Stefan R. Havik

Absence of Thrombin-Activatable Fibrinolysis Inhibitor Protects against Sepsis-Induced Liver Injury in Mice

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-α 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.

Thrombin-activatable fibrinolysis inhibitor influences disease severity in humans and mice with pneumococcal meningitis

Mortality and morbidity in patients with bacterial meningitis result from the proinflammatory response and dysregulation of coagulation and fibrinolysis. Thrombin‐activatable fibrinolysis inhibitor (TAFI) is activated by free thrombin or thrombin in complex with thrombomodulin, and plays an antifibrinolytic role during fibrin clot degradation, but also has an anti‐inflammatory role by inactivating proinflammatory mediators, such as complement activation products.

The role of thrombin-activatable fibrinolysis inhibitor in diabetic wound healing

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.

Role of isoleucine residues 182 and 183 in thrombin-activatable fibrinolysis inhibitor.

Summary.  Thrombin‐activatable fibrinolysis inhibitor (TAFI) is a procarboxypeptidase that, once activated, can attenuate fibrinolysis. The active form, TAFIa, is a labile enzyme, with a half‐life of a few minutes at 37 °C. Understanding the molecular mechanisms of TAFIa inactivation will allow the development of compounds that modulate TAFIa activity. Based on their three‐dimensional model of TAFI, Barbosa Pereira et al. [J Mol Biol (2002), vol. 321, pp. 537–547] suggested that Ile182 and Ile183 were involved in the instability of TAFIa. However, these carboxypeptidases are, unlike TAFIa, stable proteases. Therefore, we constructed, expressed and characterized a TAFI mutant in which Ile182 and Ile183 were changed into the residues found in pancreas carboxypeptidase B at corresponding positions, Arg and Glu. The active form of the mutant, TAFIa‐I182R‐I183E, had a similar half‐life as wild‐type TAFIa, showing that Ile182 and Ile183 were not involved in the regulation of TAFIa stability. Remarkably, however, TAFI‐I182R‐I183E was activated at a lower rate by thrombin–thrombomodulin (mutant: 45 ± 2 U L−1 s−1 and wild type: 103 ± 3 U L−1 s−1), thrombin (mutant: 1 ±0.1 U L−1 s−1 and wild type 3 ± 0.2 U L−1 s−1) and plasmin (mutant: 0.8 ± 0.04 U L−1 s−1 and wild type: 5.0 ±0.2 U L−1 s−1) compared with wild‐type TAFI. Accordingly, it had a sixfold reduced antifibrinolytic potential. In conclusion, analysis of TAFI‐I182R‐I183E showed that I182 and I183 are not involved in TAFIa inactivation by conformational instability but that these residues may be involved in the activation of TAFI and stabilization of the fibrin clot.

CCAAT/enhancer-binding protein δ (C/EBPδ) aggravates inflammation and bacterial dissemination during pneumococcal meningitis

BackgroundThe 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.MethodsWild-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.ResultsIn 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-1β, 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.ConclusionC/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.