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NDT Advance Access originally published online on February 27, 2007
Nephrology Dialysis Transplantation 2007 22(5):1347-1350; doi:10.1093/ndt/gfl753
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© The Author [2007]. Published by Oxford University Press on behalf of ERA-EDTA. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Association of genotypes of thrombin-activatable fibrinolysis inhibitors with thrombotic microangiopathies—a pilot study

Christoph Sucker1, Gerd Ruediger Hetzel2, Firuseh Farokhzad2, Fieras Dahhan2, Michael Schmitz2, Christine Kurschat2, Bernd Grabensee2, Beate Maruhn-Debowski1, Rainer Zotz1 and Ruediger Scharf1

1Department of Haemostasis and Transfusion Medicine and 2Department of Nephrology, Heinrich Heine University Medical Center, Duesseldorf, Germany

Correspondence and offprint requests to: Prof. Dr Rudiger E. Scharf FAHA, Department of Haemostasis and Transfusion Medicine, Heinrich Heine University Medical Center, Moorenstrasse 5, 40221 Duesseldorf, Germany. Email: rscharf{at}uni-duesseldorf.de



   Abstract
Top
 Abstract
Introduction
 Subjects and methods
 Results
 Discussion
 Acknowledgement
 References
 
Background. Thrombotic microangiopathies are characterized by microvascular thrombosis, consequently leading to microangiopathic haemolytic anaemia, thrombocytopenia and organ dysfunction. Although recent research has elucidated the pathogenesis of these rare thrombotic disorders to some extent, the determinants contributing to the manifestation remain rather unclear in the majority of affected patients.

Method. In the present pilot study, we used a case-control design, enrolling 40 patients [mean age (±SD) 35 ± 11 years] with a history of thrombotic microangiopathy and 689 control subjects to evaluate the association of gene polymorphisms of the thrombin-activatable fibrinolysis inhibitor (TAFI) with the manifestation of these rare thrombotic disorders. These polymorphisms are major determinants of TAFI plasma levels that were found to modulate the onset of venous and arterial thrombosis.

Results. As a result of our study, the prevalence of the GG genotype (adjusted OR 2.58; 95% CI 0.9–6.1, P = 0.044) and the G allele (adjusted OR 2.2; 95% CI 1.2–4.2, P = 0.017) of the C1542G polymorphism was significantly higher in patients with a history of thrombotic microangiopathy compared with controls. A higher prevalence of the GG genotype of the TAFI G505A polymorphism was also observed, but this association was not statistically significant (adjusted OR 4.97, CI 0.7–36.7, P = 0.12). Considering the established genotype–phenotype associations, our observation suggests that lower TAFI plasma levels are associated with an increased risk for the manifestation of thrombotic microangiopathies. A diminished inactivation of C3a and C5a—also known from haemolytic uraemic syndrome (HUS) associated with factor H deficiency—might be the most likely explanation.

Conclusions. The results of our pilot study indicate that the GG genotype of the C1542G polymorphism of TAFI displays risk factors for the manifestation of thrombotic microangiopathies. Our observation provides a rationale to assess genotype–phenotype relations by determination of TAFI plasma levels in various stages of disease in patients suffering from these rare thrombotic disorders.

Keywords: TAFI; thrombin-activatable fibrinolysis inhibitor; thrombotic microangiopathies



   Introduction
Top
 Abstract
 Introduction
Subjects and methods
 Results
 Discussion
 Acknowledgement
 References
 
Thrombotic microangiopathies are rare life-threatening thrombotic disorders associated with thromboses in the arterial microvasculature. The characteristic clinical triad consists of microangiopathic haemolytic anaemia, thrombocytopenia and organ dysfunction. Depending on the pattern of organ involvement, thrombotic microangiopathies are distinguished in thrombotic thrombocytopenic purpura (TTP) characterized by neurological symptoms, haemolytic-uraemic syndrome (HUS) characterized by renal insufficiency and intermediate forms (TTP–HUS) with both neurological and renal manifestations [1,2].

Although research has elucidated the development of thrombotic microangiopathies to some extent, many pathogenetic aspects remain unclear. Recently, published studies indicate that a relevant deficiency of the von Willebrand factor cleaving protease ADAMTS 13 (‘a desintegrin-like and metalloprotease with thrombospondin-1 motif’), that has been regarded as the major pathogenetic factor in TTP [3], is present only in one-third of affected patients [4], and appears to define a distinct subgroup of disease [5]. The pathogenesis of HUS is even less clear and, although defects of the complement system including factor H deficiency have been identified as contributing factors, such defects are considerably rare [6,7]. Since current knowledge does not sufficiently explain the manifestation of thrombotic microangiopathies, hitherto unknown additional pathogenetic factors appear to be relevant in this clinical setting. Considering that thrombotic microangiopathies are (i) thrombotic disorders that (ii) are almost always associated with an inflammatory stimulus, prothrombotic and proinflammatory genetic variants appear to be the most promising candidates for investigation.

In this study, we evaluated the potential role of the thrombin-activatable fibrinolysis inhibitor (TAFI), an important regulator of both fibrinolysis and inflammation, for the manifestation of thrombotic microangiopathies [8,9]. An influence of TAFI plasma levels on the onset of arterial and venous thrombosis has previously been demonstrated. Since the interindividual variability of this protein is strongly genetically controlled [10–14], we performed a study of the C1542G and the G505A (Ala147Thr) polymorphisms as important determinants of TAFI plasma levels in 40 patients with a history of thrombotic microangiopathies [15].



   Subjects and methods
Top
 Abstract
 Introduction
 Subjects and methods
Results
 Discussion
 Acknowledgement
 References
 
Subjects
From January 2003 to March 2006, 40 consecutive patients [mean age (±SD) 35 ± 11 years, range 19–66 years; 32 females, 8 males] with a history of thrombotic microangiopathy were recruited by the Department of Nephrology of the Heinrich Heine University Medical Center, Duesseldorf, Germany. The diagnosis of thrombotic microangiopathy was established by the presence of (i) microangiopathic haemolytic anaemia (haemoglobin < 12 g/dl) with negative direct Coombs test and schistocytes in the peripheral blood in combination with (ii) thrombocytopenia (platelet count < 100 000/µl), and (iii) organ dysfunction without adequate alternative explanation [1,2]. Nine patients with isolated neurological deficits were diagnosed for thrombotic thrombocytopenic purpura (TTP), 18 patients with dominating renal dysfunction were diagnosed with haemolytic uraemic syndrome (HUS) and 13 patients with combined neurological and renal symptoms were classified as TTP-HUS [16].

Healthy individuals [mean age ± (SD) 37 ± 14 years, range 13–93 years; 435 females/329 males] served as controls. These subjects were from the same geographical region as the patients, but not related to them. The study was performed according to the Helsinki Declaration and written informed consent was obtained.

Methods
Laboratory tests
Genomic DNA was extracted from peripheral blood mononuclear cells according to standard procedures using the Qiagen system (Qiagen, Hilden, Germany). Genotyping of the TAFI polymorphisms C1542G and G505A (Ala147Thr) was performed by use of sequence-specific hybridization probes following DNA amplification by polymerase chain reaction using a LightCylerTM (Roche, Germany). For this method, protocols for the determination of respective genetic polymorphisms were adapted to the LightCyclerTM by modification of previously published procedures [11–14].

Statistical analysis
From the genotype prevalences of the TAFI polymorphisms C1542G and G505A in patients with thrombotic microangiopathies and control subjects, the relative risk of patiens carrying one of the risk-associated genotypes was determined. To rule out an influence of inhomogenous gender and age distribution in patients and controls, a multiple regression analysis was used to adjust odds ratios (OR) for age and gender and calculate modified Wald intervals (CI). For statistical evaluation, the {chi}2 test was used and significance assumed at a P-value <0.05.

All statistical analyses were performed with the SAS software (version 8.2, SAS Institute Inc., Cary, NC, USA). Since there are no previously published data available regarding the prevalence of TAFI genotype frequencies in thrombotic microangiopathies, no sample size calculations were performed in this pilot study.



   Results
Top
 Abstract
 Introduction
 Subjects and methods
 Results
Discussion
 Acknowledgement
 References
 
Statistical evaluation revealed a significantly higher prevalence of the GG genotype of the TAFI C1542G polymorphisms in patients compared with controls (adjusted OR 2.58, CI 0.9–6.1, P = 0.04). According to subgroup analyses, the association obtained for the whole group of patients could be attributed to a higher prevalence of this genotype in subgroups of patients suffering from TTP (adjusted OR 4.13, CI 0.8–21, P = 0.09) and HUS (adjusted OR 2.92, CI 0.8–10.4, P = 0.1). In addition, carriers of the G allele (GG and CG genotypes) of the TAFI C1542G polymorphism were more prevalent in the patients (OR 2.2, CI 1.2–4.2, P = 0.017), particularly those suffering from HUS (OR 3.26, CI 1.2–8.8, P = 0.019). The higher prevalence of the GG genotype of the G505A polymorphism in the patients (OR 4.97, CI 0.7–36.7) was not statistically significant (Table 1).


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  		Table 1. Non-adjusted and adjusted odds ratios and confidence intervals for genotypes of the TAFI polymorphisms for TMA and subgroups of patients with TTP, HUS and TTP–HUS

 


   Discussion
Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
Acknowledgement
 References
 
This is the first pilot study dealing with the association of TAFI genotypes with thrombotic microangiopathies. As demonstrated, the GG genotype of the TAFI C1542G polymorphism is significantly more prevalent in patients suffering from these rare thrombotic disorders. In addition, the GG genotype of the other analysed TAFI polymorphism, G505A, had a higher prevalence in the patients. Along with our findings, carriers of the GG genotype of the TAFI C1542G polymorphism appear to be at an increased risk for the manifestation of thrombotic microangiopathies.

Several studies demonstrated that carriers of the G allele of the TAFI C1542G polymorphism have lower TAFI plasma levels [11–14]. To explain why genotypes that are associated with lower TAFI levels positively correlate with the occurrence of thrombotic microangiopathies, a closer look at TAFI function is required. TAFI, the ‘thrombin-activatable fibrinolysis inhibitor’, has an important role as a regulator of both fibrinolysis and inflammation (Figure 1). Activated by thrombin, TAFI inhibits fibrinolysis by cleaving residues from partially degraded fibrin that are required for interaction with plasmin, the key enzyme of fibrinolysis [8,9]. TAFIa also downregulates inflammation by inactivating important mediators such as the anaphylatoxins C3a and C5a, that are important mediators of inflammation and potent leucocyte chemoattractants, and also bradykinin [17–20]. An uncontrolled activity of C3a and C5a with consecutive damage of endothelial and subendothelial structures is thought to be the key mechanism in HUS associated with factor H deficiency. Low TAFI levels leading to an impaired clearance of C3a and C5a possibly induce the same effects as a factor H-deficient state [6,7].


Figure 1
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  		Fig. 1. TAFI function in fibrinolysis and inflammation. Activated by thrombin, TAFIa suppresses fibrinolysis by inhibition of plasmin effects on fibrin(ogen) and also inflammation by inactivation of mediators such as anaphylatoxins (C3a, C5a) and bradykinin. Thus, low TAFI activities will enhance fibrinolysis and, otherwise, promote inflammation. Notably, the effect of low TAFI levels is similar to factor H deficiency that leads to increased activation of proinflammatory complement factors.

 
The dual effect of TAFI—antithrombotic but proinflammatory—is indirectly supported by the observation that TAFI plasma levels exceeding the 90th percentile are protective for myocardial infarction that is most likely explained by the antiinflammatory effect [13]. In contrast, the association of elevated TAFI levels with an increased risk for venous thrombosis reported in the Leiden Thrombophilia Study can be explained by the inhibitory effect of TAFI on fibrinolysis [10].

Since our current genetic study provides evidence for a role of TAFI levels for the onset of thrombotic microangiopathies, we are planning to assess TAFI plasma levels in respective patients and to study genotype–phenotype relations in an ongoing trial. Acute thrombotic and inflammatory events will additionally modulate the levels of TAFI that acts as an acute phase protein [21]. Thus, our approach will crucially depend on the definition of appropriate time points for examination. Furthermore, the intraindividual and interindividual variability in TAFI plasma levels will complicate studies regarding genotype–phenotype relations in patients with thrombotic microangiopathies. Based on the results of TAFI genotyping, we speculate that carriers of risk-associated genotypes will have lower TAFI plasma levels during the initiation of thrombotic microangiopathies, enhancing the triggering effect of inflammation.

In summary, we found that TAFI genotypes encoding for lower TAFI plasma levels were associated with an increased risk for the manifestation of thrombotic microangiopathies. In analogy to its role in myocardial infarction, low TAFI levels might predispose to thrombus formation in the arterial microvasculature by impairing downregulation of local inflammation and, particularly, the clearance of C3b und C5b. In addition to standard diagnostic procedures, genotyping of TAFI polymorphisms could improve risk stratification of affected patients. Further studies including the assessment of TAFI genotype–phenotype relations in this clinical setting are required.



   Acknowledgement
Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 Acknowledgement
References
 
This work was supported by a grant of the Deutsche Forschungsgemelnschaft, Sonderforschungsbereich 612, TPB2. Additional support was provided by the Biological Medical Research Center.

Conflict of interest statement. None declared.



   References
Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 Acknowledgement
 References
 

  1. Franchini M, Zaffanello M, Veneri D. (2006) Advances in the pathogenesis, diagnosis and treatment of thrombotic thrombocytopenic purpura and hemolytic uremic syndrome. Thromb Res 118:177–184.[CrossRef][Web of Science][Medline]
  2. Lammle B, Kremer Hovinga JA, Alberio L. (2005) Thrombotic thrombocytopenic purpura. J Thromb Haemost 3:1663–1675.[CrossRef][Web of Science][Medline]
  3. Furlan M. (2003) Deficient activity of von Willebrand factor-cleaving protease in thrombotic thrombocytopenic purpura. Expert Rev Cardiovasc Ther 1:243–255.[CrossRef][Medline]
  4. Terrell DR, Williams LA, Vesely SK, Lammle B, Hovinga JA, George JN. (2005) The incidence of thrombotic thrombocytopenic purpura-hemolytic uremic syndrome: all patients, idiopathic patients, and patients with severe ADAMTS-13 deficiency. J Thromb Haemost 3:1432–1436.[CrossRef][Web of Science][Medline]
  5. Coppo P, Bengoufa D, Veyradier A, et al. (2004) Severe ADAMTS13 deficiency in adult idiopathic thrombotic microangiopathies defines a subset of patients characterized by various autoimmune manifestations, lower platelet count, and mild renal involvement. Medicine 83:233–244.[Medline]
  6. Zipfel PF, Misselwitz J, Licht C, Skerka C. (2006) The role of defective complement control in hemolytic uremic syndrome. Semin Thromb Hemost 32:146–154.[CrossRef][Web of Science][Medline]
  7. Fremeaux-Bacchi V, Kemp EJ, Goodship JA, et al. (2005) The development of atypical haemolytic-uraemic syndrome is influenced by susceptibility factors in factor H and membrane cofactor protein: evidence from two independent cohorts. J Med Genet 42:852–856.[Abstract/Free Full Text]
  8. Bajzar L, Manuel R, Nesheim ME. (1995) Purification and characterization of TAFI, a thrombin-activatable fibrinolysis inhibitor. J Biol Chem 270:14477–14484.[Abstract/Free Full Text]
  9. Bouma BN and Mosnier LO. (2003/2004) Thrombin activatible fibrinolysis inhibitor (TAFI) at the interface between coagulation and fibrinolysis. Pathophysiol Haemost Thromb 33:375–381.[CrossRef]
  10. Van Tilburg NH, Rosendaal FR, Bertina RM. (2000) Thrombin activatable fibrinolysis inhibitor and the risk for deep vein thrombosis. Blood 95:2855–2859.[Abstract/Free Full Text]
  11. Boffa MB, Nesheim ME, Koschinsky ML. (2001) Thrombin activatible fibrinolysis inhibitor (TAFI): molecular genetics of an emerging potential risk factor for thrombotic disorders. Curr Drug Targets Cardiovasc Haematol Disord 1:59–74.[CrossRef][Medline]
  12. Franco RF, Fagundes MG, Meijers JCM, et al. (2001) Identification of polymorphisms in the 5'-untranslated region of the TAFI gene: relationship with plasma TAFI levels and risk of venous thrombosis. Haematologica 86:510–517.[Abstract/Free Full Text]
  13. Juhan-Vague I, Morange PE, Aubert H, et al. (2002) Plasma thrombin-activatable fibrinolysis inhibitor antigen concentration and genotype in relation to myocardial infarction in the north and south of Europe. Arterioscler Thromb Vasc Biol 22:867–873.[Abstract/Free Full Text]
  14. Henry M, Aubert H, Morange PE, et al. (2001) Identification of polymorphisms in the promoter and the 3’ region of the TAFI gene: evidence that plasma TAFI antigen levels are strongly genetically controlled. Blood 97:2053–2058.[Abstract/Free Full Text]
  15. Morange PE, Tregouet DA, Frere C, et al. (2005) TAFI gene haplotypes, TAFI plasma levels and future risk of coronary heart disease: the PRIME Study. J Thromb Haemost 3:1503–1510.[CrossRef][Web of Science][Medline]
  16. Moake JL. (2002) Thrombotic microangiopathies. N Engl J Med 347:589–600.[Free Full Text]
  17. Campbell W and Okada H. (1989) An arginine specific carbocypeptidase generated in blood during coagulation or inflammation which is unrelated to carboxypeptidase N or its subunits. Biochem Biophys Re Commun 162:933–939.[CrossRef]
  18. Shinohara T, Sakurada C, Suzuki T, et al. (1994) Pro-carboxypeptidase R cleaves bradykinin following activation. Int Arch Allergy Immunol 103:100–104.
  19. Campbell WD, Lazoura E, Okada N, Okada H. (2002) Inactivation of C3a and C5a octapeptides by carboxypeptidase R and carboxypeptidase N. Microbiol Immunol 46:131–134.[Web of Science][Medline]
  20. Myles T, Nishimura T, Yun TH, et al. (2003) Thrombin Activatible Fibrinolysis Inhibitor, a potential regulator of of vascular inflammation. J Biol Chem 278:51059–51067.[Abstract/Free Full Text]
  21. Boffa MB, Hamill JD, Maret D, et al. (2003) Acute phase mediators modulate thrombin-activable fibrinolysis inhibitor (TAFI) gene expression in HepG2 cells. J Biol Chem 278:9250–9257.[Abstract/Free Full Text]
Received for publication: 1. 1.06
Accepted in revised form: 17.11.06


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