Department of Nephrology and Internal Medicine, Medical Academy, Bialystok, Poland
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Abstract |
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Methods. Twenty-five patients dialysed using a single injection of enoxaparin (at a mean dose of 0.68 mg/kg) were randomly assigned to either receive UFH administered as a mean bolus of 42.1 IU/kg and continuous infusion of 57.8 IU/kg (n=12) or to be maintained on enoxaparin (n=13), and were followed prospectively for 12 weeks. Plasma immunoreactive TF, TFPI and PF 1+2 were measured at the start and after 10 and 180 min of HD, and compared with values in 15 healthy controls.
Results. Pre-dialysis TF, TFPI and PF 1+2 were higher than normal (all P<0.0001). TF and PF 1+2 did not change, while TFPI levels, compared with baseline, increased at each interval in enoxaparin-anticoagulated HD patients (all P<0.0001). TFPI increments correlated inversely with pre-dialysis TFPI (both P<0.0007). In patients switched to UFH, TF levels remained unchanged compared with pre-randomization values, TFPI increased at each interval of HD sessions (all P<0.035) and PF 1+2 increased pre-dialysis (P=0.015). The over-dialysis effects of UFH resembled those of enoxaparin. In contrast, baseline TFPI and its 10-min rise correlated inversely with the UFH loading dose (both P<0.040). Pre-dialysis PF 1+2 was inversely associated with TFPI increments (both P<0.034), and directly with pre-dialysis TFPI (P=0.018) and the UFH loading dose (P=0.045).
Conclusions. Depletion of heparin-releasable stores of TFPI is an untoward effect of repeated anticoagulation during maintenance HD therapy. The traditional UFH regimen is more prothrombotic than single enoxaparin injections, with high loading doses of UFH being involved in TFPI exhaustion and subsequent hypercoagulability.
Keywords: anticoagulation; enoxaparin; haemodialysis; tissue factor; tissue factor pathway inhibitor; unfractionated heparin
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Introduction |
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In this study, we followed over-dialysis plasma levels of total TFPI and TF in relation to an activated coagulation marker, prothrombin fragment 1+2 (PF 1+2), in HD patients initially anticoagulated with LMWH enoxaparin, and then randomly assigned to receive either UFH or LMWH enoxaparin. Our aim was to determine how the type of heparin used during HD affected circulating TF and TFPI, and whether it had any impact on blood coagulation.
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Patients and methods |
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Before each HD session, the extracorporeal circuit was rinsed with 1000 ml of isotonic saline containing 2.0 IU/ml of unfractionated sodium heparin (Heparin, Biochemie, Kundl, Austria). Then, at the beginning of HD, enoxaparin was injected as a single bolus of 40 (2060) mg into the pre-dialyser arterial line. The effective dose of enoxaparin was 0.68±0.20 mg/kg dry weight (1 mg=100 IU of anti-factor Xa activity), this having been established on the basis of clinical guidelines: no visible fibrin clots in the arterial and venous bubble traps during HD, no clotted filters after HD, and no bleeding from the fistula puncture sites after compression [11,12].
Fifteen age- and sex-matched healthy non-smoking individuals served as controls for the determination of plasma levels of TF, TFPI and PF 1+2.
Study design
Plasma levels of TF, TFPI and PF 1+2 were measured at the start of and after 10 and 180 min of HD in 25 patients anticoagulated with enoxaparin. The subjects were next randomly assigned either to continue enoxaparin (n=13) or to receive UFH (n=12) during HD. The study was designed to keep the heparin doses, other HD prescriptions and pharmacological regimens constant throughout the follow-up. The approval of our institutional ethics board was obtained, and all patients and controls gave their informed consent in conformity with the Helsinki declaration.
UFH was administered as a loading dose of 2500 (15003500) IU (42.1±9.2 IU/kg) into the arterial line just prior to the start of HD, followed by a continuous infusion of 3750 (25004000) IU (57.8±12.4 IU/kg) via a syringe pump. The infusion was started at the same time as the loading dose, continued during HD and stopped 1 h before its scheduled completion. The dosage of UFH had been individually titrated, based on the whole-blood activated partial thromboplastin time (WBAPTT) and established during the first three sessions. The goal was to attain an approximately 2-fold prolongation of WBAPTT at both 30 and 120 min after the start of HD compared with baseline.
Twenty-three patients were re-examined after a 3-month interval. In 12 patients who had been switched to UFH, the over-dialysis TF, TFPI and PF 1+2 levels were compared to those obtained when they were on enoxaparin. Eleven of the 13 subjects maintained on HD treatment with enoxaparin completed the study. Two patients died out of hospital during the follow-up.
Methods
Fasting blood was drawn into Monovette vacutainers (Sarstedt, Nümbrecht, Germany) containing 0.129 M trisodium citrate (1 vol. anticoagulant and 9 vol. whole blood) by free flow from the arterial outlet of the fistula before heparinization and start of a midweek morning HD, and again after 10 and 180 min of HD. Intra-dialysis blood samples were taken after lowering the blood flow to 100 ml/min for 1 min. In healthy controls, fasting blood samples were obtained without stasis from the antecubital vein punctured with a 19-gauge needle. Cell-free plasma was prepared by centrifugation at 3000 g for 20 min at room temperature, aliquoted and stored in plastic tubes at -40°C until further processing.
Plasma antigens of TF and TFPI were measured using commercial two-antibody sandwich assay kits (Imubind, American Diagnostica Inc., Greenwich, CT, USA). The monoclonal antibodies used in the TF assay recognize both free and factor VIIa-complexed TF particles. The TFPI assay detects both intact and truncated forms of the inhibitor as well as its complexes with TF/VIIa. Plasma PF 1+2 was determined by an immunoassay (Enzygnost F 1+2 micro) purchased from Dade Behring (Marburg, Germany). The assay indirectly quantifies the amount of thrombin formed during conversion of prothrombin. The detection limits of the TF, TFPI and PF 1+2 assays are 10 pg/ml, 0.36 ng/ml and 0.04 nmol/l, respectively. The measurements were performed in duplicate on a 400 SFC photometer (SLT-Labinstruments, Gröding/Salzburg, Austria), calibrated using the supplied reference samples and standards. For calculations of the results, a computer and curve-fitting software were used. Their intra- and inter-assay coefficients of variations were <10%. The values obtained after 180 min of HD were corrected for haemoconcentration according to plasma protein levels. WBAPTT was measured using an automated coagulation system and reagents supplied by bioMérieux (Marcy-l'Etoile, France).
Statistics
The results were expressed as mean±1 SD or median (range) depending on their normal or skewed distribution provided by ShapiroWilk's W test. For intra- and inter-group comparisons, non-parametric Friedman's one-way analysis of variance (ANOVA), MannWhitney's U, Wilcoxon's and chi-squared test were used when appropriate. Bivariate correlations were assessed using Spearman's regression analysis. Every P was two-tailed, and P<0.05 was considered significant. Computations were performed using Statistica 6.0 (StatSoft, Tulsa, OK, USA).
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Results |
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The percentage increases in TFPI after both 10 min (r=-0.972, P<0.0001, Figure 2A) and 180 min of HD (r=-0.640, P=0.0006) correlated inversely with the pre-dialysis levels of the inhibitor. Neither the TFPI increments nor its pre-dialysis concentrations depended on the dose of enoxaparin (all P>0.300). Pre-dialysis PF 1+2 was not associated with baseline TFPI and TF levels, TFPI increments or dose of enoxaparin (all P>0.137). In healthy subjects, there were no relations between plasma F 1+2 concentrations and those of TF or TFPI (both P>0.178).
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Effects of a change from LMWH to UFH
As shown in Table 1, over-dialysis TF levels did not significantly change in the 12 patients changed to UFH compared with their pre-randomization values. Respective TFPI concentrations were increased at each interval (Figure 3
A), while those of PF 1+2 were higher pre-dialysis (Figure 3
B). The change of pre-dialysis TFPI did not correlate with that in PF 1+2 (P=0.914). All TF, TFPI and PF 1+2 levels in UFH-anticoagulated HD patients were higher than in healthy controls (all P<0.0001).
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TF, TFPI and PF 1+2 during UFH haemodialysis
Plasma TF and PF 1+2 levels were stable (2=1.17, P=0.558 and
2=5.50, P=0.064, respectively), while those of TFPI were markedly altered (
2=22.2, P=0.0002) also during UFH-anticoagulated HD (Table 1
). TFPI increased by 133±40% (P<0.0001) after 10 min and by 85±62% (P<0.0001) after 180 min. The percentages of the TFPI increments did not differ from those induced previously by enoxaparin in the 11 respective subjects (both P>0.530).
As for LMWH, the TFPI increases after both 10 min (r=-0.937, P<0.0001, Figure 2B) and 180 min of HD (r=-0.839, P<0.0001) correlated inversely with pre-dialysis TFPI. In contrast to enoxaparin, the 10 min TFPI rise was significantly (r=-0.615, P=0.033, Figure 2
C), while that after 180 min marginally (r=-0.545, P=0.067) inversely associated with the loading dose of UFH. The bolus (r=0.601, P=0.039) but not the infusion (r=0.315, P=0.318) dose of UFH correlated also with baseline TFPI. Pre-dialysis levels of PF 1+2 were inversely associated with both increments of TFPI at 10 min (r=-0.629, P=0.028, Figure 2
D) and 180 min (r=-0.615, P=0.033), and directly with the loading dose of UFH (r=0.587, P=0.045, Figure 2
E) and pre-dialysis TFPI (r=0.664, P=0.018).
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Discussion |
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Heparin, besides interfering with circulating haemostatic factors, causes a substantial increase in plasma TFPI levels [13]. The underlying mechanism and the vascular sites from which the molecule is released are not clear. Heparin has been found to displace TFPI from its endothelial membrane-bound complexes with glycosaminoglycans, i.e. heparan sulphate [3], as well as to mobilize TFPI from intracellular stores [19,20]. Recent comprehensive in vitro studies [19,20] have also shown that both UFH and LMWH augment TFPI synthesis by cultured endothelial cells and enhance their anti-thrombotic potential. However, circumstantial clinical evidence indicates that prolonged i.v. treatment with UFH depletes TFPI stores and results in the partial loss of its anticoagulant action [710]. This is a consequence of the endothelium-bound TFPI being more haemostatically active than the circulating lipid-bound form of the inhibitor [3].
In healthy subjects, the increase in plasma TFPI in response to a single traditional dose of i.v. UFH is more pronounced than that due to s.c. LMWH [710], which may be explained by their different bioavailability and pharmacokinetics. On the other hand, single equi-gravimetric i.v. doses of UFH and LMWH can induce comparable peak levels of circulating TFPI [1]. The causal role of heparin in the release of TFPI during HD was evidenced for the first time by Kario et al. [13]. They found a striking increase in plasma TFPI during HD procedures that used either LMWH or UFH, but no change of the inhibitor with non-heparin anticoagulants such as nafamostat mesilate or argatroban. The increase of TFPI levels during UFH-anticoagulated HD has been confirmed by others [1417]. Kario et al. [13] have also reported that the over-dialysis increase in TFPI is more marked with UFH than with LMWH. In the present report, we show that pre-dialysis plasma TFPI levels are higher than normal, and confirm that they further increase during both enoxaparin and UFH-anticoagulated HD. Our prospective study indicates that the TFPI increments after 10 and 180 min of HD are equal when the heparins are used in doses ensuring clinically adequate anticoagulation during the procedures. However, this does not exclude different behaviours of the inhibitor in between these time points. Moreover, our results demonstrate for the first time that the thrice-weekly administration of both enoxaparin and UFH results in depletion of heparin-releasable TFPI in patients on maintenance HD. This conclusion is based on the strong inverse associations between the post-heparin TFPI increments and the baseline levels of the inhibitor. A biologically similar phenomenon was previously reported by Hansen et al. [7], who found progressively lower increases in plasma TFPI during both repeated and continuous i.v. infusions of UFH in healthy subjects. This effect was accompanied by a significant loss of the anticoagulant effect of UFH. Furthermore, once-daily s.c. injections of enoxaparin have also been shown to result in transiently increased TFPI levels, but not in a rebound activation of the coagulation system after discontinuation of the therapyas evidenced by stable plasma PF 1+2 or antithrombin levels [8,10]. These studies have shown that, compared with i.v. UFH, standard regimens using s.c. LMWHs do not exhaust the endothelial pool of heparin-releasable TFPIwhich could explain previous failures of UFH treatment [3].
So far, no studies have compared the effects of repeated i.v. infusions of LMWH vs those of UFH on TFPI-related haemostasis, the situations resembling intermittent HD therapy. In our prospective study over-dialysis plasma TFPI levels increased following the switch from enoxaparin to UFH. This effect of UFH may be due to TFPI being extensively mobilized from the endothelium over a longer period of time, the haemostatic consequence of which is enhanced blood coagulation between the procedures, as evidenced by the significant 13% increase in plasma PF 1+2 before the next UFH dialysis. Although neither the time point nor the magnitude of the maximal over-dialysis increase in TFPI were established by this study, we hypothesize that UFH administration evokes greater release of the inhibitor during HD than does enoxaparin. Moreover, regression analyses of our data suggest that the loss of the long-term anticoagulant effect of UFH directly depends on its high-dose administered at the initiation of HD. The UFH bolus was found to cause a dose-dependent increase in circulating TFPI, but the association was, unexpectedly, inverse rather than direct. It points to the progressive depletion of heparin-releasable TFPI pool by repeated i.v. infusions of UFH during intermittent HD therapy. Moreover pre-dialysis levels of PF 1+2, a marker of intravascular thrombin, were associated directly with the loading dose of UFH, and inversely with the extent of TFPI release during HD. Regression analysis indicates that increasing the UFH bolus dose from 30 to 50 IU/kg could result in doubled PF 1+2 levels. Based on these findings, we suggest that a high loading dose of UFH is a risk factor for thrombotic complications in intermittent HD patients due to severe exhaustion of endothelial pool of TFPI. This, however, requires confirmation.
This study shows for the first time that enoxaparin ensures efficient anti-thrombotic protection not only during HD procedures but also in between dialysis sessionsas evidenced by stable PF 1+2 levels. The mean dose of enoxaparin used in our patients was 0.68 mg/kg (range 0.301.08 mg/kg), which was generally lower than the 1 mg/kg recommended by the manufacturer. The dosage is almost identical to that established by Saltissi et al. [11] to be clinically adequate, safe and probably more cost-effective than UFH. In contrast, an LMWH, dalteparin, at a single dose of about 5000 IU, was found to provide clinically sufficient anticoagulation but not to prevent actual activation of haemostasis during HD [12].
In conclusion, depletion of heparin-releasable stores of anticoagulant TFPI is an untoward consequence of repeated blood anticoagulation in maintenance HD therapy. The effect becomes evidently prothrombotic when the traditional UFH regimen of bolus and infusion is used instead of a single injection of LMWH enoxaparin. High loading doses of UFH may be a risk factor for severe endothelial TFPI depletion, resulting in the activation of the coagulation system in chronic HD patients. Enoxaparin administered as a single bolus of 0.7 mg/kg is haemostatically effective in this situation.
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Acknowledgments |
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Notes |
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References |
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