A possible role of thrombin-activatable fibrinolysis inhibitor in disturbances of fibrinolytic system in renal transplant recipients

Tomasz Hryszko, Jolanta Malyszko, Jacek S. Malyszko, Szymon Brzosko, Krystyna Pawlak and Michal Mysliwiec

Department of Nephrology and Internal Medicine, Medical Academy of Bialystok, Bialystok, Zurawia, Poland



   Abstract
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Background. Cardiovascular disease (CVD) is a major cause of death in renal transplant recipients (RTR). Suppression of fibrinolysis plays a role in the progression of atherosclerosis. Accelerated progression of atherosclerosis and fibrinolytic system suppression has been observed in RTR. Despite many years of intensive research, the reason for impaired fibrinolysis in this patient population is not fully understood. Thrombin-activatable fibrinolysis inhibitor (TAFI) is a recently discovered glycoprotein combining coagulation and fibrinolysis. This study was conducted to evaluate concentrations of TAFI, markers of thrombin generation, endothelial injury, and some standard laboratory parameters in RTR receiving triple immunosuppressive drug regimen.

Methods. The study was performed in 29 stable, non-diabetic kidney transplant recipients treated with cyclosporin A, azathioprine, and prednisone and in 18 age- and sex-matched healthy volunteers. Soluble thrombomodulin (sTM), prothrombin fragments F1+2 (F1+2), thrombin–antithrombin complexes (TAT), plasmin–antiplasmin complexes (PAP), and TAFI were measured with commercially available kits.

Results. The RTR group had significantly higher plasma levels of TAT, F1+2, sTM and TAFI than the healthy volunteers. There were no differences in PAP concentrations between the two groups. Plasma sTM correlated inversely with creatinine clearance, body mass index, haemoglobin, and albumin. Plasma TAT level was positively associated with total cholesterol. TAFI antigen influenced negatively PAP antigen concentration.

Conclusions. On the basis of our research, we concluded that elevated circulating TAFI antigen might be a new link in the pathogenesis of impaired fibrinolysis in RTR, and thus atherosclerosis progression. In the patient group there is also evidence of endothelial injury, followed by secondary activation of the coagulation cascade. Hypercholesterolaemia in RTR is associated with enhanced thrombin activity.

Keywords: coagulation; cyclosporin A; endothelium injury; fibrinolysis; renal transplantation; thrombin-activatable fibrinolysis inhibitor



   Introduction
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
In the general population, cardiovascular disease (CVD) is responsible for the majority of deaths. CVD is also a major death threat to renal transplant recipients (RTR) [1]. Age, gender, diabetes mellitus, dyslipidaemia, and cigarette smoking are main risk factors for vascular disease in RTR [1]. Also suppression of the fibrinolytic system is associated with increased cardiovascular morbidity [2]. As shown by Robbie et al. [3], in atherosclerotic plaque, there is an imbalance between fibrinolysis activators and inhibitors in favour of the latter. In RTR, there is strong evidence of fibrinolytic system impairment [4,5], together with enhanced coagulation [6] and endothelial injury [7]. As reported by Blann and McCollum [8] markers of endothelial injury (e.g. thrombomodulin (TM) and von Willebrand factor (vWF)) are negative predictors of coronary events. One of the major factors contributing to the above phenomena observed in RTR is attributed mainly to immunosuppressive regimen in these patients. Glucocorticoids, as well as cyclosporin A (CsA) are probably the main factors responsible for this dysfunction. Decreased fibrinolytic activity in almost two-thirds of engrafted patients is possibly attributed to the use of steroids [4]. On the contrary, other studies indicate, that CsA increases platelet aggregation [9], impairs protein C activation [10], causes endothelial injury [11] and thrombin generation, lowers tissue plasminogen activator (t-PA), and raises plasminogen activator inhibitor 1 (PAI–1) levels [7]. The negative influence of CsA is supported by the report of van den Dorpel et al. [12], who observed a recovery of fibrinolytic activity after conversion from CsA to azathioprine.

As far as we know, there is no data about thrombin-activatable fibrinolysis inhibitor (TAFI), also referred to plasma procarboxypeptidase B in RTR. TAFI is a recently discovered, 60 kDa single-chained glycoprotein produced in the liver, which combines coagulation and fibrinolysis [13]. It is present in plasma as the proenzyme, which is converted to active form (TAFIa) by thrombin. TAFIa removes COOH-terminal lysine and arginine residues from fibrin, impairing formation of t-PA, plasminogen, and fibrin complex, which makes plasmin generation less effective [14]. At high concentrations, the glycoprotein is also a plasmin inhibitor [14]. The process of TAFI activation is catalysed by the presence of soluble as well as the cellular form of thrombomodulin [14]. Also the profibrinolytic properties of activated protein C seem to depend on TAFI [14].

Taking into consideration the above-described disturbances of fibrinolytic system in RTR and the (to our knowledge) lack of data on TAFI in these patients, we decided to investigate TAFI in this group of patients and its correlations with clinical and laboratory parameters. We also took measurements to determine if there is an environment predisposed to TAFI activation:

  1. activator: thrombin (generation of thrombin was measured as a prothrombin fragments F1+2 (F1+2) and its activity as thrombin–antithrombin complexes (TAT))
  2. endogenous catalyser: thrombomodulin (measured as soluble thrombomodulin)

The activity of the fibrinolytic system was evaluated by measuring plasmin activity marker: plasmin–antiplasmin complexes (PAP).



   Subjects and methods
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Patients and controls
The study population comprised 29 renal transplant recipients with stable grafts, without any infections (C-reactive protein within the normal range), diabetes mellitus, liver dysfunction (normal prothrombin time and normal activity of alanine and asparaginine aminotransferases) and 18 sex- and age-matched healthy controls. The immunosuppressive regimen consisted of CsA (134.49±49.62 ng/ml by RIA method using monoclonal antibodies), azathioprine (100–150 mg/day) and prednisone (5–7.5 mg/day). Subjects characteristic are shown in Table 1Go.


View this table:
[in this window]
[in a new window]
 
Table 1. Subjects' characteristics

 

Study protocol
The Local Ethic Committee approved the study protocol. The collection of clinical parameters and blood samples of patients was performed in the early morning between 7 and 8 a.m. after an overnight fast. Venous blood samples were collected in 3.8% sodium citrate at 9:1 volume ratio. Citrated blood was centrifuged at 3000 r.p.m. for 20 min to yield platelet-poor plasma. Samples were aliquoted and stored at -20°C until the assay. Clinical parameters included body mass index (BMI, calculated as weight (kg) divided by the square of height (m)), mean arterial blood pressure (MAP, calculated according to the formula: diastolic blood pressure+(systolic–diastolic)/3), and time from renal transplantation. As biochemical data: serum creatinine, haemoglobin, leukocyte and platelet count, albumin, activity of alanine and asparaginine aminotransferases, fibrinogen, prothrombin time, euglobulin clot lysis time, serum lipids, C reactive protein were measured by standard laboratory methods. To make euglobulin clot lysis time (ECLT) independent of fibrinogen concentration, we expressed plasma fibrinolytic activity as a fibrinolytic activity index (FAI=(fibrinogen/ECLT)x100). Creatinine clearance was calculated according to Cockcroft–Gault formula [15].

Immunoassay of TAFI, TM, TAT, F1+2, PAP
The plasma TAFI antigen levels were measured using enzyme immunoassay (TAFI-EIA, Affinity Biologicals Inc., Canada) according to the manufacturer's instructions. A single standard curve was constructed with use of standard plasma (BioMerieux, France). All results were expressed as a percentage of standard plasma TAFI concentration. A normal range for human plasma has yet to be established.

The soluble thrombomodulin levels were measured using enzyme-linked immunoadsorbent assay (IMUBIND Thrombomodulin ELISA Kit, American Diagnostica Inc.) according to the manufacturer's instructions. A normal range for human plasma has yet to be established.

The plasma F1+2 prothrombin fragments levels were measured using enzyme immunoassay (Enzygnost F1+2 micro, Dade Behring, Germany) according to the manufacturer's instructions. According to the manufacturer reference range for F1+2 (2.5–97.5th percentile; n=140) is 0.4–1.1 nmol/l.

The plasma thrombin–antithrombin III complex levels were measured using enzyme immunoassay (Enzygnost TAT micro, Dade Behring, Germany) according to the manufacturer's instruction. Reference range for TAT (2.5–97.5th percentile; n=196) is 1–4.1 µg/l.

The plasma plasmin–antiplasmin complex levels were measured using enzyme immunoassay (Enzygnost PAP micro, Dade Behring, Germany) according to the manufacturer's instructions. Reference range for PAP (2.5–97.5th percentile; n=466) is 120–700 µg/l.

Because of manufacturers' suggestions that each laboratory should determine its own reference range, we performed each assay both in controls and in RTR.

Statistics
If possible data were logarithmically transformed to achieve normal distribution (F1+2, sTM, age, time from transplantation). Normality of variable distribution was tested using Shapiro–Wilk W-test. Measurements normally distributed are reported as mean data±SD, non-normally distributed data are expressed as a median and minimal–maximal value. Mann–Whitney U-test, Cochran–Cox test, Fisher exact test or Student's t-test was used to compare differences between groups, when appropriate. Correlations were assessed using Pearson's or Spearman's linear regression analysis, when appropriate. A P-value<0.05 was considered to be significant.



   Results
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
TAFI, sTM, F1+2, TAT, PAP, fibrinogen, euglobulin clot lysis time, and fibrinogen levels
RTR have significantly higher concentrations of TAFI, sTM, F1+2, TAT antigen levels compared to controls (Table 2Go). We did not observe any differences between RTR and control group in PAP concentrations. Concentrations of TAFI, sTM, F1+2, TAT and PAP did not significantly differ between males and females, nor in RTR and in the control group. RTR have prolonged ECLT; however, it did not reach statistical significance (P=0.09). Although we observed an elevation of fibrinogen in RTR, it was not statistically significant. There were no differences between the two examined groups in mean FAI values.


View this table:
[in this window]
[in a new window]
 
Table 2. Plasma TAFI, sTM, F1+2, TAT levels in RTR and control group

 

Correlations between TAFI, sTM, F1+2, TAT and PAP antigens levels
TAFI was inversely correlated with PAP antigen in RTR (Figure 1Go). (r=-0.4, P<0.05). F1+2 antigen correlated with TAT antigen in RTR as well as in the control group. There was no correlation between TAFI antigen and sTM, F1+2, and TAT antigen in either RTR or controls.



View larger version (11K):
[in this window]
[in a new window]
 
Fig. 1. Correlation between TAFI and PAP in RTR. r=-0.4, P<0.04.

 

Correlations between TAFI, sTM, F1+2, TAT antigens levels, and clinical–biochemical parameters
sTM correlated inversely with creatinine clearance (r=-0.51, P<0.01), BMI (r=-0.55, P<0.01), haemoglobin (r=-0.5, P<0.01), and albumin (r=-0.46, P<0.05). sTM did not correlate significantly with MAP (r=0.34, P=0.11), leukocyte count (r=-0.3, P=0.12), platelet count (r=-0.08, P=0.68), FAI (r=0.08, P=0.69), or total cholesterol (r=-0.1, P=0.8).

F1+2 correlated with creatinine clearance (r=-0.29, P=0.15), BMI (r=-0.08, P=0.72), MAP (r=-0.1, P=0.7), albumin (r=-0.3, P=0.18), haemoglobin (r=-0.07, P=0.7), leukocyte count (r=-0.07, P=0.7), platelet count (r=0.3, P=0.1), FAI (r=0.3, P=0.14), total cholesterol (r=0.34, P=0.08).

TAT was positively correlated with total cholesterol (r=0.49, P<0.01) and inversely with BMI (r=-0.46, P<0.05). The correlation between TAT and albumin almost reached statistical significance (r=-0.42, P=0.056). TAT did not correlate significantly with creatinine clearance (r=-0.25, P=0.24), MAP (r=-0.3, P=0.18), haemoglobin (r=-0.2, P=0.33), leukocyte count (r=-0.2, P=0.35), platelet count (r=0.3, P=0.13), or FAI (r=0.17, P=0.46).

TAFI did not significantly correlate with creatinine clearance (r=-0.12, P=0.6), BMI (r=0.05, P=0.8), MAP (r=-0.2, P=0.3), albumin (r=-0.2, P=0.4), haemoglobin (r=-0.3, P=0.1), leukocyte count (r=-0.02, P=0.9), platelet count (r=-0.1, P=0.6), FAI (r=-0.35, P=0.09), or total cholesterol (r=-0.09, P=0.6).

PAP antigen correlated positively with FAI (r=0.47, P<0.05). No other statistically significant correlations were found between PAP and creatinine clearance (r=-0.18, P=0.35), BMI (r=-0.09, P=0.65), MAP (r=-0.14, P=0.52), albumin (r=0.1, P=0.65), haemoglobin (r=0.05, P=0.8), leukocyte count (r=-0.04, P=0.9), platelet count (r=0.04, P=0.8), or total cholesterol (r=0.15, P=0.4).



   Discussion
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
In our study, we found significantly elevated levels of TAFI in RTR plasma. On the basis of our results we may rather exclude renal involvement in the glycoprotein metabolism (lack of correlation between creatinine clearance and TAFI antigen concentration). The cause of elevated TAFI antigen level awaits further research.

The peculiar environment (high thrombin generation and sTM concentration) that appears in RTR plasma, probably enables activation and conversion of TAFI in the presence of endogenous catalyser from the inactive to active form. To the best of our knowledge, this is the first report to describe such an abnormality in this group of patients. As found by Mosnier et al. [16], TAFI antigen concentration directly influences clot lysis in healthy volunteers, at least in vitro. In addition, a recent report by Klement et al. [17] proves the important role of TAFI in inhibition of fibrinolysis in vivo. In the animal model, they have shown that administration of TAFIa inhibitor significantly improved t-PA-induced thrombolysis. Based on the aforementioned findings we conclude that our observation may be another element in the complicated and not fully understood image of fibrinolytic system suppression in RTR, which plays an important role in the atherosclerosis progression. In addition, a negative association between TAFI antigen and PAP may support the above statement.

The lack of association between TAFI antigen concentration and FAI is probably due to the instability of TAFIa and its inactivation during performance of ECLT.

At first sight, it may seem confounding that RTR patients and the controls do not differ with regard to PAP concentration. But when we take into consideration the hyperactivation of coagulation reflected by elevated levels of thrombin generation and activity markers, we conclude that this lack of difference is another argument for impairment of fibrinolysis in RTR.

In our observations, RTR did not differ from the controls according to FAI, despite having almost significantly prolonged ECLT. This suggests that ECLT prolongation is most probably due to the elevated fibrinogen in RTR, which next to PAI–1 and t-PA [18] influences this laboratory parameter. Other researchers reporting prolongation of ECLT in this sector of the population did not make correction for elevated fibrinogen levels [7].

The elevation of sTM, a marker of endothelial cell injury [19] in RTR plasma, is probably due to CsA toxicity [11] and/or graft function impairment [20], as reflected by a positive correlation between creatinine clearance and sTM concentration. It should be noted that Blann and McCollum [8] found elevated sTM concentration as a predictor of adverse events among subjects with atherosclerosis. However, we have to take into consideration that there is no agreement of their study finding. On the other hand, the ARIC study [21] showed that high concentrations of sTM are associated with a decreased risk of coronary heart disease. Negative association between sTM and albumin confirms the protective role of the latter on endothelial cells. Albumin plays the role of a lysophosphatydylocholine binder, a toxic substance causing injury to the endothelium [22]. And Zoellner et al. [23] have found, that albumin abrogates the apoptosis of endothelial cells. The phenomenon may explain the results of Kasiske et al. [1], who reported that serum albumin concentration might serve as a vascular disease risk factor. The above findings demonstrate the need for further research in the field of endothelial injury markers and their value as prognostic markers of future cardiovascular events.

The negative relationship between BMI and sTM is difficult to explain; why overweight or obese people have a lower concentration of endothelial cell injury marker than those of normal weight. It should be noted that none of our RTR was underweight (min. BMI 19 kg/m2), thus negative association could not be attributed to the poorer nutritional status of patients with high concentration of sTM.

The pronounced coagulation enhancement reflected by elevated levels of thrombin generation and activity markers (F1+2 and TAT respectively) in RTR are in agreement with the earlier reports of others researchers [6,7]. The initiation of the coagulation cascade is most likely to be a secondary response to the toxic influence of CsA on the endothelial cell as reported by Bombeli et al. [24]. The positive association between TAT and cholesterol concentration points to the possibility that lipid abnormalities cause an enhancement of thrombin activity in RTR. Lack of a relationship between cholesterol and F1+2 may be attributed to sensitivity of this fragment to complications with venepuncture.

In conclusion, in our study we found that RTR on a triple immunosuppressive drug regimen have elevated concentration of TAFI. This is a new link in the pathogenesis of fibrinolysis suppression in this population. In that group, endothelial injury is also observed, as well as most probably secondary activation of coagulation cascade.



   Notes
 
Correspondence and offprint requests to: Tomasz Hryszko MD, Department of Nephrology and Internal Medicine, Medical Academy of Bialystok, 15-540 Bialystok, Zurawia 14, Poland. Back



   References
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 

  1. Kasiske BL, Guijarro C, Massy ZA, Wiederkehr MR, Ma JZ. Cardiovascular disease after renal transplantation. J Am Soc Nephrol 1996; 7: 158–165[Abstract]
  2. Meade TW, Ruddock V, Stirling Y, Chakrabarti R, Miller GJ. Fibrinolytic activity, clotting factors and long-term incidence of ischemic heart disease in the Northwick Park Heart study. Lancet 1993; 342: 1076–1079[ISI][Medline]
  3. Robbie LA, Booth NA, Brown AJ, Bennett B. Inhibitors of fibrinolysis are elevated in atherosclerotic plaque. Arterioscler Thromb Vasc Biol 1996; 16: 539–545[Abstract/Free Full Text]
  4. Patrassi GM, Sartori MT, Rigotti P et al. Reduced fibrinolytic potential one year after kidney transplantation. Relationship to long-term steroid treatment. Transplantation 1995; 59: 1416–1420[ISI][Medline]
  5. Verpooten GA, Cools FJ, Van der Planken MG et al. Elevated plasminogen activator inhibitor levels in cyclosporin-treated renal-allograft recipients. Nephrol Dial Transplant 1996; 11: 347–351[Abstract]
  6. Baker LRI, Tucker B, Kovacs IB. Enhanced in vitro hemostasis and reduced thrombolysis in cyclosporine-treated renal transplant recipients. Transplantation 1990; 49: 905–909[ISI][Medline]
  7. Malyszko J, Malyszko JS, Pawlak K, Mysliwiec M. The coagulo-lytic system and endothelial function in cyclosporine-treated kidney allograft recipients. Transplantation 1996; 62: 828–830[ISI][Medline]
  8. Blann AD, McCollum CN. Von Willebrand factor and soluble thrombomodulin as predictors of adrverse events among subjects with peripheral or coronary atherosclerosis. Blood Coagul Fibrinol 1999; 10: 375–380[ISI][Medline]
  9. Muraki T, Taka T, Noguchi T, Ishii H, Seki J, Yamamoto J. Effects of cyclosporine and FK506 on in vitro high shear-induced platelet reactivity in rat and human non-anticoagulated blood. Transplantation 1998; 65: 1132–1134[ISI][Medline]
  10. Garcia-Maldonaldo M, Kaufman CE, Comp PC. Decrease in endothelial cell-dependent protein C activation induced by thrombomodulin by treatment with cyclosporine. Transplantation 1991; 51: 701–705[ISI][Medline]
  11. Evans SM, Giddings JC, Muraki T, Yamamoto J. Expression of von Willebrand factor, P-selectin (CD62P) and thrombomodulin in human endothelial cells in culture modulated by cyclosporin A. Clin Lab Haematol 1997; 19: 115–122[ISI][Medline]
  12. van den Dorpel MA, Veld AJ, Levi M, ten Cate JW, Weimar W. Beneficial effects of conversion from cyclosporine to azathioprine on fibrinolysis in renal transplant recipients. Arterioscler Thromb Vasc Biol 1999; 19: 1555–1558[Abstract/Free Full Text]
  13. Bajzar L, Manuel R, Nesheim ME. Purification and characterization of TAFI, a thrombin-activable fibrinolysis inhibitor. J Biol Chem 1995; 270: 14477–14484[Abstract/Free Full Text]
  14. Nesheim M, Wang W, Boffa M, Nagashima M, Morser J, Bajzar L. Thrombin, thrombomodulin and TAFI in the molecular link between coagulation and fibrinolysis. Thromb Haemost 1997; 78: 386–391[ISI][Medline]
  15. Cockcroft DW, Gault MH. Prediction of creatinine clearance from serum creatinine. Nephron 1976; 16: 31–41[ISI][Medline]
  16. Mosnier LO, von dem Borne PA, Meijers JC, Bouma BN. Plasma TAFI levels influence the clot lysis time in healthy individuals in the presence of an intact intrinsic pathway of coagulation. Thromb Haemost 1998; 80: 829–835[ISI][Medline]
  17. Klement P, Liao P, Bajzar L. A novel approach to arterial thrombolysis. Blood 1999; 94: 2735–2743[Abstract/Free Full Text]
  18. Urano T, Sumiyoshi K, Pietraszek MH, Takada Y, Takada A. PAI–1 plays an important role in the expression of t-PA activity in the euglobulin clot lysis by controlling the concentration of free t-PA. Thromb Haemost 1991; 66: 474–478[ISI][Medline]
  19. Blann AD, Lip GYH. The endothelium in atherothrombotic disease assessment of function, mechanisms and clinical implications. Blood Coagul Fibrinol 1998; 9: 297–306[ISI][Medline]
  20. Rustom R, Leggat H, Tomura HR, Hay CR, Bone JM. Plasma thrombomodulin in renal disease: effects of renal function and proteinuria. Clin Nephrol 1998; 50: 337–341[ISI][Medline]
  21. Salomaa V, Matei C, Aleksic N et al. Soluble thrombomodulin as a predictor of incident coronary heart disease and symptomless carotid artery atherosclerosis in the Atherosclerosis Risk in Communities (ARIC) Study: a case-cohort study. Lancet 1999; 353: 1729–1734[ISI][Medline]
  22. Joles JA, Stroes ES, Rabelink TJ. Endothelial function in proteinuric renal disease. Kidney Int Suppl. 1999; 71: S57–61
  23. Zoellner H, Höfler M, Beckmann R et al. Serum albumin is a specific inhibitor of apoptosis in human endothelial cells. J Cell Sci 1996; 109: 2571–2580[Abstract/Free Full Text]
  24. Bombeli T, Muller M, Straub PW, Haeberli A. Cyclosporine-induced detachment of vascular endothelial cells initiates the intrinsic coagulation system in plasma and whole blood. J Lab Clin Med 1996; 127: 621–634[ISI][Medline]
Received for publication: 20. 7.00
Accepted in revised form: 26. 2.01





This Article
Abstract
FREE Full Text (PDF)
Alert me when this article is cited
Alert me if a correction is posted
Services
Email this article to a friend
Similar articles in this journal
Similar articles in ISI Web of Science
Similar articles in PubMed
Alert me to new issues of the journal
Add to My Personal Archive
Download to citation manager
Search for citing articles in:
ISI Web of Science (9)
Disclaimer
Request Permissions
Google Scholar
Articles by Hryszko, T.
Articles by Mysliwiec, M.
PubMed
PubMed Citation
Articles by Hryszko, T.
Articles by Mysliwiec, M.