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NDT Advance Access originally published online on March 25, 2008
Nephrology Dialysis Transplantation 2008 23(9):2911-2917; doi:10.1093/ndt/gfn137
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© The Author [2008]. Published by Oxford University Press on behalf of ERA-EDTA. All rights reserved. For Permissions, please e-mail: journals.permissions@oxfordjournals.org


Platelet activation in clinical haemodialysis: LMWH as a major contributor to bio-incompatibility?

Mareille Gritters1,2, Piet Borgdorff3, Muriel P. C. Grooteman2, Marianne Schoorl4, Margreet Schoorl4, Piet C. M. Bartels4, Geert-Jan Tangelder3 and Menso J. Nubé1,2

1 Department of Nephrology, Medical Center Alkmaar, Alkmaar 2 Department of Nephrology 3 Laboratory for Physiology, VU University Medical Centre, Amsterdam 4 Department of Clinical Chemistry, Haematology and Immunology, Medical Centre Alkmaar, Alkmaar, The Netherlands

Correspondence and offprint requests to: M. Gritters, Department of Nephrology, VU University Medical Centre, PO Box 7075, 1007 MB Amsterdam, The Netherlands. Tel: +31-20-4442673; Fax: +31-20-4442675; E-mail: m.gritters{at}vumc.nl



   Abstract
Top
 Abstract
Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Background. The sum of undesirable side effects, occurring during haemodialysis (HD), is called bio-incompatibility. Concerning platelets, both an increase in the expression of the cell surface marker P-selectin (CD62p) and release of the intracellular granule product platelet factor 4 (PF4) have been described. However, as PF4 is also abundantly present on endothelium-bound proteoglycans, it is questionable whether the HD-induced increase is exclusively attributable to release from platelets. With respect to the cause of HD-induced bio-incompatibility, interest has been focused mainly on the extracorporeal circuit (ECC), especially the dialyser, whereas only little attention has been paid to other parts of the ECC and the mode of anticoagulation applied. To address the cause and origin of platelet activation and PF4 release during clinical HD, two complementary clinical studies were performed.

Materials and methods. In study I, the relative influence of the various parts of the ECC was evaluated by measuring the expression of CD62p, platelet aggregation and levels of PF4 and serotonin at various sampling points. In study II, low-molecular-weight heparin (LMWH) was administered 10 min before the actual start of HD, in order to separate the effects from LMWH and the ECC on platelet activation.

Results. In study I, CD62p expression increased across the entire length of the ECC, including the roller pump and dialyser (median at t5 from 26% to 43%, P = 0.008; median at t30 from 28% to 48%, P = 0.007). Increments in PF4 and aggregation of platelets were relatively modest. Platelet serotonin content, which was below reference values in healthy controls, and plasma serotonin concentration, which was above reference values, did not change. In study II, PF4 levels increased markedly after the injection of LMWH (from 12 IU/ml at t–10 to 75 IU/ml at t0, P = 0.018), whereas CD62p expression remained stable until the start of HD.

Conclusions. Platelet activation, as measured by the up-regulation of CD62p, is an early process, occurring not only within the dialyser, but across the entire length of the ECC. As CD62p remained unaltered after the administration of LMWH 10 min before the actual start of HD, this kind of activation is independent of LMWH. Considering PF4 however, a sharp increment was observed after the administration of LMWH and before the start of HD. This finding suggests that the PF4 release observed early in clinical HD is largely independent from the ECC, and is probably the result of LMWH-induced detachment from the endothelium. As the platelet serotonin content was relatively reduced and the plasma serotonin levels were elevated, platelets from chronic HD patients might be depleted due to chronic repetitive activation. Based on these data, it appears first, that PF4 is an inferior marker of platelet activation in clinical HD and second, that LMWH is a major contributor to HD-induced bio-incompatibility.

Keywords: anticoagulation; bio-incompatibility; haemodialysis; platelet activation; platelet factor 4



   Introduction
Top
 Abstract
 Introduction
Subjects and methods
 Results
 Discussion
 References
 
During the course of haemodialysis (HD) various undesirable side effects occur, such as the stimulation of several protein systems in the blood and activation of circulating blood cells. The sum of these effects is termed bio-incompatibility. With respect to platelets, both an increase in the expression of the cell surface molecule P-selectin (CD62p) and the release of various intracellular granule products, such as platelet-derived growth factor (PDGF), platelet factor 4 (PF4) and β-thromboglobulin have been described [1].

Of specific interest in this respect is PF4, since it co-localizes with oxidized low-density lipoprotein (ox-LDL) in atherosclerotic plaques [2], upregulates E-selectin expression [3], lowers ox-LDL degradation and increases the uptake of ox-LDL by macrophages [4]. Hence, chronic intermittent platelet activation may contribute to oxidative stress and the extremely high risk of cardiovascular disease in chronic HD patients [5].

Concerning the cause and origin of HD-induced bio-incompatibility, interest has been focused mainly on the type of dialyser used. However, apart from the dialyser, other components of the extracorporeal circuit (ECC), such as the deflation chamber and roller pump, and the mode of anticoagulant therapy may play a role. In rats, it was found that extracorporeal circulation evoked strong platelet aggregation and serotonin release, which was attributable to pump-induced shear stress [6]. Lately, it was shown that the mode of anticoagulation also has a critical influence on the extent of platelet activation. The application of light-molecular-weight heparin (LMWH) was associated with less platelet activation than unfractionated heparin [7,8]. Moreover, both the activation and degranulation of platelets and leukocytes were almost completely absent during HD with citrate [9–11].

Recently, the origin of the HD-induced PF4 release was questioned, since this substance is not only stored in the {alpha}-granules of platelets, but also abundantly present on the surface of endothelial cells lining the blood vessels. As this proteoglycan-bound PF4 is discharged by heparin, the HD-induced rise in PF4 may result not only from platelet activation, either within the ECC or in the circulation, but also from endothelial release [12].

To address the abovementioned questions, two complementary clinical studies were performed. In the first investigation (study I), the relative influence of the various parts of the ECC on platelets was evaluated by measuring changes in the expression of CD62p, platelet aggregation, platelet serotonin content and the plasma levels of PF4 and serotonin at various sampling points. In the second study (study II), LMWH was administered 10 min before the actual start of HD, in order to separate the effects of LMWH and contact of blood with the ECC on platelet CD62p expression and plasma PF4 levels. The question whether heparin can liberate PF4 directly from platelets was addressed in a supplementary in vitro experiment.



   Subjects and methods
Top
 Abstract
 Introduction
 Subjects and methods
Results
 Discussion
 References
 
Patients
Ten stable patients [5 females and 5 males, median age 61 years (32–82)], undergoing regular HD treatment for at least 1 year [median 28 months (12–180)], were included in study I. The aetiology of renal insufficiency was hypertensive nephrosclerosis in five patients, diabetic nephropathy in one patient, chronic pyelonephritis in one patient, membranous nephropathy in one patient, Alport's syndrome in one patient and adult dominant polycystic kidney disease in one patient. Criteria for exclusion were clinical signs of infection, autoimmune disease or malignancy as well as the use of drugs known to interfere with the immune system and/or platelet function, such as immunosuppressive drugs, non-steroidal anti-inflammatory drugs, calcium antagonists, coumarines, clopidogrel and aspirin.

In study II, seven stable HD patients [3 females and 4 males, median age 61 years (48–80), median time on HD 31 months (12–71)], were included according to the same criteria. The aetiology of renal insufficiency was hypertensive nephrosclerosis in five patients and diabetic nephropathy in two patients. Written informed consent was obtained in all cases. Both protocols were approved by the local Medical Ethical Committee.

Study design and blood sampling
In study I, platelet activation, as measured by the expression of CD62p, platelet aggregation, platelet serotonin content and the plasma levels of PF4 and serotonin, was studied during a single HD session. Blood samples were taken at three different places in the ECC (Figure 1) to assess the relative contribution of the individual components (roller pump segment and dialyser segment) on platelet activation, as follows: arterial blood was drawn from the shunt before the start of HD (t0), whereas after 5 (t5), 30 (t30), 60 (t60) and 150 (t150) min, blood was drawn from three different sampling points in the ECC. The first sampling point (1) was located in the afferent line (i.e. before the roller pump), the second (2) at the first deflation chamber (i.e. in between roller pump and dialyser, the so-called pump segment) and the last (3) in the efferent line (i.e. after the dialyser, the so-called dialyser segment). With respect to sampling point 2, precautions were taken to prevent local pooling of blood. First, the deflation chamber was gently filled with air to clear its content. Thereafter, the air was removed slowly until it was completely refilled with blood after which a sample was taken.


Figure 1
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  		Fig. 1 Three different sampling points in the ECC: (1) afferent line (i.e. before the roller pump), (2) first deflation chamber (i.e. in between roller pump and dialyser), (3) efferent line (i.e. after the dialyser).

 
In study II, the influence of LMWH on CD62p expression and PF4 release was studied during and just before a single HD session. Therefore, LMWH [dalteparin, median 5000 IU (2500–7500)] was administered in the AV-shunt 10 min before the actual start of HD. Blood was taken directly from the AV-shunt 10 min before dialysis just before dalteparin injection (t–10) and at the start of HD (t0). Samples were taken from the afferent line after 5 (t5) and 30 (t30) min and from the efferent line after 1 (t1), 5 (t5) and 30 (t30) min.

All results were corrected for changes in plasma volume, based on haematocrit (Ht) measurements (corrected valuetx = valuetx*[Htt0/(1 – Htt0)]*[(1 – Httx)/Httx]).

In vitro experiment
To find out whether heparin is capable of liberating PF4 directly from platelets, blood was taken from five healthy controls, and drawn in tubes containing 1 U/ml of heparin. After storage at 37°C for 2, 4 and 8 min, blood samples were transferred to CTAD (citrate theophylline adenosine dipyridamole) tubes to stop platelet activation and for measuring PF4.

Dialysis procedure and materials
In both studies, only new (first use) high-flux polysulfone (PS) dialysers [Fresenius Medical Care, Bad Homburg, Germany; ultrafiltration (UF) factor 20–55 ml/h x mmHg, surface area 0.7–1.8 m2, steam sterilized] were used. Bicarbonate dialysate was used with a dialysate flow of 500 ml/min. For dialysate preparation, tap water, purified by reverse osmosis, was used for the dilution of a concentrated bicarbonate solution to the following concentrations (mmol/l): 138 Na+, 2.0 K+, 1.50 Ca2+, 0.50 Mg2+, 109 Cl–, 2.5 CH3COO– and 32.5 HCO–3 (SK-F 216/1; Fresenius Medical Care, Bad Homburg, Germany). All dialysers were pre-rinsed with 1000 ml 0.9% NaCl. Individual doses of dalteparin, which is the standard type of anticoagulation in our centre, were based on bodyweight (50 IU/kg) and duration of dialysis and given as a bolus injection at the beginning of the dialysis session (median 4750 IE [2000–6000]). Dialysate temperature was kept at 37°C.

Analytical methods
Platelet number.
Blood samples were collected in K2EDTA (ethylene diamine tetraacetic acid) tubes (Becton Dickinson, Plymouth, UK) and platelet numbers were determined using a Sysmex XE2100 Haematology analyser (Sysmex Corporation, Kobe, Japan).

Platelet surface markers.
The platelet surface markers CD62p (P-selectin; clone CLB Thromb 6, Beckman Coulter, Mijdrecht, The Netherlands) and CD41 (clone P2, Beckman Coulter, Mijdrecht, The Netherlands) were detected by direct labelling and flow cytometry. Blood was drawn into K2EDTA tubes and within 2 h after collection incubated with a glycoprotein-specific fluorochrome-labelled monoclonal antibody. A flowcytometer (Epics XL, Beckman Coulter, Mijdrecht, The Netherlands) was used to determine the percentage of platelets with CD62p surface expression. CD41 served as a platelet-specific label.

PF4.
Blood samples were drawn into CTAD (Vacutainer® CTAD, Becton Dickinson, Plymouth, UK) tubes, cooled on ice and centrifuged for 20 min at 2500 g. Plasma samples were stored at –70°C until measurement. PF4 was determined using a commercially available sandwich ELISA (Asserachrom PF4®, Diagnostica Stago, Asnières, France).

Platelet aggregates.
Blood samples for platelet aggregates were collected in K2EDTA tubes (Becton Dickinson Plymouth, UK). For erythrocyte lysis 20 µl of blood was diluted with 2 ml of Thrombo Plus solution (Sarstedt, Nümbrecht, Germany). Platelet aggregates were counted in a Bürker chamber under a light microscope (x200) in 60 fields each containing 0.1 µl of diluted blood.

Serotonin.
Blood samples were drawn in K2EDTA tubes (Becton Dickinson, Plymouth, UK). Platelet-rich plasma (PRP) and platelet-poor plasma (PPP) were prepared by centrifugation of blood samples at 200 g (10 min) and 4000 g (10 min), respectively. Until measurement, PRP and PPP were stored at –70°C. Serotonin was determined using an enzyme immunoassay (Serotonin EIA, DSL, Veghel, The Netherlands).

Statistical analysis
All analyses were performed with the SPSS 15 software system. Although nearly all parameters were normally distributed, non-parametric tests were used to study differences in platelet activation, as the number of observations was small. Data are expressed as median (min.–max.). Friedman's test was used for non-parametric repeated measurement comparisons. Wilcoxon-signed ranks tests were performed as post hoc analysis in the case of significant differences. Differences were considered significant at P < 0.05.



   Results
Top
 Abstract
 Introduction
 Subjects and methods
 Results
Discussion
 References
 
Study I
Platelet numbers.
The number of platelets remained stable, not only across the ECC, but also over time (data not shown).

Platelet activation: CD62p.
Compared to t0, the expression of CD62p in the ECC was already increased at t5, reaching a maximum at t30 (Figure 2). At t150, the expression of CD62p had returned to baseline. Interestingly, CD62p expression increased both over the dialysis segment and over the pump segment, as is shown in Table 1 and Figure 2.


Figure 2
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  		Fig. 2 CD62p expression (% of platelets) measured during HD (t0, t5, t30, t60 and t150) at three different sampling points in the ECC, see Figure 1. °P = 0.005–0.013 versus baseline (t0, shunt), +P = 0.008, •P = 0.038 and *P = 0.007 versus other sampling point at the same time. Boxes contain the 25th–75th percentiles, whereas the whiskers illustrate the highest and lowest value within the 1.5-interquartile ranges.

 

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  		Table 1 CD62p expression (% of platelets) and PF4 (IU/ml) measured during HD (t0, t5, t30, t60 and t150) at three different sampling points in the ECC (study I), see also Figures 2 and 3

 
Platelet degranulation: PF4.
With respect to PF4, levels increased markedly directly after the start of HD, the highest levels being observed at t5. These levels had practically returned to baseline at t150 (Figure 3 and Table 1). Increments in PF4 across the ECC were only seen at t30.


Figure 3
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  		Fig. 3 PF4 (IU/ml) measured during HD (t0, t5, t30, t60 and t150) at three different sampling points in the ECC, see Figure 1. °P = 0.005–0.013 versus baseline (t0, shunt), *P = 0.013 versus other sampling point at the same time. For interpretation of the box plots see legend of Figure 2.

 
Aggregated platelets (/250 platelets).
Aggregation of platelets had occurred already at t5, with a maximum at t30 at sampling point 2 [roller pump segment: 23 aggregates/250 platelets (2–39), P = 0.011 versus baseline, P = 0.028 versus t30 (1), Figure 4]. Interestingly, the number of aggregated platelets had decreased at sampling point 3 [dialyser segment: t30 (3) 12 aggregates/250 platelets (0–36), P = 0.007 versus t30 (2)].


Figure 4
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  		Fig. 4 Number of aggregated platelets (number/250 platelets) measured during HD (t0, t5, t30 and t60) at three different sampling points in the ECC, see Figure 1. °P < 0.05 versus baseline (t0, shunt), *P < 0.03 versus other sampling point at the same time. For interpretation of the box plots see legend of Figure 2.

 
Platelet serotonin content and plasma serotonin concentration.
Median platelet serotonin content, as measured in PRP, [t0 189 ng/10E9 platelets (24–619)] was clearly below the reference values in healthy controls (215–850 ng/10E9 platelets) and did not change over time (data not shown). By contrast, the median plasma serotonin concentration, as measured in PPP, [t0 23 ng/ml (16–57)] was obviously above the reference values (1.8–7.5 ng/ml) and did not change over time (data not shown).

Study II
Effect of LMWH on PF4 release.
After dalteparin injection 10 min before the actual start of HD (t–10), mean PF4 levels rose from 12 IU/ml (5–20) at t–10 to 75 IU/ml (50–141) at t0 (before pump start) (P = 0.018). The small increments which were observed in the ECC at t5 [from 50 IU/ml (28–138) at t5(1) to 61 IU/ml (38–151) at t5(3), P 0.028] and t30 [from 25 IU/ml (16–101) at t30(1) to 46 IU/ml (17–136) at t30(3), P 0.018] were comparable to those in study I. Of note, at t0 no increase in CD62p expression was observed as compared to t–10 (Figure 5).


Figure 5
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  		Fig. 5 CD62p expression (% of platelets) and plasma PF4 (IU/ ml) in study II. Dalteparin was injected into the shunt at t–10, HD was started at t0. •P = 0.018 versus baseline (t–10, shunt), °P = 0.018–0.028 versus start of HD (t0, shunt), *P = 0.018–0.043 versus other sampling point at the same time. For interpretation of the box plots see legend of Figure 2.

 
In vitro experiment
Except for samples that showed manifest coagulation after 4 and 8 min, PF4 release was not observed (data not shown).



   Discussion
Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
References
 
In line with previous observations, our investigation clearly showed that platelet activation, as measured by the up-regulation of the cell surface molecule CD62p, is an early process, occurring shortly after the start of HD [9]. CD62p, which is the preferable marker for platelet activation as it is rapidly exposed on the platelet surface and has a unique specificity through the double labelling with CD41a, increased steadily across the ECC both at t5 and t30, whereas at t150 its values had returned to baseline. After the separate administration of LMWH in study II, the expression of CD62p remained unaltered in the 10 min before the actual start of HD. Together, these data suggest that platelet activation, as measured by the up-regulation of CD62p, occurs early in HD across the entire length of the ECC and is independent of LMWH.

With respect to PF4 however, in study II, a sharp increment in the plasma levels was observed at t0 after the administration of LMWH 10 min before the actual start of HD. As shown in Figure 3 (study I) and Figure 5 (study II) PF4 release within the ECC was relatively modest. Therefore, it appears that PF4 release is mainly evoked by the administration of LMWH and only to a limited extent by the HD procedure itself.

With respect to the origin of PF4, both release from circulating platelets and detachment from endothelial proteoglycans [12] might play a role. To demonstrate or exclude release from platelets, blood from healthy volunteers was incubated with heparin in clinical doses. From this experiment it appeared that PF4 was not released as long as clotting did not occur, which is in line with data from others [13]. In this respect it is interesting to notice that several in vitro [14] and ex vivo studies [7] reported on a greater influence of unfractionated heparin than LMWH. Hence, as CD62p remained unaltered in the 10 min before the actual start of HD, it appears that the early rise in PF4, as observed in study II, results from LMWH-induced detachment from the endothelium and not from platelet activation. This assumption is further supported by studies in rabbits. After the intravenous injection of 131I-labelled PF4, accumulation of radioactivity occurred in the liver, disappearing after the subsequent administration of heparin. Interestingly, the loss of radioactivity in the liver was associated with the appearance of 131I-labelled PF4 in the bladder [15].

Surprisingly, and in sharp contrast to findings in rats [6], only limited platelet aggregate formation occurred in our study. Slightly increased numbers were detected at t30 at sampling point 2, indicating that some formation occurred in the segment which contains both the roller pump and the deflation chamber. As the values at sampling point 3 were similar to those at sampling point 1, it appears that these platelet aggregates are instable and disintegrate during passage through the dialyser. With respect to serotonin, both the plasma concentrations and the platelet contents did not markedly change, indicating that release from platelets into the systemic circulation did not occur. In fact, our findings on platelet aggregation and serotonin release are unexpected, as it was recently shown in non-uraemic rats that abundant platelet aggregation and marked serotonin release originated from roller pump-induced shear stress [6]. Whether the high platelet number in these animals [16], the markedly different platelet serotonin content in rats [17], the uraemic conditions in the present study [18] or intradialytical loss of released serotonin underlie the diverging results is a theoretical question.

As mentioned, PF4 release within the ECC was relatively small, despite the up-regulation of CD62p. In addition, low platelet serotonin contents and high plasma serotonin concentrations were found in our patients. In analogy to leucocytes, which exhibit signs of activation and exhaustion due to the repetitive stimulation of intermittent HD [19], these data may be explained by a state of platelet granule depletion in chronic HD patients. This assumption is supported by recent findings from our own group. Light-microscopic analysis of platelet granules in HD patients revealed severe changes in platelet volume, morphology and RNA content in comparison to healthy controls [20]. Whether intermittent platelet activation and exhaustion contribute to the signs and symptoms of uraemic thrombopathy in chronic HD patients is a matter of speculation.

A potential limitation of our study is the fact that the handling procedure itself may induce some platelet activation [21]. However, as all samples were treated uniformly and precautions were taken to avoid unnecessary delay, it is highly unlikely that the different data resulted from dissimilar technical procedures. Therefore, in our opinion, the main conclusions of this work do not result from handling artefacts, but depend on the specific design of this study.

To summarize, platelet activation, as measured by an increase in the expression of CD62p, occurs across the entire length of the ECC. This finding indicates that the contributions of the roller pump and deflation chamber are equally important as the dialyser. As far as serotonin is concerned, our data suggest that the repetitive stimulation of chronic intermittent HD results in a state of platelet depletion and exhaustion. With respect to PF4, the increase during HD seems to originate mainly from LMWH-induced detachment from the endothelium and only to a limited extent from platelets. Hence, in our opinion, PF4 is an inadequate marker of platelet activation in HD with LMWH anticoagulation. Finally, as PF4 might contribute to oxidative stress and cardiovascular disease in chronic HD patients, LMWH and probably also unfractionated heparin might play a major role in the bio-incompatibility of HD treatment.



   Acknowledgments
 
We wish to thank the patients of the Medical Center Alkmaar, Dialysis Department for their willing participation in this study and the staff for their indispensable support. This study was supported by Baxter Healthcare Corporation and Fresenius Medical Care. The sponsors were neither involved in the analysis of the results, nor in the writing of the manuscript.

Conflict of interest statement. None declared.



   References
Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 

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Received for publication: 29.10.07
Accepted in revised form: 19. 2.08


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