fibrinolysis inhibitor (TAFI) is not associated with fetal loss,
N. Folkeringa F.J. Korteweg
N.J.G.M. Veeger J.J.H.M. Erwich J. van der Meer
Thrombosis Research 2009; 123: 511-514
Introduction
Thrombin–activatable fibrinolysis inhibitor (TAFI) is a procarboxypeptidase which suppresses fibrinolysis by removing carboxy–terminal lysine residues from partially degraded fibrin1. These residues are involved in binding and activation of plasmino-gen. TAFI is activated by thrombin and the thrombin–thrombomodulin complex.
Since TAFI inhibits tissue plasminogen activator–induced fibrinolysis2, high plasma levels of TAFI may promote the development of thrombosis3.
It is hypothesized that uteroplacental thrombosis is involved in fetal loss4. In-creasing levels of pro– coagulant proteins, deIn-creasing levels of anticoagulant proteins and decreasing fibrinolysis cause a state of hypercoagulability in normal pregnancy.
TAFI may contribute to the decreased fibrinolytic potency, as its levels increase dur-ing pregnancy5. Therefore, it is possible that high TAFI levels may be associated with an increased risk of fetal loss. Thus far, no studies have been reported on this issue.
We performed a study to assess the risk of fetal loss in women with high TAFI levels.
Female subjects from four retrospective family cohort studies with an identical design were pooled6-9. These studies were designed to estimate the absolute risk of venous thromboembolism, associated with either hereditary deficiencies of an-tithrombin, protein C or protein S9, the prothrombin 20210A mutation7, elevated plasma levels of factor VIII:C6, or hyperhomocysteinemia8. Probands in each of these studies were consecutive patients with documented venous thromboembolism or premature atherosclerosis (age < 50 years), and one of these thrombophilic defects.
Relatives, who were 15 years of age or older were identified by pedigree analysis and were enrolled after informed consent was obtained. The studies were approved by the institutional review boards of the participating hospitals.
The present study addressed fetal loss in female probands and relatives. In addi-tion to above menaddi-tioned thrombophilic defects, they were tested for factor V Leiden, and TAFI activity levels were measured. Detailed information on their obstetric his-tory was obtained, using a questionnaire and reviewing medical records. Clinical data was collected prior to blood sampling. Women were evaluable if they had been pregnant before the end of study. Women with only terminated or ectopic pregnan-cies were excluded from analysis. Fetal loss was defined as early fetal loss if it had oc-curred within 22 weeks of gestation, or as late fetal loss after more than 22 weeks of gestation10.
TAFI activity was measured by a 2–step chromogenic synthetic substrate assay (Pefakit ® TAFI, Pentafarm Basel, Switzerland). In this assay TAFI is first activated by the thrombin/thrombomodulin complex followed by cleavage of a thio–derivated arginine substrate, which is monitored by colour development after reaction with Ellman's reagent (diathionitrobenzoic acid). The assay was calibrated with pooled normal plasma from 47 healthy volunteers (age 22–57 yrs; 40% women, not using oral contraceptives). Dilution in TAFI–deficient plasma (Affinity Biologicals, An-caster, Canada) instead of water did not increase the recovery of TAFI (98% versus Fetal loss is not related eith TAFI levels
106%), which confirmed the claimed specificity of the assay for TAFI. The normal range was determined in healthy individuals (95% CI, 79– 126%). TAFI levels above the upperlimit of their normal range, i.e. 126 U/dL were defined as high. Activity of antithrombin (Coatest ®, Chromogenix, Mölndal, Sweden) and protein C (Berichrom Protein C®, Dade Behring, Marburg, Germany) were measured by chromogenic sub-strate assays, protein C antigen and total and free protein S antigen levels by Enzyme Linked Immuno Sorbent Assay (ELISA) (DAKO, Glostrup, Denmark). Factor V Lei-den and the prothrombin G20210A mutation were demonstrated by polymerase chain reactions. Factor VIII:C was measured by one–stage clotting assay (Amelung GmbH, Lemgo, Germany) and was defined as high at levels above 150 IU/dL6. Fasting levels of homocysteine were measured by high performance liquid chromatography. Hy-perhomocysteinemia was defined as a fasting level above 18.5 µmol/L, as described in the Dutch population.
Table 1 Characteristics and fetal loss rates in women with high TAFI levels and normal
High TAFI Normal TAFI P or adjusted levels (n=87) levels (n=756) relative risk
(95%CI)
Venous thromboembolism, n (%) 4 (5) 59 (8) 0.39
Age at onset, median (range), yr 32 (23-40) 33 (17-45) 0.68
TAFI levels, U/dL, mean (SD) 138 (11) 100 (14)
Age at 1st pregnancy, median (range), yr 24 (17-37) 25 (11-42) 0.68 Thrombophilic defects, % (n tested)
Antithrombin deficiency 2 (86) 2 (748) 0.68
Protein C deficiency 4 (86) 4 (752) 1.00
Protein S deficiency type I 4 (84) 4 (737) 1.00
Prothrombin G20210A 13 (87) 12 (750) 1.00
Factor V Leiden 12 (87) 12 (750) 0.86
Factor VIII:CN150 IU/dL 53 (86) 36 (739) 0.003
Hyperhomocysteinemia 38 (78) 30 (644) 0.16
Pregnancies, n 249 2223 0.81
Total fetal loss, n (%) 19 (8) 307 (14) 0.82
Early fetal loss, n (%) 17 (7) 271 (12) 0.83
Late fetal loss, n (%) 2 (1) 32 (1) 0.89
Not classified, n (%) 0 4 (1)
Women, n 87 756
Total fetal loss, n (%) 18 (21) 211 (28) 0.66 (0.38–1.16)
Early fetal loss, n (%) 16 (18) 181 (24) 0.74 (0.42–1.33)
Late fetal loss, n (%) 2 (2) 29 (4) 0.54 (0.14–2.09)
Not classified, n (%) 0 1
Adjusted for trombophilic defects and for clustering of pregnancies in women.
Fetal loss rates were expressed as percentages of pregnancies ending in fetal loss and percentages of women with fetal loss. Risk of fetal loss in women with high TAFI levels was compared to that in women with normal TAFI levels. Pregnancies after prior VTE were excluded from analysis, considering that thromboprophylaxis might have influenced outcome of these pregnancies. Continuous variables were expressed as mean values and standard deviations and categorical data as counts and percent-ages. Differences between groups were evaluated by the Student t test or Mann–
Whitney U test, depending on the normality of data for continuous data, and by Fisher exact test for categorical data. Relative risks were adjusted for clustering of pregnancies in women by random effects logistic regression. A two–tailed p–value of less than 0.05 was considered to indicate statistical significance. Statistical analy-ses were performed using SAS software, version 9.1 (SAS–Institute inc., Cary, North Carolina, USA).
Of 1557 women, 175 were excluded because they were younger than 15 years of age, had deceased or did not consent. Another 444 women were not evaluable because they had never been pregnant or had had only terminated pregnancies, and 95 women because of missing TAFI measurements. The remaining 843 women (in-cluding probands) were analysed, of whom 87 had high TAFI levels. Characteristics of these 843 women are summarized in Table 1. Age at time of first pregnancy was comparable in women with high TAFI levels and women with normal TAFI levels.
Fetal loss is not related eith TAFI levels
Figure 1.
Cumulative distribution curve of fetal loss related to TAFI levels. The vertical dashed line represents the 95th percentile of TAFI levels. Fetal loss (p=0.39) was equally distrib-uted among quartiles (hori-zontal dashed lines).
Venous tromboembolism had occurred in 4 women (5%) with high TAFI levels and in 59 women (8%) with normal TAFI levels. Thrombophilic defects were equally dis-tributed among both groups, except for high factor VIII levels which were more fre-quently observed in women with high TAFI levels. Total numbers of pregnancies were 219 and 2223 in women with high TAFI levels and women with normal TAFI lev-els, respectively, and mean numbers were 2.9 per woman in each group.
Eighteen women (21%) with high TAFI levels had experienced fetal loss, compared to 211 women (28%) with normal TAFI levels; adjusted relative risk 0.66 (95% CI, 0.38–
1.16) (Table 1). Relative risks of early and late fetal loss were 0.74 (95% CI, 0.42–1.33) and 0.54 (95% CI, 0.14–2.09), respectively. Age did not affect the TAFI related relative risk of fetal loss. A cumulative distribution curve did not show a relationship between fetal loss and TAFI levels (Figure 1). Comparing quartiles of TAFI levels showed no relationship between fetal loss and TAFI levels; Q1, 29.2%; Q2, 25.1%; Q3, 29.1%; Q4, 22.9% (p=0.39).
Of 37 women who were excluded because they had venous thromboembolism prior to their first pregnancy, 0/2 with high TAFI levels had experienced fetal loss, compared to 8/35 women (23%) with normal TAFI levels.
In our study, high TAFI levels were not identified as a risk factor for fetal loss. In fact, fetal loss rates in women with high TAFI levels and in women with normal TAFI levels were comparable to those in the normal population; 8% versus 14% of preg-nancies (normal population, 10–15%)10. Of women, 21% versus 28% had fetal loss (normal population, no data available). Therefore, it is not likely that this finding can be explained by the high prevalence of concomitant thrombophilic defects. Only the prevalence of high factor VIII:C levels showed a difference between women with high TAFI levels and women with normal TAFI levels, but it did not influence fetal loss rates (p=0.37).
It has been suggested that thrombophilic defects are mainly associated with late fetal loss rather then early fetal loss4. Most women in our study experienced early fetal loss. The number of women with late fetal loss was too small to allow proper analysis. This may have influenced our negative finding. Although it is assumed that fetal loss in women with a thrombophilic defect is due to placental thrombosis, other mechanisms may also be involved, whereas the effects of various thrombophilic de-fects may be different. Experiments in mice provided evidence that fibrin degradation products, induced by the subsequent generation of thrombin and fibrin, cause cell death of throphoblasts13. This effect was reversed by anticoagulant and antifibri-nolytic drugs, and by depletion of fibrinogen. Increasing levels of TAFI during normal pregnancy in humans, especially in women with increased baseline levels, may sim-ilarly protect against fetal loss. Our observation that the risk of fetal losswas lower rather than higher at high TAFI levels might be explained in this way.
Previous studies on the association of thrombosis with high TAFI levels provided no consistent results, maybe due to differences between applied TAFI assays. These differences concern the sensitivity of enzyme linked immunosorbent assays (ELISA)
for various isoforms of TAFI12. TAFI activity assays may be influenced by thermal in-stability of TAFIa and may be sensitive for other carboxypeptidases in plasma11. We measured TAFI activity with an assay without these disadvantages.12
A limitation of this study is its retrospective design. Selection bias may have been introduced by excluding pregnancies after prior VTE, and consequently women at potentially higher risk of fetal loss. However, this is unlikely as only 2 of 37 excluded women had high TAFI levels, while they had no fetal loss.
In conclusion, our data suggests that high TAFI levels are not associated with an increased risk of early fetal loss.
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Fetal loss is not related eith TAFI levels