Background

Uremic patients have a bleeding tendency associated with a platelet dysfunction. We evaluated the impact of a repeated hemodialysis procedure on primary hemostasis by analyzing different aspects of platelet activation in uremic patients.

Methods

Studies were performed in (1) eight patients with end-stage renal disease before the hemodialysis program was initiated and after initiating hemodialysis treatment, and in (2) eight patients on maintenance hemodialysis who were transferred to continuous ambulatory peritoneal dialysis. Studies included routine platelet aggregations and evaluation of platelet–subendothelium interactions under flow conditions. Contractile proteins and tyrosine phosphorylation associated with the cytoskeleton were analyzed, before and after thrombin activation of platelets, by electrophoresis after Triton X-100 extraction.

Results

No changes in the clinical parameters analyzed were observed among the different study groups. Aggregation and platelet adhesion only improved when patients were shifted from hemodialysis to continuous ambulatory peritoneal dialysis (P < 0.05 for both percentage of surface covered by platelets and aggregate formation). The association of cytoskeletal proteins in platelets from patients under hemodialysis treatment was statistically decreased with respect to the corresponding values in platelets from patients not subjected to dialysis (P < 0.01 for actin). However, after two months on peritoneal dialysis, these values increased to almost control values (P < 0.001 for actin, vs. hemodialysis). Similarly, translocation of tyrosine-phosphorylated proteins to the cytoskeletal fraction was impaired in platelets from hemodialyzed patients, and it recovered partially after the patients transferred to continuous ambulatory peritoneal dialysis.

Conclusions

Our present data support the concept that repeated platelet stress during hemodialysis has a deleterious effect on the organization of platelet cytoskeleton, which seems to impair the translocation of signal transduction proteins within platelets compromising the platelet function in uremia.

METHODS

Patients

Our study, approved by the Human Experimental Committee of the Hospital Clinic, was carried out according to the principles of the Declaration of Helsinki. Informed consent was obtained from all the participants. Eight patients with end-stage renal disease (clearance creatinine < 10 mL/min) were evaluated before starting HD and after two months on maintenance HD. There were five men and three women from the age of 38 to 62 years. The causes of renal failure were nephrosclerosis (3), chronic glomerulonephritis (3), diabetic nephropathy (1), and unknown (1). Eight uremic patients on maintenance HD (6 women and 2 men, age range of 42 to 75 years, time on HD from 6 to 171 months) were included in this study. The causes of renal failure were polycystic kidney disease (2), diabetic nephropathy (2), nephroangiosclerosis (1), chronic interstitial nephropathy (1), hemolytic uremic syndrome (1), and unknown (1). Patients included in the study were chosen on the basis that they were moved from HD to continuous ambulatory peritoneal dialysis (CAPD) for various reasons, including the lack of vascular access for HD (5), ischemic heart disease (2), and personal decision of the patient (1). Patients performed HD for four hours three time per week, and hollow fiber dialyzers with cellulose acetate membranes (CA 140; Baxter, Deerfield, IL, USA) were used. None of the patients complained of severe bleeding or were hypertensive at the time that they were enrolled in the study. The KT/V in all hemodialyzed patients was higher than 1.1. No patient had received blood related products for two months, and none had taken aspirin or other drugs that affect platelet function for at least two weeks prior to the study.

Experimental design

Studies were carried out on (1) eight patients with ESRD before the HD program was initiated (pre-HD) and after undergoing HD (post-HD), and (2) eight patients while on maintenance HD and at least two months after starting CAPD treatment. Blood samples from healthy volunteers were also obtained for control experiments. Studies included the evaluation of certain clinical parameters, including bleeding times and platelet aggregation using turbidimetric techniques, and the evaluation of platelet–subendothelium interactions under flow conditions, using an annular perfusion chamber. The association of contractile and phosphotyrosine proteins with the platelet cytoskeleton after thrombin stimulation was also investigated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in both groups of patients under the different treatments.

In each set of experiments, studies were performed using blood from two uremic patients and from a control donor. This design was applied in studies performed while patients were in pre-treatment and post-treatment, considering that blood samples were always obtained from different donors. Therefore, the number for each group of patients was 8, and the number for the control group was 16.

Blood sampling

Blood samples were obtained from uremic patients (1) before initiating HD and after at least two months of starting HD and (2) while under HD and after two months of being transferred to CAPD. In those patients under HD treatment, blood samples were always obtained just before the HD session. Aliquots of citrated blood samples were reserved for perfusion studies. Platelet-rich plasma (PRP) was obtained by centrifugation of citrate-anticoagulated blood for 20 minutes at 100 g. Aliquots of PRP were used in aggregation studies.

Platelets were also isolated from PRP and were washed twice with equal volumes of CCD (93 mmol/L sodium citrate, 7 mmol/L citric acid, and 140 mmol/L dextrose), pH 6.5, containing 5 mmol/L adenosine and 3 mmol/L theophylline²⁷. The final pellet was resuspended in Hanks' balanced salt solution (HBSS) and incubated for 20 minutes at 37°C. Aliquots of washed uremic platelet suspensions were used to electrophoretically analyze any changes in the association of contractile proteins to the cytoskeleton before and after activation with 0.1 U/mL of thrombin.

Aggregation studies

Platelet aggregation studies were carried out in a Hitachi-Aggrecorder aggregometer. Samples of PRP from the uremic patients included in the study were placed in 6 mm wide siliconized cuvettes. Platelet counts were normalized to the same value (2 10⁵ platelets/L). The minimum and maximum amplitudes of the recorder were adjusted with PRP (0% transmission) and platelet poor plasma (PPP) (100% transmission), respectively. Arachidonic acid (1.2 mmol/L), adenosine diphosphate (ADP; 4 mol/L), collagen (2.5 mg/mL), epinephrine (10 mol/L), and ristocetin (1 mg/mL) were used as inductors, under stirring. Results were expressed as percentages of maximum aggregation obtained after five minutes of stimulation^28,29.

Perfusion studies and morphometric evaluation

Perfusion experiments were performed in annular chambers, as previously described^30,31. De-endothelialized rabbit aorta segments were exposed to citrated whole blood at a shear rate of 800 s^-1 at 37°C. After 10 minutes of perfusion, segments were fixed, dehydrated with alcohols, embedded in JB-4, thin sectioned for light microscopy, and stained with toluidine blue.

Platelets interacting with subendothelium were evaluated according to the morphometric criteria described by Baumgartner and Muggli³⁰. A semiautomated method was used to divide platelets into different classes of interaction³². Platelets or groups of platelets were classified as follows: contact (C), platelets that were attached but not spread on the subendothelium; adhesion (A), platelets that were spread on subendothelium or form layers of less than 5 m in height; and thrombi (T), platelet aggregates of 5 m or more in height. All of these basic parameters were expressed as a percentage of the total length of the vessel screened. The total covered surface (CS) was obtained by adding the previous basic parameters (C + A + T).

Obtaining cytoskeletal proteins

Platelet cytoskeletons were obtained according to the procedure described by Jennings et al³³ with minor modifications³⁴. Samples of resuspended platelets were adjusted to 1.2 10⁶ platelets/L. Before activation of platelets, aliquots of platelet suspensions were treated to quantitate the protein content, which was always comparable among the samples obtained (around 2 g/L). Platelet suspensions were divided into two aliquots, one remaining undisturbed at 37°C and one being subjected to stimulation with 0.1 U/mL thrombin. Samples were mixed by gentle inversion every 30 seconds. After 90 seconds, all of the samples were treated with an equal volume of ice-cold lysis buffer (final pH 7.4) containing 2% Triton X-100, 100 mmol/L Tris-HCl, 10 mmol/L ethylene glycol bis(b-aminoethylether)-N,N,N',N'-tetraacetic acid (EGTA), 4 mmol/L ethylenediaminetetraacetic acid (EDTA), 2 mmol/L phenylmethylsulfonyl fluoride (PMSF), 1 mmol/L benzamidine, 2 g/mL leupeptin, 2 g/mL pepstatin, and 2 mmol/L sodium orthovanadate. The low-speed Triton-insoluble residues, corresponding to the polymerized cytoskeletal fraction, were isolated by sedimentation at 12,000 g for five minutes at 4°C in a microfuge. After collection of the supernatants, residues were washed twice with washing buffer, without Triton X-100, at 4°C, solubilized with a SDS-containing buffer, and heated at 100°C for five minutes. Platelet membrane cytoskeletons were sedimented by centrifugation of the supernatants, recovered at low speed, at 100,000 g for three hours. The corresponding pellets (high-speed residues) were solubilized as mentioned before. Samples were frozen at -40°C until electrophoretical evaluation was performed.

Analysis of cytoskeletal proteins

Triton-insoluble fractions from an equal number of thrombin nonactivated and activated platelets were obtained from control donors and from the uremic patients enrolled in the study. Cytoskeletal proteins present in both the low- and high-speed fractions were separated by 7 to 12% SDS-PAGE³⁵. To evaluate the contractile proteins associated with the cytoskeleton, gels were stained with Coomassie brilliant blue R250 and densitometrically quantitated as previously described¹⁶. Basically, stained protein bands were densitometrically analyzed using digital-video technology provided by a computerized image analyzer running specific software (SigmaGel, Jandel GmbH, Erkrath, Germany). After selection of the bands on the monitor screen, the software automatically analyzed the color density of each protein band and integrated areas beneath densitometric peaks. Values of protein peak areas in the lanes containing Triton-insoluble residues from nonactivated platelets were considered as 100%. The association of certain protein with the thrombin-activated cytoskeleton was expressed as the percentage of increase over the amount of the same protein found in the respective lane corresponding to nonactivated platelets.

Analysis of tyrosine-phosphorylated proteins

Phosphotyrosine proteins associated with both the low- and high-speed cytoskeletal fractions were analyzed on 8% SDS-polyacrylamide gels. Proteins present in the gels were transferred to nitrocellulose membranes (BioRad, Hercules, CA, USA). After blocking nonspecific binding, Western blots were probed with a horseradish peroxidase-antiphosphotyrosine recombinant antibody (Transduction Laboratories, Lexington, KY, USA). The excess of antibody was removed by extensive washing, and blots were developed by enhanced chemiluminiscence (ECL) method (Amersham Pharmacia Biotek, Essex, UK).

Statistics

Data are expressed as mean SEM. Student's t-test for paired data was used for statistical comparisons between data obtained pretreatment and posttreatment in each group. Student's t-test was used to compare data from patients versus controls. A P level < 0.05 was considered statistically significant.

RESULTS

Clinical parameters

Table 1 shows changes in blood cell counts and laboratory parameters. Hematocrit and hemoglobin levels of the uremic patients enrolled in the study were measured in pre-HD, post-HD, HD, and CAPD situations of the study. These values showed practically the same levels during the different treatments in both groups of patients. Platelet counts and coagulation tests (prothrombin time, partial thromboplastin time, and fibrinogen levels) were within the normal range in both groups under the different periods of the study. Plasma levels of nitrogen-retention products and creatinine levels did not change after initiating HD (post-HD) or while on HD, and they were similar or increased slightly after starting CAPD. No significant differences were observed when comparing bleeding times among the different treatments in both groups of patients.

Table 1 - Changes in blood cell counts and laboratory parameters.

Full table

Aggregation studies

Aggregation results for control platelets reached values of approximately 100% of aggregation five minutes after the addition of the agonists at the concentrations employed. Platelet suspensions from all the patients included in the study showed a reduced aggregating response to the agonists used, when compared with control values (P < 0.05; Table 2).

Table 2 - Bleeding time and platelet aggregation studies.

Full table

After five minutes of PRP stimulation with arachidonic acid (1.2 mmol/L), collagen (2.5 mg/mL), ADP (4 mmol/L), and ristocetin (1 mg/mL), percentages of maximal aggregation were 68.5 6.5%, 76 3.5%, 69.1 5.3%, and 72.9 4.0%, respectively (mean SEM, N = 8) in patients with pre-HD ESRD. In the same group of patients, aggregation patterns did not significantly differ after two months of starting HD. Aggregation values were 61.8 3.0%, 78.8 3.6%, 64.9 4.8%, and 66.4 2.5%, respectively.

Aggregation responses of platelets from the second group of uremic patients, while on HD, to arachidonic acid, collagen, ADP, and ristocetin at the concentrations indicated before were 55.8 6.8%, 60.2 8.3%, 56.2 7.8%, and 65.7 8.9%, respectively (mean SEM, N = 8). Under the same experimental conditions, all of the aggregation patterns statistically improved (P < 0.05) when patients were moved to CAPD, changing to 78.9 6.9%, 81.2 7.5%, 79.1 7.6%, and 89.2 7.1%, respectively Table 2.

Perfusion studies

Blood samples were recirculated for 10 minutes through the annular chamber at 800 s^-1 of shear rate. Values of surface covered by platelets, expressed as percentages (%SC) obtained with control blood samples were 34.5 3.4% with an aggregate formation of 20.4 2.5% (mean SEM, N = 16).

Studies of uremic patients before and after two months of initiating HD treatment.

The surface covered by platelets obtained after perfusing denuded vascular segments with blood samples from patients with ESRD was 24.1 1.9% (mean SEM, N = 8), with an aggregate formation of 15.7 0.5%. After two months of initiating HD treatment, perfusion experiments were carried out with blood samples from the same patients. Results of %SC and percentage aggregate formation were slightly inferior (21.7 2% and 13.4 2.3%, respectively), but did not statistically vary from pre-HD values. Values of surface coverage observed in both situations were statistically reduced with respect to those observed in control experiments (P < 0.05; Figure 1a).

Figure 1.

Platelet deposition after 10 minutes of perfusion of denuded rabbit aorta. Perfusates consisted of blood samples from (A) eight patients with end-stage renal disease before (pre-HD) and after (post-HD) two months of initiating HD treatment, and (B) eight patients while on HD and after being transferred to CAPD. Bar diagrams represent morphometric parameters obtained in the different perfusion experiments. Bars represent the percentage of the vessel surface covered with platelets. The open portion of the bar corresponds to the surface covered by adhesive platelets (adhesion), and the dashed inserts indicate the surface covered with groups of platelets forming aggregates of more than 5 m in height (thrombi). The results are expressed as percentages of the total surface of the vessel screened. *P < 0.05 vs. control values and ^aP < 0.05 CAPD vs. HD. N = 16 for control experiments and N = 8 for each group of uremic patients.

Full figure and legend (48K)

Studies of HD patients who were transferred to CAPD.

The %SC using blood from the uremic patients while on HD was 18.6 4.5% (mean SEM, N = 8), which was significantly below that obtained in control experiments (P < 0.05). After CAPD began, platelets from the same uremic patients displayed a higher surface coverage, increasing statistically to 24.6 3.3% (P < 0.05). This increase in the surface coverage paralleled a statistically significant improvement of the aggregate formation: from 12.3 4.4% when patients were on HD, to 17.6 5% after two months of CAPD treatment (P < 0.05; Figure 1b).

Changes in the distribution of platelet cytoskeletal proteins

Profiles corresponding to the low-speed Triton X-100–insoluble residues of resting platelets and platelets activated with thrombin from control donors and from both groups of uremic patients included in the study who underwent the different treatments Figure 2 were analyzed densitometrically. The degree of association of the different proteins with the cytoskeleton was expressed as the percentage of incorporation of each protein in thrombin stimulated versus resting platelet samples.

Figure 2.

Coomassie brilliant blue-stained SDS polyacrylamide gels showing protein profiles corresponding to the low-speed cytoskeletal fraction. Platelet suspensions were obtained from control donors (lanes 1 and 2); uremic patients undergoing HD treatment (lanes 3 and 4) and after being moved to CAPD treatment (lanes 5 and 6). Cytoskeletal assembly was evaluated before (lanes 1, 3, and 5) and after activation with 0.1 U/mL of thrombin for 90 seconds (lanes 2, 4, and 6). Arrowheads indicate defective association of contractile proteins after thrombin activation of platelets from patients under HD. Profiles shown correspond to one experiment and are representative of the eight different experiments performed.

Full figure and legend (104K)

Profiles corresponding to resting platelets from the control donors and from all of the uremic patients included in the study showed no significant qualitative differences for the presence of the major cytoskeletal proteins recovered in the low-speed cytoskeletal fraction (Figure 2, lanes 1, 3, and 5). However, proteins in profiles from uremic patients on HD treatment densitometrically appeared to be decreased (Figure 2, lane 3).

Thrombin activation of control platelets resulted in an augmented incorporation of contractile proteins (Figure 2, lane 2). Incorporations of ABP, myosin, -actinin, and actin to the low-speed cytoskeletal fraction after thrombin stimulation were 140 5%, 50 11.4%, 61.4 7.9%, and 120 4.6%, respectively Figure 3.

Figure 3.

Percent increase in the incorporation of cytoskeletal proteins after activation with 0.1 U/mL of thrombin. Values express percentages of increase (mean SEM) over the amount of the same protein in cytoskeletons of nonactivated platelets. Experiments were performed with platelet suspensions from the same patient (A) before (pre-HD) and after (post-HD) two months of initiating HD treatment, and (B) while on HD and after transfer to CAPD. Control results are also represented. *P < 0.01; **P < 0.001. N = 16 for the control group and N = 8 for each group of uremic patients. Symbols are: () control; () HD; () CAPD.

Full figure and legend (44K)

Studies of uremic patients before and after two months of initiating HD treatment.

Proteins recovered at the low-speed cytoskeletal fraction corresponding to thrombin-activated platelets from eight uremic patients with ESRD, pre-HD, were also analyzed. After activation of platelets with 0.1 U/mL of thrombin, there was an incorporation of contractile proteins of 87.5 4.3%, 45.0 5.3%, 45.7 7%, and 74.8 4.2% for ABP, myosin, -actinin, and actin, respectively.

In studies performed post-HD, blood samples from the same patients were collected, and the same experimental conditions were applied. When platelets were activated with thrombin, incorporation of contractile proteins to the low-speed cytoskeletal fraction was significantly decreased (P < 0.01 vs. pre-HD for all the proteins studied). Results in terms of protein incorporation were 60.6 6.3%, 35.9 5.6%, 25.6 5.0%, and 34.4 8% for ABP, myosin, -actinin and actin, respectively Figure 3a.

Studies of patients on HD who transferred to CAPD treatment.

Amounts of proteins found in cytoskeletons from resting platelets obtained from the patients undergoing CAPD were slightly greater to those observed in platelets from the same patients while they were on HD. After thrombin stimulation, an incorporation of contractile proteins to the cytoskeleton of platelets from the same patients was observed during both the HD and CAPD treatments, although the rate of incorporation was much higher in platelets obtained while patients were on CAPD Figure 2.

Densitometric evaluation Figure 3b of protein bands corresponding to Triton-insoluble residues of thrombin-activated platelets from patients under CAPD showed a statistically significant increase in the association of ABP, -actinin, and actin versus those obtained when patients were undergoing HD treatment [%protein incorporation of 110 6% vs. 42 2% for ABP (P < 0.01), 45.8 8.0% vs. 35.0 6.0% for myosin, 53.6 3.2% vs. 18.0 4% for -actinin (P < 0.01), and 96 1.2% vs. 36 3.2% for actin (P < 0.01)]. The association of myosin after thrombin activation was not significantly different in the two situations.

Figure 4 shows SDS-PAGE profiles corresponding to the high-speed cytoskeletal fraction from resting and thrombin-activated platelets. Profiles correspond to an equal number of platelets from the control donors and the uremic patients included in the study. No significant differences were observed among the profiles from resting platelets obtained from control donors and uremic patients after initiating CAPD (Figure 4, lanes 1 and 5). However, lane 3 in Figure 4 shows that the amount of proteins associated with the high-speed cytoskeletal fraction in platelets from patients undergoing HD was increased, correlating to a decrease in the density of those proteins associated with the low-speed cytoskeletal fraction (Figure 2, lane 3).

Figure 4.

Coomassie brilliant blue-stained SDS polyacrylamide gels showing protein profiles corresponding to the high-speed cytoskeletal fraction. Platelet suspensions were obtained from control donors (lanes 1 and 2); uremic patients under HD treatment (lanes 3 and 4) and after transfer to CAPD treatment (lanes 5 and 6). Cytoskeletal assembly was evaluated before (lanes 1, 3, and 5) and after activation with 0.1 U/mL of thrombin for 90 seconds (lanes 2, 4, and 6). Profiles shown correspond to one experiment and are representative of the eight different experiments performed.

Full figure and legend (97K)

After thrombin activation, the contractile proteins associated with this fraction decreased in control platelets (46 2.8% decrease of actin). This was actually expected since polymerized contractile proteins were recovered in the low-speed fraction (Figure 4, lane 2 vs. lane 1), due to the cytoskeleton assembly. Nearly similar results were observed in profiles corresponding to the platelets from patients who were pre-HD and those after CAPD treatment (percentage of decrease of 62 0.9% and 50 1.8%, respectively; Figure 4, lane 6 vs. lane 5).

However, when analyzing the protein profiles corresponding to platelets obtained from patients in both groups under HD treatment, either post-HD or HD, we did not observe significant differences among the profiles from nonactivated and thrombin-activated platelets (Figure 4, lanes 3 and 4, respectively). After thrombin activation, actin decreased in 16 2% and 14 3% in profiles corresponding to platelets from patients undergoing post-HD and HD treatments, respectively.

Distribution of phosphotyrosine proteins within platelets

Thrombin-induced changes in the tyrosine-phosphorylation patterns of proteins were analyzed in platelets from control donors and uremic patients under HD and after being transferred to CAPD. Studies were performed in order to evaluate changes in the association of these proteins with both the low- and high-speed cytoskeletal fractions after thrombin activation in the situations analyzed.

Figure 5 shows profiles corresponding to the low-speed cytoskeletal fraction from control and uremic platelets from HD patients and those who were transferred to CAPD. Under nonactivating conditions, almost undetectable phosphorylation was present. After thrombin activation for 90 seconds, some of the proteins associated with the low-speed pellet from control platelets (proteins p120, p85, p78, p75, pp62, pp60, and p54) were phosphorylated.

Figure 5.

Tyrosine-phosphorylated proteins associated with the low-speed cytoskeletal fraction from platelets before (lanes 1, 3, and 5) and after activation with 0.1 U/mL of thrombin (lanes 2, 4, and 6). Platelets were obtained from control donors (lanes 1 and 2); uremic patients under HD treatment (lanes 3 and 4) and after transfer to CAPD treatment (lanes 5 and 6). After electrophoresis of the low-speed Triton-insoluble cytoskeletal fractions through SDS polyacrylamide gels, proteins were transferred to nitrocellulose membranes. Blots were incubated with a peroxidase-conjugated antibody to phosphotyrosine residues and detected by ECL. The profiles shown correspond to one experiment and are representative of the eight different experiments performed.

Full figure and legend (67K)

When analyzing the profiles corresponding to platelets from uremic patients under HD, we did not observe detectable phosphorylation after thrombin activation. Only in two of the patients studied could traces of tyrosine-phosphorylated proteins be detected. However, phosphorylation patterns recovered to almost control levels when patients were transferred to CAPD.

Tyrosine phosphorylation associated with the high-speed fraction obtained from control platelets was low under both basal and stimulating conditions Figure 6. Only p100 and pp60 appeared slightly phosphorylated. However, phosphorylation levels in profiles from nonactivated platelets from uremic patients under HD were significantly higher than in controls. Proteins p100, pp62, and pp60 were initially phosphorylated and remained at similar or even higher levels after thrombin activation. Transfer to CAPD induced a normalization of the phosphorylation patterns associated with this cytoskeletal fraction.

Figure 6.

Tyrosine-phosphorylated proteins associated with the high-speed cytoskeletal fraction from platelets before (lanes 1, 3, and 5) and after activation with 0.1 U/mL of thrombin (lanes 2, 4, and 6). Platelets were obtained from control donors (lanes 1 and 2); uremic patients under HD treatment (lanes 3 and 4) and after transfer to CAPD treatment (lanes 5 and 6). After electrophoresis of the high-speed Triton-insoluble cytoskeletal fractions through SDS polyacrylamide gels, proteins were transferred to nitrocellulose membranes. Blots were incubated with a peroxidase-conjugated antibody to phosphotyrosine residues and detected by ECL. Profiles shown correspond to one experiment and are representative of the eight different experiments performed.

Full figure and legend (66K)

DISCUSSION

Data from our present study suggest that repeated platelet stress during current HD procedures has a deleterious effect on platelet function. This negative effect would be characterized by an impairment of platelet cytoskeletal assembly and in an abnormal translocation of the signaling molecules studied in response to activation. Moreover, CAPD would be less aggressive on these functional and biochemical mechanisms.

Patients with chronic renal failure have a hemorrhagic tendency^3,4. This bleeding disorder seems to have a multifactorial origin and has been associated with a platelet dysfunction. Our group reported that uremic patients with a clinical history of hemorrhagic disorders showed a defective platelet adhesion with vessel subendothelium in experiments under flow conditions⁷. Further studies have confirmed the functional impairment of uremic platelets, demonstrated by a limitation of the platelet-spreading capabilities⁸. Other authors have also found a defect in the adhesive and cohesive platelet functions in uremic patients, attributed to factors in plasma³⁶.

The present study was designed to evaluate how repeated stress by HD could have a deleterious effect on primary hemostasis. Results from our studies in the group of patients with ESRD who had just started HD treatment indicate that this procedure does not imply an improvement of platelet function. Moreover, cytoskeletal assembly in response to thrombin seems to be compromised as early as two months after starting HD treatment. Further evidence was obtained from studies on patients who were switched from HD to CAPD. Our present observations in this group of patients reinforce the findings obtained by other authors in terms of aggregating platelet responses^6,17,37, indicating a recovery of the cohesive functions of uremic platelets in patients undergoing CAPD. Furthermore, the use of the Baumgartner perfusion method in our present study has demonstrated that not only were the aggregating responses of uremic platelets better preserved, but that the adhesive functions improved in the same uremic patients while on CAPD. Interestingly, those improvements were associated with an amelioration of the cytoskeletal assembly in response to thrombin, suggesting that this might be the underlying mechanism of the platelet function recovery.

The molecular assembly of actin into a filamentous network and the organization of other structural proteins of the cytoskeleton are of critical importance for platelet shape change and internal contraction^38,39. During these events, the contractile proteins that constitute both membrane and cytoplasmic cytoskeletons rearrange themselves through polymerization and depolymerization processes. Previous studies performed in uremic patients on maintenance HD who had a history of bleeding showed a platelet-spreading defect on vascular^7,8 and artificial surfaces¹⁶. Moreover, the biochemical evaluation by electrophoresis of cytoskeletal assembly after thrombin stimulation of uremic platelets under HD treatment showed a defective incorporation of the contractile proteins when compared with that observed in control platelets^16,40. Our present results confirm those observations.

Platelet cytoskeleton also plays a role in localizing signaling molecules. In platelets from healthy individuals, some of the proteins susceptible to be phosphorylated at tyrosine residues are known to be localized at a submembrane level in resting conditions²⁶. After thrombin activation and as we have observed in control platelets, they appeared phosphorylated and associated with the cytoplasmic cytoskeleton (recovered at the low-speed cytoskeletal fraction). In platelets from uremic patients under HD treatment, in which the cytoskeletal assembly was defective, a certain degree of tyrosine phosphorylation was detected but not properly associated with the polymerized cytoskeletal matrix (low-speed fraction). In our study, the shift from HD to CAPD treatment resulted in a partial recovery of the cytoskeletal response of uremic platelets. Interestingly, signaling through tyrosine phosphorylation in platelets from patients under CAPD treatment showed similar patterns to those observed in control platelets. In this group of patients, the nitrogen retention products and creatinine levels did not change or even increase slightly under the CAPD therapy. Therefore, this improvement seems to be unrelated to a correction of the uremic status of the patients. The better preservation of platelet cytoskeletal functions may be in agreement with the improved hemostasis observed by previous authors in patients with CAPD treatment^24,25,37.

Several reports have pointed out that HD induces platelet activation^41,42,43, which mainly depends on the geometry of the dialyzer and the biocompatibility of the dialysis membrane used⁴⁴. Until now, HD-induced platelet activation has been indirectly estimated by measuring increases in plasma levels of different intraplatelet substances or by the detection of P-selectin (GMP-140) expression on platelet membrane by flow cytometry⁴⁵. It is a fact that platelets can circulate even after mild activation⁴⁶; thus, the a relative storage pool deficiency described in uremic platelets¹² may be a result of a partial degranulation due to platelet activation by the HD procedure. Recent studies on platelet RNA contents suggest that current HD procedures alter the platelet life span probably through elimination of the younger and biologically more active platelets⁴⁷. In any case, the possibility of a deleterious effect of mechanical stress caused by HD on platelet function, being compensated by the beneficial action of HD clearing plasma toxins^3,6,17, deserves full consideration.

Our results show that several parameters of platelet function, specifically signal transduction mechanisms, improved when ESRD patients on regular HD were switched to CAPD. The recovery of platelet function was not related to an increase in platelet or erythrocyte counts, nor to an improvement of the uremic milieu. In our opinion, the repeated platelet activation induced by HD procedures would lead to platelet exhaustion by compromising signal-transduction mechanisms. Moreover, our results suggest that derangement of the cytoskeletal organization or a lack of synchronization between the cytoskeletal assembly and the signal transduction processes may lead to impaired platelet function.

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Acknowledgments

This work was partially supported by grants FIS 98/321 from Fondo de Investigaciones Sanitarias de la Seguridad Social and SGR97-133 from CIRIT.