Department of Nephrology and Internal Medicine, Medical Academy of Bialystok, Bialystok, Zurawia, Poland
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Abstract |
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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), thrombinantithrombin complexes (TAT), plasminantiplasmin 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
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Introduction |
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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:
The activity of the fibrinolytic system was evaluated by measuring plasmin activity marker: plasminantiplasmin complexes (PAP).
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Subjects and methods |
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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+(systolicdiastolic)/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 CockcroftGault 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 (2.597.5th percentile; n=140) is 0.41.1 nmol/l.
The plasma thrombinantithrombin III complex levels were measured using enzyme immunoassay (Enzygnost TAT micro, Dade Behring, Germany) according to the manufacturer's instruction. Reference range for TAT (2.597.5th percentile; n=196) is 14.1 µg/l.
The plasma plasminantiplasmin complex levels were measured using enzyme immunoassay (Enzygnost PAP micro, Dade Behring, Germany) according to the manufacturer's instructions. Reference range for PAP (2.597.5th percentile; n=466) is 120700 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 ShapiroWilk W-test. Measurements normally distributed are reported as mean data±SD, non-normally distributed data are expressed as a median and minimalmaximal value. MannWhitney U-test, CochranCox 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.
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Results |
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Correlations between TAFI, sTM, F1+2, TAT and PAP antigens levels
TAFI was inversely correlated with PAP antigen in RTR (Figure 1). (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.
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Correlations between TAFI, sTM, F1+2, TAT antigens levels, and clinicalbiochemical 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).
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Discussion |
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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 PAI1 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.
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Notes |
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References |
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