Evidence for an enhanced fibrinolytic capacity in cirrhosis as measured with two different global fibrinolysis tests.

BACKGROUND AND OBJECTIVES: It has been known for a long time that cirrhosis is associated with hyperfibrinolysis, which might contribute to an increased risk and severity of bleeding. However, recent papers have questioned the presence of a hyperfibrinolytic state in cirrhotic patients and postulated a rebalanced system owing to concomitant changes in both pro- and anti-fibrinolytic factors. Therefore we re-investigated the fibrinolytic state of cirrhotic patients using two different overall tests including a recently developed test for global fibrinolytic capacity (GFC) using whole blood. PATIENTS AND METHODS: Blood was collected from 30 healthy controls and 75 patients with cirrhosis of varying severity (34 Child-Pugh A, 28 Child-Pugh B and 13 Child-Pugh C). The plasma clot lysis time (CLT), which is inversely correlated with fibrinolysis, was determined as well as the GFC. RESULTS: The mean CLT was 74.5 min in the controls and decreased significantly to 66.9 min in Child-Pugh class A patients, 59.3 min in class B patients and 61.0 min in class C patients, and hyperfibrinolysis existed in 40% of the patients. The median GFC was 1.7 µg mL(-1) in the controls and increased significantly to 4.0 µg mL(-1) in Child-Pugh class A patients, 11.1 µg mL(-1) in class B patients and 22.5 µg mL(-1) in class C patients, and hyperfibrinolysis existed in 43% of the patients. Taken together, 60% of the patients showed hyperfibrinolysis in at least one of the two global assays. CONCLUSION: A rebalanced fibrinolytic system may occur, but hyperfibrinolysis is found in the majority of patients with cirrhosis.

The role of thrombin activatable fibrinolysis inhibitor in arterial thrombosis at a young age: the ATTAC study.

BACKGROUND AND OBJECTIVES: Thrombin activatable fibrinolysis inhibitor (TAFI) attenuates fibrinolysis and may therefore contribute to the pathophysiology of arterial thrombosis. The aim of the present study was to elucidate the pathogenetic role of TAFI levels and genotypes in young patients with arterial thrombosis. PATIENTS AND METHODS: In a case-control study, 327 young patients with a recent first-ever event of coronary heart disease (CHD subgroup) or cerebrovascular disease (ischemic stroke subgroup) and 332 healthy young controls were included. TAFI levels [intact TAFI, activation peptide (TAFI-AP) and (in)activated TAFI (TAFIa(i)] and TAFI activity were measured and genetic variations in the TAFI gene (-438G/A, 505G/A and 1040C/T) were determined. RESULTS: In the total group of patients, TAFIa(i) levels were higher (145.1 +/- 37.5%) than in controls (137.5 +/- 31.3%, P = 0.02). Plasma levels of intact TAFI, TAFI-AP and TAFI activity were similar in patients and controls. In the CHD subgroup (n = 218), intact TAFI levels were higher (109.4 +/- 23.0%) than in controls (102.8 +/- 20.7%, P = 0.02). In 325Ile/Ile homozygotes, lower TAFI levels and a decreased risk of arterial thrombosis were observed (OR 0.58, 95% CI 0.34-0.99) compared with patients with the common 325Thr/Thr genotype. This association was most evident in CHD patients (OR 0.48, 95% CI 0.26-0.90). Haplotype analyses supported a role for the Thr325Ile polymorphism. CONCLUSIONS: TAFIa(i) levels were higher in patients with cardiovascular disease. Furthermore, the TAFI 325Thr/Ile polymorphism was associated with lower TAFI levels and with the risk of cardiovascular disease in young patients, especially in CHD.

Thrombin-activatable fibrinolysis inhibitor is associated with severity and outcome of severe meningococcal infection in children.

BACKGROUND AND OBJECTIVES: In pediatric meningococcal sepsis, an imbalance between coagulation and fibrinolysis and proinflammatory action play major roles. We hypothesized that thrombin activatable fibrinolysis inhibitor (TAFI) and/or TAFI activation markers are involved in the pathogenesis of meningococcal sepsis. PATIENTS AND METHODS: Children with severe meningococcal sepsis (n = 112) previously included in Rotterdam-based trials participated in this study. Clinical and laboratory parameters and severity scores were assessed. TAFI and TAFI activation markers were determined: TAFI activation peptide (TAFI-AP) and (in)activated TAFI [TAFIa(i)]. The -438G/A, Ala147Thr, and Thr325Ile polymorphisms were genotyped. RESULTS: TAFI levels were significantly decreased in patients with meningococcal disease at admission compared to the convalescence state. TAFI was decreased in patients with septic shock vs. those with no shock. TAFI-AP levels were increased in patients with disseminated intravascular coagulation (DIC) vs. patients without DIC. TAFI-AP and TAFIa(i) were significantly increased in non-survivors vs. survivors. TAFI-AP levels and the TAFI-AP/TAFI ratio were also strongly correlated to severity scores and laboratory parameters. The TAFI 325Ile/Ile genotype was overrepresented in patients with DIC. CONCLUSIONS: Activation markers of TAFI were associated with the occurrence of DIC and mortality in meningococcal sepsis patients. A determination of TAFI, TAFI-AP, and TAFIa(i) is required to enable coherent interpretation of the role of TAFI in disease.

High functional levels of thrombin-activatable fibrinolysis inhibitor are associated with an increased risk of first ischemic stroke.

BACKGROUND AND OBJECTIVE: Several studies have suggested that thrombin-activatable fibrinolysis inhibitor (TAFI) levels are associated with the risk of arterial thrombosis, but results have been contradictory. We studied functional TAFI levels and TAFI gene polymorphisms in 124 patients with a recent ischemic stroke and 125 age- and sex-matched controls to establish the role of TAFI in ischemic stroke. METHODS AND RESULTS: Functional TAFI levels, defined as TAFI-related retardation (RT), the difference in clot lysis time (LT) in the absence or presence of a specific activated TAFI inhibitor (potato carboxypeptidase inhibitor [PCI]), were higher in patients than controls (19.5 +/- 4.2 vs. 17.7 +/- 3.7 min, P < 0.005). Clot LTs in the presence of PCI, which were independent of TAFI, were also increased in ischemic stroke patients. This indicates that in these patients fibrinolysis is impaired not only by high TAFI levels, but also by other mechanisms. Individuals with functional TAFI levels in the highest quartile had an increased risk of ischemic stroke compared with the lowest quartile [odds ratio (OR) 4.0, 95% confidence interval (CI): 1.6-9.8]. In an unselected group of 36 of the 125 stroke patients functional TAFI levels were also measured at 3 months, and were persistently high. This indicates that increased functional TAFI levels after stroke are not caused by an acute phase reaction. No difference was found between patients and controls with respect to TAFI genotype distribution. CONCLUSIONS: Increased functional TAFI levels, resulting in decreased fibrinolysis, are associated with an increased risk of ischemic stroke.

A new functional assay of thrombin activatable fibrinolysis inhibitor.

New thrombin activatable fibrinolysis inhibitor (TAFI) assays are necessary for studying the role of this fibrinolysis inhibitor in cardiovascular disease. The identification of a functional single nucleotide polymorphism (SNP) (1040C/T) leading to a TAFI-variant with increased stability but lower antigen levels has made the determination of functional activity even more essential. Therefore, we developed a new assay for the functional activity of TAFI in citrated plasma samples. This assay is based on the retardation of plasma clot lysis by TAFIa. TAFI activation was induced simultaneously with fibrin formation and lysis was mediated by rt-PA. The variability of other plasma components was minimized by a 20-fold dilution of the samples in TAFI-depleted plasma. Lysis times (-/+ potato carboxypeptidase inhibitor) and the TAFI-related retardation of clot lysis, the functional parameter of the assay, were determined in a group of 92 healthy volunteers, as well as TAFI antigen levels (electroimmunoassay) and two TAFI SNPs (-438A/G and 1040C/T). TAFI-related retardation was 19.8 +/- 5.6 min (mean +/- SD) and was correlated with the antigen level. The specific antifibrinolytic activity of TAFI was associated with the -438A/G and 1040C/T genotypes. Individuals with the 325Ile-variant had on average a 34% higher TAFI-specific antifibrinolytic activity than individuals with the 325Thr-isoform. The TAFI-related retardation in the two groups of individuals did not differ, as a lower level compensated for the higher specific antifibrinolytic activity of the 325Ile-isoform. This assay provides valuable information about the performance of different TAFI isoforms and constitutes a new method for studying the role of TAFI in cardiovascular disease.

Migration of the activation peptide of thrombin-activatable fibrinolysis inhibitor (TAFI) during SDS-polyacrylamide gel electrophoresis.

Thrombin-activatable fibrinolysis inhibitor (TAFI) is a plasma zymogen, which upon activation is capable of delaying fibrinolysis. We investigated the migration and detection of the activation peptide of TAFI during SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Purified TAFI before and after activation by thrombin/thrombomodulin was electrophoresed on 4-20% polyacrylamide gels and stained with Coomassie blue as well as Western blotting. Before activation, Coomassie blue staining resulted in one main band of TAFI. After activation, a sharp band corresponding to TAFIa was observed. No distinct activation peptide was detected, in agreement with the literature. Western blotting using a polyclonal anti-TAFI antibody, on the other hand, showed one additional broad band with an Mr of about 33 000 after TAFI activation. N-terminal sequence analysis confirmed that this band represented the activation peptide of TAFI. In addition, we tested the reactivity of two anti-TAFI monoclonal antibodies (MA-T3D8 and MA-T18A8) towards TAFI before and after activation by Western blotting. Both monoclonal antibodies recognized TAFI. After activation of TAFI, MA-T3D8 reacted with TAFIa, while MA-T18A8 reacted with the activation peptide. We identify the 33 000 band as the activation peptide of TAFI and exemplify the use of this information for the characterization of monoclonal antibodies against TAFI.

Association between thrombin activatable fibrinolysis inhibitor genotype and levels in plasma: comparison of different assays.

Thrombin activatable fibrinolysis inhibitor (TAFI) antigen levels exhibit a large interindividual variability in which genetic control seems to play a major role. However, recent reports have questioned the association between TAFI concentration and genotype, suggesting that variable antibody reactivity towards TAFI isoforms, particularly the Thr325Ile polymorphism (1040C/T), may lead to artefacts in TAFI antigen levels. In order to compare assay outcome we determined plasma TAFI levels in 92 healthy individuals, using an enzyme-linked immunosorbent assay (ELISA) (commercial antibodies), an electroimmunoassay (in-house antibodies) and a commercial chromogenic assay (Actichrome TAFI). Each individual was genotyped for the -438A/G and 1040C/T polymorphisms in the TAFI gene. TAFI levels were significantly associated with genotype in both antigen and chromogenic assays. All assays displayed significant correlations with each other. Linear regression and Bland-Altman agreement analysis in the genotype subgroups showed that neither the genotype nor the concentration affected the relationship between the Actichrome TAFI and the electroimmunoassay. In contrast, the ELISA/Actichrome TAFI and the ELISA/electroimmunoassay relationships were concentration- and genotype-dependent. Our results demonstrate that artefacts may arise when measuring TAFI antigen levels by ELISA. Nevertheless, the electroimmunoassay and the Actichrome TAFI assay support a genotype-related variation of TAFI concentration.