RESUMO
BACKGROUND: Oxidized phospholipids play a key role in the atherogenic potential of lipoprotein(a) (Lp[a]); however, Lp(a) is a complex particle that warrants research into additional proinflammatory mediators. We hypothesized that additional Lp(a)-associated lipids contribute to the atherogenicity of Lp(a). METHODS: Untargeted lipidomics was performed on plasma and isolated lipoprotein fractions. The atherogenicity of the observed Lp(a)-associated lipids was tested ex vivo in primary human monocytes by RNA sequencing, ELISA, Western blot, and transendothelial migratory assays. Using immunofluorescence staining and single-cell RNA sequencing, the phenotype of macrophages was investigated in human atherosclerotic lesions. RESULTS: Compared with healthy individuals with low/normal Lp(a) levels (median, 7 mg/dL [18 nmol/L]; n=13), individuals with elevated Lp(a) levels (median, 87 mg/dL [218 nmol/L]; n=12) demonstrated an increase in lipid species, particularly diacylglycerols (DGs) and lysophosphatidic acid (LPA). DG and the LPA precursor lysophosphatidylcholine were enriched in the Lp(a) fraction. Ex vivo stimulation with DG(40:6) demonstrated a significant upregulation in proinflammatory pathways related to leukocyte migration, chemotaxis, NF-κB (nuclear factor kappa B) signaling, and cytokine production. Functional assessment showed a dose-dependent increase in the secretion of IL (interleukin)-6, IL-8, and IL-1ß after DG(40:6) and DG(38:4) stimulation, which was, in part, mediated via the NLRP3 (NOD [nucleotide-binding oligomerization domain]-like receptor family pyrin domain containing 3) inflammasome. Conversely, LPA-stimulated monocytes did not exhibit an inflammatory phenotype. Furthermore, activation of monocytes by DGs and LPA increased their transendothelial migratory capacity. Human atherosclerotic plaques from patients with high Lp(a) levels demonstrated colocalization of Lp(a) with M1 macrophages, and an enrichment of CD68+IL-18+TLR4+ (toll-like receptor) TREM2+ (triggering receptor expressed on myeloid cells) resident macrophages and CD68+CASP1+ (caspase) IL-1B+SELL+ (selectin L) inflammatory macrophages compared with patients with low Lp(a). Finally, potent Lp(a)-lowering treatment (pelacarsen) resulted in a reduction in specific circulating DG lipid subspecies in patients with cardiovascular disease with elevated Lp(a) levels (median, 82 mg/dL [205 nmol/L]). CONCLUSIONS: Lp(a)-associated DGs and LPA have a potential role in Lp(a)-induced monocyte inflammation by increasing cytokine secretion and monocyte transendothelial migration. This DG-induced inflammation is, in part, NLRP3 inflammasome dependent.
Assuntos
Lisofosfolipídeos , Monócitos , Proteína 3 que Contém Domínio de Pirina da Família NLR , Humanos , Diglicerídeos/metabolismo , Inflamassomos/metabolismo , Inflamação/metabolismo , Interleucina-1beta/metabolismo , Interleucina-6/metabolismo , Lipoproteína(a)/metabolismo , Monócitos/metabolismo , NF-kappa B/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismoRESUMO
Hyperglycemia and type 2 diabetes (T2D) are caused by failure of pancreatic beta cells. The role of the gut microbiota in T2D has been studied, but causal links remain enigmatic. Obese individuals with or without T2D were included from two independent Dutch cohorts. Human data were translated in vitro and in vivo by using pancreatic islets from C57BL6/J mice and by injecting flagellin into obese mice. Flagellin is part of the bacterial locomotor appendage flagellum, present in gut bacteria including Enterobacteriaceae, which we show to be more abundant in the gut of individuals with T2D. Subsequently, flagellin induces a pro-inflammatory response in pancreatic islets mediated by the Toll-like receptor (TLR)-5 expressed on resident islet macrophages. This inflammatory response is associated with beta-cell dysfunction, characterized by reduced insulin gene expression, impaired proinsulin processing and stress-induced insulin hypersecretion in vitro and in vivo in mice. We postulate that increased systemically disseminated flagellin in T2D is a contributing factor to beta-cell failure in time and represents a novel therapeutic target.
Assuntos
Diabetes Mellitus Tipo 2 , Flagelina , Microbioma Gastrointestinal , Células Secretoras de Insulina , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Diabetes Mellitus Tipo 2/microbiologia , Flagelina/genética , Flagelina/metabolismo , Humanos , Inflamação/metabolismo , Insulina , Células Secretoras de Insulina/metabolismo , CamundongosRESUMO
OBJECTIVE: Increasing evidence indicates that intestinal microbiota play a role in diverse metabolic processes via intestinal butyrate production. Human bariatric surgery data suggest that the gut-brain axis is also involved in this process, but the underlying mechanisms remain unknown. METHODS: We compared the effect of fecal microbiota transfer (FMT) from post-Roux-en-Y gastric bypass (RYGB) donors vs oral butyrate supplementation on (123I-FP-CIT-determined) brain dopamine transporter (DAT) and serotonin transporter (SERT) binding as well as stable isotope-determined insulin sensitivity at baseline and after 4 weeks in 24 male and female treatment-naïve metabolic syndrome subjects. Plasma metabolites and fecal microbiota were also determined at these time points. RESULTS: We observed an increase in brain DAT after donor FMT compared to oral butyrate that reduced this binding. However, no effect on body weight and insulin sensitivity was demonstrated after post-RYGB donor feces transfer in humans with metabolic syndrome. Increases in fecal levels of Bacteroides uniformis were significantly associated with an increase in DAT, whereas increases in Prevotella spp. showed an inverse association. Changes in the plasma metabolites glycine, betaine, methionine, and lysine (associated with the S-adenosylmethionine cycle) were also associated with altered striatal DAT expression. CONCLUSIONS: Although more and larger studies are needed, our data suggest a potential gut microbiota-driven modulation of brain dopamine and serotonin transporters in human subjects with obese metabolic syndrome. These data also suggest the presence of a gut-brain axis in humans that can be modulated. NTR REGISTRATION: 4488.
Assuntos
Transplante de Microbiota Fecal/métodos , Síndrome Metabólica/microbiologia , Síndrome Metabólica/terapia , Idoso , Butiratos/farmacologia , Córtex Cerebral/metabolismo , Proteínas da Membrana Plasmática de Transporte de Dopamina/metabolismo , Método Duplo-Cego , Fezes/microbiologia , Feminino , Microbioma Gastrointestinal , Humanos , Insulina/metabolismo , Resistência à Insulina/fisiologia , Masculino , Síndrome Metabólica/metabolismo , Microbiota , Pessoa de Meia-Idade , Obesidade/metabolismo , Proteínas da Membrana Plasmática de Transporte de Serotonina/metabolismoRESUMO
BACKGROUND: Genetic factors partly determine the risk for premature myocardial infarction (MI). OBJECTIVES: We report the identification of a novel rare genetic variant in a kindred with an autosomal dominant trait for premature MI and atherosclerosis and explored the association of a common nonsynonymous variant in the same gene with the risk of ischemic heart disease (IHD) in a population-based study. METHODS: Next-generation sequencing was performed in a small pedigree with premature MI or subclinical atherosclerosis. A common variant, rs8141797 A>G (p.Asn466Ser), in sushi domain-containing protein 2 (SUSD2) was studied in the prospective Copenhagen General Population Studies (N = 105,408) for association with IHD. RESULTS: A novel heterozygous nonsense mutation in SUSD2 (c.G583T; p.Glu195Ter) was associated with the disease phenotype in the pedigree. SUSD2 protein was expressed in aortic specimens in the subendothelial cell layer and around the vasa vasorum. Furthermore, the minor G-allele of rs8141797 was associated with per allele higher levels of SUSD2 mRNA expression in the heart and vasculature. In the Copenhagen General Population Study, hazard ratios for IHD were 0.92 (95% CI: 0.87-0.97) in AG heterozygotes and 0.86 (0.62-1.19) in GG homozygotes vs noncarrriers (P-trend = .002). Finally, in meta-analysis including 73,983 IHD cases and 215,730 controls, the odds ratio for IHD per G-allele vs A-allele was 0.93 (0.90-0.96) (P = 4.6 × 10-7). CONCLUSIONS: The identification of a truncating mutation in SUSD2, which was associated with premature MI and subclinical atherosclerosis, combined with the finding that a common missense variant in SUSD2 was strongly associated with a lower risk of IHD, suggest that SUSD2 may alter the risk of atherosclerosis.
Assuntos
Predisposição Genética para Doença/genética , Glicoproteínas de Membrana/genética , Isquemia Miocárdica/genética , Adulto , Idoso , Estudos de Casos e Controles , Códon sem Sentido , Feminino , Heterozigoto , Homozigoto , Humanos , Masculino , Metanálise como Assunto , Pessoa de Meia-IdadeRESUMO
GPIHBP1 is a protein localized at the endothelial cell surface that facilitates triglyceride (TG) lipolysis by binding lipoprotein lipase (LPL). Whether Glycosyl Phosphatidyl Inositol high density lipoprotein binding protein 1 (GPIHBP1) function is impaired and may underlie the hyperTG phenotype observed in type 2 diabetes is not yet established. To elucidate the mechanism underlying impaired TG homeostasis in insulin resistance state we studied the effect of insulin on GPIHBP1 protein expression in human microvascular endothelial cells (HMVEC) under flow conditions. Next, we assessed visceral adipose tissue GPIHBP1 protein expression in type 2 diabetes Lepr db/db mouse model as well as in subjects with ranging levels of insulin resistance. We report that insulin reduces the expression of GPIHBP1 protein in HMVECs. Furthermore, GPIHBP1 protein expression in visceral adipose tissue in Lepr db/db mice is significantly reduced as is the active monomeric form of GPIHBP1 as compared to Leprdb/m mice. A similar decrease in GPIHBP1 protein was observed in subjects with increased body weight. GPIHBP1 protein expression was negatively associated with insulin and HOMA-IR. In conclusion, our data suggest that decreased GPIHBP1 availability in insulin resistant state may hamper peripheral lipolysis capacity.
Assuntos
Diabetes Mellitus Tipo 2/genética , Hipertrigliceridemia/genética , Gordura Intra-Abdominal/metabolismo , Receptores de Lipoproteínas/genética , Animais , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/patologia , Modelos Animais de Doenças , Células Endoteliais/metabolismo , Regulação da Expressão Gênica , Humanos , Hipertrigliceridemia/metabolismo , Hipertrigliceridemia/patologia , Insulina/genética , Insulina/metabolismo , Resistência à Insulina/genética , Lipólise/genética , Lipase Lipoproteica/genética , Camundongos , Camundongos Endogâmicos NOD , Microvasos/metabolismo , Triglicerídeos/genética , Triglicerídeos/metabolismoRESUMO
BACKGROUND AND AIMS: Proteoglycan 4 (Prg4) has a high structural similarity with the established atherosclerosis-modulating proteoglycan versican, but its role in atherogenesis is still unknown. Therefore, the impact of Prg4 deficiency on macrophage function in vitro and atherosclerosis susceptibility in vivo was investigated. METHODS: The presence and localization of Prg4 was studied in atherosclerotic lesions. Furthermore, the effect of Prg4 deficiency on macrophage foam cell formation, cholesterol efflux and lipopolysaccharide (LPS) response was determined. Finally, susceptibility for atherosclerotic lesion formation was investigated in bone marrow-specific Prg4 knockout (KO) mice. RESULTS: Prg4 mRNA expression was induced 91-fold (p<0.001) in murine initial atherosclerotic lesions and Prg4 protein co-localized with human lesional macrophages. Murine Prg4 KO macrophages showed increased foam cell formation (+2.1-fold, p<0.01). In parallel, the expression of the cholesterol efflux genes ATP-binding cassette transporter A1 and scavenger receptor type B1 was lower (-35%, p<0.05;-40%, p<0.05) in Prg4 KO macrophages. This translated into an impaired cholesterol efflux to high-density lipoprotein (-13%, p<0.001) and apolipoprotein A1 (-8%, p<0.05). Furthermore, Prg4 KO macrophages showed an impaired LPS-induced rise in TNFα secretion as compared to wild-type controls (-31%, p<0.001), indicating a reduced inflammatory response. Combined, these pro- and anti-atherogenic effects did not translate into a significant difference in atherosclerotic lesion formation upon bone marrow-specific deletion of Prg4 in low-density lipoprotein receptor KO mice. CONCLUSIONS: Prg4 is present in macrophages in both murine and human atherosclerotic lesions and critically influences macrophage function, but deletion of Prg4 in bone marrow-derived cells does not affect atherosclerotic lesion development.
Assuntos
Aterosclerose/metabolismo , Células da Medula Óssea/metabolismo , Artérias Carótidas/metabolismo , Doenças das Artérias Carótidas/metabolismo , Células Espumosas/metabolismo , Macrófagos Peritoneais/metabolismo , Placa Aterosclerótica , Proteoglicanas/metabolismo , Transportador 1 de Cassete de Ligação de ATP/metabolismo , Animais , Aterosclerose/genética , Aterosclerose/patologia , Células da Medula Óssea/patologia , Transplante de Medula Óssea , Artérias Carótidas/patologia , Doenças das Artérias Carótidas/genética , Doenças das Artérias Carótidas/patologia , Células Cultivadas , Colesterol/metabolismo , Modelos Animais de Doenças , Células Espumosas/efeitos dos fármacos , Células Espumosas/patologia , Células Espumosas/transplante , Humanos , Mediadores da Inflamação/metabolismo , Lipopolissacarídeos/farmacologia , Macrófagos Peritoneais/efeitos dos fármacos , Macrófagos Peritoneais/patologia , Macrófagos Peritoneais/transplante , Masculino , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Knockout para ApoE , Fenótipo , Proteoglicanas/deficiência , Proteoglicanas/genética , Receptores Depuradores Classe B/metabolismo , Fator de Necrose Tumoral alfa/metabolismoRESUMO
An altered intestinal microbiota composition has been implicated in the pathogenesis of metabolic disease including obesity and type 2 diabetes mellitus (T2DM). Low grade inflammation, potentially initiated by the intestinal microbiota, has been suggested to be a driving force in the development of insulin resistance in obesity. Here, we report that bacterial DNA is present in mesenteric adipose tissue of obese but otherwise healthy human subjects. Pyrosequencing of bacterial 16S rRNA genes revealed that DNA from the Gram-negative species Ralstonia was most prevalent. Interestingly, fecal abundance of Ralstonia pickettii was increased in obese subjects with pre-diabetes and T2DM. To assess if R. pickettii was causally involved in development of obesity and T2DM, we performed a proof-of-concept study in diet-induced obese (DIO) mice. Compared to vehicle-treated control mice, R. pickettii-treated DIO mice had reduced glucose tolerance. In addition, circulating levels of endotoxin were increased in R. pickettii-treated mice. In conclusion, this study suggests that intestinal Ralstonia is increased in obese human subjects with T2DM and reciprocally worsens glucose tolerance in DIO mice.
Assuntos
Intolerância à Glucose/complicações , Intolerância à Glucose/microbiologia , Infecções por Bactérias Gram-Negativas/microbiologia , Intestinos/microbiologia , Obesidade/complicações , Obesidade/microbiologia , Ralstonia pickettii/fisiologia , Idoso , Animais , DNA Bacteriano/análise , Diabetes Mellitus Tipo 2/complicações , Diabetes Mellitus Tipo 2/microbiologia , Dieta Hiperlipídica , Fezes/microbiologia , Feminino , Infecções por Bactérias Gram-Negativas/patologia , Humanos , Inflamação/complicações , Inflamação/patologia , Intestinos/patologia , Gordura Intra-Abdominal/microbiologia , Gordura Intra-Abdominal/patologia , Masculino , Camundongos Endogâmicos C57BLRESUMO
Studies on the role of B lymphocytes in atherosclerosis development, have yielded contradictory results. Whereas B lymphocyte-deficiency aggravates atherosclerosis in mice; depletion of mature B lymphocytes reduces atherosclerosis. These observations led to the notion that distinct B lymphocyte subsets have different roles. B1a lymphocytes exert an atheroprotective effect, which has been attributed to secretion of IgM, which can be deposited in atherosclerotic lesions thereby reducing necrotic core formation. Tumor necrosis factor (TNF)-family member 'A Proliferation-Inducing Ligand' (APRIL, also known as TNFSF13) was previously shown to increase serum IgM levels in a murine model. In this study, we investigated the effect of APRIL overexpression on advanced lesion formation and composition, IgM production and B cell phenotype. We crossed APRIL transgenic (APRIL-Tg) mice with ApoE knockout (ApoE-/-) mice. After a 12-week Western Type Diet, ApoE-/-APRIL-Tg mice and ApoE-/- littermates showed similar increases in body weight and lipid levels. Histologic evaluation showed no differences in lesion size, stage or necrotic area. However, smooth muscle cell (α-actin stain) content was increased in ApoE-/-APRIL-Tg mice, implying more stable lesions. In addition, increases in both plaque IgM deposition and plasma IgM levels were found in ApoE-/-APRIL-Tg mice compared with ApoE-/- mice. Flow cytometry revealed a concomitant increase in peritoneal B1a lymphocytes in ApoE-/-APRIL-Tg mice. This study shows that ApoE-/-APRIL-Tg mice have increased oxLDL-specific serum IgM levels, potentially mediated via an increase in B1a lymphocytes. Although no differences in lesion size were found, transgenic ApoE-/-APRIL-Tg mice do show potential plaque stabilizing features in advanced atherosclerotic lesions.
Assuntos
Placa Aterosclerótica/imunologia , Placa Aterosclerótica/metabolismo , Membro 13 da Superfamília de Ligantes de Fatores de Necrose Tumoral/metabolismo , Animais , Contagem de Células , Expressão Ectópica do Gene , Humanos , Imunoglobulina M/sangue , Camundongos , Miócitos de Músculo Liso/patologia , Peritônio/imunologia , Placa Aterosclerótica/sangue , Placa Aterosclerótica/patologia , Linfócitos T/citologia , Membro 13 da Superfamília de Ligantes de Fatores de Necrose Tumoral/genéticaRESUMO
BACKGROUND: The prognosis of bacterial meningitis largely depends on the severity of the inflammatory response. The transcription factor CAAT/enhancer-binding protein δ (C/EBPδ) plays a key role in the regulation of the inflammatory response during bacterial infections. Consequently, we assessed the role of C/EBPδ during experimental meningitis. METHODS: Wild-type and C/EBPδ-deficient mice (C/EBPδ(-/-)) were intracisternally infected with Streptococcus pneumoniae and sacrificed after 6 or 30 h, or followed in a survival study. RESULTS: In comparison to wild-type mice, C/EBPδ(-/-) mice showed decreased bacterial loads at the primary site of infection and decreased bacterial dissemination to lung and spleen 30 h after inoculation. Expression levels of the inflammatory mediators IL-10 and KC were lower in C/EBPδ(-/-) brain homogenates, whereas IL-6, TNF-α, IL-1ß, and MIP-2 levels were not significantly different between the two genotypes. Moreover, C/EBPδ(-/-) mice demonstrated an attenuated systemic response as reflected by lower IL-10, IL-6, KC, and MIP-2 plasma levels. No differences in clinical symptoms or in survival were observed between wild-type and C/EBPδ(-/-) mice. CONCLUSION: C/EBPδ in the brain drives the inflammatory response and contributes to bacterial dissemination during pneumococcal meningitis. C/EBPδ does, however, not affect clinical parameters of the disease and does not confer a survival benefit.
Assuntos
Proteína delta de Ligação ao Facilitador CCAAT/metabolismo , Citocinas/metabolismo , Regulação Bacteriana da Expressão Gênica/genética , Inflamação/etiologia , Meningite Pneumocócica/complicações , Streptococcus pneumoniae/fisiologia , Alanina Transaminase/sangue , Animais , Aspartato Aminotransferases/sangue , Carga Bacteriana , Proteína delta de Ligação ao Facilitador CCAAT/genética , Citocinas/genética , Modelos Animais de Doenças , Progressão da Doença , L-Lactato Desidrogenase/sangue , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , RNA Mensageiro/metabolismo , Fatores de TempoRESUMO
INTRODUCTION: One of the major complications in patients with diabetes mellitus is impaired wound healing. The fibrinolytic system is involved in parts of the wound healing process and deficiency of thrombin-activatable fibrinolysis inhibitor (TAFI) results in delayed wound closure. Moreover, levels of TAFI are affected by diabetes mellitus. The aim of this study was to elucidate the effect of hyperglycaemia on TAFI and to determine the effect of deficiency of TAFI on wound healing under hyperglycaemic conditions. MATERIALS AND METHODS: Hyperglycaemia was induced with streptozotocin (STZ) and used as a model for diabetes mellitus. TAFI plasma levels and TAFI gene expression in the liver were determined. Incisional and excisional wound healing were studied in non-treated and STZ-treated wild-type and TAFI-deficient mice. Wound closure was scored daily as open or closed. RESULTS: Mice treated with STZ showed hyperglycaemia, and TAFI plasma levels and TAFI gene expression were increased in diabetic mice. TAFI-deficient mice and diabetic wild-type and diabetic TAFI-deficient mice showed delayed wound healing of incisional wounds. No differences were observed between diabetic and non-diabetic TAFI-deficient mice and between diabetic wild-type and diabetic TAFI-deficient mice. CONCLUSIONS: This study illustrated that TAFI was affected by hyperglycaemia and confirmed that TAFI is involved in wound healing. No additional effect was observed under hyperglycaemic conditions, indicating that deficiency of TAFI did not have an additive or synergistic effect in diabetic wound healing. Further research has to elucidate if TAFI and hyperglycemia affect wound healing via similar mechanisms.
Assuntos
Carboxipeptidase B2/sangue , Diabetes Mellitus/sangue , Diabetes Mellitus/patologia , Animais , Modelos Animais de Doenças , Fibrinólise , Hiperglicemia/sangue , Hiperglicemia/patologia , Camundongos , Camundongos Endogâmicos C57BL , CicatrizaçãoRESUMO
Thrombin-activatable fibrinolysis inhibitor (TAFI), also known as carboxypeptidase R, has been implicated as an important negative regulator of the fibrinolytic system. In addition, TAFI is able to inactivate inflammatory peptides such as complement factors C3a and C5a. To determine the role of TAFI in the hemostatic and innate immune response to abdominal sepsis, TAFI gene-deficient (TAFI-/-) and normal wild-type mice received an i.p. injection with Escherichia coli. Liver TAFI mRNA and TAFI protein concentrations increased during sepsis. In contrast to the presumptive role of TAFI as a natural inhibitor of fibrinolysis, TAFI-/- mice did not show any difference in E. coli-induced activation of coagulation or fibrinolysis, as measured by plasma levels of thrombin-anti-thrombin complexes and D-dimer and the extent of fibrin depositions in lung and liver tissues. However, TAFI-/- mice were protected from liver necrosis as indicated by histopathology and clinical chemistry. Furthermore, TAFI-/- mice displayed an altered immune response to sepsis, as indicated by an increased neutrophil recruitment to the peritoneal cavity and a transiently increased bacterial outgrowth together with higher plasma TNF-alpha and IL-6 levels. These data argue against an important part for TAFI in the regulation of the procoagulant-fibrinolytic balance in sepsis and reveals a thus far unknown role of TAFI in the occurrence of hepatic necrosis.
Assuntos
Carboxipeptidase B2/deficiência , Fígado/lesões , Sepse/prevenção & controle , Animais , Carboxipeptidase B2/genética , Carboxipeptidase B2/fisiologia , Quimiocinas/metabolismo , Citocinas/metabolismo , Infecções por Escherichia coli/enzimologia , Infecções por Escherichia coli/genética , Infecções por Escherichia coli/imunologia , Infecções por Escherichia coli/patologia , Fibrinólise , Fígado/enzimologia , Fígado/imunologia , Fígado/patologia , Pulmão/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neutrófilos/patologia , Cavidade Peritoneal/patologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Sepse/sangue , Sepse/enzimologia , Sepse/patologia , Componente Amiloide P Sérico/metabolismo , Regulação para CimaRESUMO
Activated thrombin-activable fibrinolysis inhibitor (TAFIa) is a carboxypeptidase B that can down-regulate fibrinolysis. TAFIa is a labile enzyme that can be inactivated by conformational instability or proteolysis. TAFI is approximately 40% identical to pancreatic carboxypeptidase B (CPB). In contrast to TAFIa, pancreatic CPB is a stable protease. We hypothesized that regions or residues that are not conserved in TAFIa compared with pancreatic CPB play a role in the conformational instability of TAFIa and that replacement of these non-conserved residues with residues of pancreatic CPB would lead to a TAFIa molecule with an increased stability. Therefore, we have expressed, purified, and characterized two TAFI-CPB chimeras: TAFI-CPB-(293-333) and TAFI-CPB-(293-401). TAFI-CPB-(293-333) could be activated by thrombin-thrombomodulin, but not as efficiently as wild-type TAFI. After activation, this mutant was unstable and was hardly able to prolong clot lysis of TAFI-deficient plasma. Binding of TAFI-CPB-(293-333) to both plasminogen and fibrinogen was normal compared with wild-type TAFI. TAFI-CPB-(293-401) could be activated by thrombin-thrombomodulin, although at a lower rate compared with wild-type TAFI. The activated mutant displayed a markedly prolonged half-life of 1.5 h. Plasmin could both activate and inactivate this chimera. Interestingly, this chimera did not bind to plasminogen or fibrinogen. TAFI-CPB-(293-401) could prolong the clot lysis time in TAFI-deficient plasma, although not as efficiently as wild-type TAFI. In conclusion, by replacing a region in TAFI with the corresponding region in pancreatic CPB, we were able to generate a TAFIa form with a highly stable activity.