Platelet Factor 4 Antibodies and Tissue Factor Pathway Inhibitor Plasma Antigen Level in Chronic Dialysis

Thrombosis Research 89 (1998) 115–122

REGULAR ARTICLE

Platelet Count, Anti-Heparin/Platelet Factor 4 Antibodies and Tissue Factor Pathway Inhibitor Plasma Antigen Level in Chronic Dialysis Guido Luzzatto1, Massimo Bertoli2, Giuseppe Cella1, Fabrizio Fabris1, Barbara Zaia1 and Antonio Girolami1 2 Department of Medicine and 2Division of Nephrology and Dialysis, Padua University Hospital, Padova, Italy.

1 nd

(Received 30 September 1997 by Editor M. Cattaneo; revised/accepted 24 November 1997)

Abstract We studied 50 chronic dialysis patients with endstage renal disease. Mean platelet count was within normal limits. An inverse linear correlation was observed between pre-dialysis platelet count and serum creatinine (r50.304, p50.038). Dialysis caused a decrease in platelet count (2166803109/ L, pre; 198668, post; p50.0001), and the higher the pre-dialysis platelet count, the greater the decrease (r50.623, p50.0001). Post-dialysis triglyceride decreased (1.6761.27 mmol/L, pre; 1.2360.96, post; p50.0001). Tissue factor pathway inhibitor (TFPI) antigen plasma level was higher in uremic patients than in controls (114642 ng/ml vs. 64612, p5 0.0001). TFPI increased 2.3 times following dialysis and such an increase was directly correlated with post-dialysis plasma heparin concentration (r5 0.571, p50.0002) and inversely correlated with postdialysis triglyceride variation (r50.407, p50.005). Six of fifty patients (12%) had anti-heparin/platelet factor 4 antibodies (Hab), 3 IgG, and 3 IgM. Female sex and the use of cuprophane membranes were more frequent among Hab-positive patients (p5 0.0001), while a lower percentage of them were on anti-aggregating drugs as compared to Hab-negative Abbreviations: Hab, anti-heparin/platelet factor 4 antibodies; HIT, heparin-induced thrombocytopenia; PF4, platelet factor 4; TFPI, tissue factor pathway inhibitor. Corresponding author: Guido Luzzatto, Patologia Medica 2, 88 piano Monoblocco, via Niccolo` Giustiniani 2, 35128 Padova, Italy; Tel 139 49 821 2668; Fax 139 49 821 2661.

patients (p50.002). Only one Hab-positive patient was slightly thrombocytopenic and none showed bleeding or thrombotic manifestations. Serum albumin and g globulin decreased following dialysis in Hab-positive patients, while the opposite was seen in those Hab-negative (22.4761.72 g/L, vs. 0.2161.77, p50.001 and 20.4860.60 g/L vs. 0.6460.97, p5 0.007, respectively). In vivo factors other than Hab are involved in the development of heparin-induced thrombocytopenia. Besides a blunted immunological response, increased levels of TFPI, the use of anti-aggregating drugs, and the observed behavior of serum proteins might play a role in this regard.  1998 Elsevier Science Ltd. Key Words: Platelet count; Thrombocytopenia; Heparin; Protease inhibitors; Chronic kidney failure; Dialysis

T

he immunological form of heparin-induced thrombocytopenia (HIT) is the most severe side effect of heparin therapy [1]. In fact, it is unpredictable and may lead to paradoxical thrombotic events with relevant morbidity and mortality [2]. The syndrome is due to antibody-mediated platelet activation and the main antigen is represented by the heparin/platelet factor 4 (PF4) complex [3]. It is still debated whether heparin/PF4 complexes form on the surface of platelets to provide a site for subsequent antibody binding [4], or immune complexes are formed first in plasma and then bind to platelet Fc receptors [5]. Other heparin-associated antigens and so far unidentified in vivo factors are also implied in the pathogenesis

0049-3848/98 $19.00 1 .00  1998 Elsevier Science Ltd. Printed in the USA. All rights reserved. PII S0049-3848(97)00301-0

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of the syndrome [6]. Particular lots of the drug have been implicated in the syndrome [7], depending on the degree of sulfation and molecular weight [8]. Despite repeated exposure to heparin, the literature is relatively scarce about HIT in dialysis patients and articles usually deal with occlusion of the arteriovenous access, while more severe complications rarely occur [9,10]. A prevalence of antiheparin/PF4 antibodies (Hab) of 0–3.9% was only recently reported in dialysis patients [11–13]. The exact incidence and prevalence of Hab in patients without HIT receiving heparin is still unknown and several questions remain about such issues [5]. Besides several platelet defects [14] and the immune dysfunction [15] characteristic of uremic patients, other in vivo factors may be implicated in the rarity of heparin-related complications in chronic dialysis. Tissue factor pathway inhibitor (TFPI) is a protease inhibitor with strong anticoagulant properties, due to activity against the factor VIIa/tissue factor complex, with factor Xa acting as a cofactor and being also directly inhibited [16]. TFPI is bound to lipoproteins in plasma, especially low-density lipoprotein (LDL), and to endothelial cell surface; about 10% is stored in platelets [17]. Heparin injection causes a 2–4-fold increase in TFPI plasma level, mainly due to mobilization from the endothelial pool [17]. TFPI, measured by functional assay, has been reported to be increased in uremic patients [18]. However, potential discrepancies may occur between the results of functional and antigenic assay of TFPI [19]. We then found it useful to study the main parameters related to the dialytic procedure, together with TFPI antigen plasma level and the prevalence of Hab, in a group of patients with end-stage renal disease.

1. Patients and Methods 1.1. Subjects and Dialysis We studied all hepatitis B- and C-negative patients undergoing chronic dialysis on antemeridian sessions at the Division of Nephrology and Dialysis of Padua General Hospital. The group consisted of 50 patients with end-stage renal disease (M529; F521), 38–88 years old (mean 66.8611.9 SD),

36–93 kg post-dialysis weight (65.3613.1). Etiology of renal failure was unknown in 11 cases, nephrosclerosis and polycystic kidney in nine cases each, chronic glomerulonephritis in seven, diabetic nephropathy in six, interstitial nephritis in three, tuberculosis in two, and Alport’s syndrome, multiple myeloma and chronic pyelonephritis in one case each. They were on chronic dialysis for 1–263 months (54.0658.8), 49 three times a week, and one twice weekly. In all patients an arteriovenous fistula was used as vascular access. The dialysate and blood flow rates were 250–350 ml/min21 and 500 ml/min21, respectively. Cuprophane membranes were used in 26 cases, polysulfone in eight, cuprophane and polysulfone in six, ethylvinylalcohol in five, polyacrilonitryle in four, and polymetacrylate in one case. All membranes were disposable, as well as polyvinylchloride connecting lines. Dialysis time was 3–4 hours (3.3660.28). After pre-washing dialyzers with 2000 ml of saline containing heparin 5 IU/ml, heparin was given, without any priming, at the rate of 250–4000 IU/ hour (18816681 SD), for a total dose of 750–16,000 IU/session (641962617). Unfractioned heparin with a molecular weight of 13–16 kD was from seven different lots extracted from porcine hog mucosa (Biologici Italia Laboratories, Milan, Italy). Thirty-three patients chronically received erythropoietin subcutaneously at the dose of 2000–18,000 U/ weekly (480064915) and 16 were on anti-aggregating agents (nine aspirin, 100 mg daily, six ticlopidine, 250–500 mg daily, and one indobufen, 200 mg daily). Patients gave informed consent to the study.

1.2. Blood Collection Blood samples were collected into vacutainer disposable tubes immediately before and at the end of each dialysis session directly from the fistula. We concurrently collected 10 ml of 3.8% Na citrate blood (1:10 v/v) for assay of plasma heparin concentration, tissue factor pathway inhibitor (TFPI) plasma antigen and anti-heparin/platelet factor 4 (PF4) antibodies (Hab). Samples were promptly centrifuged at 1500g315 minutes at room temperature and platelet poor plasma was immediately separated and stored at 2808C until assay.

1.3. Assay Procedures Hematocrit (Ht), white blood cell (WBC) and platelet count, blood urea nitrogen (BUN), serum

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creatinine, sodium (Na), potassium (K), calcium (Ca), phosphorus (P), cholesterol, triglyceride, lactate dehydrogenase (LDH), osmolarity, albumin, and g globulin were determined at the Clinical Pathology Department of our hospital by automated techniques [Coulter counter STKS (Miami, FL) for hematology; AU 5032 analyzer (Olympus Co., Japan) for biochemical profile and Paragon CZE 2000 analyzer (Beckman, Brea, CA) for serum proteins]. Plasma heparin concentration was determined by a chromogenic substrate assay, using a commercially available kit (Berichrom Heparin, Behring, Marburg, Germany). TFPI antigen plasma level was determined by an ELISA method using a commercially available kit (Imubind, American Diagnostica Inc., Greenwich, CT), as previously described [20], and results were expressed in ng/ml. The fasting levels of TFPI in 20 sex and age-matched patients with no sign of renal or liver failure were used as controls. To overcome the influence of hemoconcentration, the post-dialysis values of WBC and platelet count, BUN, creatinine, cholesterol, triglyceride, LDH, albumin, g globulin, and TFPI were corrected according to Keber [21]. The assay of Hab was performed by an ELISA method according to Visentin et al. [22], as previously described [23]. In short, 50 mL of heparin/ PF4 solution were added to the test wells of a 96well flat bottom microtiter plate (Costar, Cambridge, MA) and heparin alone was added to the control wells. Sera from patients and controls were diluted 1:100 in 0.05 M phosphate buffered saline/ 2% adult bovine serum and incubated with heparin/PF4 bound to the solid phase. After screening by a polyvalent antiserum, bound IgG or IgM was detected by alkaline phosphatase-conjugated goat anti-human IgG or IgM (Sigma, St. Louis, MO). Plasma of a proven HIT patient was used as a positive control. Samples from apparently healthy volunteers never exposed to heparin were used as negative controls. Results were expressed as the difference in optical density at 405 nm between each test well (heparin/PF4) and the respective control (heparin alone). Samples with optical density difference straying by .2 SD from the mean difference of negative samples were considered positive. In order to quantify positivity, we further expressed

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the results as the ratio between each positive and the cut-off value.

1.4. Statistical Analysis Delta was defined as the difference between any post-dialysis determination and its respective predialysis value. Data were analyzed by Student’s t-test for paired and unpaired data, Pearson’s correlation (r), and chi-square test.

2. Results A significant linear correlation was observed between patients’ weight and the total amount of heparin administered per dialysis session (r50.361, p50.01, not shown). Dialysis time and the hourly amount of heparin administered per session were significantly higher in patients treated with cuprophane-containing membranes than in those treated with membranes not containing cuprophane (3.446 0.28 hours vs. 3.2460.25; t52.56, p50.014 and 20366564 IU/hour vs. 16076795; t52.22, p50.03, respectively). Results of hematology, biochemical profile, serum protein determination, plasma heparin concentration, and TFPI are summarized in Table 1. All patients, but two females, were slightly anemic. WBC count was within normal limits and did not significantly change following dialysis; however, when sub-grouping patients according to the type of membrane utilized, post-dialysis WBC count was significantly higher in patients treated with cuprophane membranes than in the others. Mean platelet count was within the normal range. Six patients were thrombocytopenic, but platelet count was below 503109/L only in one of them, who was also suffering from liver cirrhosis, while it was 87–1243109/L in the other five thrombocytopenic patients. None showed bleeding or thrombotic manifestations. Pre-dialysis values of BUN, creatinine, K, and P were above normal limits. A significant postdialysis decrease in platelet count, BUN, creatinine, K, P, triglyceride, and osmolarity was observed, as well as a significant increase in serum Ca and g globulin. Basal TFPI in dialysis patients was significantly higher than in controls (114642

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Table 1. Mean values of hematology, biochemical profile, serum protein, plasma heparin, and tissue factor pathway inhibitor (TFPI) antigen plasma level before (Pre) and after (Post) dialysis

Ht WBC Cuprophane Noncuprophane Platelets BUN Creatinine Na K Ca P Cholesterol Triglyceride LDH Osmolarity Albumin g globulin Plasma heparin TFPI a

L/L 3106/L 3109/L mmol/L mmol/L mmol/L mmol/L mmol/L mmol/L mmol/L mmol/L U/L mosm/L g/L g/L U/L ng/ml

Pre

Post

0.31260.06 7.15562.218 7.33362.096 6.81262.472 216680 24.765.0 7086160 138.463.0 5.260.83 2.360.22 1.5860.48 4.9461.33 1.6761.27 3546134 29869 38.463.6 10.163.0 NA 114642

0.34460.07 7.16762.967 7.794b63.020 5.993b62.553 198668 7.562.3 262669 139.162.7 3.660.55 2.660.27 0.8160.47 4.8161.23 1.2360.96 3686120 28267 38.263.8 10.663.1 5126353 2616128

t NAa 0.01 26.58 229.58 226.29 1.27 212.42 9.61 28.15 20.86 25.58 1.04 28.07 20.53 3.17 NA 6.94

p NA NS

0.0001 0.0001 0.0001 NS 0.0001 0.0001 0.0001 NS 0.0001 NS 0.0001 NS 0.002 NS 0.0001

NA, not applicable; NS, nonsignificant. b t52.028, p50.048.

ng/ml vs. 64612, t55.15, p50.0001) and increased 2.3 times following dialysis. As shown in Figure 1, we observed a significant inverse linear correlation between the pre-dialysis values of creatinine and platelet count. As shown in Figure 2, we also observed a significant inverse linear correlation between pre-dialysis platelet count and its post-dialysis variation. Post-dialysis

Fig. 1. Inverse linear correlation between the pre-dialysis values of serum creatinine and platelet count (3109/L).

plasma heparin concentration was inversely correlated with post-dialysis triglyceride variation (r5 0.509, p50.0003, not shown) and directly correlated with the change in TFPI plasma antigen level following dialysis (r50.571, p50.0002, not shown). Figure 3 shows the significant inverse linear corre-

Fig. 2. Inverse linear correlation between the pre-dialysis value of platelet count (3109/L) and its change following dialysis (Delta platelets).

2.961.4 ,1 20.4860.60b 0.6460.97b Hab1 Hab2 Percent

(n56) (n544) Hab1 Hab2 Mean6SD

F583.3%d F536.4%d

Yes516.7%c Cp583.3%d Yes534.1%c Cp561.4%d

212684 217681

188668 200668

22.4761.72a 0.2161.77a

3.961.7 ,1

Negative 2.76 4.25 Negative Negative 1.55 0.31 20.85 21.07 21.2 0.14 20.18 No Yes No No No No 12(F) 19(F) 25(F) 30(M) 33(F) 48(F)

Cp Cp1Ps Cp Cp Ps Cp

181 232 177 124 189 369

157 206 148 127 173 317

20.26 22.94 23.82 24.82 21.02 21.97

5.11 Negative Negative 4.61 1.97 Negative

Hab Igme Hab IgGe Delta g g/L Delta alb g/L Post Pre AAD

Dialyzer

Platelets3109/L

Patient #(sex)

lation between the change in serum triglyceride following dialysis and post-dialysis TFPI plasma antigen values. The results of the assay for anti-heparin/PF4 antibodies (Hab) are summarized in Table 2. Three patients (M50, F53) were positive for Hab IgG and three patients (M51, F52) were positive for Hab IgM. The cut-off ratio was 3.961.7 for IgG and 2.961.4 for IgM, while it was around 10 for the HIT positive control. The higher prevalence of females among Hab-positive patients, as compared to those Hab-negative, was statistically significant (5/65 83.3% vs. 16/44536.4%; x2521.1, p50.0001). In Hab-positive patients, heparin was from five different lots out of total seven. The use of dialyzers containing cuprophane, alone or in association with polysulfone, was significantly higher in Hab-positive patients, as compared to those Hab-negative (5/6583.3% vs. 27/44561.4%; x2521.9, p50.0001). The percentage of patients on anti-aggregating agents was lower in Hab-positive than in Hab-negative patients (1/6516.7% vs. 15/ 44534.1%; x259.96, p50.002). In Hab-positive patients we observed a slight decrease in serum albumin and g globulin concentration following dialysis, while the opposite was seen in Hab-negative patients (22.4761.72 g/L, vs. 0.216 1.77, t53.46, p50.001 and 20.4860.60 g/L vs. 0.646 0.97, t52.83, p50.007, respectively). None of the other investigated parameters was significantly dif-

Table 2. Main parameters related to anti-heparin/platelet factor 4 antibodies

Fig. 3. Inverse linear correlation between the change in serum triglyceride following dialysis (Delta triglyceride) and post-dialysis tissue factor pathway inhibitor antigen (TFPI) plasma level.

AAD, antiaggregating drugs; Cp, cuprophane; Ps, polysulfone; Delta alb; Delta g: change in serum albumin and gamma gobulin levels following dialysis. Hab: anti-heparin/platelet factor 4 antibodies. a p50.001; bp50.007; cp50.002; dp50.0001 when comparing Hab-positive (Hab1) to Hab-negative (Hab2) patients. e Hab results were expressed as the ratio between their optical density and the cut-off value.

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ferent in Hab-positive patients as compared to Habnegative ones. In particular, no difference was observed in duration of uremia and, as a consequence, the number of exposures to heparin between Habpositive and Hab-negative patients.

3. Discussion Slight thrombocytopenia may occur in uremia, mainly as a result of impaired thrombopoiesis [14]. In accord with this, we found that only a minority of chronic dialysis patients suffered from a mild decrease in platelet count. We then observed an inverse linear relationship between pre-dialysis serum creatinine and platelet count. The existence of a correlation between such two variables is not by itself a proof of a cause/effect relationship. However, a negative effect of creatinine on platelet count, due either to an inhibitory effect on thrombopoiesis or to direct cytotoxicity, might be hypothesized. We also found that dialysis caused a significant decrease in platelet count, properly corrected for hematocrit, and the higher the platelet count, the greater the decrease. These data are in keeping with a platelet activation state and mechanical damage caused by the extracorporeal circulation [24]. In accord with previous data by Kario et al. [18], basal TFPI also was increased in our chronic dialysis patients, likely due to impaired TFPI clearance or vascular damage with increased TFPI release [18]. We also confirmed that, even using an antigenic assay instead of a functional one, there is a strong correlation between plasma heparin concentration and the increase in TFPI following dialysis. Our evidence may also suggest that while TFPI in plasma is reported to be mainly associated to lowdensity lipoprotein in normals [17], triglyceride could be a major source of TFPI in chronic dialysis patients. In fact, we found a strong correlation between the change in triglyceride following dialysis and post-dialysis TFPI levels. In contrast, we saw no correlation between TFPI and cholesterol. We found that 12% of our chronic dialysis patients had anti-heparin/PF4 antibodies, with a higher prevalence among females and patients treated with cuprophane-containing membranes. The influence of genetic factors may be at the root of the gender influence, as elsewhere proposed [5]. The role of

cuprophane membranes may be related to the higher amounts of heparin they require, which could be more relevant than the number of exposures to such drug. Furthermore, cuprophane membranes are known to be less biocompatible [25] and postdialysis WBC count was actually higher in patients using such membranes. Hab prevalence in our patients was higher than elsewhere reported in dialysis patients [11–13]. This could be explained by the use in other series also of low molecular weight heparin, which may be less antigenic than the unfractioned form [26], by a different ELISA [27] or a more stringent cut-off point for assessing Hab positivity. However, similar [28, 29] or even higher [30] presence of Hab have been reported in non-uremic patients receiving heparin. A protective role of anti-aggregating drugs could be suggested by their higher use in our Hab-negative patients. In spite of Hab presence, we observed no signs or symptoms of heparin-induced thrombocytopenia (HIT), nor related thrombotic complications. This has been recently reported also in different categories of patients [29,30] and it has been proposed that only high-titer antibodies—triggered by other in vivo cofactors—could be pathogenic [30]. Although dilutions and optical density are not directly interchangeable, the lower optical density in our Habpositive asymptomatic patients, as compared to the HIT positive control, seems in accord with such an hypothesis. Moreover, besides the immune dysfunction [15] and several platelet defects [14] characteristic of uremic patients, increased levels of TFPI, which has strong anticoagulant properties, might play a protective role toward the development of HIT. Finally, in Hab-positive patients serum albumin and g globulin decreased following dialysis, while they increased in Hab-negative patients. These data might be at the root of either a causative factor in Hab development, or a protective role against their clinical manifestations.

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