Normalization of haemoglobin concentration with recombinant erythropoietin has minimal effect on blood haemostasis

Anders G. Christensson,1, Bo G. Danielson2 and Stefan R. Lethagen3

1 Department of Vascular and Renal Diseases, 3 Department for Coagulation Disorders, Malmö University Hospital, Malmö and 2 Department of Internal Medicine, Uppsala University Hospital, Uppsala, Sweden



   Abstract
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Background. Elevation of haemoglobin (Hb) with recombinant erythropoietin (rHuEpo) in patients with chronic renal failure has raised concern of increased risk of thromboembolic diseases. In this study, a substudy of the Scandinavian multicentre trial, we examined the influence on haemostatic parameters of normalization of Hb levels from subnormal levels in patients with chronic renal failure.

Methods. Twenty-six patients, 17 males (before study start Hb 113±6 g/l) and nine females (Hb 111±8 g/l), with end-stage renal disease were included. Both dialysis and predialysis patients were included. After 3 months of rHuEpo therapy Hb levels reached 136±14 g/l for males and 128±13 g/l for females, and after 1 year 142±11 g/l and 126±14 g/l respectively. The increase in Hb was significant both at 3 months and 1 year, compared to baseline. At baseline, after 3 months and 1 year haemostatic and prothrombotic parameters were measured, including prothrombin complex test, activated partial thromboplastin time, platelet aggregation and retention, von Willebrand factor antigen, antithrombin, protein C, total and free protein S, activated protein C resistance, FV–Leiden mutation, D-dimers, plasminogen activator inhibitor-1 and prothrombin fragments 1+2 (PF 1+2).

Results. The only statistically significant change was a transient decrease in total levels of protein S at 3 months from 131 to 120% (P=0.0093). The free and active form of protein S showed no significant change. After 1 year the difference was not seen.

Conclusions. Apart from a transient and clinically insignificant decrease in total protein S, we found no prothrombotic changes after normalization of Hb from subnormal levels. Our findings indicate that rHuEpo treatment may aim at normalizing Hb levels without significant effects on haemostatic parameters in patients with chronic renal failure compared to patients with subnormal Hb levels.

Keywords: coagulation; erythropoietin; haemostasis; protein S; renal anaemia; thrombosis



   Introduction
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Blood haemostasis is carefully controlled by a complex interaction between platelets, the coagulation cascade, coagulation inhibitors, fibrinolysis, and the vascular endothelium. Patients with chronic renal failure often exhibit a bleeding tendency and multiple haemostatic disorders [1]. The changes in haemostatic conditions are evident as glomerular filtration rate (GFR) falls below 30 ml/min. The increased bleeding tendency has been regarded useful in preventing thrombosis of AV fistulae and clotting in the dialysis system.

Introduction of recombinant erythropoietin (rHuEpo) in the therapy of renal anaemia in the 1980s has dramatically changed the quality of life of patients with chronic renal failure. Among the adverse effects possibly associated with the use of rHuEpo in patients with chronic renal failure, a risk of thrombosis of the vascular access has been reported [2,3]. Shortening of bleeding time [4], a rise in whole-blood viscosity [5], increased platelet count [6], improvement of platelet function [7, 8], spontaneous platelet aggregation [9], impairment of fibrinolysis [10], and a decrease in natural coagulation inhibitors [11,12] have been reported after rHuEpo therapy. Although rHuEpo may induce these prothrombotic changes, it is unclear whether it results in clinically significant thrombotic events [13].

Furthermore, the increased risk of thrombosis of AV fistulae remains controversial [14], but in a recent study, [15], a higher incidence of fistula thrombosis was shown in the normal-haematocrit (Hct) group compared to the low-Hct group. Another study could not find any rheological changes that could contribute to an increased risk of access thrombosis in haemodialysis (HD) patients with normal initial Hct before HD [16].

Because of the risk of side-effects, hypertension, and thrombosis, rHuEpo was initially used with restriction and for many years a subnormal haemoglobin (Hb) level around 100–110 g/l was considered to be optimal. The present study is a substudy of the Scandinavian multicentre trial. The aim of this study was to establish if normalization of Hb from subnormal values with rHuEpo in patients with end-stage renal disease caused any prothrombotic changes in haemostatic parameters.



   Subjects and methods
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
This is a substudy of a randomized, prospective, open-labelled, multicentre study comprising 416 patients (age 18–80 years) with end-stage renal disease in Scandinavia, which started in 1995.

Inclusion criteria
Included were: Patients with end-stage renal disease defined as predialysis patients with creatinine clearance <30 ml/min not yet on dialysis, patients undergoing regular HD, and patients on continuous ambulatory peritoneal dialysis (CAPD). All patients on dialysis regimen were considered for inclusion. Predialysis patients were included consecutively.

Exclusion criteria
These were: anaemia of causes other than chronic renal failure, B12 and folic acid deficiency, uncontrolled hypertension (diastolic blood pressure >100 mmHg), uncontrolled diabetes mellitus, clinically relevant abnormal liver function, severe hyperparathyroidism, and uncontrolled overhydration. Predialysis patients likely to become dialysis-dependent within 1 year were excluded. Salicylic acid and non-steroidal anti-inflammatory drugs were postponed 10 days prior to blood tests.

Twenty-six patients (mean age 54 years, range 22–79) at three centres in Sweden (Malmö, Lund, and Uppsala) were included. Seventeen males with a mean age of 57 years (range 22–79) and nine females with a mean age of 50 years (range 36–71) participated. Eleven patients were being treated by HD, two were on CAPD, and 13 were predialysis patients (Table 1Go). Predialysis patients and those on CAPD were recruited from Uppsala and patients on HD from Malmö and Lund. The predialysis patients presented a mean GFR of 16 ml/min (range 6–24). All patients, except five of the predialysis patients, were on rHuEpo treatment at the start of the study. Furthermore, all patients were evaluated by liver tests every 12 months and revealed normal values at the start of the study and no significant changes during the study. None of the patients suffered from viral hepatitis according to tests for hepatitis B and C. Dialysis patients (HD and CAPD) in our study fulfilled the criterion of adequate dialysis treatment (Kt/V >1.2 for HD and a weekly creatinine clearance >55 ml/min for CAPD). None of the CAPD patients and only two of the predialysis patients had a vascular access at the start of the study.


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Table 1. Patient characteristics

 

Study protocol
Before inclusion the patients demonstrated stable Hb in the subnormal range for at least 3 months with or without rHuEpo treatment. Inclusion criteria was Hb in the range 90–110 g/l for females and 100–120 g/l for males during the run-in period. Iron deficiency, serum ferritin <100 µg/l and/or transferrin saturation <20%, was treated with intravenous iron sucrose (Venofer®) or oral iron supplementation. Planned study time was 1 year. The regimen of rHuEpo administration remained patient-specific, i.e. intravenously (2–3 times weekly) or subcutaneously (1–3 times weekly), according to the route of administration at baseline. The weekly rHuEpo dose was increased to reach the new steady-state Hb within 2–3 months. The dose escalation was tailored to a smooth Hb increase of 10–15 g/l per month. The initial dose increment was in the range of 50%. The goal for rHuEpo treatment was a Hb level of 125–140 g/l in females and 145–160 g/l in males, 2–3 months after study start and during the rest of the study.

Evaluations
In HD patients all laboratory investigations were performed predialysis and mid-week. Anticoagulation requirements during dialysis were recorded. All adverse events, hospitalizations, coronary events and treatments, thromboses at vascular access sites, or procedures involving vascular access site, transfusions, and deaths were recorded.

All haemostatic parameters were analysed at the Department for Coagulation Disorders at Malmö University Hospital. Platelet aggregation, platelet retention, and bleeding time were only performed in patients (n=10) from Malmö and Lund, as these tests had to be done while the patients visited the laboratory.

In addition to Hb, Hct, and platelet count, the following haemostatic parameters were measured before the study start, at 3 months, and at 1 year; prothrombin complex test, activated partial thromboplastin time (APTT), platelet aggregation and retention, bleeding time (Ivy), von Willebrand factor antigen (VWF.Ag), multimeric structure of VWF, fibrinogen, antithrombin (AT), protein C, total and free protein S, activated protein C resistance (APC resistance), FV–Leiden mutation analysis, D-dimers, F VII, plasminogen activator inhibitor-1 (PAI-1), and prothrombin fragments 1+2.

Three patients on medication with vitamin K antagonists were excluded from evaluation of APTT, prothrombin complex test, F VII, protein C, protein S, and prothrombin fragments 1+2.

Methods
Blood sampling
Venous blood for plasma samples was collected in silicone-coated Vacutainer® tubes (Becton Dickinson) with a volume of 5 ml, containing 0.5 ml of 0.129 mol/l sodium citrate. Platelet-poor plasma (PPP) for coagulation analyses was obtained after centrifugation at 2000 g for 20 min. Plasma was frozen immediately and kept at -70°C until assessed. Samples from Uppsala were transported by express service to Malmö at -70°C. When arriving at Malmö the samples were immediately taken care of at the Department for Coagulation Disorders and kept at -70°C until analysed. No delay was accepted. This transportation routine has been approved by the Department for Coagulation Disorders in Malmö. Blood tubes for analysis of platelet retention were not centrifuged. They were placed in a test tube rack in room temperature for a period of 45–120 min, after which platelet retention was measured. Venous blood for DNA extraction was collected in EDTA tubes. Venous blood for platelet aggregation studies was collected in 10-ml polystyrene tubes containing 1 ml 0.129 mol/l sodium citrate. Platelet-rich plasma (PRP) was obtained after centrifugation at 275 g for 10 min.

Assays

  1. Prothrombin complex test was performed in a Thrombolyzer by a prothrombin–proconvertin method using a reagent containing thromboplastin, calcium2+ ions, and bovine plasma lacking coagulation factors II, VII, and X. The coagulation time is proportional to the levels of factors II, VII, and X in the patient plasma. The coagulation time is compared with dilutions of a reference sample and results are given as %. Normal range 70–130%.
  2. The activated partial thromboplastin time (APTT) was tested in a Thrombolyzer according to a standard method. Normal range 24–37 s.
  3. Platelets were counted visually by phase microscopy. Normal range 125–340x109/l.
  4. Platelet aggregation was measured in a Lumiaggregometer (Chrono-Log, Haverton, PA, USA) as described earlier [17].
    Platelet retention was measured on citrated venous whole blood with a modified Adeplat S test (Semmelweis, Milan, Italy), as described earlier [17], (normal range 16–27%). The intra-individual coefficient of variation (CV) of 10 blood samples taken on one occasion in one healthy volunteer has been found to be 7.1% (mean platelet retention 17.7%, SD 1.25%). The intra-individual day-to-day variation tested in two healthy volunteers has been found to be 8 and 13% respectively [18].
  5. Bleeding times were measured with a Simplate-II-R device. Normal range 230–630 s.
  6. von Willebrand factor antigen (VWF.Ag) in plasma was analysed by enzyme-linked immunosorbent assay (ELISA) using a polyclonal anti-human VWF antibody (Dakopatts, Stockholm, Sweden) as the primary antibody and a polyclonal peroxidase-conjugated immunoglobulin to human VWF as the secondary antibody (Dakopatts, Stockholm, Sweden). Normal range 0.60–2.73 IU/ml.
  7. The multimeric structure of VWF in plasma was analysed with sodium dodecyl sulphate (SDS)–agarose gel electrophoresis (1.9% agarose concentration) as described elsewhere [19,20].
  8. Fibrinogen in plasma was measured by a turbidimetric method with Baxtrobin as activator. The formation rate of fibrin is measured spectrophotometrically at 340 nm and is considered to be proportional to the initial concentration of fibrinogen in the sample. Normal range 2.0–4.3 g/l.
  9. Antithrombin (AT) in plasma was measured spectrophotometrically with a chromogenic substrate method according to the manufacturer's description (Coamatic Antithrombin, Chromogenix, Mölndal, Sweden). Normal range 0.82–1.11 IU/ml.
  10. Protein C in plasma was measured spectrophotometrically with a chromogenic substrate method according to the manufacturer's description (Coamatic Protein C, Chromogenix, Mölndal, Sweden). Normal range 0.70–1.30 IU/ml.
  11. Total and free protein S in plasma was measured as a competitive radioimmunoassay (RIA). Normal range total protein S, 70–130%, free protein S, 18–58% [21].
  12. APC resistance was measured according to the manufacturers description (Coatest APC-resistance kit and Coatest APC resistance V kit, Chromogenix, Mölndal, Sweden). Normal range for the APC-resistance ratio was >2.4, and >1.9 after dilution with FV-free plasma.
  13. The F V–Leiden mutation (G1691A) was measured as described earlier [22] in patients with pathological APC-resistance ratio.
  14. D-dimers in plasma was measured with a NycoCardTM D-dimer immunoassay (Nycomed AB, Lidingö, Sweden). Normal range <0.5 mg/l.
  15. Factor VII (F VII) in plasma was measured with a one-stage clotting assay using Neoplastin and F VII-deficient plasma (Diagnostika Stago). Normal range 0.60–1.60 IU/ml.
  16. Plasminogen activator inhibitor 1 (PAI-1) in plasma was measured with a two-stage indirect enzymatic analysis according to the manufacturers description (Spectrolyse (pL) PAI, Biopool, Umeå, Sweden). Normal range 0–16 U/ml.
  17. Prothrombin fragments 1+2 in plasma was measured with an ELISA (Enzygnost F1+2®, Behring, Germany). Normal range 0.44–1.10 nmol/l

Calibration against an international standard
Standard plasma from about 40 healthy subjects was calibrated against a calibrator substance (international standard) for analysis of VWF.Ag, antithrombin, protein C and F VII. Results were expressed as IU/ml.

Statistical methods
Each patient served as his/her own control. Changes from baseline in each patient were evaluated after 3 months and 1 year using non-parametric statistics (Wilcoxon signed rank). In order to avoid mass significance due to multiple variables, we have not chosen to define statistical significance as P<0.05 but rather as P<0.01. All P values are presented. Correlation between different variables was tested using the Spearman rank correlation test.

Ethics
The investigation was approved by the Ethics Committee of all centres.



   Results
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 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Before inclusion mean Hb was 113±7 g/l (range 95–123) in both males and females (Table 2Go). Hb-levels after increased doses of rHuEpo are expressed in Table 2Go. The increases in Hb levels were significant (P<0.001) at 3 months and 1 year, compared to baseline.


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Table 2. Haemoglobin levels before study start, at 3 months, and at 1 year after study start

 
To achieve desired Hb levels, rHuEpo requirements were increased by about 200% at 3 months and 130% at 1 year after study start, compared to baseline doses. Three patients did not respond to increased doses of rHuEpo. In total, seven patients did not reach target Hb at 3 months or 1 year.

Two patients of 26 did not fulfil the 1-year study. One man was excluded after 10 months due to deterioration of his general condition and one woman was excluded after 8 months when pregnancy was detected. These two patients are analysed only at baseline and at 3 months.

Intravenous iron sucrose was administered in 21 of the 26 patients. During the study the administered dose of iron was increased at 3 months by about 150% and 60% at 1 year, compared to baseline.

At baseline four patients were heterozygous for FV-Leiden mutation. None of these exhibited thrombotic events. No other prothrombotic coagulation disorders were found at baseline.

The only significant change was a slight and transient decrease in total protein S occurring 3 months after study start (Table 3Go). Total protein S diminished from 131±27 to 120±24% (P=0.0093). However, the level at baseline was slightly above reference level, and after 3 months was within normal range. After 1 year there was no significant difference compared to baseline levels regarding this parameter. All other haemostatic parameters were unchanged during this 1-year study. We found no significant changes in platelet count and platelet retention during the study. High levels of prothrombin fragments 1+2 were found at all three occassions for blood sampling. After 3 months there was a decrease in these fragments (3.0±2.11 nmol/l to 2.4±1.07) but the difference only reached a P value of 0.0496.


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Table 3. Haemostatic parameters at baseline, at 3 months, and at 1 year after normalization of Hb with rHuEpo

 
In this rather small group of patients (n=26) we observed no vascular access thromboses or other thrombotic events. However, the patients on CAPD had no vascular access and among the predialysis patients only two had vascular access at the start of the study.

In order to establish if the observed change in levels of total protein S were related to the dose of the drug, correlation test between doses of rHuEpo and levels of total protein S was done (not shown). There was no such correlation.

We also excluded those seven patients who did not reach target Hb, 125 g/l for women and 145 g/l for men, at either 3 months or at 1 year. After this exclusion 19 patients remained. Hb levels are expressed in Table 4Go. In this group of 19 patients we found a slight decrease in total protein S from 127±25 to 115±19% after 3 months (P=0.028). However, our limit for statistical significance was less than 0.01 due to multiple variables. Also after 1 year there was no statistically significant decrease in protein S.


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Table 4. Patient data of those who responded to increased rHuEpo doses

 



   Discussion
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
The introduction of rHuEpo treatment of uraemic anaemia has markedly improved the quality of life of patients with end-stage renal disease. rHuEpo was used with a restriction due to the fear of side-effects such as hypertension and thrombosis and for many years a subnormal Hb level around 100–110 g/l was considered to be optimal. Several studies have addressed the potential effect of rHuEpo treatment on haemostatic variables, but in most studies Hb levels only reached subnormal values [4,68]. Many of these found a transient increase in the number of platelets and improved platelet function, but for other haemostatic parameters the results have been contradictory [23]. It should be emphasized that most of these studies compared baseline values without rHuEpo vs post-treatment values aiming at Hb levels of 100–110 g/l.

The aim of our study was to establish if normalization of Hb from subnormal values with rHuEpo in patients with end-stage renal disease caused any prothrombotic changes in haemostatic parameters. Therefore this study is different from most other studies with respect to baseline Hb and target Hb.

We included both dialysis and predialysis patients with end-stage renal disease that were anaemic. These two categories may have different haemostatic conditions because the increased bleeding tendency, caused by uraemia, is more pronounced in severe uraemia compared to moderate uraemia, although the exact influence on bleeding parameters at different uraemic levels is hard to address. It has been shown by Remuzzi et al. [24] that dialysis only partially corrects abnormal bleeding time and platelet retention. It has also been shown that there are no differences between efficient haemodialysis and peritoneal dialysis in improving the renal bleeding tendency [25]. The predialysis patients in this study presented a GFR around 16 ml/min and the dialysis patients were adequately dialysed, which means that the range in uraemic level was limited. Furthermore, this study was designed with each patient being his/her own control, which diminishes the effect of mixing predialysis and dialysis patients. In order to evaluate if these two groups of patients were comparable in haemostatic conditions we compared baseline characteristics of coagulation parameters (except platelet aggregation and retention) in the two groups. However, we found no statistically significant changes between dialysis and predialysis patients. Although the patients were treated in different ways (HD, CAPD, and conservative treatment) they should be comparable with respect to haemostatic disorder.

We included patients in two regions at three hospitals. Investigation of platelet function (platelet aggregation and retention and bleeding time) could only be performed in one region. All other haemostatic parameters were analysed in all patients. Thus predialysis patients and those on CAPD (from Uppsala) were not analysed for platelet function, whereas HD patients (from Malmö and Lund) were analysed for all haemostatic parameters.

Except for a small decrease in total protein S levels at 3 months, we did not find any prothrombotic changes in plasma levels of haemostatic parameters, including platelets. This observed change probably has no clinical significance since the change is small, within normal ranges, and affects only the total amount of protein S. The free and active form of protein S did not change significantly. We speculated that the decrease of protein S at 3 months could be related to relatively high doses of rHuEpo, about 200% above baseline dosage at 3 months compared to 130% at 1 year. However, the lack of correlation between rHuEpo dosage and protein S levels did not support this hypothesis.

To our knowledge there are only limited data from other studies on the effects of normalization of Hb levels with rHuEpo treatment. An American study of patients with clinically evident congestive heart failure or ischaemic heart disease receiving haemodialysis demonstrated higher mortality in the normal-Hct group compared to the low-Hct group. The difference in mortality was not due to increased venous thromboembolism [15]. However, both in the normal- and low-Hct groups the mortality rate decreased at higher Hct values.

In contrast to several other studies, we found no changes in platelet levels or function. One reason for this discrepancy could be that we only measured platelet function in a subset of patients (n=10). Another reason may be that most other studies did not aim at normalizing Hb and compared those patients treated with rHuEpo with those without treatment. Our observation of a transient decrease of total protein S at 3 months is in parallel with the observation made by Macdougall and co-workers [12] who also found a decrease at 3–4 months.

Other studies have found divergent results concerning haemostatic parameters. A transient reduction of antithrombin, free protein S, plasminogen, and tissue type plasminogen activator in plasma was shown by Vaziri and co-workers [26]. Lai et al. [27] found no significant changes in immunological or functional activities of protein C, protein S, or antithrombin in uraemic patients on CAPD when Hb was increased from 69±13 to 96±13 g/l. They found a small but significant change of prothrombin time and APTT within normal range. Clyne and co-workers [8] found no changes of total and free protein S after elevation of Hb from 82±9 to 111±12 g/l.

Our findings indicate that Hb levels can be normalized with rHuEpo without significant prothrombotic changes compared to patients with Hb at subnormal levels. These results were based on a 1-year follow-up of patients being their own controls. But this study does not evaluate the difference in haemostatic parameters between patients with severe, untreated anaemia and patients with normalized Hb. Results in this study are in accordance with Tang et al. [23], who made a literature review and concluded that the effects of rHuEpo on the coagulation cascade are of minimal clinical importance.

Our findings are also supported by the larger Scandinavian multicentre study, of which our work is a substudy, where there was no significant difference in number of vascular access thromboses or other thrombovascular events between subnormal and normal Hb groups. This is also supported by a recently published German study [16]. From our investigation, we conclude that elevation of Hb from subnormal to normal values in patients with uraemic anaemia is achievable without causing significant changes in haemostatic parameters.



   Acknowledgments
 
Kerstin Wiklund at Clinical Data Care, Uppsala, contributed with generous statistical support and know-how. The assistance of Margareta Forsman of Uppsala University Hospital, Helene Ohlsson of Malmö University Hospital, Jörgen Hegbrant of Park Dialys, Lund, and Jan Kurkus of Lund University Hospital is greatly appreciated.



   Notes
 
Correspondence and offprint requests to: Anders Christensson, Department of Vascular and Renal Diseases, Malmö University Hospital, S-205 02 Malmö, Sweden. Back



   References
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 

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Received for publication: 22. 9.99
Revision received 30. 8.00.