1 Department of Internal Medicine, 2 Research Institute for Internal Medicine, National Hospital, Oslo, Norway
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Abstract |
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Methods. The effect of tapered doses of dalteparin during 84 HD sessions (44.5 h) was prospectively examined in 12 patients. Six of the patients were treated with warfarin. The initial dalteparin dose was reduced to 50% if no clotting was observed. Clinical clotting was evaluated by inspection of the air trap every hour and by inspection of the dialyser after each session. Anti-FXa activity was measured for assessment of dalteparin activity. Markers of activated plasma coagulation, (thrombin-antithrombin (TAT) and prothrombin fragment 1+2 (PF1+2)) and a marker of platelet activation (ß-thromboglobulin, ß-TG), were measured before the start of and after 3 and 4 h of dialysis. Ten pre-dialytic patients with chronic renal failure served as a control group. A total sof 230 measurements of each parameter were performed.
Results. An anti-FXa activity above 0.4 IU/ml at the end of HD inhibits overt clot formation for 4 h. This was obtained by an intravenous dalteparin dose of about 5000 IU. TAT and PF1+2 correlated to clinical clotting episodes (r=0.50 and 0.47, P<0.001). ß-TG was not significantly correlated to clinical clotting. All parameters increased during the sessions (TAT, PF1+2, ß-TG, P<0.001). When measurements during clinical clotting episodes were disregarded, all parameters were still markedly increased. Warfarin-treated patients had lower TAT and PF1+2. Dialysis patients had higher ß-TG values than pre-dialytic patients.
Conclusion. Despite clinically effective anticoagulation, obtained by dalteparin administration, platelets and coagulation are activated by HD, resulting in a potentially thrombophilic state. Warfarin treatment reduces clinical clot formation and subclinical activation of coagulation.
Keywords: dalteparin; haemodialysis; plasmin antiplasmin; prothrombin fragment 1+2 (PF1+2); thrombin-antithrombin; ß-thromboglobulin
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Introduction |
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Platelet activation is important for the initiation of the thrombogenic process, and ß-thromboglobulin (ß-TG) released from alpha-granules of activated platelets after contact with biomaterials is a sensitive marker of platelet activation [4]. The fibrinolytic system was evaluated by measuring plasmin-antiplasmin (PAP), a marker of activated fibrinolysis [5]. Dalteparin anticoagulant effect was measured as anti-FXa activity.
Traditionally, anticoagulation during haemodialysis (HD) is obtained by giving an intravenous (i.v.) bolus of unfractionated heparin followed by continuous i.v. infusion or another bolus during dialysis. However, low molecular weight heparins are frequently used having several potential advantages over unfractionated heparin. They are dialysable and less frequently associated with bleeding complications and heparin-induced thrombocytopenia [68]. In addition, the administration is practical, and the effect on blood lipids may be favourable [9,10].
The aim of the present study was to examine activation of platelets and the coagulation system during HD in patients with and without warfarin and to see if such activation may occur also in the absence of clinical clotting episodes.
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Patients and methods |
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Dialysis procedure
Dalteparin was given as a single bolus dose into the arterial line at the start of dialysis. The mean applied dalteparin dose at the start of the study was 60.9±19.3 IU/kg in the patient group receiving warfarin and 69.5±11.5 IU/kg in those without oral anticoagulation. The dialysis sessions were 4 h three times per week in seven patients and 4.5 and 5 h three times per week in five patients. A polysulphone hollow fibre dialyser (F6 HPS, Fresenius, Germany), was used during the study. Bicarbonate dialysate was used in all patients. The dialyser and the bubble trap were checked at the end of each dialysis session to ensure that there was no clot formation and the dalteparin dose was kept unchanged. The blood flow rate varied between 230 and 300 ml/min, but was kept close to constant within each patient. Blood flow was recorded by the dialysis machine flowmeter. The dialysate flow was kept constant at 500 ml/min.
Study design
The dalteparin dose was gradually tapered in subsequent dialysis sessions to examine the relationship between anti-FXa activity, markers of coagulation and platelet activation versus clinical clotting events during dialysis in each of the 12 patients. Nine HD sessions over 3 weeks were studied in eight of the patients and three sessions over 1 week in four patients. The regular dose of dalteparin was given in the first session and then reduced by 25% for each session down to 50% if no clotting was observed. Clinical clotting was evaluated by visual inspection after blood draining of the air trap every h (1=no clotting in the trap, 2=fibrinous ring, 3=clot formation and 4=coagulated system) and by visual inspection of the dialyser at the end of each session (1=clean filter, 2=a few blood stripes (affecting less than 5% of the fibres seen at the surface of the dialyser), 3=many blood stripes (affecting more than 5% of the fibres) and 4=coagulated filter). If clinical signs of clotting grade 3 or 4 were observed, the dalteparin dose was increased by one step (25% of the initial dose). Clinical clotting was compared with simultaneously measured anti-FXa activity, TAT, PF1+2, PAP, AT, ß-TG, use of oral warfarin and dialysis time. The study was designed to keep Hb, HCT, platelet count, blood flow and ultrafiltration rate constant from 2 weeks before and during the study.
Blood sampling
Blood specimens were taken from the arterial line after lowering the blood flow to 100 ml/min for 1 min. Fibrinogen, Activated partial thromboplastin time (APTT), D-dimer, Thrombin clotting time and ethanol gelation test were taken at the start and at the end of the first treatment. Hb, HCT, platelets, white cell count, thrombin time and urea were measured at the start and at the end of each dialysis session.
Samples for TAT, PF1+2, PAP, ß-TG and AT were drawn before start without venous compression and after 3 and 4 h of each dialysis session. Blood for TAT, PF1+2, PAP and AT was collected into tubes containing citrate and centrifuged at 1500 g for 10 min at room temperature. For ß-TG assay blood was collected into Diatube H® tubes and immediately cooled in a crushed ice-water mix for 15 min. Within 1 h after collection the blood sample was prepared by centrifugation at 2500 g for 30 min at 4°C. Anti-FXa activity was measured after 1, 3 and 4 h in each of the dialysis sessions. Anti-FXa activity in the dialysis group was not measured before start of dialysis because the pre-dialytic activity was expected to be below the detection limit (0.1 IU/ml). This is supported by the fact that five of the patients in this study had no detectable anti-FXa activity after 4 h of dialysis, being in accord with the findings by other investigators [6,10,11]. Blood for anti-FXa activity was collected into tubes containing citrate and cooled in an ice-water mix before centrifugation in the same way as ß-TG. In the control group of pre-dialysis patients creatinine, PAP, TAT, PF1+2, ß-TG and AT were analysed only once, and the blood specimens were collected without stasis and processed as described above.
Laboratory methods
After centrifugation, one-third the volume of the plasma supernatant from the middle region of the liquid portion was collected and stored at -70°C for later determination of ß-TG. ß-TG was measured with an ELISA (EIA) procedure (Asserachrom® ß-TG kit). AT was measured with a chromogenic assay (Coamatic®, Chromogenix AB, Mølndal, Sweden). PAP was measured with an ELISA procedure (Enzygnost® PAP micro, Behring). PF1+2 and TAT were also measured with an ELISA, (Enzygnost® F1+2 micro and Enzygnost® TAT micro, Behring).
Anti-FXa activity was measured with a chromogenic assay (Coatest®, Chromogenix AB, Mölndal, Sweden).
Statistics
For non-parametric distributions, statistical comparisons between two different groups of patients were done by the MannWhitney two-sample rank test and paired comparisons by the Wilcoxon matched-pairs signed ranks test. For normal distributions parametric t-tests were used. Parameters not normally distributed were logarithmically transformed before linear regression analysis.
These statistical analyses were performed by use of SPSS and by a computer based statistical soft-ware program, Graph Pad prism, CA, US.
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Results |
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In a regression analysis between anti-FXa activity and each of the parameters TAT, PF1+2 and ß-TG TAT and PF1+2, but not ß-TG, were negatively and significantly correlated to anti-FXa (P<0.001). This means that tapering of the dalteparin dose results in increased levels of TAT and PF1+2.
Table 2 summarizes the mean values for coagulation markers (TAT, PF1+2, AT), fibrinolytic parameter (PAP) plus platelet activation (ß-TG) in 84 HD sessions in all patients at baseline and at the end of each session. All markers showed a significant increase during 4 h of HD.
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There were striking differences between the patients receiving warfarin and those who did not. Figure 1 shows the values of TAT, PF1+2 and PAP during HD. The values of TAT and PF1+2 rose to levels more than three times as high in the non-warfarin-treated patients (P<0.001). The coagulation markers TAT and PF1+2 were low and within normal levels in the warfarin-treated patients in contrast to PAP which was higher and above upper normal limits with warfarin treatment. The difference in PAP between the two groups at baseline and during dialysis was not statistically significant. Figure 2
shows that all patients had increasing levels of ß-TG throughout the dialysis sessions (P<0.0001), the levels being higher than normal values and significantly higher in the warfarin-treated group at baseline (P<0.0001) and after 3 h of dialysis, (P=0.02).
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Discussion |
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However, clinical clotting is a crude measure of activation of coagulation. Activation of platelets and coagulation may take place without clinical clotting episodes. Subclinical activation of platelet and coagulation was evaluated by excluding measurements performed with simultaneous clinical clotting. We then found that TAT and PF1+2 values were significantly increased in non-warfarin-treated patients compared to the warfarin-treated patients, P<0.00001 (Figure 3). This indicates thrombin generation during HD in spite of a clinically adequate dosage of dalteparin.
In the present study ß-TG was increased before the start of HD in all the patients, the highest values were seen in the warfarin-treated patients (Figure 2). Increased levels of ß-TG in non-dialysed patients with impaired renal function have previously been reported by others, and the renal excretion of ß-TG is reported to explain this [2,11,12]. The present study confirmed that the levels of ß-TG were higher than normal in pre-dialytic patients with chronic renal failure. Adler et al. found increased levels of ß-TG before dialysis in patients on regular HD, but in non-dialysed patients with chronic renal failure ß-TG was not statistically significantly different from normal [13]. The molecular weight of ß-TG is 36000 Da and the half life is 100 min, and ß-TG pass the glomerular basement membrane and is reabsorbed by the tubuli [13,14]. It has been suggested by many authors that ß-TG cannot be used as a marker of platelet release reaction in vivo in patients with renal insufficiency since ß-TG is excreted in the urine and its plasma level rises steeply with decreasing GFR [15]. Deficient catabolism of ß-TG in the diseased kidney rather than deficient excretion is also suggested to account for the elevated levels of ß-TG in patients with chronic renal insufficiency [16]. In addition to decreased clearance by the kidneys and deficient catabolism, another possible explanation for the increased baseline levels of ß-TG may be that the platelet activation does not normalize until the next dialysis session. Moreover, the increase in ß-TG during the HD sessions in the present study was statistically significant, and this can only be explained by platelet release with secretion of ß-TG from the ß-granulae. Increased levels of ß-TG during HD have also been found by others [8,17,18].
In conclusion, the levels of ß-TG were reduced before the next dialysis session, but not to normal levels, while TAT was reduced to normal levels before the next dialysis sessions in all the patients. The normalization of TAT before the next HD sessions in our patients is in contrast to the reports of Ambühl et al. who found TAT levels at baseline three times higher than the upper normal limit in 39 HD patients [3]. Ambühl et al. did not study pre-dialytic, uraemic patients, as was done in the present study. The present study shows more than three times higher levels of TAT in pre-dialytic uraemic patients compared with the dialysis patients before start of dialysis, P=0.0001. This is in contrast to the findings of Sagripanti et al. who found that baseline levels of TAT in 22 HD patients were higher than in patients on conservative treatment [2]. However, the normal range of TAT was not given in the latter report, and with our upper normal limit of 4.1 mg/l the reported mean level is just slightly increased compared with the normal range. There are few studies reporting the levels of coagulation parameters in pre-dialytic, uraemic patients compared with dialysis patients. There is no rational explanation why TAT in the present study is three times higher in pre-dialytic patients compared to dialysis patients. The results indicate that pre-dialytic patients with chronic renal failure as well as dialysis patients have an activated coagulation system. In conclusion, the previous reports of the baseline levels of TAT in HD patients differ. The normal baseline levels of TAT in all the dialysis patients as well as the increased baseline levels of PF1+2 in the non-warfarin-treated dialysis patients in the present study are in accordance with the findings of Cella et al. [19,20]. The different half-lives of TAT and PF1+2 are suggested to account for this discrepancy [19]. However, activation of coagulation in chronic renal insufficiency also between the dialysis sessions is another possible explanation. The rise in TAT and PF1+2 during HD reflects an activation of the coagulation system, and this increase is in accordance with previous reports [3,19].
Warfarin treatment did not inhibit the increase of ß-TG during HD, as was the case with TAT and PF1+2, which were kept in the normal range almost without increment with warfarin treatment. The fact that ß-TG levels were significantly higher in the patients on warfarin treatment both before and during dialysis is in contrast to previously reported lower levels of ß-TG in patients with normal renal function who received standard warfarin therapy [21]. This difference in observations cannot easily be explained.
It is well known that HD patients have an increased rate of cardiovascular disease, and cardiovascular complications account for more than 50% of all deaths in HD patients [2224]. Activation of coagulation may be associated with cardiovascular events and platelet activation also with growth factors with potential adverse effects on heart and vessel structure [25]. The long-term consequences may be accelleration of the atherosclerotic process. Abnormalities in the coagulation system are associated with an increased rate of cardiovascular complications, and PF1+2 is reported to correlate strongly with risk factors for cardiovascular disease [26]. As expected, the level of TAT and PF1+2 were lowered by warfarin in the present study. Thus, warfarin therapy reduces the tendency towards hypercoagulability in dialysis patients.
In conclusion, we previously found that a single dose of dalteparin effectively inhibits significant intradialytic clinical clotting for at least 4 h during HD [1]. However, activation markers of platelets and the coagulation system may be prominent even without clinical clotting episodes, and these markers increased during HD. Regression analysis showed that anti-Fxa activity was negatively and significantly correlated to TAT and to PF1+2. This suggests that higher doses of dalteparin may reduce the activation of plasma coagulation, but our study was not designed to investigate this issue. However, our data also show that the activation of coagulation is reduced with simultaneous oral warfarin treatment. Activation of coagulation and platelets may be associated with cardiovascular complications. Assessments of this aspect of anticoagulation in HD patients seems warranted.
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Acknowledgments |
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Notes |
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References |
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