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ABSTRACT: Emerging evidence indicates that various haemostatic components can regulate the progression of liver disease. Thrombin-activatable fibrinolysis inhibitor (TAFI) possesses anti-inflammatory properties besides its anti-fibrinolytic function. Here, we investigated the contribution of TAFI to the progression of disease in murine models of chronic and acute liver failure. Chronic carbon tetrachloride (CCL4) administration induced liver damage and fibrosis both in TAFI knockout (TAFI-/-) mice and wild-type controls. Smooth muscle actin-α (α-SMA) content of liver tissue was significantly increased after 1 and 3 weeks, and pro-collagen α1 expression was significantly increased after 3 and 6 weeks in TAFI-/- mice. TAFI-/- mice showed significantly elevated levels of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) after 3 weeks of CCL4. Neutrophil influx was significantly increased in TAFI-/- mice after 6 weeks of CCL4. No difference in hepatic fibrin deposition between TAFI-/- and wild-types was observed. After acetaminophen intoxication, necrosis was significantly increased in TAFI-/- mice at 24 hours (h) after injection. AST and ALT levels were decreased at 2 and 6 h after acetaminophen injection in TAFI-/- mice, but were significantly higher in the TAFI-/- mice at 24 h. Similarly, hepatic fibrin deposition was decreased at 6 h in TAFI-/- mice, but was comparable to wild-types at 24 h after injection. In conclusion, TAFI deficiency results in accelerated fibrogenesis and increased liver damage in murine models of chronic and acute liver disease, which may be related to increased inflammation.
Thrombosis and Haemostasis 03/2013; 109(5). · 5.04 Impact Factor
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ABSTRACT: The protein C (PC) system is an important regulator of both coagulation and inflammation. Activated PC (APC), together with its receptor the endothelial protein C receptor (EPCR), has anticoagulant and anti-inflammatory properties. During tuberculosis (TB), a devastating chronic pulmonary disease caused by Mycobacterium (M.) tuberculosis, both a local inflammatory reaction characterised by the recruitment of mainly mononuclear cells and the formation of pulmonary granulomas as well as activation of coagulation occurs as part of the host immune response. We investigated the role of EPCR and APC in a mouse model of TB using mice overexpressing EPCR (Tie2-EPCR), mice deficient for EPCR (EPCR -/- ), mice treated with APC-inhibiting antibodies and mice overexpressing APC (APC high ) and compared them with wild-type (WT) mice. Blood and organs were harvested to quantify bacterial loads, cellular influxes, cytokines, histopathology and coagulation parameters. Additionally observation studies were performed. Lung EPCR expression was upregulated during experimental TB. No significant differences in bacterial growth were seen between WT and Tie2-EPCR mice. However, Tie2-EPCR mice had decreased pulmonary coagulation activation, displayed an increased influx of macrophages 2 and 6 weeks after infection, but no increase in other proinflammatory markers. On the other hand, in EPCR -/- -mice coagulation activation was decreased 6 weeks post-infection, with little impact on other inflammation markers. APC-overexpression or treatment with anti-(A)PC antibodies displayed minimal effects during experimental TB. In conclusion, EPCR and APC play a limited role in the host response during experimental pulmonary TB.
Thrombosis and Haemostasis 01/2013; 109(4). · 5.04 Impact Factor
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ABSTRACT: Patients with venous-thromboembolism (VTE) and myocardial infarction (MI) have elevated prothrombin fragment 1+2 (F1+2) levels. In patients with postoperative VTE, urinary F1+2 (uF1+2) was higher than in individuals without VTE. To explore the relationship between plasma and uF1+2 we performed a pilot study in patients with thrombotic events and healthy controls. In 40 patients with VTE or MI, and 25 age- and sex-matched healthy controls, F1+2 and D-dimer levels were measured in urine and plasma within 48 h after diagnosis. In addition, in all subjects renal function was assessed. Plasma and uF1+2 levels were positively correlated. Compared to controls, patients with VTE had higher levels of both plasma F1+2 (271 vs 160 pmol L(-1), p < 0.05) and uF1+2 levels (38 vs 28 pmol L(-1)), the latter, however, was not statistically significant. Patients with acute MI had similar F1+2 levels as controls in both plasma and urine. Differences in urinary F1+2 levels could not be attributed to differences in concentrations of creatinine or albumin in spot urine samples. Overall, D-dimer and F1+2 levels in urine were extremely low in all groups.
Journal of Thrombosis and Thrombolysis 12/2012; · 1.48 Impact Factor
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ABSTRACT: BACKGROUND: Activated protein C (APC) exerts anticoagulant effects via inactivation of factors Va and VIIIa and cytoprotective effects via protease activated receptor (PAR)1. Inhibition of endogenous APC in endotoxemia and sepsis results in exacerbation of coagulation and inflammation, with consequent enhanced lethality. OBJECTIVES: We here sought to dissect the distinct roles of the anticoagulant and cytoprotective functions of endogenous APC in severe Gram-negative pneumonia derived sepsis (melioidosis). Methods: We infected wildtype (WT) mice with Burkholderia pseudomallei, a common sepsis pathogen in Southeast-Asia, and treated them with antibodies inhibiting both the anticoagulant and cytoprotective functions of APC (MPC1609) or the anticoagulant functions of APC (MAPC1591) only. Additionally, we administered SEW2871 (stimulating the S1P1-pathway downstream from PAR1) to control and MPC1609-treated mice. RESULTS: MPC1609, but not MAPC1591 significantly worsened survival, increased coagulation activation, facilitated bacterial growth and dissemination and enhanced the inflammatory response. The effects of MPC1609 could not be reversed by SEW2871 suggesting that S1P1 does not play a major role in this model. CONCLUSIONS: These results suggest that the mere inhibition of the anticoagulant function of APC does not interfere with its protective role during Gram-negative pneumosepsis, suggesting a more prominent role for cytoprotective effects of APC herein. © 2012 International Society on Thrombosis and Haemostasis.
Journal of Thrombosis and Haemostasis 12/2012; · 5.73 Impact Factor
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ABSTRACT: Endocrine disorders affect both the coagulation and fibrinolytic systems, and have been associated with the development of cardiovascular diseases. Thrombin-activatable fibrinolysis inhibitor (TAFI) is a link between coagulation and the fibrinolytic system. The aim of this study was to determine the effect of thyroid hormone excess and deficiency on TAFI levels and function. The effect of hyperthyroxinemia on TAFI was studied in healthy volunteers who were randomised to receive levothyroxine or no medication for 14 days in a crossover design. The effect of hypothyroidism on TAFI was studied in a multicentre observational cohort study. Blood was drawn before treatment of patients with newly diagnosed hypothyroidism and when euthyroidism was achieved. Plasma clot-lysis times, activated TAFI (TAFIa)-dependent prolongation of clot-lysis and TAFI levels were measured. Thyroid hormone excess resulted in a hypofibrinolytic condition and in an enhanced TAFIa-dependent prolongation of clot lysis. A trend towards decreased plasma TAFI levels was observed in healthy volunteers who used levothyroxine. Hypothyroidism resulted in hyperfibrinolysis and a reduced TAFIa-dependent prolongation of clot lysis. In conclusion, alterations of TAFIa-dependent prolongation of clot lysis in patients with thyroid disorders may cause an impaired haemostatic balance. The disturbed haemostatic balance in patients with hyperthyroidism might make them prone to thrombosis, while the risk for bleeding may increase in patients with hypothyroidism.
Thrombosis and Haemostasis 11/2012; 109(2). · 5.04 Impact Factor
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ABSTRACT: Background: Mice with single gene deficiency of Thrombin Activatable Fibrinolysis Inhibitor (TAFI) or Plasminogen Activator Inhibitor-1 (PAI-1) have an enhanced fibrinolytic capacity. Objectives: To unravel the respective function and relevance of both antifibrinolytic proteins through the generation and characterization of mice with combined TAFI and PAI-1 gene deficiency. Results: Mating of TAFI knock-out (KO) with PAI-1 KO mice resulted in the production of TAFI/PAI-1 double KO mice that were viable, fertile and developed normally. In a tail vein bleeding model, the bleeding time and hemoglobin content of the TAFI/PAI-1 double KO mice did not deviate significantly from that of the single KO mice nor of the wild-type (WT) counterparts. Interestingly, in ex vivo rotational thromboelastometry (ROTEM®) measurements using whole blood samples, TAFI KO mice and TAFI/PAI-1 double KO mice were more sensitive to fibrinolytic activation with tPA as compared to blood samples of WT or PAI-1 KO mice. This enhanced fibrinolytic capacity was confirmed in vivo in a mouse thromboembolism model as evidenced by the decreased fibrin deposition in the lungs of TAFI KO mice and TAFI/PAI-1 double KO mice as compared to WT or PAI-1 KO mice. Conclusions: TAFI gene inactivation predominantly contributes to the increased fibrinolytic capacity of TAFI and PAI-1 double gene deficient mice, as observed in some basic thrombosis models. © 2012 International Society on Thrombosis and Haemostasis.
Journal of Thrombosis and Haemostasis 10/2012; · 5.73 Impact Factor
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ABSTRACT: Viral infections are associated with coagulation disorders. All aspects of the coagulation cascade, primary hemostasis, coagulation, and fibrinolysis, can be affected. As a consequence, thrombosis and disseminated intravascular coagulation, hemorrhage, or both, may occur. Investigation of coagulation disorders as a consequence of different viral infections have not been performed uniformly. Common pathways are therefore not fully elucidated. In many severe viral infections there is no treatment other than supportive measures. A better understanding of the pathophysiology behind the association of viral infections and coagulation disorders is crucial for developing therapeutic strategies. This is of special importance in case of severe complications, such as those seen in hemorrhagic viral infections, the incidence of which is increasing worldwide. To date, only a few promising targets have been discovered, meaning the implementation in a clinical context is still hampered. This review discusses non-hemorrhagic and hemorrhagic viruses for which sufficient data on the association with hemostasis and related clinical features is available. This will enable clinicians to interpret research data and place them into a perspective.
Journal of Medical Virology 10/2012; 84(10):1680-96. · 2.82 Impact Factor
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ABSTRACT: It has been well established that hemostatic potential in patients with chronic liver disease is in a rebalanced status due to a concomitant decrease in pro- and antihemostatic drivers. The hemostatic changes in patients with acute liver injury/failure (ALI/ALF) are similar but not identical to the changes in patients with chronic liver disease and have not been studied in great detail.
To assess thrombin generation and fibrinolytic potential in patients with ALI/ALF.
We performed thrombin generation tests and clot lysis assays in platelet-poor plasma from 50 patients with ALI/ALF. Results were compared with values obtained in plasma from 40 healthy volunteers.
The thrombin generation capacity of plasma from patients with ALI/ALF sampled on the day of admission to hospital was indistinguishable from that of healthy controls, provided thrombomodulin was added to the test mixture. Fibrinolytic capacity was profoundly impaired in patients with ALI/ALF on admission (no lysis in 73.5% of patients, compared with 2.5% of the healthy controls), which was associated with decreased levels of the plasminogen and increased levels of plasminogen activator inhibitor type 1. The intact thrombin generating capacity and the hypofibrinolytic status persisted during the first week of admission. Patients with ALI/ALF have a normal thrombin generating capacity and a decreased capacity to remove fibrin clots. These results contrast with routine laboratory tests such as the PT/INR, which are by definition prolonged in patients with ALI/ALF and suggest a bleeding tendency.
Journal of Thrombosis and Haemostasis 05/2012; 10(7):1312-9. · 5.73 Impact Factor
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ABSTRACT: The antiphospholipid syndrome is an autoimmune disease that manifests clinically as recurrent thrombotic complications or foetal losses and serologically with elevated levels of antiphospholipid antibodies in the plasmas of these patients. The term 'antiphospholipid syndrome' is confusing, because the auto-antibodies are not directed against phospholipids but towards a plasma protein, β(2) -glycoprotein I. For many years, the reason why auto-antibodies against β(2) -glycoprotein I were pro-thrombotic was unclear, because β(2) -glycoprotein I seems to be an obsolete protein in our circulation. Human and mice deficient in this protein do not express a clear phenotype. Recent studies on the structure and function of β(2) -glycoprotein I have provided novel insights into the importance of this protein in physiology and its role in the pathology of the antiphospholipid syndrome.
International journal of laboratory hematology 03/2012; 34(3):223-31. · 1.30 Impact Factor
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ABSTRACT: Protein Z (PZ)-dependent protease inhibitor (ZPI) is a serine protease inhibitor which efficiently inactivates activated factor X, when ZPI is complexed with PZ in plasma. Reduced plasma levels of ZPI and PZ have been reported in association with thrombosis. It has also been reported that PZ increases during pregnancy and that its partial deficiency is related to early pregnancy loss or recurrent miscarriage (RM). However, until now there has been no report on ZPI in pregnancy. To explore the possible role(s) of ZPI in the maintenance of pregnancy, we studied 42 non-pregnant normal women, 32 women with normal pregnancies, and 134 cases of unexplained RM in Japan, as well as 64 non-pregnant normal German females. Plasma ZPI was measured by in-house ELISA. There were significantly higher concentrations of plasma ZPI in normal pregnancies compared to non-pregnant women. The present study also confirmed that both factor X, the major target of ZPI, and protein Z increased during normal pregnancies. This increased ZPI and PZ may counteract the increased activated factor X, which may in turn contribute to the maintenance of normal placental circulation. Plasma ZPI levels were unchanged in non-pregnant RM women, while the plasma PZ level was slightly reduced, a finding consistent with existing reports. The exact relationship between RM and this unaltered ZPI with mild PZ reduction relative to normal pregnancies warrants further investigation.
Thrombosis and Haemostasis 03/2012; 107(3):507-12. · 5.04 Impact Factor
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G M A Van O S, J C M Meijers,
Ç Agar,
M V Seron,
J A Marquart,
P Åkesson,
R T Urbanus,
R H W M Derksen,
H Herwald,
M Mörgelin,
P G D E Groot
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ABSTRACT: The antiphospholipid syndrome (APS) is characterized by the persistent presence of anti-β(2) -glycoprotein I (β(2) -GPI) autoantibodies. β(2) -GPI can exist in two conformations. In plasma it is a circular protein, whereas it adopts a fish-hook conformation after binding to phospholipids. Only the latter conformation is recognized by patient antibodies. β(2) -GPI has been shown to interact with Streptococcus pyogenes.
To evaluate the potential of S. pyogenes-derived proteins to induce anti-β(2) -GPI autoantibodies.
Four S. pyogenes surface proteins (M1 protein, protein H, streptococcal collagen-like protein A [SclA], and streptococcal collagen-like protein B [SclB]) were found to interact with β(2) -GPI. Only binding to protein H induces a conformational change in β(2) -GPI, thereby exposing a cryptic epitope for APS-related autoantibodies. Mice were injected with the four proteins. Only mice injected with protein H developed antibodies against the patient antibody-related epitope in domain I of β(2) -GPI. Patients with pharyngotonsillitis caused by S. pyogenes who developed anti-protein H antibodies also generated anti-β(2) -GPI antibodies.
Our study has demonstrated that a bacterial protein can induce a conformational change in β(2) -GPI, resulting in the formation of antiβ(2) -GPI autoantibodies. This constitutes a novel mechanism for the formation of anti-β(2) -GPI autoantibodies.
Journal of Thrombosis and Haemostasis 12/2011; 9(12):2447-56. · 5.73 Impact Factor
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ABSTRACT: β₂-Glycoprotein I (β₂GPI) is a highly abundant plasma protein and the major antigen for autoantibodies in the antiphospholipid syndrome. Recently, we have described a novel function of β₂GPI as scavenger of lipopolysaccharide (LPS). With this in mind we investigated the conservation of β₂GPI in vertebrates and set out to identify the binding site of LPS within β₂GPI. The genome sequences of 42 species were surveyed. Surface plasmon resonance (SPR) was performed with peptides to characterise the binding site of β₂GPI for LPS. β₂GPI could be identified in most tested vertebrates with a high overall amino acid homology of 80% or more in mammals. SPR revealed that a synthesised peptide (LAFWKTDA) from domain V of β₂GPI was able to compete for binding of β₂GPI to LPS. The AFWKTDA sequence was completely conserved in all mammals. The peptide containing the LPS binding site attenuated the inhibition by β₂GPI in a cellular model of LPS-induced tissue factor expression. Other important sites, such as the binding site for anionic phospholipids and the antiphospholipid antibody binding epitope, were also preserved. β₂GPI is highly conserved across the animal kingdom, which suggests that the function of β₂GPI may be more important than anticipated.
Thrombosis and Haemostasis 09/2011; 106(6):1069-75. · 5.04 Impact Factor
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ABSTRACT: Melioidosis is a frequent cause of sepsis in Southeast Asia caused by the Gram-negative bacterium Burkholderia pseudomallei. Patients with melioidosis have elevated circulating levels of plasminogen activator inhibitor type 1 (PAI-1), an important regulator of inflammation and fibrinolysis.
In this study, we aimed to investigate the role of PAI-1 during melioidosis.
Wild-type (WT) and PAI-1-deficient (PAI-1-/1(-/-) ) mice were intranasally infected with B. pseudomallei. Mice were killed after 24, 48 or 72 h. Lungs, liver and blood were harvested for measurement of bacterial loads, cytokines, clinical chemistry, histopathology, and coagulation parameters. Additionally, survival studies were performed.
PAI-1(-/-) mice demonstrated enhanced susceptibility to B. pseudomallei infection, as shown by a strongly increased mortality rate (100% vs. 58% among WT mice, P < 0.001), associated with enhanced bacterial loads in lungs, liver, and blood. Additionally, PAI-1(-/-) mice showed elevated levels of proinflammatory cytokines in lungs and plasma, accompanied by enhanced local and systemic coagulation activation (thrombin-antithrombin complexes and D-dimer), increased hepatocellular injury (plasma aspartate aminotransferase and alanine aminotransferase), and renal failure (plasma creatinine and urea).
PAI-1 has a protective role during severe Gram-negative sepsis caused by B. pseudomallei by limiting bacterial growth, inflammation, and coagulation, and probably, as a consequence thereof, distant organ injury.
Journal of Thrombosis and Haemostasis 08/2011; 9(10):2020-8. · 5.73 Impact Factor
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ABSTRACT: Several hemostatic abnormalities have been reported in hyperthyroidism, but the overall effect of thyroid hormone excess on coagulation and fibrinolysis is unclear.
Our aim was to assess whether the use of supraphysiological doses of levothyroxine leads to coagulation activation and inhibition of fibrinolysis.
Healthy volunteers were randomized to receive levothyroxine or no medication for 14 days with a washout period of at least 28 days in a crossover design. To study the effects of different degrees of thyroid hormone excess, 16 participants received levothyroxine in a dose of 0.3 mg per day, and 12 received levothyroxine 0.45 or 0.6 mg per day depending on body weight. Several variables of coagulation and fibrinolysis were measured.
Levels of von Willebrand factor activity (VWF:RiCo) and antigen (VWF:Ag), factor (F) VIII, plasminogen activator inhibitor-1 (PAI-1) and clot-lysis time were slightly higher after levothyroxine 0.3 mg per day than after the control situation, but only levels of VWF showed a significant increase from baseline. After levothyroxine 0.45 or 0.6 mg per day, levels of fibrinogen increased by 17%, VWF activity by 24%, VWF antigen by 26%, FVIII by 19%, FIX by 14%, FX by 7%, PAI-1 by 116% and clot-lysis time by 14%, and activated partial thromboplastin time decreased by 3%; all were significant changes compared with the control situation. We did not observe clear evidence of coagulation activation.
Our data suggest that thyroid hormone excess increases coagulation factor levels and inhibits fibrinolysis in a dose-dependent fashion. This implies an increased risk of venous thrombosis during hyperthyroidism.
Journal of Thrombosis and Haemostasis 07/2011; 9(9):1816-24. · 5.73 Impact Factor
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Journal of Thrombosis and Haemostasis 06/2011; 9(8):1659-61. · 5.73 Impact Factor
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Journal of Thrombosis and Haemostasis 06/2011; 9(8):1657-9. · 5.73 Impact Factor
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Journal of Thrombosis and Haemostasis 05/2011; 9(7):1432-4. · 5.73 Impact Factor
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ABSTRACT: β(2) -Glycoprotein I (β(2) -GPI) is a protein that circulates in blood at high concentrations. The function of β(2) -GPI has long been an enigma. More than 20 years ago, it was discovered that β(2) -GPI is the major antigen for the circulating antibodies in the antiphospholipid syndrome. However, this knowledge has not advanced our understanding of the physiologic role of the protein. In recent years, new insights have suggested an important function of this protein in innate immunity. β(2) -GPI was found to scavenge lipopolysaccharide and was able to clear unwanted anionic cellular remnants such as microparticles from the circulation. The function of β(2) -GPI seems to depend on the structural conformation of the protein, and it has been established that β(2) -GPI can exist in at least two conformations. In this review, we will highlight and summarize the current knowledge on this protein.
Journal of Thrombosis and Haemostasis 05/2011; 9(7):1275-84. · 5.73 Impact Factor
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ABSTRACT: Genetic determinants of plasma levels of protein C (PC) are poorly understood. Recently, we identified a locus on chromosome 20 determining high PC levels in a large Dutch pedigree with unexplained thrombophilia. Candidate genes in the LOD-1 support interval included FOXA2, THBD and PROCR.
To examine these candidate genes and their influence on plasma levels of PC.
Exons, promoter and 3'UTR of the candidate genes were sequenced in 12 family members with normal to high PC levels. Four haplotypes of PROCR, two SNPs in the neighboring gene EDEM2 and critical SNPs encountered during resequencing were genotyped in the family and in a large group of healthy individuals (the Leiden Thrombophilia Study (LETS) controls). Soluble endothelial protein C receptor (sEPCR) and soluble thrombomodulin (sTM) plasma levels were measured in the family.
PROCR haplotype 3 (H3) and FOXA2 rs1055080 were associated with PC levels in the family but only PROCR H3 was also associated with plasma levels in the healthy individuals. Carriers of both variants had higher PC levels than carriers of only PROCR H3 in the family but not in healthy individuals, suggesting that a second determinant is present. EDEM2 SNPs were associated with PC levels, but their effect was small. PC and sEPCR levels were associated in both studies. sTM was not associated with variations of THBD or PC levels.
Chromosome 20 harbors genetic determinants of PC and sEPCR levels and the analysis of candidate genes suggests that the PROCR locus is responsible.
Journal of Thrombosis and Haemostasis 03/2011; 9(5):969-76. · 5.73 Impact Factor
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ABSTRACT: Aortic stenosis patients often show bleeding complications. Previously, a prolonged platelet function analyzer (PFA-100) closure time was observed with plasma of severe aortic stenosis patients. To elucidate a possible role of circulating preactivated von Willebrand factor (VWF), we determined the level of VWF in its active, platelet-binding conformation in plasma of patients with aortic stenosis.
Sixty-two aortic stenosis patients were included in this study. VWF and related parameters were measured, and the results were related to severity of aortic stenosis.
VWF activation factor, indicating the proportion of circulating VWF able to bind to platelets, correlated negatively with peak transvalvular gradient and PFA-100 closure time. No correlation was observed between ADAMTS13 activity and peak transvalvular gradient or PFA-100 closure time. Both VWF antigen and VWF propeptide levels were significantly higher in patients with mild and moderate aortic stenosis, but not in those with severe stenosis.
Our data demonstrate that the aortic pressure gradient is inversely associated with VWF activation factor, but not with VWF antigen or VWF multimerization in patients with aortic stenosis. These findings might have implications for the bleeding observed in patients with aortic stenosis.
Journal of Thrombosis and Haemostasis 02/2011; 9(5):953-8. · 5.73 Impact Factor
