Ana Guimarães

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

Summary.  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.

Hypofibrinolysis is a risk factor for arterial thrombosis at young age

The relationship between defective fibrinolysis and arterial thrombosis is uncertain. The evaluation of the plasma fibrinolytic potential might provide stronger evidence linking fibrinolysis to arterial thrombosis than the evaluation of the individual fibrinolytic factors. We determined the plasma fibrinolytic potential of 335 young survivors of a first arterial thrombosis, including coronary artery disease (n = 198), ischaemic stroke (n = 103) and peripheral artery disease (n = 34), enrolled in a population‐based case–control study and of 330 healthy individuals. Patients had significantly higher clot lysis times (CLTs) than the controls. Odds ratios (ORs) were calculated as a measure of relative risk. The OR for arterial thrombosis was determined in these subjects who had a CLT above the 60th, 70th, 80th, 90th and 95th percentiles of the values found in the control subjects. We found a progressive increase in risk of arterial thrombosis in subjects with hypofibrinolysis (OR: 1·7, 2·0, 2·3, 2·3 and 2·9, respectively). Relative risk estimates obtained in the whole group were comparable those obtained in the event‐subgroups. In conclusion, a low plasma fibrinolytic potential, found in 10% of the population, increases the relative risk of arterial thrombosis twofold. This points to an important contribution of hypofibrinolysis to the burden of arterial thrombosis.

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

Summary.  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.

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

Summary.  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.

Impaired fibrinolysis as a risk factor for Budd-Chiari syndrome

In Budd-Chiari syndrome (BCS), thrombosis develops in the hepatic veins or inferior vena cava. To study the relationship between hypofibrinolysis and BCS, we measured plasma levels of fibrinolysis proteins in 101 BCS patients and 101 healthy controls and performed a plasma-based clot lysis assay. In BCS patients, plasminogen activator inhibitor 1 (PAI-1) levels were significantly higher than in controls (median, 6.3 vs 1.4 IU/mL, P < .001). Thrombin-activatable fibrinolysis inhibitor and plasmin inhibitor levels were lower than in controls (13.8 vs 16.9 microg/mL and 0.91 vs 1.02 U/L, both P < .001). Median plasma clot lysis time (CLT) was 73.9 minutes in cases and 73.0 minutes in controls (P = .329). A subgroup of cases displayed clearly elevated CLTs. A CLT above the 90th or 95th percentile of controls was associated with an increased risk of BCS, with odds ratios of 2.4 (95% confidence interval, 1.1-5.5) and 3.4 (95% confidence interval, 1.2-9.7), respectively. In controls, only PAI-1 activity was significantly associated with CLT. Analysis of single nucleotide polymorphisms of fibrinolysis proteins revealed no significant differences between cases and controls. This case-control study provides the first evidence that an impaired fibrinolytic potential, at least partially caused by elevated PAI-1 levels, is related to the presence of BCS.

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

Summary.  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.

A new functional assay of thrombin activatable fibrinolysis inhibitor

Summary.  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.

Thrombin activatable fibrinolysis inhibitor (TAFI) affects fibrinolysis in a plasminogen activator concentration-dependent manner Study of seven plasminogen activators in an internal clot lysis model

TAFIa was shown to attenuate fibrinolysis. In our in vitro study, we investigated how the inhibitory effect of TAFIa depended on the type and concentration of the plasminogen activator (PA). We measured PA-mediated lysis times of plasma clots under conditions of maximal TAFI activation by thrombin-thrombo-modulin in the absence and presence of potato carboxypeptidase inhibitor. Seven different PAs were compared comprising both tPA-related (tPA, TNK-tPA, DSPA), bacterial PA-related (staphylokinase and APSAC) and urokinase-related (tcu-PA and k2tu-PA) PAs. The lysis times and the retardation factor were plotted against the PA concentration. The retardation factor plots were bell-shaped. At low PA concentrations, the retardation factor was low, probably due to the limited stability of TAFIa. At intermediate PA concentrations the retardation factor was maximal (3-6 depending on the PA), with TNK-tPA, APSAC and DSPA exhibiting the strongest effect. At high PA concentrations, the retardation factor was again low, possibly due to inactivation of TAFIa by plasmin or to a complete conversion of glu-plasminogen into lys-plasminogen. Using individual plasmas with a reduced plasmin inhibitor activity (plasmin inhibitor Enschede) the bell-shaped curve of the retardation factor shifted towards lower tPA and DSPA concentrations, but the height did not decrease. In conclusion, TAFIa delays the lysis of plasma clots mediated by all the plasminogen activators tested. This delay is dependent on the type and concentration of the plasminogen activator, but not on the fibrin specificity of the plasminogen activator. Furthermore, plasmin inhibitor does not play a significant role in the inhibition of plasma clot lysis by TAFI.

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

Summary.  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.

High thrombin activatable fibrinolysis inhibitor levels are associated with an increased risk of premature peripheral arterial disease

BACKGROUND Previous studies suggested that hypofibrinolysis is associated with increased risk of peripheral arterial disease. Thrombin activatable fibrinolysis inhibitor (TAFI) has been identified as an important inhibitor of fibrinolysis. The aim of our study was to assess the role of TAFI in young patients with peripheral arterial disease. METHODS In a single-center case-control study we measured plasma TAFI antigen levels and functional TAFI in consecutive young patients (men 18-45 years and women 18-55 years) with a first manifestation of peripheral arterial disease and compared these with a population-based control group. RESULTS A total of 47 peripheral arterial disease patients and 141 controls (mean age 43) were included. Intact TAFI antigen levels were significantly higher in patients with peripheral arterial disease (112.4±21.1%) than in controls (104.9±19.9%, p=0.03). The risk of peripheral arterial disease increased with 18% (OR 1.18; CI 1.01-1.34) per 10% increase of TAFI antigen. Functional TAFI levels were slightly higher in patients compared to controls, however this difference was not significant. For individuals with the highest functional TAFI levels, above the 90th percentile, the increased risk for peripheral arterial disease was most pronounced (OR 3.1; CI 1.02-9.41). CONCLUSION High TAFI levels are associated with increased risk of premature peripheral arterial disease.