibidor 1 de Activador Plasminogénico/inmunología , Accidente Cerebrovascular/tratamiento farmacológico , Animales , Anticuerpos Monoclonales/farmacología , Modelos Animales de Enfermedad , RatonesRESUMEN
BACKGROUND: Therapeutic drug monitoring of adalimumab (ADM) has been introduced recently. When no detectable ADM serum concentrations can be found, the formation of antidrug antibodies (ADA) should be investigated. A variety of assays to measure the occurrence of ADA have been developed. Results are expressed as arbitrary units or as a titration value. The aim was to develop a monoclonal antibody (MA) that could serve as a universal calibrator to quantify the amount of ADA in ADM-treated patients. METHODS: Hybridoma technology was used to generate a MA toward ADM. The functionality of the MA was tested in a bridging enzyme linked immunosorbent assay (ELISA) setup and in a cell-based assay. Sera from 25 anti-tumor necrosis factor naive patients with inflammatory bowel disease were used to determine the cutoff values. Sera from 9 ADM-treated patients with inflammatory bowel disease, with undetectable serum concentrations of ADM were used to quantify the ADA response. RESULTS: In this study, MA-ADM6A10, an IgG1 that can be used as a calibrator in both an ELISA to quantify the amount of binding antibodies and in a cell-based assay to quantify the amount of neutralizing antibodies, was generated. Combining the results of both assays showed that the sera with high concentrations of anti-ADM binding antibodies also had the highest neutralizing capacity. CONCLUSIONS: The availability of a universal calibrator could facilitate the interlaboratory harmonization of antibody titers in patients who develop anti-adalimumab antibodies.
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Anticuerpos Monoclonales Humanizados/sangre , Anticuerpos/sangre , Anticuerpos/inmunología , Inmunoensayo/métodos , Inmunoglobulina G/inmunología , Laboratorios/normas , Adalimumab , Antiinflamatorios/sangre , Antiinflamatorios/inmunología , Anticuerpos Monoclonales Humanizados/inmunología , Línea Celular Tumoral , Monitoreo de Drogas/métodos , Fibrosarcoma/metabolismo , Regulación de la Expresión Génica/fisiología , Humanos , Hibridomas/inmunología , Interleucina-6/genética , Interleucina-6/metabolismoRESUMEN
INTRODUCTION: Chronic inflammatory diseases, including psoriasis, are associated with development of venous thromboembolism (VTE). The clot lysis profile (CLP) provides information on both the clotting tendency and fibrinolysis activity. We hypothesized that CLP in uncontrolled psoriasis patients is disturbed towards more clotting/less lysis compared to healthy controls (HC) and that successful psoriasis treatment could normalize the CLP. In this project, we aim to compare the CLP in patients with uncontrolled psoriasis with age- and sex-matched HC and investigate the effect of anti-inflammatory treatment on CLP. METHODS: Patients with uncontrolled psoriasis [psoriasis area severity index (PASI) or body surface area (BSA) > 10] (n = 87) and HC (n = 87) were recruited at a tertiary dermatology department. Samples from patients were obtained before treatment and when disease control was obtained (PASI < 3). Amplitude, area under the curve (AUC) and 50% clot lysis time were determined. RESULTS: At baseline, psoriasis patients had higher median amplitude and AUC compared with HC (p < 0.0001). After correction for possible confounders (BMI, smoking behavior, psoriatic arthritis, arterial hypertension, diabetes and coronary artery disease), the increased amplitude in psoriasis patients compared to HC remained significant. Successful anti-inflammatory treatment resulted in a significant decrease in amplitude (p = 0.0365). CONCLUSION: This is the first prospective study comparing the CLP of psoriasis patients with that of HC. A significant increase in both amplitude and area under the curve, indicative of a hypercoagulable CLP, was observed in psoriasis patients compared to HC. After successful anti-inflammatory treatment, amplitude significantly decreased.
RESUMEN
The enhancement of fibrinolysis constitutes a promising approach to treat thrombotic diseases. Activated thrombin activatable fibrinolysis inhibitor (TAFIa) attenuates fibrinolysis and is an attractive target to develop profibrinolytic drugs. TAFI can be activated by thrombin, thrombin/thrombomodulin, or plasmin, but the in vivo physiologic TAFI activator(s) are unknown. Here, we generated and characterized MA-TCK26D6, a monoclonal antibody raised against human TAFI, and examined its profibrinolytic properties in vitro and in vivo. In vitro, MA-TCK26D6 showed a strong profibrinolytic effect caused by inhibition of the plasmin-mediated TAFI activation. In vivo, MA-TCK26D6 significantly decreased fibrin deposition in the lungs of thromboembolism-induced mice. Moreover, in the presence of MA-TCK26D6, plasmin-α(2)-antiplasmin complexes in plasma of thromboembolism-induced mice were significantly increased compared with a control antibody, indicative of an acceleration of fibrinolysis through MA-TCK26D6. In this study, we show that plasmin is an important TAFI activator that hampers in vitro clot lysis. Furthermore, this is the first report on an anti-TAFI monoclonal antibody that demonstrates a strong profibrinolytic effect in a mouse thromboembolism model.
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Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/farmacología , Carboxipeptidasa B2/inmunología , Fibrinólisis/inmunología , Tromboembolia/terapia , Animales , Afinidad de Anticuerpos/inmunología , Antitrombina III/inmunología , Carboxipeptidasa B2/metabolismo , Reacciones Cruzadas/inmunología , Modelos Animales de Enfermedad , Humanos , Técnicas In Vitro , Pulmón/irrigación sanguínea , Pulmón/inmunología , Ratones , Ratones Mutantes , Péptido Hidrolasas/inmunología , Especificidad de la Especie , Tromboembolia/inmunología , Tromboembolia/metabolismoRESUMEN
Therapeutic drug monitoring, which is the measurement of drug concentrations in the blood, is a useful tool to guide clinical decision-making and treatment adjustments, on the condition that drug concentrations are correlated with treatment response. For guselkumab, an anti-IL-23 monoclonal antibody for the treatment of moderate-to-severe psoriasis, such a concentration-response relationship could not yet be determined as no commercial assays for the quantification of this drug or antibodies against this drug are available. Therefore, the aim of this study was to develop and validate immunoassays for the quantification of guselkumab and anti-guselkumab antibodies according to the guidelines of the European Medicines Agency (EMA). A diverse panel of 20 highly specific anti-guselkumab monoclonal antibodies (MA-GUS) was generated of which eight revealed a neutralizing capacity of ≥65 %. At least seven different antibody clusters were identified based on their epitope binning profile. Using MA-GUS9F6 as the capture antibody and MA-GUS12G12 as the detection antibody, an ELISA was developed with a dose-response curve ranging from 0.08 to 5 ng/mL. The assay was specific, selective and could accurately and precisely quantify guselkumab concentrations in spiked healthy control serum and serum from guselkumab-treated psoriasis patients with a cut-off for quantification of 0.014 µg/mL. The presence of IL-23 in physiological concentrations or of non-neutralizing antibodies did not impact the quantification of guselkumab, while the presence of neutralizing antibodies did. Using MA-GUS12A9 as a calibrator, two anti-guselkumab antibody assays were developed to detect anti-guselkumab antibodies, which differ in the threshold for detection and quantification and the tolerance to the presence of guselkumab. Together, these validated immunoassays are essential to establish a concentration-response relationship and will allow the future implementation of therapeutic drug monitoring in moderate-to-severe psoriasis patients receiving guselkumab treatment.
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Anticuerpos Monoclonales Humanizados , Psoriasis , Anticuerpos Monoclonales , Humanos , Inmunoensayo , Psoriasis/tratamiento farmacológicoRESUMEN
Around 70% of circulating alpha-2-antiplasmin (α2AP), the main natural plasmin inhibitor, is N-terminally cleaved between residues Pro12 and Asn13 by antiplasmin-cleaving enzyme. This converts native Met-α2AP into the more potent fibrinolysis inhibitor Asn-α2AP. The Arg6Trp (R6W) polymorphism affects the N-terminal cleavage rate of Met-α2AP in a purified system, with ~8-fold faster conversion of Met(R6)-α2AP than Met(W6)-α2AP. To date, assays to determine N-terminally intact Met-α2AP in plasma have been limited to an ELISA that only measures Met(R6)-α2AP. The aim of this study was to generate and characterize monoclonal antibodies (mAbs) against Met(R6)-α2AP, Met(W6)-α2AP and all α2AP forms (total-α2AP) in order to develop specific Met(R6)-α2AP and Met(W6)-α2AP ELISAs. Recombinant Met(R6)-α2AP, Met(W6)-α2AP and Asn-α2AP were expressed in Drosophila S2 cells. Using hybridoma technology, a panel of 25 mAbs was generated against a mixture of recombinant Met(R6)-α2AP and Met(W6)-α2AP. All mAbs were evaluated for their specific reactivity using the three recombinant α2APs in one-site non-competitive ELISAs. Three mAbs were selected to develop sandwich-type ELISAs. MA-AP37E2 and MA-AP34C4 were selected for their specific reactivity against Met(R6)-α2AP and Met(W6)-α2AP, respectively, and used for coating. MA-AP15D7 was selected for its reactivity against total-α2AP and used for detection. With the novel ELISAs we determined Met(R6)-α2AP and Met(W6)-α2AP levels in plasma samples and we showed that Met(R6)-α2AP was converted faster into Asn-α2AP than Met(W6)-α2AP in a plasma milieu. In conclusion, we developed two specific ELISAs for Met(R6)-α2AP and Met(W6)-α2AP, respectively, in plasma. This will enable us to determine N-terminal heterogeneity of α2AP in plasma samples.
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Anticuerpos Monoclonales/química , Ensayo de Inmunoadsorción Enzimática/normas , alfa 2-Antiplasmina/análisis , alfa 2-Antiplasmina/inmunología , Sustitución de Aminoácidos , Animales , Anticuerpos Monoclonales/biosíntesis , Anticuerpos Monoclonales/aislamiento & purificación , Especificidad de Anticuerpos , Arginina/genética , Arginina/inmunología , Clonación Molecular , Drosophila/citología , Ensayo de Inmunoadsorción Enzimática/métodos , Fibrinólisis/efectos de los fármacos , Expresión Génica , Humanos , Hibridomas/química , Hibridomas/inmunología , Ratones , Ratones Endogámicos BALB C , Dominios Proteicos , Proteolisis , Proteínas Recombinantes/sangre , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/farmacología , Triptófano/genética , Triptófano/inmunología , alfa 2-Antiplasmina/genética , alfa 2-Antiplasmina/farmacologíaRESUMEN
OBJECTIVE: To date, quantitation of TAFI antigen levels has been mainly focused on "total" antigen levels and has been shown to yield ambiguous results because of the existence of different isoforms and various degrees of activation. Our objective was to develop assays that allow measuring the extent of TAFI activation. METHODS AND RESULTS: A variety of enzyme-linked immunosorbent assays (ELISAs) were evaluated for their preferential reactivity toward TAFI before and after activation, and toward the recombinantly expressed activation peptide. Three ELISAs with distinct reactivities were selected: recognizing either exclusively nonactivated TAFI, the released activation peptide, or exclusively TAFIa (activated TAFI). Evaluation of TAFI activation during clot lysis revealed that decreases of TAFI levels are associated with increases of the released activation peptide and TAFIa levels. In addition, antigenic measurement of TAFIa parallels activity measured by chromogenic assay. Analyzing plasma samples revealed that subjects with hyperlipidemia had significantly higher plasma levels of both the activation peptide (109.2 versus 95.5; P<0.001) and TAFIa (112.1 versus 103.3; P=0.03), and not of TAFI antigen (92.5 versus 87.9; P=0.07) (results in % of plasma pooled from normolipidemic subjects). CONCLUSIONS: ELISAs that allow to measure the extent of TAFI activation were developed. These ELISAs constitute more sensitive markers in studies on the relationship between TAFI and cardiovascular diseases.
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Carboxipeptidasa B2/sangre , Ensayo de Inmunoadsorción Enzimática/métodos , Isoenzimas/sangre , Trombosis/sangre , Trombosis/enzimología , Biomarcadores , Carboxipeptidasa B2/análisis , Activación Enzimática , Humanos , Hiperlipidemias/sangre , Hiperlipidemias/enzimología , Isoenzimas/análisis , Fragmentos de Péptidos/análisis , Fragmentos de Péptidos/sangre , Sensibilidad y Especificidad , SueciaRESUMEN
BACKGROUND: Vedolizumab (VDZ) has been approved for the treatment of patients with moderate-to-severe Crohn's disease and ulcerative colitis. The GEMINI trials reported a low prevalence of anti-VDZ antibodies (AVA). However, the AVA assays used in GEMINI were drug sensitive, resulting in a possible underestimation of the rate of AVA formation. This study aimed to monitor immunogenicity of VDZ in a real-life cohort using a drug-resistant AVA assay. METHODS: Using a combination of VDZ/AVA complex precipitation and acid dissociation, a drug-resistant assay for AVA detection in the presence of high VDZ concentrations has been developed and analytically validated. The assay was applied on serum samples of 179 VDZ-treated patients with inflammatory bowel disease to evaluate the prevalence and time course of AVA. RESULTS: A dose-response curve ranging from 25 to 1600 ng/mL using 1/125 diluted serum was obtained, allowing the detection of AVA concentrations up to 200 µg/mL of MA-VDZ19C11 equivalents, a calibrator antibody to VDZ. This assay was highly AVA specific and drug resistant. Four of 179 VDZ-treated patients (2.2%) were AVA positive and AVA were detected already after the first VDZ infusion. AVA were all transient in these patients without need for any dosage optimization. There was no correlation between VDZ and AVA concentrations in the AVA-positive samples. CONCLUSIONS: AVA appear from the first VDZ infusion onward and disappear over time. The low prevalence of AVA suggests that immunogenicity does not influence response to treatment.
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Anticuerpos Antiidiotipos/inmunología , Anticuerpos Monoclonales Humanizados/inmunología , Anticuerpos Monoclonales Humanizados/farmacología , Resistencia a Medicamentos/inmunología , Fármacos Gastrointestinales/inmunología , Fármacos Gastrointestinales/farmacología , Enfermedades Inflamatorias del Intestino/tratamiento farmacológico , Anticuerpos Antiidiotipos/sangre , Estudios de Casos y Controles , Estudios de Cohortes , Femenino , Estudios de Seguimiento , Humanos , Enfermedades Inflamatorias del Intestino/sangre , Enfermedades Inflamatorias del Intestino/inmunología , MasculinoRESUMEN
OBJECTIVE: A Thr/Ile polymorphism at position 325 in the coding region of proCPU has been reported. Immunological assays, fully characterized (including genotype dependency), are required for the quantitation of proCPU levels. METHODS AND RESULTS: We have generated a panel of monoclonal antibodies against human, plasma-derived proCPU. Two combinations exhibiting distinct reactivities were selected for measurement of proCPU in plasma. T12D11/T28G6-HRP yielded values of 10.1+/-3.1 microg/mL (mean+/-SD, n=86; normal donors), and T32F6/T9G12-HRP yielded values of 5.4+/-3.0 microg/mL. Grouping according to the 325 genotype demonstrated that T12D11/T28G6-HRP was independent to this polymorphism whereas T32F6/T9G12-HRP revealed a complete lack of reactivity with the Ile/Ile genotype (ie, 0.0+/-0.0, 4.2+/-1.7, and 7.3+/-2.9 microg/mL for the Ile/Ile, Ile/Thr, and Thr/Thr isoforms, respectively). Commercially available antigen assays appeared to be partially dependent on the 325 genotype (eg, 44+/-8.9% and 100+/-30% for the Ile/Ile and Thr/Thr isoforms, respectively). CONCLUSIONS: Our data demonstrate that great care should be taken when evaluating proCPU antigen values as a putative causative agent or as a diagnostic risk marker for cardiovascular events.
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Carboxipeptidasa B2/análisis , Carboxipeptidasa B2/genética , Ensayo de Inmunoadsorción Enzimática , Isoenzimas/genética , Animales , Anticuerpos Monoclonales/inmunología , Especificidad de Anticuerpos , Biomarcadores , Carboxipeptidasa B2/inmunología , Enfermedades Cardiovasculares/epidemiología , Estudios de Casos y Controles , Activación Enzimática , Genotipo , Humanos , Isoenzimas/inmunología , Ratones , Ratones Endogámicos BALB C , Fragmentos de Péptidos/inmunología , Factores de Riesgo , Trombina/metabolismo , Trombomodulina/metabolismoRESUMEN
BACKGROUND: Inflammatory bowel disease (IBD) is recognized as an independent risk factor for thrombosis. First, we investigate whether the concentration of fibrinolysis inhibitors is increased in patients with IBD. Second, we investigate the effect of infliximab induction therapy on the hemostatic profile. METHODS: This prospective study included 103 patients with IBD starting infliximab therapy and 113 healthy controls. Plasma was collected before the first infliximab infusion (wk 0) and after induction therapy (wk 14). Patients not showing a clinical response on induction were considered as primary nonresponders. Fibrinolysis inhibitors were measured by enzyme-linked immunosorbent assay. Using a clot lysis assay, the area under the curve (global marker for coagulation/fibrinolysis), 50% clot lysis time (marker for fibrinolytic capacity), and amplitude (indicator for clot formation) were determined. RESULTS: Patients with IBD selected for infliximab treatment have higher area under the curve (median 29 [interquartile range, 20-38]) and amplitude (0.4 [0.3-0.5]) compared with healthy controls (18 [13-24] and 0.3 [0.2-0.3], respectively, P < 0.001). Primary nonresponders showed a decrease neither in inflammatory markers nor in hemostatic parameters, whereas in primary responders, a decrease in inflammatory markers was associated with a decrease in both area under the curve (29 [20-38] (wk 0) to 20 [14-28] (wk 14), P < 0.001) and amplitude (0.4 [0.3-0.5] (wk 0) to 0.3 [0.3-0.4] (wk 14), P < 0.001). CONCLUSIONS: This is the first prospective study demonstrating that the clot lysis profile differs between patients with IBD and healthy individuals. On infliximab induction treatment, this clot lysis profile normalizes in responders suggesting that infliximab treatment is advisable for patients with IBD with an activated hemostatic profile.
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Colitis Ulcerosa/complicaciones , Enfermedad de Crohn/complicaciones , Fibrinólisis/efectos de los fármacos , Fármacos Gastrointestinales/uso terapéutico , Infliximab/uso terapéutico , Trombosis/tratamiento farmacológico , Adolescente , Adulto , Anciano , Estudios de Casos y Controles , Colitis Ulcerosa/patología , Enfermedad de Crohn/patología , Femenino , Humanos , Masculino , Persona de Mediana Edad , Pronóstico , Estudios Prospectivos , Trombosis/sangre , Trombosis/etiología , Adulto JovenRESUMEN
BACKGROUND: The occurrence of thromboembolic events (TE) is an important extraintestinal manifestation in patients with inflammatory bowel disease (IBD). The aim of this study was to compare fibrinolysis and clot lysis parameters between (1) patients with IBD and healthy controls and (2) patients with IBD with TE (IBD + TE) and without TE (IBD - TE). METHODS: One hundred thirteen healthy controls and 202 patients with IBD, of which 84 patients with IBD + TE and 118 patients with IBD - TE, were included in this case-control study. Three clot lysis parameters (area under the curve, 50% clot lysis time, and amplitude) were determined using a clot lysis assay. Plasminogen activator inhibitor 1 (PAI-1) and thrombin activatable fibrinolysis inhibitor concentrations were determined by enzyme-linked immunosorbent assay. RESULTS: PAI-1 antigen, active PAI-1, and intact thrombin activatable fibrinolysis inhibitor concentrations, as well as 50% clot lysis time and area under the curve, were significantly associated with the presence of IBD (all P < 0.05). The median time between TE and plasma collection was 5.0 (1.8-11.0) years. Comparing IBD + TE versus IBD - TE, active to total PAI-1 ratio (0.36 [0.24-0.61] versus 0.24 [0.13-0.40]), area under the curve (31 [24-49] versus 22 [13-31]), 50% clot lysis time (110 [64-132] versus 95 [70-126] minutes), and amplitude (0.295 [0.222-0.436] versus 0.241 [0.168-0.308]) were significantly higher in IBD + TE (all P <0.05) and remained higher after adjustment for age, gender, C-reactive protein, type of disease, presence of comorbidities, and disease activity. CONCLUSIONS: Patients with IBD have an altered clot lysis profile compared with healthy controls. Clot lysis parameters differ significantly between patients with IBD with and without a history of TE and should be included in the risk assessment.
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Carboxipeptidasa B2/sangre , Enfermedades Inflamatorias del Intestino/complicaciones , Inhibidor 1 de Activador Plasminogénico/sangre , Tromboembolia/sangre , Trombosis/sangre , Adulto , Área Bajo la Curva , Estudios de Casos y Controles , Ensayo de Inmunoadsorción Enzimática , Femenino , Humanos , Masculino , Persona de Mediana EdadRESUMEN
BACKGROUND: Both activated Thrombin Activatable Fibrinolysis Inhibitor (TAFI) and active Plasminogen Activator Inhibitor-1 (PAI-1) attenuate fibrinolysis and may therefore contribute to the pathophysiology of Venous ThromboEmbolism (VTE). Whether increased TAFI and/or PAI-1 concentrations are associated with VTE is unclear. OBJECTIVE: To study an association of impaired fibrinolysis and VTE using a comprehensive panel of in-house developed assays measuring intact TAFI, activation peptide of TAFI (AP-TAFI), PAI-1 antigen, endogenous PAI-1:t-PA complex (PAI-1:t-PA) and active PAI-1 levels in 102 VTE patients and in 113 healthy controls (HC). RESULTS: Active PAI-1 was significantly higher in VTE patients compared to HC (20.9 [9.6-37.8] ng/ml vs. 6.2 [3.5-9.7] ng/ml, respectively). Active PAI-1 was the best discriminator with an area under the ROC curve and 95% confidence interval (AUROC [95%CI]) of 0.84 [0.79-0.90] compared to 0.75 [0.68-0.72] for PAI-1:t-PA, 0.65 [0.58-0.73] for PAI-1 antigen, 0.62 [0.54-0.69] for AP-TAFI and 0.51 [0.44-0.59] for intact TAFI. Using ROC analysis, we defined an optimal cut-off of 12.8 ng/ml for active PAI-1, with corresponding sensitivity of 71 [61-79] % and specificity of 89 [82-94] %. A lack of association with the time between VTE event and sample collection or with the intake of anticoagulant treatment suggests that active PAI-1 levels are sustainable high in VTE patients. CONCLUSIONS: This case-control study emphasizes the clinical importance of measuring active PAI-1 instead of PAI-1 antigen and identifies active PAI-1 as a potential marker of VTE. Prognostic studies will need to address the clinical significance of active PAI-1 as biomarker.
Asunto(s)
Carboxipeptidasa B2/metabolismo , Inhibidor 1 de Activador Plasminogénico/metabolismo , Tromboembolia Venosa/sangre , Tromboembolia Venosa/diagnóstico , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Carboxipeptidasa B2/sangre , Estudios de Casos y Controles , Femenino , Fibrinólisis , Humanos , Masculino , Persona de Mediana Edad , Inhibidor 1 de Activador Plasminogénico/sangre , Tromboembolia Venosa/metabolismo , Adulto JovenRESUMEN
Activated thrombin activable fibrinolysis inhibitor (TAFIa), generated upon activation of TAFI, exerts an antifibrinolytic effect. TAFIa is a thermolabile enzyme, inactivated through a conformational change. The objective of the current study was to generate a stable variant of human TAFIa. Using a site-directed as well as a random mutagenesis approach to generate a library of TAFI mutants, we identified two mutations that increase TAFIa stability, i.e. a Ser305 to Cys and a Thr329 to Ile mutation, respectively. Combining these mutations in TAFI-Ala147-Ile325, the most stable isoform of TAFIa (half-life of 9.4 +/- 0.4 min), revealed a TAFIa half-life of 70 +/- 3.1 min (i.e. an 11-fold increase versus 6.3 +/- 0.3 min for TAFIa-Ala147-Thr325, the most frequently occurring isoform of TAFI in humans) at 37 degrees C. Moreover, clot lysis (induced by tissue plasminogen activator) experiments in which TAFI-Ala147-Cys305-Ile325-Ile329 was added to TAFI-depleted plasma revealed a 50% clot lysis time of 313 +/- 77 min (i.e. a 3.0-fold increase versus 117 +/- 10 min for TAFI-Ala147-Thr325). The availability of a more stable TAFIa variant will facilitate the search for inhibitors and allow further structural analysis to elucidate the mechanisms of the instability of TAFIa.