Differential effects of dialysis and ultrafiltrate from individuals with CKD, with or without...

Dialysis and Platelet PS Externalisation 1

THE DIFFERENTIAL EFFECTS OF DIALYSIS AND ULTRAFILTRATE

FROM INDIVIDUALS WITH CKD, WITH OR WITHOUT DIABETES, ON

PLATELET PHOSPHATIDYLSERINE EXTERNALISATION.

Yingjie Wang1, Werner Beck2, Reinhold Deppisch2,

Sally M. Marshall1, Nicholas A. Hoenich1 and Michael G. Thompson1.

1Faculty of Medical Sciences, Framlington Place, Newcastle University,

Newcastle-Upon-Tyne, United Kingdom, NE2 4HH.

2Gambro Corporate Research,

Hechingen, Germany.

Correspondence and Reprint Requests To: Dr. M.G.Thompson

TEL: +44 191 222 8112

FAX: +44 191 222 0723

E-MAIL: [email protected]

Short title: Dialysis and Platelet PS Externalisation.

Keywords: Cell Signalling, Thrombosis, 5-HT2A/2C receptors.

Page 1 of 39Articles in PresS. Am J Physiol Renal Physiol (August 1, 2007). doi:10.1152/ajprenal.00279.2007

Copyright © 2007 by the American Physiological Society.


ABSTRACT

Individuals with chronic kidney disease (CKD) and/or diabetes mellitus (DM) are at

increased risk of cardiovascular events and have elevated externalisation of

phosphatidylserine (PS), (which propagates thrombus formation), in a small, sub-

population of platelets. The aim of this study was to examine the effect of (i)

removing uraemic toxins by hemodialysis on PS externalisation in patients with either

CKD or CKD and DM and (ii) ultrafiltrate (UF) from these individuals on PS

externalisation in healthy platelets. PS externalisation was quantified by a

fluorescence-activated cell sorter using Annexin V in platelet rich plasma. PS

externalisation was elevated 3-fold in CKD patients and returned to basal values

during 3 hrs hemodialysis. In contrast, it was elevated 5-fold in individuals with CKD

and DM and was still 3-fold above control after 3 hrs treatment. UF significantly

increased PS externalisation in a small, sub-population of platelets from healthy

controls. The effect of UF from individuals with CKD and DM was significantly

greater than that from patients with CKD alone and the responses were partially

inhibited by the protein kinase Cδ (PKCδ) inhibitor, rottlerin, and the 5-

hydroxytryptamine (5-HT)2A/2C receptor antagonist, ritanserin. The data suggest that

uremic toxins present in UF mediate PS externalisation in a small, sub-population of

platelets, at least in part, via the 5-HT2A/2C receptor and PKCδ and demonstrate that

DM further enhances platelet PS externalisation in CKD patients undergoing

hemodialysis. This may explain, at least in part, the additional increase in vascular

damage observed in CKD patients when DM is present.

Page 2 of 39


INTRODUCTION

Cardiovascular disease, in particular atherothrombosis, is a common cause of death

in patients receiving dialysis and co-morbid conditions such as diabetes mellitus (DM)

further influence the incidence of vascular events (13, 19, 20). Progression is

associated with vascular endothelial cell injury, atherosclerotic plaque fissuring and

rupture (33). An early key event in such progression is increased adherence of

monocytes to the damaged vascular endothelium resulting in fatty streak/atheroma

development with platelets subsequently attaching firmly to the site of the lesion (29).

In the plasma membrane of resting platelets, the phospholipid, phosphatidylserine

(PS), resides in the inner leaflet (5) but, upon activation of Scramblase by Protein

Kinase Cδ (PKCδ), PS translocates to the outer leaflet of the plasma membrane (5,

15). It has become increasingly clear that chronically elevated or prolonged exposure

of PS on the cell surface both increases vascular damage and results in the formation

of a hypercoagulable environment by stimulating adherence of inflammatory cells to

the vascular endothelium (e.g. 22) and providing a catalytic surface for assembly of

the prothrombinase complex (29) that accelerates the generation of thrombin (32, 49).

Evidence now suggests that classic platelet agonists such as thrombin and ADP

elicit PS externalisation in a small, sub-population of these cells (45, 48) and that the

size of this population correlates with prothrombinase activity (48). In addition,

increased PS externalisation in a sub-population of platelets has been observed in

individuals with chronic kidney disease (CKD) and DM (45), although the factor(s)

responsible for this response is/are unknown.

Advanced glycation end products (AGE) arise by covalent modification of cellular

and plasma proteins and form a series of heterogeneous compounds (21). AGE are

substantially increased in individuals with either CKD or DM when compared to the

Page 3 of 39


general population (25, 44) and may contribute to the development and progression of

cardiovascular disease in these patient groups (35, 39).

In a recent study, we showed that Human serum albumin-AGE (HSA-AGE),

prepared in vitro, elicits PS externalisation in a small, sub-population of human

platelets. Moreover, this response was completely blocked by both the 5-

hydroxytryptamine (5-HT)2A/2C receptor antagonist, ritanserin, and the PKCδ

inhibitor, rottlerin (45).

In order to translate this work towards the in vivo situation, we have now explored

firstly, the effect of hemodialysis on the elevated PS externalisation we observed in

patients with CKD/DM and secondly, the ability of ultrafiltrate from these individuals

to elicit PS externalisation in platelets from healthy controls and the mechanism(s)

involved.

Page 4 of 39


METHODS

Materials and Reagents

Annexin V-fluorescein isothiocyanate (FITC) was purchased from Immunotech

(Marseille, France) and the FluoroSpheres for FACScan calibration were obtained

from DakoCytomation (Glostrup, Denmark). The fluorescence-activated cell-scanner,

CD61 – PerCP and the Caspase 3 inhibitor, Z-DEVD-FMK, were purchased from

Becton Dickinson (Cowley, Oxford, UK). Rottlerin and Bisindolylmaleimide 1 were

from Merck Biosciences Ltd (Beeston, Nottingham, UK). The 5-HT2A2C receptor

antagonist, ritanserin, and all general chemicals were obtained from Sigma-Aldrich

(Poole, Dorset, UK). The low-flux Fresenius polysulfone® hemodialysis membrane

was purchased from Fresenius Medical Care (Bad Homburg, Germany) and the

ENDOSAFE® LAL Gel Clot Test was from Charles River (Charleston, USA).

Patients

26 stable individuals (age range 32-85 years) who had been receiving low flux

conventional hemodialysis using a Fresenius Polysulfone membrane 3 times per week

for at least 3 months gave their informed consent to participate in the study. 8 (6 male

and 2 female) of these also had Type 2 DM, whilst the remaining 18 (13 male and 5

female) did not.

The effect of a single, low-flux hemodialysis treatment on platelet PS externalization

in individuals with either CKD or CKD and DM

Hemodialysis patients at Newcastle undergo three sessions a week for 4 hours at a

time. The individuals selected for this study were treated on Monday, Wednesday and

Page 5 of 39


Friday. There was no preference for any particular day when examining the effect of a

single treatment.

A 2.5 ml blood sample was withdrawn pre-dialysis and then 1, 2 and 3 hours after

the commencement of hemodialysis from patients with either CKD or CKD and DM

using a standard 16G hypodermic needle. This was then immediately added to a tube

containing acid citrate dextrose (ACD) and centrifuged at 100g for 15 minutes at

20°C (Beckman J6-MC). The upper phase containing the Platelet Rich Plasma (PRP)

was removed and the extent of PS externalisation was determined as described below.

The effect of three low-flux hemodialysis treatments during a seven day period on

platelet PS externalisation in individuals with either CKD or CKD and DM

Pre- and 3 hours post-dialysis blood samples (2.5 ml) were withdrawn on Monday,

Wednesday and Friday from patients with either CKD or CKD and DM using a

standard 16G hypodermic needle. PRP was then prepared as above and the extent of

PS externalisation was determined as described below.

Preparation of PRP from healthy control subjects

Blood was obtained from 6 healthy control subjects (2 male and 4 female, age

range 28-52 years) who had no history of cardiovascular disease or hypertension and

were not using any medication. PRP was then prepared as above and subsequently

incubated with ultrafiltrate under various treatment conditions as described below.

Collection of Ultrafiltrate (UF) from individuals with either CKD or CKD and DM

Studies using urea as a candidate molecule to understand solute kinetics in

hemodialysis have revealed that it is rapidly removed during the first 30 minutes, but

Page 6 of 39


that its removal rate then gradually declines throughout the rest of dialysis (e.g. 24).

We have therefore utilised two time points, 20 minutes and 180 minutes, to

investigate effects on PS externalisation in these two different kinetic phases.

To collect pure ultrafiltrate, the dialysis fluid was disconnected from the dialyser

for a short period both 20 minutes (UF20) and 180 minutes (UF180) after the start of

treatment. The dialysis fluid pathway was evacuated, ultrafiltrate was allowed to flow

for 2-3 minutes and 3 mls of ultrafiltrate was collected into an endotoxin-free tube.

The system was then reconnected and the treatment continued.

Effect of ultrafiltrate from individuals with CKD or CKD and DM on PS

externalisation

PRP was prepared from healthy controls as described above and then incubated

with increasing volumes (2.5 – 20 µl) of either UF20 or UF180 from individuals with

CKD or CKD and DM at 37°C for 10 minutes. Samples were immediately placed on

ice and PS externalisation was determined as described below.

Effect of ADP on PS externalisation in healthy controls and individuals with CKD or

CKD and DM during hemodialysis

PRP was prepared from individuals with either CKD or CKD and DM at 20

minutes and 180 minutes after the commencement of hemodialysis, or from healthy

controls. ADP (100 nM) was then added for 1 minute. The extent of PS

externalisation was then determined as described below.

Page 7 of 39


Effect of inhibition of Caspase 3 on PS externalisation mediated by ultrafiltrate from

individuals with CKD or CKD and DM

PRP prepared from healthy controls was pre-incubated with 20 µM of the Caspase

3 inhibitor, Z-DEVD-FMK, or its negative control, Z-FAD-FMK (17), for 30 minutes

and then 10 µl of either UF20 or UF180 from individuals with CKD or CKD and DM

was added for 10 minutes at 37°C. The extent of PS externalisation was determined as

described below.

Effect of inhibition of Protein Kinase Cδ on PS externalisation mediated by

ultrafiltrate from individuals with CKD or CKD and DM

PRP prepared from healthy controls was pre-incubated with 10 µM of the PKCδ

inhibitor, rottlerin, for 5 minutes. This concentration has been reported to block PKCδ

activity by approx. 80-90% whilst having no effect on representative examples of

either the classical e.g. PKCα or atypical e.g. PKCζ isoforms (15). 10 µl of either

UF20 or UF180 from individuals with CKD or CKD and DM was then added for 10

minutes at 37°C. The extent of PS externalisation was determined as described below.

Effect of a 5-HT2A/2C receptor antagonist on PS externalisation mediated by

ultrafiltrate from individuals with CKD or CKD and DM

PRP prepared from healthy controls was pre-incubated for 30 minutes with 1 µM of

the 5-HT2A/2C receptor antagonist, ritanserin (26). 10 µl of either UF20 or UF180 from

individuals with CKD or CKD and DM was then added for 10 minutes at 37°C. The

extent of PS externalisation was determined as described below.

Page 8 of 39


Measurement of PS externalisation in PRP

PS externalisation was determined using the cell membrane-impermeant, PS-

specific, Annexin V-fluorescein isothiocyanate (FITC) conjugate and CD61 – PerCP

as a platelet-specific marker with subsequent quantification by a fluorescence-

activated cell-scanner (FACScan) supporting Lysis II software as described

previously (45).

Statistical analysis

Statistical analysis was undertaken using Minitab 14 (Minitab Inc, State College,

PA, USA). Studies with the Caspase 3 / PKCδ inhibitors and the 5-HT2A/2C receptor

antagonist were analysed by use of Student’s t-test and are presented as Means ±

s.e.m. where *p<0.05, **p<0.01 and ***p<0.001 with respect to the relevant control.

Serial measurements with ultrafiltrate from individuals with CKD or CKD and DM

were analysed as summary measures (27), i.e. the areas under the concentration or

time curves (AUC). All experiments were performed on at least three occasions.

Page 9 of 39


RESULTS

The analysis of PRP prepared from individuals with CKD or CKD and DM pre-

dialysis revealed a sub-population of platelets (approx 3 % and 10% of the total

population respectively) with increased PS externalisation when compared to healthy

controls (Figs. 1 and 2). We have therefore characterised this small population in

detail and all subsequent data describing the effects of dialysis relate to this fraction.

The degree of pre-dialysis PS externalisation in platelets derived from individuals

with CKD and DM was significantly higher than that from patients with CKD alone

(161 ± 1 vs 99 ± 5 x 102 total fluorescent binding sites, p<0.001; n=3). Analysis of PS

externalisation during low flux (Fresenius Polysulfone) dialysis for 3 hours in

individuals with CKD showed that, whilst still slightly elevated after 60 minutes, this

parameter had returned to basal values after 2 hours (Figs. 1 and 3). In contrast, PS

externalization in the platelet sub-population derived from patients with CKD and

DM, whilst significantly reduced, was still well above basal values (78 ± 1 vs 30 ± 1 x

102 total fluorescent binding sites, p<0.001; n=3) after 3 hours dialysis (Figs. 2 and 3).

When patients undergo dialysis on Monday, Wednesday and Friday, there is a 2

day interval between treatments Monday – Wednesday and Wednesday – Friday, but

a 3 day interval between Friday – Monday. We were therefore interested to establish

if the 3 day interval, with its likely greater accumulation of metabolites, resulted in

increased PS externalization on a Monday when compared with Wednesday/Friday.

Analysis showed that the degree of pre-dialysis PS externalization in patients with

CKD or CKD and DM was significantly higher on Monday than the two other days of

the week when dialysis took place (Fig. 4). Moreover, in spite of the increased PS

externalization seen in CKD patients on a Monday, this had returned to basal values

following dialysis treatment (Fig. 4a).

Page 10 of 39


When increasing volumes of ultrafiltrate from individuals with either CKD or CKD

and DM were added to PRP from healthy controls, statistically significant (p<0.001 in

all cases; n=4) increases in PS externalization in a sub-population of platelets (approx

3 % and 5 % of the total population respectively), were observed (Fig. 5). In contrast,

equivalent material prepared from healthy controls using a 30 kDa cut-off filter had

no effect (data not shown). A small, but highly significant stimulation (p<0.001, n=4)

was also seen when hemodialysis fluid, which had been used to rinse the dialyser, was

added to PRP (Fig. 5). We have therefore characterised this small population in detail

and all subsequent data relate to this fraction. Optimal responses were observed with

10 µl of ultrafiltrate (data not shown) and analysis demonstrated that the levels of

endotoxin present were below the minimum value (0.03 EU / ml) detectable by the

test. The effects of UF20 and UF180 taken from individuals with CKD and DM were

significantly greater than the corresponding samples generated by patients with CKD

alone and, interestingly, UF180 from both patient groups appeared as potent in eliciting

PS externalisation as UF20 (Fig. 5).

At this point in our investigation, it was not clear why, with the CKD data for

example, PS externalisation had returned to basal values after 180 minutes dialysis

(Figs 1 and 2), yet ultrafiltrate removed at the same time point elicited a substantial

increase in PS externalisation when added to PRP from healthy individuals (Fig. 5). In

order to address this question, we firstly examined the ability of UF20 and UF180 from

individuals with CKD to elicit PS externalisation on PRP from both healthy controls

and that taken from patients with CKD after 180 minutes hemodialysis. Whilst UF20

and UF180 elicited substantial increases in PS externalisation in PRP from healthy

controls, they had no effect on that generated from individuals with CKD following

180 minutes dialysis (Fig. 6a). Similar data were obtained when ultrafiltrate and PRP

Page 11 of 39


from patients with CKD and DM were used (Fig. 6b). This insensitivity could be due

to either the uremic status per se of the individuals and / or the hemodialysis process

which in some way desensitizes platelets to the active component(s) present in

ultrafiltrate.

In order to investigate these observations further, we then examined the ability of

ADP, a classic physiological activator of human platelets (16), to elicit PS

externalisation in PRP from either healthy controls or individuals with CKD or CKD

and DM after 20 or 180 minutes hemodialysis. In platelets obtained from patients with

CKD after 20 minutes, the magnitude of the response to ADP was reduced (Fig. 7a)

compared to healthy controls (although PS externalisation in the untreated sample was

still significantly elevated as reported in Fig. 3a). After 180 minutes hemodialysis,

platelets from individuals with CKD were unresponsive to ADP (Fig 7a). Platelets

obtained from patients with CKD and DM were unresponsive to ADP at both time

points although, as we have shown in Fig 3b, PS externalisation in the untreated

samples obtained following both 20 and 180 minutes hemodialysis had not returned to

basal values (Fig. 7b). This suggests that hemodialysis results in both (i) the removal

of one or more factors able to elicit PS externalisation for at least 180 minutes after

the commencement of treatment and (ii) a time-dependent desensitization of the small

platelet sub-population to both physiological agonists such as ADP and uremic toxins.

In addition to its role in thrombosis, PS externalisation is also implicated in

apoptosis via Caspase 3 – mediated cleavage of PKC δ (11, 30). When PRP from

healthy controls was pre-incubated for 30 minutes with the Caspase 3 inhibitor, Z-

DVED-FMK (20 µM) and then subsequently challenged for 10 minutes with UF20 or

UF180 from individuals with either CKD or CKD and DM, their ability to elicit PS

externalisation was unaffected (data not shown) suggesting that the pathway(s)

Page 12 of 39


employed by the active component(s) did not involve Caspase 3. We have previously

shown that when used under identical conditions, Z-DVED-FMK completely

abolished the effect of ADP (100 nM) added for 1 minute whilst the negative control,

Z-FAD-FMK (20 µM), had no effect (45).

The pre-incubation of PRP from healthy controls for 10 minutes with

bisindolylmaleimide 1 (10 nM), an inhibitor of both the classical (α, β, γ) and novel

(δ, ε) isoforms of PKC (40, 46) resulted in a partial inhibition of the increase in PS

externalisation in the small platelet sub-population elicited by the addition of either

UF20 or UF180 from individuals with CKD or CKD and DM for 10 minutes (data not

shown).

Similarly, pre-incubation of PRP from healthy controls for 5 minutes with the

PKCδ inhibitor, rottlerin (10 µM), partially prevented (57-73 % inhibition) the

increase in PS externalisation elicited by the addition of either UF20 or UF180 from

both patient groups after 10 minutes incubation (Fig. 8) suggesting a role for PKCδ in

this response. In all cases, the effects of ultrafiltrate and inhibitor were significantly

different from inhibitor alone (p<0.001, n=3).

The pre-incubation of PRP from healthy controls for 30 minutes with ritanserin (1

µM), a 5-HT2A/2C receptor antagonist, also resulted in a partial inhibition (35-73 %) of

PS externalisation elicited by 10 minutes incubation with either UF20 or UF180 from

both patient groups (Fig. 9). In all cases, the effects of ultrafiltrate and inhibitor were

significantly different from inhibitor alone. This suggests that a significant fraction of

the effects of ultrafiltrate from individuals with CKD or CKD and DM on PS

externalisation in the small sub-population of platelets are mediated via 5-HT2A/2C

receptors.

Page 13 of 39


DISCUSSION

Cardiovascular disease, particularly atherosclerosis, is a major cause of morbidity

and mortality in patients undergoing current haemodialysis therapies (13, 19, 20). It is

clear from our study that, with regard to platelet PS externalisation, there is a distinct

difference between the effect of hemodialysis on individuals with CKD and DM when

compared to CKD alone. In the former group, PS externalisation in the platelet sub-

population had essentially reached a nadir after 3 hours hemodialysis at a value well

above that observed in healthy controls whilst, in the latter, it had returned to normal

values. It remains to be established if the failure of PS externalisation to return to

normal values in individuals with co-morbid conditions such as DM plays a role in the

increased vascular damage observed in this patient group (13, 19, 20).

A primary cause of vascular disease in individuals undergoing hemodialysis is the

inadequate removal of uremic toxins (3) and AGE, which are amongst the best studied

of these toxins (42), may contribute to the development and progression of

cardiovascular disease in these patients (35, 39). In support of this hypothesis, we

have recently provided evidence suggesting that HSA-AGE elicits PS externalisation,

a key step in the generation of thrombin (32, 49), in a small, sub-population of human

platelets via the 5-HT2A/2C receptor and PKCδ (45).

Our observation that the effects of UF20 / UF180 from both patient groups on PS

externalisation could also be partially blocked by the same inhibitors we employed in

our previous study suggests that AGE, acting through a similar mechanism, may also

play a role in the responses we report here. With regard to AGE in plasma, most are

modified amino acid residues of plasma protein, whilst glycation-free adducts

normally comprise less than 5% of the total. The latter increase substantially in

uraemia e.g. Nε-carboxymethy-lysine (CML), Nε-carboxyethyl-lysine (CEL), glyoxal–

Page 14 of 39


HI (G-H1), methylglyoxal (MG-H1) and 3-deoxyglucosone (3DG-H) are elevated

many fold and, at the end of a 4 hour dialysis session with a polysulfone membrane,

these increases in plasma (and ultrafiltrate) concentrations were reversed to varying

degrees, but not normalised (1).

When considering the plasma protein content of AGE, most are not eliminated

efficiently by hemodialysis e.g.G-H1 residues, CEL, CML and pentosidine, which are

elevated 2-, 3- and 4-fold respectively prior to dialysis, did not change during

treatment (1). The potential role / identity of glycation-free adducts and plasma

protein AGE in the responses we report here requires further investigation.

In addition to AGE, another potential receptor-mediated mechanism which

requires consideration involves release of the contents of both platelet-dense granules

and α-granules which occurs when blood comes into contact with artificial material

during hemodialysis. The factors released include ADP, Serotonin, Platelet-derived

growth factor (PDGF), platelet factor-4 (PF4) and β-thromboglobulin (βTG), some of

which do not return to basal values until 20 hours after the end of the hemodialysis

session (4, 8). It is possible that autocrine / paracrine loops play a role in our

observations.

As well as a possible role for receptor-mediated pathways in the stimulation of PS

externalisation we report here, other mechanisms also require consideration. Protein-

bound uremic solutes (42, 43) such as p-cresol and indoxyl sulphate, at concentrations

commonly found in uremia, have been shown to have biological effects (9). The

involvement of such agents is worthy of future study.

Perhaps the most interesting possibility involves a direct effect of one or more

uremic toxins on the activity of Scramblase and / or Aminophospholipid Translocase

(APLT), which opposes the role of Scramblase and is responsible for the inward

Page 15 of 39


translocation of aminophospholipids such as PS (6). There is increasing evidence that

oxidative stress is an important complication in hemodialysis (12) and it is now clear

that the activity of both Scramblase and APLT can be modified by alterations to

critical –SH groups (7, 10) e.g. the activity of Scramblase is enhanced by oxidative

modification of one or more sulphydryl groups and is suppressed by the reducing

agent, dithiothreitol (23). In addition to oxidative stress, there is a growing interest in

nitrosative stress (14) and recent evidence suggests that this is present in patients

undergoing hemodialysis (1, 28) e.g. 3-nitrotyrosine levels in plasma proteins have

been reported to be elevated 3-fold prior to treatment (1). Moreover, a very recent

investigation has shown that nitrosative stress both activates Scramblase and inhibits

APLT resulting in PS externalisation (41). Intriguingly, in preliminary experiments

with dithiothreitol, which both reduces disulphides and de-nitrosylates S-nitrosylated

proteins in live cells (38), we observed that this agent was able to partially reverse the

increased PS externalisation seen in the sub-population of platelets from individuals

with CKD (Wang Y and Thompson MG, unpublished observation). Clearly, a

possible link between uremic toxins inducing oxidative / nitrosative stress and the

effects on PS externalisation we describe in this manuscript requires further

examination.

Regardless of the mechanism(s) involved, our data suggest that, in both patient

groups, PS externalisation in the small sub-population of platelets either is, or

becomes, desensitized to uremic toxins / ADP during a 3 hour hemodialysis session.

A number of other studies have also observed decreased platelet activation following

hemodialysis (2, 31, 36, 37). For example, individuals with CKD undergoing

hemodialysis (with either a cellulose acetate or polysulfone membrane) had a lower

percentage of platelets expressing the adhesion molecule, P-Selectin, in response to

Page 16 of 39


ADP (0-1 µM) at the end of dialysis than at the start (2). In contrast, increased platelet

activation following hemodialysis has also been reported (18). The mechanism

through which PS externalisation becomes desensitized is not yet clear, but a better

understanding of these observations may potentially facilitate the development of

novel treatments aimed at manipulating platelet responses in pathophysiological

states.

We have previously reported that the effect of HSA-AGE on platelet PS

externalisation was independent of Caspase 3 activity (45) and demonstrate similar

findings here with UF20 and UF180 from individuals with CKD or CKD and DM. In

nucleated cells, these observations correlate with the efficient propagation and control

of coagulation and thrombosis (49), rather than a signal for apoptotic cell removal

(11). Although recent studies have identified the presence of Caspase activity

(including Caspase 3) in human platelets (34, 47), their precise role in these cells

remains unclear.

In conclusion, we have demonstrated distinct differences both between (i) the

effects of hemodialysis on PS externalisation in a small, sub-population of platelets in

individuals with CKD and those with CKD and DM and (ii) responses of healthy

platelets to ultrafiltrate derived from these two patient groups. The metabolites /

mechanisms involved and their consequences in relation to the increased vascular

damage observed in these individuals requires further investigation.

Page 17 of 39


ACKNOWLEDGEMENTS

This work was supported by scientific grants from the HOSPAL Cardiovascular

Research Programme and the Northern Counties Kidney Research Fund.

Page 18 of 39


REFERENCES

1. Agalou S, Ahmed N, Babaei-Jadidi R, Dawnay A, and Thornalley PJ. Profound

mishandling of protein glycation degradation products in uremia and dialysis. J Am

Soc Nephrol 16: 1471-1485, 2005.

2. Aggarwal A, Kabbani SS, Rimmer JM, Gennari J, Taatjes DJ, Sobel BE, and

Schneider DJ. Biphasic effects of hemodialysis on platelet reactivity in patients with

end-stage renal disease: a potential contributor to cardiovascular risk. Am J Kidney

Dis 40: 315-322, 2002.

3. Cheung AK, Sarnak MJ, Yan G, Dwyer JT, Heyka RJ, Rocco MV, Teehan

BP, and Levey ES. Atherosclerotic cardiovascular disease risks in chronic

hemodialysis patients. Kidney Int 58: 353-362, 2000.

4. Cianciolo G, Stefoni S, Donati G, De Pascalis A, Iannelli S, Manna C, Coli L,

Bertuzzi V, La Manna G, Raimondi C, Boni P, and Stefoni V. Intra- and post-

dialytic platelet activation and PDGF-AB release: cellulose diacetate vs polysulfone

membranes. Nephrol Dial Transplant 16: 1222-1229, 2001.

5. Daleke DL. Regulation of transbilayer plasma membrane phospholipid asymmetry.

J Lipid Res 44: 233-242, 2003.

6. Daleke DL, and Lyles JV. Identification and purification of aminophospholipid

flippases. Biochim Biophys Acta 1486: 108-127, 2000.

Page 19 of 39


7. de Jong K, and Kuypers FA. Sulphydryl modifications alter scramblase activity

in murine sickle cell disease. Brit J Haematol 133: 427-432, 2006.

8. Donati G, Cianciolo G, D’Addio F, Coli L, La Manna G, Feliciangeli G, and

Stefoni S. Platelet activation and PDGF-AB release during dialysis. Int J Artif Organs

25: 1128-1136, 2002.

9. Dou L, Bertrand E, Cerini C, Faure V, Sampol J, Vanholder R, Berland Y,

and Brunet P. The uremic solutes p-cresol and indoxyl sulfate inhibit endothelial

proliferation and wound repair. Kidney Int 65: 442-451, 2004.

10. Fabisiak JP, Tyurin VA, Tyurina YY, Sedlov A, Lazo JS, and Kagan VE.

Nitric oxide dissociates lipid oxidation from apoptosis and phosphatidylserine

externalisation during oxidative stress. Biochemistry 39: 127-138, 2000.

11. Fadok VA, Voelker DR, Campbell PA, Cohen JJ, Bratton DL, and Henson

PM. Exposure of phosphatidylserine on the surface of apoptotic lymphocytes triggers

specific recognition and removal by macrophages. J Immunol 148: 2207-2216, 1992.

12. Floccari F, Aloisi C, Crosci E, Sofi T, Campo S, Tripodo D, Criseo M, Frisina

N, Buemi M. Oxidative stress and uremia. Med Res Rev 25: 473-486, 2005.

Page 20 of 39


13. Foley RN, Wang C, and Collins AJ. Cardiovascular risk profiles and kidney

function stage in the US general population: the NHANES III study. Mayo Clin Proc

80: 1270-1277, 2005.

14. Foster MW, McMahon TJ, and Stamler JS. S-nitrosylation in health and

disease. Trends Mol Med 9: 160-168, 2003.

15. Frasch SC, Henson PM, Kailey JM, Richter DA, Janes MS, Fadok VA, and

Bratton DL. Regulation of phospholipids scramblase activity during apoptosis and

cell activation by protein kinase Cδ. J Biol Chem 275: 23065-23073, 2000.

16. Gaarder A, Jonsen J, Laland S, Hellem A, and Owren PA. Adenosine

diphosphate in red cells as a factor in the adhesiveness of human blood platelets.

Nature 192: 531-532, 1961.

17. Garcia-Calvo M, Petersen EP, Leiting B, Ruel R, Nicholson DW, and

Thornberry NA. Inhibition of human caspases by peptide-based and macromolecular

inhibitors. J Biol Chem 273: 22608-22613, 1998.

18. Gawaz M, and Bogner C. Changes in platelet membrane glycoproteins and

platelet-leucocyte interaction during hemodialysis. Clin Invest 72: 424-429, 1994.

19. Go AS, Chertow GM, Fan D, McCulloch CE, and Hsu CY. Chronic kidney

disease and the risks of death, cardiovascular events, and hospitalization. N Engl J

Med 351: 1296-1305, 2004.

Page 21 of 39


20. Goodkin DA, Bragg-Gresham JL, Koenig KG, Wolfe RA, Akiba T,

Andreucci VE, Saito A, Rayner HC, Kurokawa K, Port FK, Held PJ, and Young

EW. Association of comorbid conditions and mortality in hemodialysis patients in

Europe, Japan, and the United States: the Dialysis Outcomes and Practice Patterns

Study (DOPPS) J Am Soc Nephrol 14: 3270-3277, 2003.

21. Horvat S, and Jakas A. Peptide and amino acid glycation: New insights into the

Maillard reaction. J Pept Sci 10: 119-137, 2004.

22. Kalayoglu MV, and Perkins BN. Chlamydia pneumoniae-infected monocytes

exhibit increased adherence to human aortic endothelial cells. Microbes Infect 3: 963-

969, 2001.

23. Kamp D, Sieberg T, and Haest CW. Inhibition and stimulation of phospholipid

scrambling activity. Consequences for lipid asymmetry, echinocytosis and

microvesiculation of erythrocytes. Biochemistry 40: 9438-946, 2001.

24. Kemp HJ, Parnham A, and Tomson CR. Urea kinetic modeling: a measure of

dialysis adequacy. Ann Clin Biochem 38: 20-27, 2001.

25. Kilhovd BK, Berg TJ, Birkeland KI, Thorsby P, and Hanssen KF. Serum

levels of advanced glycation end products are increased in patients with type 2

diabetes and coronary heart disease. Diabetes Care 22: 1543-1548, 1998.

Page 22 of 39


26. Leysen JE, Gommeren W, Van Gompel P, Wynants J, Janssen PFM, and

Laduran PM. Receptor-binding properties in vitro and in vivo of ritanserin: A very

potent and long acting Serotonin-S2 antagonist. Mol Pharmacol 27: 600-611, 1985.

27. Matthews JN, Altman DG, Campbell MJ, and Royston P. Analysis of serial

measurements in medical research. Brit Med J 330: 230-235, 1990.

28. Mitrogianni Z, Barbouti A, Galaris D, and Siamopoulos KC. Tyrosine

nitration in plasma proteins from patients undergoing hemodialysis. Am j Kidney Dis

44: 286-293, 2004.

29. Monroe DM, Hoffman M, and Roberts HR. Platelets and thrombin generation.

Arterioscler Thromb Vasc Biol 22: 1381-1389, 2002.

30. Pongracz J, Webb P, Want K, Deacon E, Lunn OJ, and Lord JM.

Spontaneous neutrophil apoptosis involves caspase 3-mediated activation of protein

kinase C-delta. J Biol Chem 274: 37329-37334, 1999.

31. Reverter JC, Escolar G, Sanz C, Cases A, Villamor N, Nieuwenhuis HK,

Lopez S, and Ordinas A. Platelet activation during hemodialysis measured through

exposure of P-selectin: Analysis of flow cytometric and ultrastructural techniques. J

Lab Clin Med 124: 79-85, 1994.

32. Rosing J, Bevers EM, Comfurious P, Henker HC, van Dieijen G, Weiss HJ,

and Zwaal RF. Impaired factor X and prothrombin activation associated with

Page 23 of 39


decreased phospholipid exposure in platelets from a patient with a bleeding disorder.

Blood 65: 1557-1561, 1985.

33. Ross R. Atherosclerosis – An inflammatory disease. N Engl J Med 340: 115-126,

1999.

34. Shcherbina A, and Remold-O’Donnell E. Role of caspase in a subset of human

platelet activation responses. Blood 93: 4222-4231, 1999.

35. Shlipak MG, Fried LF, Cushman M, Manolio TA, Peterson D, Stehman-

Breen C, Bleyer A, Newman A, Siscovick D, and Psaty B. Cardiovascular mortality

risk in chronic kidney disease: comparison of traditional and novel risk factors. JAMA

293: 1737-1745, 2005.

36. Sloand JA, and Sloand EM. Studies on platelet membrane glycoproteins and

platelet function during hemodialysis. J Am Soc Nephrol 8: 799-803, 1997.

37. Sreedhara R, Itagaki I, Lynn B, and Hakim RM. Defective platelet aggregation

in uremia is transiently worsened by hemodialysis. Am J Kidney Dis 25: 555-563,

1995.

38. Stoyanovsky DA, Tyurina YY, Tyurina VA, Anand D, Mandavia DN, Gius D,

Ivanova J, Pitt B, Billar TR, and Kagan VE. Thioredoxin and lipoic acid catalyze

the denitrosation of low molecular weight and protein S-nitrothiols. J Am Chem Soc

127: 15815-15823, 2005.

Page 24 of 39


39. Thomas MC, Baynes JW, Thorpe SR, and Cooper ME. The role of AGEs and

AGE inhibitors in diabetic cardiovascular disease. Curr Drug Targets 6: 453-474,

2005.

40. Toullec D, Pianetti P, Coste H, Bellevergue P, Grand-Perrett T, Ajakane M,

Baudet V, Boissin P, Boursier E, Loriolle F, Duhamel L, Charon D, and

Kirilovsky J. The bisindolylmaleimide GF 109203X is a potent and selective

inhibitor of protein kinase C. J Biol Chem 266: 15771-15781, 1991.

41. Tyurina YY, Basova LV, Konduru NV, Tyurin VA, Potapovich AL, Cai P,

Bayir H, Stoyanovsky D, Pitt BR, Shvedova AA, Fadeel B, and Kagan VE.

Nitrosative stress inhibits the aminophospholipid translocase resulting in

phosphatidylserine externalization and macrophage engulfment. Implications for the

resolution of inflammation. J Biol Chem 282:8498-8509, 2007.

42. Vanholder R, De Smet R, Glorieux G, Argiles A, Baurmeister U, Brunet P,

Clark W, Cohen G, De Deyn PP, Deppisch R, Descamps-Latscha B, Henle T,

Jorres A, Lemke HD, Massy ZA, Passlick-Deetjen J, Rodriguez M, Stegmayr B,

Stenvinkel P, Tetta C, Wanner C, and Zidek W. Review on uremic toxins:

Classification, concentration and interindividual variability. Kidney Int 63: 1934-

1943, 2003.

43. Vanholder R, De Smet R, and Lameire N. Protein-bound uremic solutes: The

forgotten toxins. Kidney Int 59 (Suppl 78): S266-S270, 2001.

Page 25 of 39


44. Walter R, Mischak H, and Haller H. Haemodialysis, atherosclerosis and

inflammation – Identifying molecular mechanisms of chronic vascular disease in

ESRD patients. Nephrol Dial Transplant 17: 24-29, 2002.

45. Wang Y, Beck W, Deppisch R, Marshall SM, Hoenich NA, and Thompson

MG. Advanced glycation end products elicit externalization of phosphatidylserine in

a sub-population of platelets via 5-HT2A/2C receptors. Am J Physiol Cell Physiol in

press.

46. Wilkinson SE, Parker PJ, and Nixon JS. Isoenzyme specificity of

bisindolylmaleimides, selective inhibitors of protein kinase C. Biochem J 294: 335-

337, 1993.

47. Wolf BB, Goldstein JC, Stenmicke HR, Beere H, Amarante-Mendes GP,

Salvesen GS, and Green DR. Calpain functions in a caspase-independent manner to

promote apoptosis-like events during platelet activation. Blood 94: 1683-1692, 1999.

48. Wolfs JLN, Comfurious P, Rasmussen JT, Keuren JFW, Lindhout T, Zwaal

RFA, and Bevers EM. Activated scramblase and inhibited aminophospholipid

translocase cause phosphatidylserine exposure in a distinct platelet fraction. Cell Mol

Life Sci 62: 1514-1525, 2005.

49. Zwaal RF, and Schroit AJ. Pathophysiologic implications of membrane

phospholipid asymmetry in blood cells. Blood 89: 1121-1132, 1997.

Page 26 of 39


FIGURE LEGENDS

Fig. 1. The effect of a single, low-flux hemodialysis treatment on PS externalization

in a sub-population of human platelets in individuals with CKD.

PRP was prepared from individuals with CKD either pre-dialysis or 1, 2 and 3

hours after the commencement of dialysis. PS externalisation was then assessed using

Annexin V-FITC and CD61-PerCP with subsequent quantification by FACScan. For

comparison purposes, a healthy control is also shown and data are presented both as

(a) a histogram and (b) a dot-plot.


in a sub-population of human platelets in individuals with CKD and DM.

PRP was prepared from individuals with CKD and DM either pre-dialysis or 1, 2

and 3 hours after the commencement of dialysis. PS externalisation was then assessed

using Annexin V-FITC and CD61-PerCP with subsequent quantification by

FACScan. For comparison purposes, a healthy control is also shown and data are

presented both as (a) a histogram and (b) a dot-plot.


in a sub-population of human platelets in individuals with either CKD or CKD and

DM.

PRP was prepared from individuals with (a) CKD or (b) CKD and DM either pre-

dialysis or 1, 2 and 3 hours after the commencement of dialysis. PS externalisation

was then assessed using Annexin V-FITC and CD61-PerCP with subsequent

quantification by FACScan. For comparison purposes, a healthy control is also

Page 27 of 39


shown. Data are expressed as total fluorescent binding sites and are shown as Mean ±

s.e. of three independent experiments with statistical analysis by Student’s t-test.

Fig.4. The effect of three low-flux hemodialysis treatments during a seven day period

on PS externalisation in a sub-population of human platelets in individuals with either

CKD or CKD and DM

PRP was prepared from pre- (■) and post-dialysis (□) blood samples withdrawn on

Monday, Wednesday and Friday from patients with either (a) CKD or (b) CKD and

DM. PS externalisation was then assessed using Annexin V-FITC and CD61-PerCP

with subsequent quantification by FACScan. Data are expressed as total fluorescent

binding sites and are shown as Mean ± s.e. of three independent experiments with

statistical analysis by Student’s t-test.

Fig. 5 The effect of ultrafiltrate from individuals with CKD or CKD and DM

undergoing low-flux hemodialysis on PS externalisation in a sub-population of human

platelets

PRP from healthy controls was incubated for 10 minutes with increasing

concentrations (2.5 – 20 µl)) of either unused hemodialysis fluid (HF), hemodialysis

fluid used to rinse the dialyser (RS) or ultrafiltrate taken at 20 minutes (UF20) or 180

minutes (UF180) from individuals with either CKD or CKD and DM. PS

externalisation was determined using Annexin V-FITC and CD61-PerCP with

subsequent quantification by FACScan. Data are expressed as total fluorescent

binding sites and statistical analysis was determined as AUC (27). The effects of

ultrafiltrate vs HF was significant in all instances (p<0.001) and the response elicited

Page 28 of 39


by both UF20 (p<0.001) and UF180 (p<0.01) from individuals with CKD and DM was

significantly greater than that seen in individuals with CKD alone.

Fig. 6. The effect of ultrafiltrate from individuals with CKD or CKD and DM on PS

externalisation in a sub-population of human platelets from both healthy controls and

individuals with either CKD or CKD and DM after 180 minutes hemodialysis.

PRP from healthy controls (HP) and individuals with either CKD (a) or CKD and

DM (b) following 180 minutes hemodialysis (UP180) was incubated for 10 minutes

with 10 µl of ultrafiltrate taken at 20 minutes (UF20) or 180 minutes (UF180) from

individuals with either CKD (a) or CKD and DM (b). PS externalisation was

determined using Annexin V-FITC and CD61-PerCP with subsequent quantification

by FACScan. Data are expressed as total fluorescent binding sites and are shown as

Mean ± s.e. of 3 independent experiments with statistical analysis by Student’s t-test.

Fig.7. The effect of ADP on PS externalisation in a sub-population of human platelets

from both healthy controls and individuals with either CKD or CKD and DM after 20

or 180 minutes hemodialysis.

PRP from either healthy controls or that obtained from individuals with either CKD

(a) or CKD and DM (b) following 20 or 180 minutes hemodialysis were incubated

with (■) or without (□) 100 nM ADP for 1 minute. PS externalisation was determined

using Annexin V-FITC and CD61-PerCP with subsequent quantification by

FACScan. Data are expressed as total fluorescent binding sites and are shown as

Mean ± s.e. of 3 independent experiments with statistical analysis by Student’s t-test.

Page 29 of 39


Fig. 8. Effect of the PKCδ inhibitor, Rottlerin, on PS externalisation in a sub-

population of human platelets in response to ultrafiltrate taken from individuals with

either CKD or CKD and DM after 20 or 180 minutes hemodialysis.

PRP from healthy controls was pre-incubated with or without 10 µM PKCδ

inhibitor, rottlerin (Rott), for 5 minutes at 37ºC. Ultrafiltrate taken from individuals

with (a) CKD or (b) CKD and DM after either 20 (UF20) or 180 (UF180) minutes

hemodialysis was then added and the incubation continued for a further 10 minutes.

PS externalisation was determined using Annexin V-FITC and CD61-PerCP with

subsequent quantification by FACScan. Data are expressed as total fluorescent

binding sites and are shown as Mean ± s.e. of 3 independent experiments with


Fig. 9. Effect of the 5-HT2A/2C receptor antagonist, Ritanserin, on PS externalisation in

a sub-population of human platelets in response to ultrafiltrate taken from individuals

with either CKD or CKD and DM after 20 or 180 minutes hemodialysis.

PRP from healthy controls was pre-incubated with or without 1 µM 5-HT2A/2C

receptor antagonist, Ritanserin (Rit), for 30 minutes at 37ºC. Ultrafiltrate taken from

individuals with (a) CKD or (b) CKD and DM after either 20 (UF20) or 180 (UF180)

minutes hemodialysis was then added and the incubation continued for a further 10

minutes. PS externalisation was determined using Annexin V-FITC and CD61-PerCP

with subsequent quantification by FACScan. Data are expressed as total fluorescent

binding sites and are shown as Mean ± s.e. of 3 independent experiments with


Page 30 of 39

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Page 31 of 39

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Page 32 of 39

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Page 33 of 39

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Page 35 of 39

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Page 36 of 39

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Page 37 of 39

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Page 38 of 39

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Page 39 of 39

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