Kidney International, Vol. 57 (2000), pp. 1905–1914

Abnormal platelet cytoskeletal assembly in hemodialyzedpatients results in deficient tyrosine phosphorylation signaling

MARIBEL DIAZ-RICART, EVA ESTEBANELL, ALEIX CASES, JORDI CALLS, JOSE LOPEZ-PEDRET,MARTA CARRETERO, RICARDO CASTILLO, ANTONIO ORDINAS, and GINES ESCOLAR

Servicios de Hemoterapia-Hemostasia and Nefrologıa, Hospital Clınic, Fundacio Clınic (IDIBAPS), Universitat de Barcelona,Barcelona, Spain

Abnormal platelet cytoskeletal assembly in hemodialyzed pa- Extensive literature exists describing the bleeding dis-tients results in deficient tyrosine phosphorylation signaling. order suffered by uremic patients. This tendency to bleed

Background. Uremic patients have a bleeding tendency as- observed in uremia is multifactorial: It has been associ-sociated with a platelet dysfunction. We evaluated the impactated with a platelet dysfunction, as there are no majorof a repeated hemodialysis procedure on primary hemostasisalterations in the coagulation system, but its origin re-by analyzing different aspects of platelet activation in uremic

patients. mains obscure [1–6]. Platelets from uremic patients pres-Methods. Studies were performed in (1) eight patients with ent an impaired adhesion [7], which is manifested by

end-stage renal disease before the hemodialysis program was defective spreading on subendothelial surfaces in experi-initiated and after initiating hemodialysis treatment, and in (2)ments performed with flowing blood [8]. However, ure-eight patients on maintenance hemodialysis who were trans-mic patients have normal or even increased plasma levelsferred to continuous ambulatory peritoneal dialysis. Studies in-

cluded routine platelet aggregations and evaluation of platelet– of von Willebrand factor (vWF) [9], the adhesive proteinsubendothelium interactions under flow conditions. Contractile with a major role in platelet adhesion [10]. The presenceproteins and tyrosine phosphorylation associated with the cy-

and binding functions of platelet membrane receptorstoskeleton were analyzed, before and after thrombin activation(GPIb and GPIIb-IIIa) in these patients have been foundof platelets, by electrophoresis after Triton X-100 extraction.

Results. No changes in the clinical parameters analyzed were to be generally preserved [11–13], although some minorobserved among the different study groups. Aggregation and abnormalities have been reported in patients with end-platelet adhesion only improved when patients were shifted stage renal disease [14, 15].from hemodialysis to continuous ambulatory peritoneal dial-

Recent studies have suggested a cytoskeletal abnor-ysis (P , 0.05 for both percentage of surface covered by plate-mality of uremic platelets to fully spread under staticlets and aggregate formation). The association of cytoskeletal

proteins in platelets from patients under hemodialysis treat- conditions [16]. This limitation of adhesive capabilitiesment was statistically decreased with respect to the correspond- was found to be associated with an impaired organizationing values in platelets from patients not subjected to dialysis of contractile proteins into the cytoskeleton of suspen-(P , 0.01 for actin). However, after two months on peritoneal

sion-activated platelets. The removal of uremic toxinsdialysis, these values increased to almost control values (P ,by dialysis only partially corrects the abnormal platelet0.001 for actin, vs. hemodialysis). Similarly, translocation of

tyrosine-phosphorylated proteins to the cytoskeletal fraction function observed in uremic patients [3, 6, 17]. However,was impaired in platelets from hemodialyzed patients, and it hemodialysis (HD) procedures are known to producerecovered partially after the patients transferred to continuous profound activation, which adversely affects plateletambulatory peritoneal dialysis.

function [18–23]. Repeated activation of platelets duringConclusions. Our present data support the concept that re-maintenance HD may result in consecutive cycles ofpeated platelet stress during hemodialysis has a deleterious

effect on the organization of platelet cytoskeleton, which seems polymerization and depolymerization of the cytoskeletalto impair the translocation of signal transduction proteins proteins, leading to the development of a certain degreewithin platelets compromising the platelet function in uremia. of platelet refractoriness. On the other hand, peritoneal

dialysis is more effective in improving platelet functionand bleeding times [24, 25].Key words: hemodialysis, peritoneal dialysis, cytoskeleton, phospho-

tyrosine proteins, signal transduction, uremia. Two main components of the platelet cytoskeleton arecurrently considered: the cytoplasmic portion composedReceived for publication February 16, 1999of actin filaments (F-actin) and the membrane cytoskele-and in revised form October 8, 1999

Accepted for publication December 15, 1999 ton consisting of shorter actin filaments, actin-bindingprotein (ABP), spectrin, vinculin, and other noncharac- 2000 by the International Society of Nephrology

1905

Dıaz-Ricart et al: Signal transduction defect in uremic platelets1906

terized proteins. In resting platelets, the membrane cy- including the lack of vascular access for HD (5), ischemicheart disease (2), and personal decision of the patienttoskeleton acts to stabilize the plasma membrane and

regulate the platelet shape. While some signaling mole- (1). Patients performed HD for four hours three time perweek, and hollow fiber dialyzers with cellulose acetatecules in resting platelets have been found to be associated

with the membrane skeleton, tightly related to the extra- membranes (CA 140; Baxter, Deerfield, IL, USA) wereused. None of the patients complained of severe bleedingcellular receptors, after activation of platelets they be-

come associated with the cytoplasmic cytoskeleton. An or were hypertensive at the time that they were enrolledin the study. The KT/V in all hemodialyzed patients wasassociation between membrane and cytoplasmic cyto-

skeletons results in platelet motion and internal contrac- higher than 1.1. No patient had received blood relatedproducts for two months, and none had taken aspirin ortion necessary for release. Recent investigations have

demonstrated that the cytoskeleton plays a role in localiz- other drugs that affect platelet function for at least twoweeks prior to the study.ing signaling molecules, facilitating their function [26].

Therefore, platelet cytoskeleton may act as a connectionExperimental designbetween the extracellular receptors and the cytoplasmic

signaling proteins. Derangement of the cytoskeletal or- Studies were carried out on (1) eight patients withESRD before the HD program was initiated (pre-HD)ganization thus may lead to impaired signal transduction

processes. and after undergoing HD (post-HD), and (2) eight pa-tients while on maintenance HD and at least two monthsWe have evaluated the impact of standard HD on the

platelet dysfunction observed in uremia. Studies were after starting CAPD treatment. Blood samples fromhealthy volunteers were also obtained for control experi-developed for two groups of uremic patients: (1) eight

patients with end-stage renal disease (ESRD) before ments. Studies included the evaluation of certain clinicalparameters, including bleeding times and platelet aggre-initiating renal replacement therapy and two months

after starting HD treatment, and (2) eight uremic pa- gation using turbidimetric techniques, and the evaluationof platelet–subendothelium interactions under flow con-tients on maintenance HD who were transferred to peri-

toneal dialysis for clinical reasons. Studies included ditions, using an annular perfusion chamber. The associ-ation of contractile and phosphotyrosine proteins withbleeding times, platelet aggregations, and evaluation of

platelet–subendothelium interactions under flow condi- the platelet cytoskeleton after thrombin stimulation wasalso investigated by sodium dodecyl sulfate-polyacryl-tions. Changes induced in the association of contractile

and tyrosine-phosphorylated proteins with the cytoskele- amide gel electrophoresis (SDS-PAGE) in both groupsof patients under the different treatments.ton, before and after thrombin stimulation, were evalu-

ated by electrophoretic techniques. In each set of experiments, studies were performedusing blood from two uremic patients and from a controldonor. This design was applied in studies performed while

METHODSpatients were in pre-treatment and post-treatment, con-

Patients sidering that blood samples were always obtained fromdifferent donors. Therefore, the number for each groupOur study, approved by the Human Experimental

Committee of the Hospital Clinic, was carried out ac- of patients was 8, and the number for the control groupwas 16.cording to the principles of the Declaration of Helsinki.

Informed consent was obtained from all the participants.Blood samplingEight patients with end-stage renal disease (clearance

creatinine , 10 mL/min) were evaluated before starting Blood samples were obtained from uremic patients(1) before initiating HD and after at least two monthsHD and after two months on maintenance HD. There

were five men and three women from the age of 38 to of starting HD and (2) while under HD and after twomonths of being transferred to CAPD. In those patients62 years. The causes of renal failure were nephrosclerosis

(3), chronic glomerulonephritis (3), diabetic nephropa- under HD treatment, blood samples were always ob-tained just before the HD session. Aliquots of citratedthy (1), and unknown (1). Eight uremic patients on main-

tenance HD (6 women and 2 men, age range of 42 to blood samples were reserved for perfusion studies. Plate-let-rich plasma (PRP) was obtained by centrifugation of75 years, time on HD from 6 to 171 months) were in-

cluded in this study. The causes of renal failure were citrate-anticoagulated blood for 20 minutes at 100 3 g.Aliquots of PRP were used in aggregation studies.polycystic kidney disease (2), diabetic nephropathy (2),

nephroangiosclerosis (1), chronic interstitial nephropa- Platelets were also isolated from PRP and werewashed twice with equal volumes of CCD (93 mmol/Lthy (1), hemolytic uremic syndrome (1), and unknown

(1). Patients included in the study were chosen on the sodium citrate, 7 mmol/L citric acid, and 140 mmol/Ldextrose), pH 6.5, containing 5 mmol/L adenosine andbasis that they were moved from HD to continuous am-

bulatory peritoneal dialysis (CAPD) for various reasons, 3 mmol/L theophylline [27]. The final pellet was resus-

Dıaz-Ricart et al: Signal transduction defect in uremic platelets 1907

pended in Hanks’ balanced salt solution (HBSS) and remaining undisturbed at 378C and one being subjectedto stimulation with 0.1 U/mL thrombin. Samples wereincubated for 20 minutes at 378C. Aliquots of washed

uremic platelet suspensions were used to electrophoreti- mixed by gentle inversion every 30 seconds. After 90seconds, all of the samples were treated with an equalcally analyze any changes in the association of contractile

proteins to the cytoskeleton before and after activation volume of ice-cold lysis buffer (final pH 7.4) containing2% Triton X-100, 100 mmol/L Tris-HCl, 10 mmol/L eth-with 0.1 U/mL of thrombin.ylene glycol bis(b-aminoethylether)-N,N,N9,N9-tetraace-

Aggregation studies tic acid (EGTA), 4 mmol/L ethylenediaminetetraaceticacid (EDTA), 2 mmol/L phenylmethylsulfonyl fluoridePlatelet aggregation studies were carried out in a Hi-

tachi-Aggrecorder aggregometer. Samples of PRP from (PMSF), 1 mmol/L benzamidine, 2 mg/mL leupeptin, 2mg/mL pepstatin, and 2 mmol/L sodium orthovanadate.the uremic patients included in the study were placed in

6 mm wide siliconized cuvettes. Platelet counts were The low-speed Triton-insoluble residues, correspondingto the polymerized cytoskeletal fraction, were isolatednormalized to the same value (2 3 105 platelets/mL).

The minimum and maximum amplitudes of the recorder by sedimentation at 12,000 3 g for five minutes at 48Cin a microfuge. After collection of the supernatants, resi-were adjusted with PRP (0% transmission) and platelet

poor plasma (PPP) (100% transmission), respectively. dues were washed twice with washing buffer, withoutTriton X-100, at 48C, solubilized with a SDS-containingArachidonic acid (1.2 mmol/L), adenosine diphosphate

(ADP; 4 mmol/L), collagen (2.5 mg/mL), epinephrine buffer, and heated at 1008C for five minutes. Plateletmembrane cytoskeletons were sedimented by centrifuga-(10 mmol/L), and ristocetin (1 mg/mL) were used as

inductors, under stirring. Results were expressed as per- tion of the supernatants, recovered at low speed, at100,000 3 g for three hours. The corresponding pelletscentages of maximum aggregation obtained after five

minutes of stimulation [28, 29]. (high-speed residues) were solubilized as mentioned be-fore. Samples were frozen at 2408C until electrophoreti-

Perfusion studies and morphometric evaluation cal evaluation was performed.Perfusion experiments were performed in annular

Analysis of cytoskeletal proteinschambers, as previously described [30, 31]. De-endothe-lialized rabbit aorta segments were exposed to citrated Triton-insoluble fractions from an equal number of

thrombin nonactivated and activated platelets were ob-whole blood at a shear rate of 800 s21 at 378C. After 10minutes of perfusion, segments were fixed, dehydrated tained from control donors and from the uremic patients

enrolled in the study. Cytoskeletal proteins present inwith alcohols, embedded in JB-4, thin sectioned for lightmicroscopy, and stained with toluidine blue. both the low- and high-speed fractions were separated

by 7 to 12% SDS-PAGE [35]. To evaluate the contractilePlatelets interacting with subendothelium were evalu-ated according to the morphometric criteria described by proteins associated with the cytoskeleton, gels were

stained with Coomassie brilliant blue R250 and densito-Baumgartner and Muggli [30]. A semiautomated methodwas used to divide platelets into different classes of inter- metrically quantitated as previously described [16]. Basi-

cally, stained protein bands were densitometrically ana-action [32]. Platelets or groups of platelets were classifiedas follows: contact (C), platelets that were attached but lyzed using digital-video technology provided by a

computerized image analyzer running specific softwarenot spread on the subendothelium; adhesion (A), plate-lets that were spread on subendothelium or form layers (SigmaGel, Jandel GmbH, Erkrath, Germany). After

selection of the bands on the monitor screen, the soft-of less than 5 mm in height; and thrombi (T), plateletaggregates of 5 mm or more in height. All of these basic ware automatically analyzed the color density of each

protein band and integrated areas beneath densitometricparameters were expressed as a percentage of the totallength of the vessel screened. The total covered surface peaks. Values of protein peak areas in the lanes con-

taining Triton-insoluble residues from nonactivated(CS) was obtained by adding the previous basic parame-ters (C 1 A 1 T). platelets were considered as 100%. The association of

certain protein with the thrombin-activated cytoskeletonObtaining cytoskeletal proteins was expressed as the percentage of increase over the

amount of the same protein found in the respective lanePlatelet cytoskeletons were obtained according to theprocedure described by Jennings et al [33] with minor corresponding to nonactivated platelets.modifications [34]. Samples of resuspended platelets were

Analysis of tyrosine-phosphorylated proteinsadjusted to 1.2 3 106 platelets/mL. Before activation ofplatelets, aliquots of platelet suspensions were treated Phosphotyrosine proteins associated with both the

low- and high-speed cytoskeletal fractions were analyzedto quantitate the protein content, which was always com-parable among the samples obtained (around 2 mg/mL). on 8% SDS-polyacrylamide gels. Proteins present in the

gels were transferred to nitrocellulose membranes (Bio-Platelet suspensions were divided into two aliquots, one

Dıaz-Ricart et al: Signal transduction defect in uremic platelets1908

Table 1. Changes in blood cell counts and laboratory parameters

Pre-HD Post-HD HD CAPD

Hemoglobin g/L 84.265.6 87.364.1 88.7611.5 91.1613.6Hematocrit % 25.561.6 26.860.9 28.665.3 29.465.4Platelet count 3 103/mL 193.8620 201624 309.7654 323.1669BUN mg/dL 108.5610 93.569.5 78.1614.6 77.2614.6Creatinine mg/dL 9.061 8.760.9 10.662.2 11.562.1

Hemoglobin, hematocrit, platelet count, BUN and creatinine values were measured in (1) patients with end-stage renal disease before (pre-HD) and after twomonths of initiating hemodialysis (post-HD), and in (2) patients undergoing hemodialysis (HD) and after transferring to continuous ambulatory peritoneal dialysis(CAPD). No significant changes were observed in the parameters analyzed. Data are expressed as mean 6 SEM. N 5 8 for both groups.

Rad, Hercules, CA, USA). After blocking nonspecific sponse to the agonists used, when compared with controlvalues (P , 0.05; Table 2).binding, Western blots were probed with a horseradish

After five minutes of PRP stimulation with arachi-peroxidase-antiphosphotyrosine recombinant antibodydonic acid (1.2 mmol/L), collagen (2.5 mg/mL), ADP(Transduction Laboratories, Lexington, KY, USA). The(4 mmol/L), and ristocetin (1 mg/mL), percentages ofexcess of antibody was removed by extensive washing,maximal aggregation were 68.5 6 6.5%, 76 6 3.5%, 69.1 6and blots were developed by enhanced chemiluminis-5.3%, and 72.9 6 4.0%, respectively (mean 6 SEM, N 5cence (ECL) method (Amersham Pharmacia Biotek, Es-8) in patients with pre-HD ESRD. In the same group ofsex, UK).patients, aggregation patterns did not significantly differ

Statistics after two months of starting HD. Aggregation valueswere 61.8 6 3.0%, 78.8 6 3.6%, 64.9 6 4.8%, and 66.4 6Data are expressed as mean 6 SEM. Student’s t-test2.5%, respectively.for paired data was used for statistical comparisons be-

Aggregation responses of platelets from the secondtween data obtained pretreatment and posttreatment ingroup of uremic patients, while on HD, to arachidoniceach group. Student’s t-test was used to compare dataacid, collagen, ADP, and ristocetin at the concentrationsfrom patients versus controls. A P level , 0.05 was con-indicated before were 55.8 6 6.8%, 60.2 6 8.3%, 56.2 6sidered statistically significant.7.8%, and 65.7 6 8.9%, respectively (mean 6 SEM, N 58). Under the same experimental conditions, all of the

RESULTS aggregation patterns statistically improved (P , 0.05)when patients were moved to CAPD, changing to 78.9 6Clinical parameters6.9%, 81.2 6 7.5%, 79.1 6 7.6%, and 89.2 6 7.1%,Table 1 shows changes in blood cell counts and labora-respectively (Table 2).tory parameters. Hematocrit and hemoglobin levels of

the uremic patients enrolled in the study were measured Perfusion studiesin pre-HD, post-HD, HD, and CAPD situations of the

Blood samples were recirculated for 10 minutesstudy. These values showed practically the same levels through the annular chamber at 800 s21 of shear rate.during the different treatments in both groups of pa- Values of surface covered by platelets, expressed as per-tients. Platelet counts and coagulation tests (prothrom- centages (%SC) obtained with control blood samplesbin time, partial thromboplastin time, and fibrinogen were 34.5 6 3.4% with an aggregate formation of 20.4 6levels) were within the normal range in both groups 2.5% (mean 6 SEM, N 5 16).under the different periods of the study. Plasma levels Studies of uremic patients before and after two monthsof nitrogen-retention products and creatinine levels did of initiating HD treatment. The surface covered by plate-not change after initiating HD (post-HD) or while on lets obtained after perfusing denuded vascular segmentsHD, and they were similar or increased slightly after with blood samples from patients with ESRD was 24.1 6starting CAPD. No significant differences were observed 1.9% (mean 6 SEM, N 5 8), with an aggregate formationwhen comparing bleeding times among the different of 15.7 6 0.5%. After two months of initiating HD treat-treatments in both groups of patients. ment, perfusion experiments were carried out with blood

samples from the same patients. Results of %SC andAggregation studies percentage aggregate formation were slightly inferior

Aggregation results for control platelets reached val- (21.7 6 2% and 13.4 6 2.3%, respectively), but did notues of approximately 100% of aggregation five minutes statistically vary from pre-HD values. Values of surfaceafter the addition of the agonists at the concentrations coverage observed in both situations were statisticallyemployed. Platelet suspensions from all the patients in- reduced with respect to those observed in control experi-

ments (P , 0.05; Fig. 1A).cluded in the study showed a reduced aggregating re-

Dıaz-Ricart et al: Signal transduction defect in uremic platelets 1909

Table 2. Bleeding time and platelet aggregation studies

Pre-HD Post-HD HD CAPD Normal range

Bleeding time min 8.262.3 7.062.8 9.363.2 8.363.8 3–8AA 1.2 mmol/L 68.566.5 61.863.0 55.866.8 78.966.9a 75–100Col 2.5 lg/mL 76.063.5 78.863.6 60.268.3 81.267.5a 68–100ADP 4 lmol/L 69.165.3 64.964.8 56.267.8 79.167.6a 75–100Risto 1 mg/mL 72.964.0 66.462.5 65.768.9 89.267.1a 70–100

Bleeding time (min) and aggregation results (% of maximal aggregation) after 5 minutes of stimulation of platelet-rich plasma (PRP) obtained from two groupsof uremic patients: (1) eight patients with end-stage renal disease before (pre-HD) and after two months of initiating hemodialysis (post-HD), and (2) eight patientsundergoing hemodialysis (HD) and after transferring to continuous ambulatory peritoneal dialysis (CAPD). Aggregation studies were performed with the agonistsarachidonic acid (AA), collagen (Col), adenosine diphosphate (ADP), and ristocetin (Risto). No differences were observed among pre-HD and post-HD results.Transfer to CAPD treatment resulted in a statistically significant increase in the aggregation profiles (aP , 0.05). Data are expressed as mean 6 SEM, N 5 8 forboth groups.

Studies of HD patients who were transferred to CAPD.The %SC using blood from the uremic patients whileon HD was 18.6 6 4.5% (mean 6 SEM, N 5 8), whichwas significantly below that obtained in control experi-ments (P , 0.05). After CAPD began, platelets fromthe same uremic patients displayed a higher surface cov-erage, increasing statistically to 24.6 6 3.3% (P , 0.05).This increase in the surface coverage paralleled a statisti-cally significant improvement of the aggregate forma-tion: from 12.3 6 4.4% when patients were on HD, to17.6 6 5% after two months of CAPD treatment (P ,0.05; Fig. 1B).

Changes in the distribution of plateletcytoskeletal proteins

Profiles corresponding to the low-speed Triton X-100–insoluble residues of resting platelets and platelets acti-vated with thrombin from control donors and from bothgroups of uremic patients included in the study whounderwent the different treatments (Fig. 2) were ana-lyzed densitometrically. The degree of association of thedifferent proteins with the cytoskeleton was expressedas the percentage of incorporation of each protein inthrombin stimulated versus resting platelet samples.

Profiles corresponding to resting platelets from thecontrol donors and from all of the uremic patients in-cluded in the study showed no significant qualitativedifferences for the presence of the major cytoskeletalproteins recovered in the low-speed cytoskeletal fraction(Fig. 2, lanes 1, 3, and 5). However, proteins in profilesfrom uremic patients on HD treatment densitometricallyappeared to be decreased (Fig. 2, lane 3).

Fig. 1. Platelet deposition after 10 minutes of perfusion of denudedThrombin activation of control platelets resulted inrabbit aorta. Perfusates consisted of blood samples from (A) eight

patients with end-stage renal disease before (pre-HD) and after (post- an augmented incorporation of contractile proteins (Fig.HD) two months of initiating HD treatment, and (B) eight patients 2, lane 2). Incorporations of ABP, myosin, a-actinin,while on HD and after being transferred to CAPD. Bar diagrams

and actin to the low-speed cytoskeletal fraction afterrepresent morphometric parameters obtained in the different perfusionexperiments. Bars represent the percentage of the vessel surface covered thrombin stimulation were 140 6 5%, 50 6 11.4%, 61.4 6with platelets. The open portion of the bar corresponds to the surface 7.9%, and 120 6 4.6%, respectively (Fig. 3).covered by adhesive platelets (adhesion), and the dashed inserts indicate

Studies of uremic patients before and after two monthsthe surface covered with groups of platelets forming aggregates of morethan 5 mm in height (thrombi). The results are expressed as percentages of initiating HD treatment. Proteins recovered at theof the total surface of the vessel screened. *P , 0.05 vs. control values low-speed cytoskeletal fraction corresponding to throm-and aP , 0.05 CAPD vs. HD. N 5 16 for control experiments andN 5 8 for each group of uremic patients. bin-activated platelets from eight uremic patients with

Dıaz-Ricart et al: Signal transduction defect in uremic platelets1910

Fig. 2. Coomassie brilliant blue-stained SDSpolyacrylamide gels showing protein profilescorresponding to the low-speed cytoskeletalfraction. Platelet suspensions were obtainedfrom control donors (lanes 1 and 2); uremicpatients undergoing HD treatment (lanes 3and 4) and after being moved to CAPD treat-ment (lanes 5 and 6). Cytoskeletal assemblywas evaluated before (lanes 1, 3, and 5) andafter activation with 0.1 U/mL of thrombinfor 90 seconds (lanes 2, 4, and 6). Arrowheadsindicate defective association of contractileproteins after thrombin activation of plateletsfrom patients under HD. Profiles shown corre-spond to one experiment and are representa-tive of the eight different experiments per-formed.

ESRD, pre-HD, were also analyzed. After activation corporation of 110 6 6% vs. 42 6 2% for ABP (P ,0.01), 45.8 6 8.0% vs. 35.0 6 6.0% for myosin, 53.6 6of platelets with 0.1 U/mL of thrombin, there was an

incorporation of contractile proteins of 87.5 6 4.3%, 3.2% vs. 18.0 6 4% for a-actinin (P , 0.01), and 96 61.2% vs. 36 6 3.2% for actin (P , 0.01)]. The association45.0 6 5.3%, 45.7 6 7%, and 74.8 6 4.2% for ABP,

myosin, a-actinin, and actin, respectively. of myosin after thrombin activation was not significantlydifferent in the two situations.In studies performed post-HD, blood samples from the

same patients were collected, and the same experimental Figure 4 shows SDS-PAGE profiles corresponding tothe high-speed cytoskeletal fraction from resting andconditions were applied. When platelets were activated

with thrombin, incorporation of contractile proteins to thrombin-activated platelets. Profiles correspond to anequal number of platelets from the control donors andthe low-speed cytoskeletal fraction was significantly de-

creased (P , 0.01 vs. pre-HD for all the proteins studied). the uremic patients included in the study. No significantdifferences were observed among the profiles from rest-Results in terms of protein incorporation were 60.6 6

6.3%, 35.9 6 5.6%, 25.6 6 5.0%, and 34.4 6 8% for ing platelets obtained from control donors and uremicpatients after initiating CAPD (Fig. 4, lanes 1 and 5).ABP, myosin, a-actinin and actin, respectively (Fig. 3A).

Studies of patients on HD who transferred to CAPD However, lane 3 in Figure 4 shows that the amountof proteins associated with the high-speed cytoskeletaltreatment. Amounts of proteins found in cytoskeletons

from resting platelets obtained from the patients under- fraction in platelets from patients undergoing HD wasincreased, correlating to a decrease in the density ofgoing CAPD were slightly greater to those observed in

platelets from the same patients while they were on HD. those proteins associated with the low-speed cytoskeletalfraction (Fig. 2, lane 3).After thrombin stimulation, an incorporation of contrac-

tile proteins to the cytoskeleton of platelets from the After thrombin activation, the contractile proteins as-sociated with this fraction decreased in control plateletssame patients was observed during both the HD and

CAPD treatments, although the rate of incorporation (46 6 2.8% decrease of actin). This was actually expectedsince polymerized contractile proteins were recoveredwas much higher in platelets obtained while patients

were on CAPD (Fig. 2). in the low-speed fraction (Fig. 4, lane 2 vs. lane 1), dueto the cytoskeleton assembly. Nearly similar results wereDensitometric evaluation (Fig. 3B) of protein bands

corresponding to Triton-insoluble residues of thrombin- observed in profiles corresponding to the platelets frompatients who were pre-HD and those after CAPD treat-activated platelets from patients under CAPD showed

a statistically significant increase in the association of ment (percentage of decrease of 62 6 0.9% and 50 61.8%, respectively; Fig. 4, lane 6 vs. lane 5).ABP, a-actinin, and actin versus those obtained when

patients were undergoing HD treatment [%protein in- However, when analyzing the protein profiles corre-

Dıaz-Ricart et al: Signal transduction defect in uremic platelets 1911

after being transferred to CAPD. Studies were per-formed in order to evaluate changes in the associationof these proteins with both the low- and high-speed cy-toskeletal fractions after thrombin activation in the situa-tions analyzed.

Figure 5 shows profiles corresponding to the low-speedcytoskeletal fraction from control and uremic plateletsfrom HD patients and those who were transferred toCAPD. Under nonactivating conditions, almost unde-tectable phosphorylation was present. After thrombinactivation for 90 seconds, some of the proteins associatedwith the low-speed pellet from control platelets (proteinsp120, p85, p78, p75, pp62, pp60, and p54) were phosphor-ylated.

When analyzing the profiles corresponding to plateletsfrom uremic patients under HD, we did not observe de-tectable phosphorylation after thrombin activation. Onlyin two of the patients studied could traces of tyrosine-phosphorylated proteins be detected. However, phos-phorylation patterns recovered to almost control levelswhen patients were transferred to CAPD.

Tyrosine phosphorylation associated with the high-speed fraction obtained from control platelets was lowunder both basal and stimulating conditions (Fig. 6). Onlyp100 and pp60 appeared slightly phosphorylated. How-ever, phosphorylation levels in profiles from nonactivatedplatelets from uremic patients under HD were signifi-cantly higher than in controls. Proteins p100, pp62, andpp60 were initially phosphorylated and remained at simi-lar or even higher levels after thrombin activation. Trans-fer to CAPD induced a normalization of the phosphory-Fig. 3. Percent increase in the incorporation of cytoskeletal proteins

after activation with 0.1 U/mL of thrombin. Values express percentages lation patterns associated with this cytoskeletal fraction.of increase (mean 6 SEM) over the amount of the same protein incytoskeletons of nonactivated platelets. Experiments were performedwith platelet suspensions from the same patient (A) before (pre-HD) DISCUSSIONand after (post-HD) two months of initiating HD treatment, and (B)while on HD and after transfer to CAPD. Control results are also Data from our present study suggest that repeatedrepresented. *P , 0.01; **P , 0.001. N 5 16 for the control group and platelet stress during current HD procedures has a dele-N 5 8 for each group of uremic patients. Symbols are: (h) control;

terious effect on platelet function. This negative effect( ) HD; ( ) CAPD.would be characterized by an impairment of plateletcytoskeletal assembly and in an abnormal translocationof the signaling molecules studied in response to activa-

sponding to platelets obtained from patients in both tion. Moreover, CAPD would be less aggressive on thesegroups under HD treatment, either post-HD or HD, we functional and biochemical mechanisms.did not observe significant differences among the profiles Patients with chronic renal failure have a hemorrhagicfrom nonactivated and thrombin-activated platelets (Fig. tendency [3, 4]. This bleeding disorder seems to have a4, lanes 3 and 4, respectively). After thrombin activation, multifactorial origin and has been associated with a plate-actin decreased in 16 6 2% and 14 6 3% in profiles let dysfunction. Our group reported that uremic patientscorresponding to platelets from patients undergoing with a clinical history of hemorrhagic disorders showedpost-HD and HD treatments, respectively. a defective platelet adhesion with vessel subendothelium

in experiments under flow conditions [7]. Further studiesDistribution of phosphotyrosine proteins have confirmed the functional impairment of uremicwithin platelets platelets, demonstrated by a limitation of the platelet-

Thrombin-induced changes in the tyrosine-phosphor- spreading capabilities [8]. Other authors have also foundylation patterns of proteins were analyzed in platelets a defect in the adhesive and cohesive platelet functions

in uremic patients, attributed to factors in plasma [36].from control donors and uremic patients under HD and

Dıaz-Ricart et al: Signal transduction defect in uremic platelets1912

Fig. 4. Coomassie brilliant blue-stained SDSpolyacrylamide gels showing protein profilescorresponding to the high-speed cytoskeletalfraction. Platelet suspensions were obtainedfrom control donors (lanes 1 and 2); uremicpatients under HD treatment (lanes 3 and 4)and after transfer to CAPD treatment (lanes5 and 6). Cytoskeletal assembly was evaluatedbefore (lanes 1, 3, and 5) and after activationwith 0.1 U/mL of thrombin for 90 seconds(lanes 2, 4, and 6). Profiles shown correspondto one experiment and are representative ofthe eight different experiments performed.

The present study was designed to evaluate how re- and artificial surfaces [16]. Moreover, the biochemicalpeated stress by HD could have a deleterious effect on evaluation by electrophoresis of cytoskeletal assemblyprimary hemostasis. Results from our studies in the after thrombin stimulation of uremic platelets under HDgroup of patients with ESRD who had just started HD treatment showed a defective incorporation of the con-treatment indicate that this procedure does not imply tractile proteins when compared with that observed inan improvement of platelet function. Moreover, cyto- control platelets [16, 40]. Our present results confirmskeletal assembly in response to thrombin seems to be those observations.compromised as early as two months after starting HD Platelet cytoskeleton also plays a role in localizingtreatment. Further evidence was obtained from studies signaling molecules. In platelets from healthy individu-on patients who were switched from HD to CAPD. Our als, some of the proteins susceptible to be phosphory-present observations in this group of patients reinforce lated at tyrosine residues are known to be localized atthe findings obtained by other authors in terms of aggre- a submembrane level in resting conditions [26]. Aftergating platelet responses [6, 17, 37], indicating a recovery thrombin activation and as we have observed in controlof the cohesive functions of uremic platelets in patients platelets, they appeared phosphorylated and associatedundergoing CAPD. Furthermore, the use of the Baum- with the cytoplasmic cytoskeleton (recovered at the low-gartner perfusion method in our present study has dem-

speed cytoskeletal fraction). In platelets from uremiconstrated that not only were the aggregating responses of

patients under HD treatment, in which the cytoskeletaluremic platelets better preserved, but that the adhesiveassembly was defective, a certain degree of tyrosinefunctions improved in the same uremic patients whilephosphorylation was detected but not properly associ-on CAPD. Interestingly, those improvements were asso-ated with the polymerized cytoskeletal matrix (low-ciated with an amelioration of the cytoskeletal assemblyspeed fraction). In our study, the shift from HD to CAPDin response to thrombin, suggesting that this might be thetreatment resulted in a partial recovery of the cytoskele-underlying mechanism of the platelet function recovery.tal response of uremic platelets. Interestingly, signalingThe molecular assembly of actin into a filamentousthrough tyrosine phosphorylation in platelets from pa-network and the organization of other structural proteinstients under CAPD treatment showed similar patternsof the cytoskeleton are of critical importance for plateletto those observed in control platelets. In this group ofshape change and internal contraction [38, 39]. Duringpatients, the nitrogen retention products and creatininethese events, the contractile proteins that constitute bothlevels did not change or even increase slightly under themembrane and cytoplasmic cytoskeletons rearrangeCAPD therapy. Therefore, this improvement seems tothemselves through polymerization and depolymeriza-be unrelated to a correction of the uremic status of thetion processes. Previous studies performed in uremicpatients. The better preservation of platelet cytoskeletalpatients on maintenance HD who had a history of bleed-

ing showed a platelet-spreading defect on vascular [7, 8] functions may be in agreement with the improved he-

Dıaz-Ricart et al: Signal transduction defect in uremic platelets 1913

Fig. 6. Tyrosine-phosphorylated proteins associated with the high-speedcytoskeletal fraction from platelets before (lanes 1, 3, and 5) and afterFig. 5. Tyrosine-phosphorylated proteins associated with the low-speedactivation with 0.1 U/mL of thrombin (lanes 2, 4, and 6). Platelets werecytoskeletal fraction from platelets before (lanes 1, 3, and 5) and afterobtained from control donors (lanes 1 and 2); uremic patients underactivation with 0.1 U/mL of thrombin (lanes 2, 4, and 6). Platelets wereHD treatment (lanes 3 and 4) and after transfer to CAPD treatmentobtained from control donors (lanes 1 and 2); uremic patients under(lanes 5 and 6). After electrophoresis of the high-speed Triton-insolubleHD treatment (lanes 3 and 4) and after transfer to CAPD treatmentcytoskeletal fractions through SDS polyacrylamide gels, proteins were(lanes 5 and 6). After electrophoresis of the low-speed Triton-insolubletransferred to nitrocellulose membranes. Blots were incubated with acytoskeletal fractions through SDS polyacrylamide gels, proteins wereperoxidase-conjugated antibody to phosphotyrosine residues and de-transferred to nitrocellulose membranes. Blots were incubated with atected by ECL. Profiles shown correspond to one experiment and areperoxidase-conjugated antibody to phosphotyrosine residues and de-representative of the eight different experiments performed.tected by ECL. The profiles shown correspond to one experiment and

are representative of the eight different experiments performed.

switched to CAPD. The recovery of platelet functionwas not related to an increase in platelet or erythrocytemostasis observed by previous authors in patients withcounts, nor to an improvement of the uremic milieu. InCAPD treatment [24, 25, 37].our opinion, the repeated platelet activation induced bySeveral reports have pointed out that HD inducesHD procedures would lead to platelet exhaustion byplatelet activation [41–43], which mainly depends on thecompromising signal-transduction mechanisms. More-geometry of the dialyzer and the biocompatibility of theover, our results suggest that derangement of the cytoskel-dialysis membrane used [44]. Until now, HD-inducedetal organization or a lack of synchronization between theplatelet activation has been indirectly estimated by mea-cytoskeletal assembly and the signal transduction pro-suring increases in plasma levels of different intraplateletcesses may lead to impaired platelet function.substances or by the detection of P-selectin (GMP-140)

expression on platelet membrane by flow cytometry [45].ACKNOWLEDGMENTSIt is a fact that platelets can circulate even after mild

activation [46]; thus, the a relative storage pool deficiency This work was partially supported by grants FIS 98/321 from Fondode Investigaciones Sanitarias de la Seguridad Social and SGR97-133described in uremic platelets [12] may be a result of afrom CIRIT.

partial degranulation due to platelet activation by the HDprocedure. Recent studies on platelet RNA contents sug- Reprint requests to Maribel Dıaz-Ricart, Ph.D., Servicio de Hemoter-

apia y Hemostasia, Hospital Clınic, Villarroel, 170, 08036 Barcelona,gest that current HD procedures alter the platelet life spanSpain.

probably through elimination of the younger and biologi- E-mail: mdiaz@medicina.ub.escally more active platelets [47]. In any case, the possibilityof a deleterious effect of mechanical stress caused by HD REFERENCESon platelet function, being compensated by the beneficial

1. Lewis J, Zucker M, Ferguson J: Bleeding tendency in uremia.action of HD clearing plasma toxins [3, 6, 17], deserves Blood 11:1073–1076, 1956

2. Andrassy K, Ritz E: Uremia as a cause of bleeding. Am J Nephrolfull consideration.5:313–318, 1985Our results show that several parameters of platelet

3. Di Minno G, Martinez J, McKean ML, Delarosa J, Burke JF,function, specifically signal transduction mechanisms, Murphy S: Platelet dysfunction in uremia: Multifaceted defect

partially corrected by dialysis. Am J Med 79:552–559, 1985improved when ESRD patients on regular HD were

Dıaz-Ricart et al: Signal transduction defect in uremic platelets1914

4. Gordge MP, Neild GH: Platelet function in uraemia. Platelets kidney transplantation on blood platelet function. Nephron 23:287–292, 19792:115–123, 1991

5. Ibels LS, Stewart JH, Mahony JF, Neale FC, Sheil AGR: Occlu- 26. Fox JEB, Lipfert L, Clark EA, Reynolds CC, Austin CD,Brugge JS: On the role of the platelet membrane skeleton insive arterial disease in uraemic and haemodialysis patients and

renal transplant recipients. Q J Med 46:197–214, 1997 mediating signal transduction. J Biol Chem 268:25973–25984, 199327. Rao GH, Escolar G, White JG: Epinephrine reverses the inhibi-6. Remuzzi G, Livio M, Marchiaro G, Mecca G, de Gaetano G:

Bleeding in renal failure: Altered platelet function in chronic urae- tory influence of aspirin on platelet-vessel wall interactions.Thromb Res 44:65–74, 1986mia only partially corrected by haemodialysis. Nephron 22:347–353,

1978 28. Born GVR: The aggregation of blood platelets by adenosine di-phosphate and its reversal. Nature 194:927–929, 19627. Castillo R, Lozano T, Escolar G, Revert L, Lopez J, Ordinas

A: Defective platelet adhesion on vessel subendothelium in uremic 29. Escolar G, Cases A, Vinas M, Calls J, Cirera I, Ordinas A:Evaluation of acquired platelet dysfunctions in uremic and cirrhoticpatients. Blood 68:337–342, 1986

8. Escolar G, Cases A, Bastida E, Garrido M, Lopez J, Revert patients using patient function analyzer (PFA-100E): Influence ofhematocrit elevation. Haematologica 84:614–619, 1999L, Castillo R, Ordinas A: Uremic platelets have a functional

defect affecting the interaction of von Willebrand factor with glyco- 30. Baumgartner HR, Muggli R: Adhesion and aggregation: Mor-phological demonstration and quantitation in vivo and in vitro, inprotein IIb-IIIa. Blood 76:1336–1340, 1990

9. Castaman G, Rodeghiero F, Lattuada A, Lagreca G, Mannucci Platelets in Biology and Pathology, edited by Gordon JL, Amster-dam, North Holland Biomedical Press, 1976, pp 23–60PM: Multimeric pattern of plasma and platelet von Willebrand

factor is normal in uremic patients. Am J Hematol 44:266–269, 1993 31. Dıaz-Ricart M, Tandon NN, Gomez-Ortiz G, Carretero M,Escolar G, Ordinas A, Jamieson GA: Antibodies to CD3610. Meyer D, Baumgartner HR: Role of von Willebrand factor in

platelet adhesion to the subendothelium. Br J Haematol 54:1–9, (GPIV) inhibit platelet adhesion to subendothelial surfaces underflow conditions. Arterioscler Thromb Vasc Biol 16:883–888, 19961983

11. Escolar G, Monteagudo J, Castillo R, Cases A, Garrido M, 32. Escolar G, Bastida E, Castillo R, Ordinas A: Development ofa computer program to analyze the parameters of platelet-vesselOrdinas A: Ultrastructural immunolocalization and morphomet-

ric quantification of platelet membrane GPIb and GPIIb-IIIa in wall interaction. Haemostasis 16:8–14, 198633. Jennings LK, Fox JEB, Edwards HH, Phillips DR: Changes inuremic patients. Prog Clin Biol Res 283:197–201, 1988

12. Gralnick HR, McKeown LP, Williams SB, Shafer B: Plasma the cytoskeletal structure of human platelets following thrombinactivation. J Biol Chem 256:6927–6932, 1981and platelet von Willebrand factor defects in uremia. Am J Med

85:806–810, 1989 34. Kometani M, Sato T, Fujii T: Platelet cytoskeletal componentsinvolved in shape change and secretion. Thromb Res 41:801–809,13. Zwaginga JJ, Ijsseldijk MJ, Beeser-Visser N, de Groot PG, Vos

J, Sixma JJ: High von Willebrand factor concentration compensates 198635. Laemmli UK: Cleavage of structural proteins during the assemblya relative adhesion defect in uremic blood. Blood 75:1498–1508,

1990 of the head of bacteriophage. Nature 227:680–685, 197036. Zwaginga JJ, Ijsseldijk MJ, de Groot PG, Vos J, de Bos Kuil14. Benigni A, Boccardo P, Galbusera M, Monteagudo J, De

Marco L, Remuzzi G, Ruggeri ZM: Reversible activation defect RL, Sixma JJ: Defects in platelet adhesion and aggregate formationin uremic bleeding disorder can be attributed to factors in plasma.of the platelet glycoprotein IIb-IIIa complex in patients with ure-

mia. Am J Kidney Dis 22:668–676, 1993 Arterioscler Thromb 11:733–744, 199137. Roger SD, Piper J, Tucker B, Raine AEG, Baker LRI, Kovacs15. Gawaz MP, Dobos G, Spath M, Schollmeyer P, Gurland HJ,

Mujais SK: Impaired function of platelet membrane glycoprotein IB: Comparison of haemostatic activity in haemodialysis and peri-toneal dialysis patients with a novel technique, haemostatometry.IIb-IIIa in end-stage renal disease. J Am Soc Nephrol 5:36–46, 1994

16. Escolar G, Dıaz-Ricart M, Cases A, Castillo R, Ordinas A, Nephron 62:422–428, 199238. White JG: An overview of platelet structural physiology. ScanningWhite JG: Abnormal cytoskeletal assembly in platelets from ure-

mic patients. Am J Pathol 143:823–831, 1993 Microscopy 1:1677–1700. 198739. Hartwig JH: Mechanisms of actin rearrangements mediating17. Lindsay RM, Friesen M, Aronstam A, Andrus F, Clark WF,

Linton AL: Improvement of platelet function by increased fre- platelet activation. J Cell Biol 118:1421–1442, 199240. Estebanell E, Dıaz-Ricart M, Lozano M, Mazzara R, Escolarquency of haemodialysis. Clin Nephrol 10:67–70, 1978

18. Lindsay RM, Prentice CRM, Davidson JF, Burton JA, McNicol G, Ordinas A: Cytoskeletal reorganization after preparation ofplatelet concentrates, using the buffy coat method, and during theirGP: Haemostatic changes during dialysis associated with thrombus

formation on dialysis membranes. Br Med J 4:454–458, 1972 storage. Haematologica 83:112–117, 199841. Adler AJ, Berlyne GM: b-Thromboglobulin and platelet factor-419. Sultan Y, London GM, Goldfarb B, Toulon P, Marchais SJ:

Activation of platelets coagulation and fibrinolysis in patients on levels during hemodialysis with polyacrylonitrile. Trans Am SocArtif Intern Organs 4:100–102, 1981long-term haemosialysis: Influence of cuprophan and polyacryloni-

trile membranes. Nephrol Dial Transplant 5:362–368, 1990 42. Hakim RM, Schater AI: Hemodialysis-associated platelet activa-tion and thrombocytopenia. Am J Med 78:575–580, 198520. Wardle EN, Piercy DA: Studies of contact activation of blood

in haemodialysis. J Clin Pathol 25:1045–1049, 1972 43. Gawaz MP, Ward RA: Effects of hemodialysis on platelet-derivedthrombospondin. Kidney Int 40:257–265, 199121. Schmitt GW, Moake JL, Rudy CK, Vicks SL, Hamburger RJ:

Alterations in hemostatic parameters during hemodialysis with 44. Cases A, Reverter JC, Escolar G, Sanz C, Lopez-Pedret J,Revert L, Ordinas A: Platelet activation on hemodialysis: Influencedialyzers of different membrane composition and flow design:

Platelet activation and factor VIII-related von Willebrand factor of dialysis membranes. Kidney Int 43(Suppl 41):S217–S220, 199345. Reverter JC, Escolar G, Sanz C, Cases A, Villamor N, Nieu-during hemodialysis. Am J Med 83:411–418, 1987

22. Docci D, Turci F, del Vecchio C, Bilancioni R, Cencioni L, wenhuis HK, Lopez J, Ordinas A: Platelet activation during he-modialysis measured through exposure of p-selectin: Analysis byPretolani E: Hemodialysis-associated platelet loss: Study on the

relative contribution of dialyzer membrane composition and geom- flow cytometric and ultrastructural techniques. J Lab Clin Med124:79–85, 1994etry. Int J Artif Organs 7:337–340, 1986

23. Sloand JA, Sloand EM: Studies on platelet membrane glycopro- 46. Harker LA, Malpass TW, Branson HE, Hassell EA II, SlichterSJ: Mechanism of abnormal bleeding in patients undergoing cardio-teins and platelet function during hemodialysis. J Am Soc Nephrol

8:799–803, 1997 pulmonary bypass: Acquired transient platelet dysfunction associ-ated with selective a-granule release. Blood 56:824–834, 198024. Lindsay RM, Friesen M, Koens F, Linton AL, Oreopoulos D,

de Veber G: Platelet function in patients on long term peritoneal 47. Tassies D, Reverter JC, Cases A, Escolar G, Villamor N, Lo-pez-Pedret J, Castillo R, Ordinas A: Reticulated platelets indialysis. Clin Nephrol 6:335–339, 1976

25. Nenci GG, Berrettini M, Agnelli G, Parise P, Buoncristiani uremic patients: Effect of hemodialysis and continuous ambulatoryperitoneal dialysis. Am J Hematol 50:161–166, 1995U, Ballatori E: Effect of peritoneal dialysis, hemodialysis and