Plasma transforming growth factor ß1 and platelet activation: implications for studies in transplant recipients

Beatrice M. Coupes, Shelley Williams, Ian S. D. Roberts1, Colin D. Short and Paul E. C. Brenchley

Renal Research Laboratories, Manchester Institute of Nephrology and Transplantation, The Royal Infirmary, Manchester, and 1 Laboratory Medicine Academic Group, University of Manchester, UK



   Abstract
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Background. Evidence from animal models supports the hypothesis that dysregulated transforming growth factor ß1 (TGFß1) expression plays a role in chronic allograft rejection, the progression of diabetic nephropathy and fibrotic glomerulopathies. However, more evidence is required to support this hypothesis in man, and the current literature concerning blood TGFß1 levels in clinical studies is highly confused. We have investigated: (i) the hypothesis that the widespread practice of activating clinical samples prior to measurement of TGFß1 is detecting the platelet-released pool of TGFß1, artefactually generated on venepuncture and unrepresentative of the real circulating in vivo TGFß1 pool; and (ii) the effect of different immunosuppressive drugs on apparent TGFß1 plasma levels.

Methods. The effect of two different venepuncture procedures on plasma TGFß1 was compared in 10 healthy volunteers, one procedure designed to minimize platelet activation and the other representing standard venepuncture practice in a clinic situation. Blood samples from 52 renal transplant recipients on either cyclosporine or tacrolimus immunosuppression were taken by standard venepuncture to investigate the effect of immunosuppressive drugs on plasma TGFß1. Plasma TGFß1 and ß thromboglobulin were measured by ELISA.

Results. Among 10 healthy volunteers who underwent two different methods of venepuncture, eight of 10 had undetectable levels of TGFß1 (<100 pg/ml) under conditions that minimize platelet activation. In contrast, all 10 paired plasma samples collected by vacutainer had measurable TGFß1 (median 7.70 ng/ml, interquartile range 5.87–13.64 ng/ml) following acid/ urea activation. The median ßTG level (a measure of platelet degranulation) was 0.71 µg/ml (interquartile range 0.53–1.19 µg/ml) in the special collections compared with 3.39 µg/ml (interquartile range 2.27–4.33 µg/ml) in the vacutainer samples (P=0.0029).

Among 52 allograft recipients there was a significantly higher mean TGFß1 level in plasma from patients on cyclosporine therapy compared with patients on tacrolimus (28 090±26 860 pg/ml vs 7173±10 610 pg/ml, respectively; P<0.002). Mean plasma ßTG levels were also significantly higher during cyclosporine therapy compared with tacrolimus (8.14±5.54 µg/ml vs 3.66±3.32 µg/ml, respectively; P<0.002). However, when TGFß1 values were corrected for the degree of platelet activation (by factoring with ßTG) there was no significant difference between TGFß1 levels on cyclosporine or tacrolimus (4117±2993 pg/µg ßTG vs 2971±658 pg/µg ßTG, respectively; P=0.294).

Conclusions. To avoid erroneous hypotheses concerning TGFß1 and perpetuating confusion in the literature over levels in health and disease, it is imperative that proper internal controls for platelet activation are used. The effects of experimental treatments and drugs on platelet biology must be rigorously controlled when attempting to measure and interpret plasma levels of TGFß1 in clinical practice.

Keywords: immunosuppression; platelets; renal allografts; TGFß1; immunoassay



   Introduction
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
There is little doubt that TGFß1 is profibrotic in many disease conditions. In an experimental model, persistent expression of active TGFß1 in the vasculature promotes fibrosis in the kidney [1]. Neutralizing the effects of TGFß1 in experimental models with natural antagonist [2] protects against the development of tissue fibrosis.

Clinical studies of fibrotic disease have measured circulating blood levels of TGFß1 in patients and healthy individuals to investigate the link between TGFß1 expression and the clinical presentation and prognosis of disease. A highly confused literature on blood levels of TGFß1 has developed with claims that values in normal controls are low [3] or high [4], that certain drugs induce high levels of circulating TGFß1 [5], and that patients in end-stage renal failure (ESRF), in particular African Americans [6], have high levels of circulating TGFß1.

Attention has been drawn recently to the difficulties involved in interpretation of data on TGFß1 blood levels [7]. The complex interaction of factors including the choice of blood sample (serum or plasma), the deliberate sample activation, the technical measurement of TGFß1 by ELISA, and the presence of latent and active pools of TGFß1 have a profound influence on the interpretation of TGFß1 levels in a clinical context. The fundamental problem in estimating a real in vivo circulating level of TGFß1 is how to avoid the enormous platelet pool of readily releasable TGFß1. TGFß1 was originally isolated from platelets [8], where it is stored at high concentration as a latent complex. Most of the confusion in the literature relates to whether or not researchers adopt a strategy to avoid, minimize or control for platelet-released TGFß1 occurring in the sample ex vivo.

The choice of serum as the medium in which to quantitate TGFß1 seems particularly inappropriate for two reasons. First, there is a significant correlation between the total platelet count and TGFß1 levels [9] (measured after activation of the latent molecule, see below), which is common to other platelet-stored proteins e.g. ß-thromboglobulin [10]. Secondly, it has been demonstrated that 95% of the TGFß1 in serum is artefactually derived from platelets on blood clotting [3]. This accounts for serum TGFß1 levels being reported as 10- to 20-fold [11], 20-fold [3] and 3.84-fold [9] higher than plasma levels.

TGFß1 levels in plasma (measured after activation of the latent molecule and often referred to as total level) have also been reported by workers in clinical studies of TGFß1 expression as representing a circulating blood level [3]. The recommendation that plasma TGFß1 levels should be related to the level of a recognized platelet release protein (ß thromboglobulin or platelet factor 4) has unfortunately not been adopted as standard practice [3]. Workers who recognize the danger of platelet-released TGFß1 recommend the use of platelet poor plasma [12], but this does not address or control for the variable platelet release of proteins that occurs during the trauma of venepuncture.

This study is therefore designed (i) to illustrate the effect of venepuncture on the non-specific release of TGFß1 from platelets at the point of blood collection, and (ii) to demonstrate how a failure to correct for platelet activation can lead to erroneous conclusions about the effect of different immunosuppressive drugs on plasma TGFß1 expression. This study is not designed to recommend the optimum method of venepuncture or quantitation of plasma TGFß1, but is investigating the effects of two important modulators of platelet activation (trauma at venepuncture and drug-platelet effects) on the subsequent measurement of plasma TGFß1 after deliberate sample activation and how these impact on our interpretation of TGFß1 biology in renal transplantation.



   Subjects and methods
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
The effect of venepuncture: the healthy volunteer group
Ten healthy volunteers (mean age±SEM 34.4±2.3 years; 7 males, 3 females; 9 Caucasions, 1 non-Caucasion) were subjected to two venepunctures from two different sites on the same occasion by both the special procedure and the standard procedure. Informed consent was obtained for venepuncture.

Special venepuncture procedure
This procedure is recommended to minimize platelet activation during venepuncture [12]. Blood samples were collected from healthy volunteers without the use of a tourniquet by introducing a wide bore (16 gauge needle) into the antecubital vein with minimal trauma and allowing blood to flow slowly without suction from syringe or vacutainer into a pre-chilled EDTA bottle. Samples were separated immediately by centrifugation at 1200 g for 10 min and the plasma aliquots were stored at -70°C.

Standard venepuncture
Blood samples were collected from healthy volunteers using a tourniquet by venepuncture with a 21 gauge needle and vacutainer blood collection system (Becton and Dickenson, Oxford, UK). Blood was collected into EDTA tubes and plasma was immediately separated by centrifugation at 1200 g for 10 min, and plasma aliquots were stored frozen at -70 °C. Thus, trauma during venepuncture was the only difference in the procedure for obtaining plasma samples from normal volunteers.

The effect of immunosuppressive drugs: the allograft recipients
Blood samples from 52 patients (mean age±SEM 44.2±2.0 years; 35 males, 17 females; 44 Caucasion, 8 non-Caucasians) undergoing renal transplantation at Manchester Royal Infirmary were obtained by standard venepuncture procedure as part of normal clinical follow up and monitoring of graft function. Samples were separated within 3 h by centrifugation at 1200 g and storage at -70°C. Thirty-two patients were receiving cyclosporine-based immunosuppression with a mean drug trough level of 156 ng/ml, range 64–275 ng/ml (monotherapy or combined with steroids and azathioprine), and 20 patients received tacrolimus monotherapy with a mean drug trough level of 13 ng/ml, range 8–36 ng/ml. In the total patient group, the methods of venepuncture and sample preparation were kept constant (and were typical of those employed widely in clinical practice) to allow comparison of the effects of immunosuppressive drugs on TGFß1 levels.

Acid/urea activation of plasma TGFß1
The method was based on the R&D Systems (Abingdon, UK) procedure from the Quantikine Immunoassay kit. To 100 µl of plasma, 100 µl of 2.5 N acetic acid/10 M urea were added, mixed and incubated for 10 min at room temperature. The samples were neutralized by adding 100 µl of 2.7 N NaOH/1 M HEPES and added to the assay plate within 15 min of activation.

Acid activation of plasma TGFß1
One-and-a-half microlitres of 6 M HCl was added to 100 µl of plasma and incubated for 15 minutes at room temperature. Samples were neutralized to pH 7.0–7.4 with ~3.0 µl of neutralizing buffer (50% vol/vol 1 M HEPES/6 M NaOH).

Immunoassay for TGFß1
The in-house assay has been described in detail [13]. Briefly, the capture antibody was a monoclonal to TGFß1,2,3. (Genzyme, Framingham, USA, clone 1D11) and the detection antibody was a chicken polyclonal antibody specific to TGFß1 (R&D Systems), followed by peroxidase conjugated donkey anti-chicken Ig (Jackson Labs, Stratch, Luton, UK). Signal was generated by the addition of Amerlite substrate (Johnson and Johnson Clinical Diagnostics, Ascot, UK) and read on a Microlumat LB96P luminometer (EG&G Berthold, Leeds, UK). Data was analysed using Mikrowin software.

Assay for ß thromboglobulin (ßTG)
An ELISA for ßTG was employed using the principle of competition between soluble ßTG in plasma and solid phase ßTG coated onto an ELISA plate for a limited amount of anti-ßTG antibody added to the samples. Purified ßTG (Novabiochem Ltd, Nottingham, UK) was coated onto ELISA plates at 200 ng/ml in 100 µl of 0.1 M sodium bicarbonate/carbonate buffer, pH 9.6 for 16 h. After washing, the plates were blocked with 5% bovine serum albumin (BSA) for 1 h. Plasma samples were diluted 1:25 with assay buffer (5% BSA in PBS with 0.05% Tween 20) and mixed with an equal volume of rabbit anti-ßTG antibody (at a dilution of 1:2000) and added to the plate. ßTG standards covering the range 3.0–600 ng/ml were included and the assay was calibrated against the WHO 1st International Standard Preparation for ßTG (83/501). After incubation for 4 h, the plates were washed and incubated with peroxidase-conjugated anti-rabbit IgG (Jackson Labs) (dilution 1:8000) for 2 h. Following washing, ABTS substrate was added and the absorbance was measured at 405 nm in a multiwell spectrophotometer and results calculated using Softmax software.

Statistical methods
Non-parametric data sets were analysed by Mann–Whitney U-test for differences in median. Data sets showing a normal distribution were analysed for differences in mean by students t-test or paired t-test. A correlation between TGFß1 and ßTG was assessed using linear regression and Pearson's correlation test. Significance was attributed to analyses with P<0.05.



   Results
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
The effect of venepuncture
Ten healthy normal individuals volunteered to undergo two venepunctures on the same occasion using the special procedure (to avoid platelet activation) applied to one arm and the standard method with vacutainer (to replicate normal trauma causing platelet activation) to the other. The subsequent processing of the samples was identical to allow investigation of the effect of venepuncture trauma only. The samples were analysed for the presence of TGFß1 and ßTG, and the results are shown in Table 1Go and Figure 1Go. Eight of 10 samples taken under conditions that avoid platelet activation showed undetectable levels of TGFß1 (<100 pg/ml) following acid/urea activation by the in-house assay (Figure 1aGo). In contrast, all the paired samples taken by standard venepuncture had measurable levels of TGFß1 (median 7.70 ng/ml, interquartile range 5.87–13.64 ng/ml). In parallel measurements of ßTG (Figure 1bGo) in the same samples, the median value of ßTG in the special samples was 0.71 µg/ml (interquartile range 0.53–1.19 µg/ml) compared with a median value of 3.39 µg/ml (interquartile range 2.27–4.33 µg/ml) in the standard samples, a significant 4.8-fold increase (P=0.0029 by the Mann–Whitney U-test). However, these values for ßTG clearly indicate that despite the precautions taken with the special venepuncture procedure, the ßTG values at 0.71 µg/ml represent a >20-fold increase above the level deemed to be present in plasma where no platelet activation has occurred, but ~20-fold less than is found in serum [12].


View this table:
[in this window]
[in a new window]
 
Table 1. TGFß1 and ßTG results on 10 normal blood samples taken on the same occasion by two different venepuncture methods

 


View larger version (16K):
[in this window]
[in a new window]
 
Fig. 1. The effect of venepuncture procedure on the detection of (a) TGFß1 and (b) ßTG in paired plasma samples from normal subjects following deliberate sample activation. Values are represented as mean±1 SD, with values below detection limit designated as zero.

 
The two special samples that had detectable TGFß1 also had the highest levels of ßTG (4.98 and 1.70 µg/ml) and their paired standard samples showed the lowest increases in ßTG (Figure 1bGo), indicating either a significant in vivo platelet activation in these normal individuals or a greater sensitivity to platelet activation on venepuncture. By expressing the results from the standard venepuncture as TGFß1:ßTG ratio, the spuriously high TGFß1 levels are corrected for degree of platelet activation (Table 1Go).

The effect of immunosuppressive drugs on platelet-released TGFß1 and ßTG
TGFß1 and ßTG were measured in plasma samples taken by standard venepuncture from renal transplant patients on either cyclosporine- or tacrolimus-based immunosuppressive therapy and the results are shown in Figure 2Go. There was a significant relationship between TGFß1 and ßTG in the sample population when analysed by Pearson's correlation test, r=0.751, P<0.0001.



View larger version (10K):
[in this window]
[in a new window]
 
Fig. 2. Correlation of TGFß1 levels and ßTG levels in plasma samples from 52 transplant patients after deliberate sample activation. Pearson's correlation test, r=0.751, P<0.0001.

 
There was no correlation between the platelet count and either TGFß1 (Pearson correlation r=-0.067, P=0.634) or ßTG (r=0.025, P=0.959) in these samples (data not shown).

There was a significantly higher mean concentration of TGFß1 in samples from patients on cyclosporine therapy compared with patients on tacrolimus (28 090±26 860 pg/ml vs 7173±10 610 pg/ml, respectively; students t-test P<0.002) (Figure 3aGo). Mean ßTG values were also higher in samples from patients on cyclosporine therapy compared with those on tacrolimus (8.14±5.45 µg/ml vs 3.66±3.32 µg/ml, respectively, students t-test P<0.002) (Figure 3bGo). However, when the TGFß1 values were corrected for the degree of platelet activation by factoring with ßTG, there was no significant difference in plasma TGFß1 between patients on cyclosporine or tacrolimus (4117±2993 pg/µg ßTG vs 2971±658 pg/µg ßTG, respectively; P=0.294) (Figure 3cGo).



View larger version (24K):
[in this window]
[in a new window]
 
Fig. 3. Plasma values (mean±SD) in patients on cyclosporin () or tacrolimus (). (a) TGFß1; P<0.002 students t-test. (b) ßTG; P<0.002 students t-test. (c) TGFß1 per µg ßTG; P=0.294, not significant, students t-test. (d) Platelet count P=0.113, not significant, students t-test.

 
There was no difference in the mean absolute platelet count between patients on cyclosporine- or tacrolimus-based therapy (295±84 vs 258±72x109/l, respectively; P=0.113) (Figure 3dGo).



   Discussion
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
This report documents a median plasma TGFß1 level of 7.7 ng/ml (interquartile range 5.87–13.64 ng/ml) following acid/urea activation of the plasma samples taken by standard venepuncture from healthy controls. This is consistent with an aggregated mean of 8.1 ng/ml from other reported normal plasma values of 3.8 ±2.9 ng/ml [11], 12.2 ng/ml (2.2–19.3 ng/ml) [9] and 8.2 ng/ml (4.0–18.9 ng/ml interquartile range) [14], where standard venepuncture has been used.

The one study carefully established to avoid platelet activation during venepuncture reports 4.1±2.0 ng/ml (range 2.0–12.0 ng/ml) [3]. However, we have demonstrated that this plasma TGFß1 level is artefactually generated by activating platelets during the standard venepuncture procedure and therefore does not represent the in vivo circulating pool of TGFß1. Eight of 10 paired samples from healthy volunteers show <100 pg/ml TGFß1 when precautions to reduce platelet activation are introduced. The two normal samples with the detectable level of TGFß1 have the highest level of ßTG, an accepted measure of platelet {alpha} granule secretion [12], consistent with either in vivo platelet activation or activation despite the special procedure. Furthermore, dual measurement of TGFß1 and ßTG in 52 plasma samples from allograft recipients taken by standard venepuncture shows a significant positive correlation, indicating that the degree of platelet activation either in vivo or during venepuncture determines the level of detectable TGFß1 following activation of the sample.

Thus, deliberate sample activation prior to measurement of TGFß1 simply reveals the quantity of latent TGFß1 released from platelets during venepuncture. For each subject, the TGFß1 level represents the product of their intrinsic platelet stability, the degree of trauma that these platelets suffer during venepuncture and their total platelet count. In addition to these variables, further modulation of platelet biology in vivo by disease, drug therapy, surgery, haemodialysis or ethnic influences may need to be controlled. We contend that the evidence we have provided in this study supports the hypothesis that normal plasma TGFß1 levels are undetectable (<100 pg/ml) if procedures are taken to minimize platelet activation.

It is valid to ask whether immunosuppressive drugs used in transplantation, in particular cyclosporine and tacrolimus, promote the expression of TGFß1. It is conceivable that part of their immunosuppressive action may be mediated through the potent immunosuppressive properties of TGFß1. Additionally, their chronic nephrotoxicity may be driven through the profibrotic expression of TGFß1. Therefore, we quantitated the levels of plasma TGFß1 in transplant patients on cyclosporine or tacrolimus using routine venepuncture and deliberate activation of the samples prior to measurement. Under these conditions, patients on cyclosporine appear initially to have higher plasma levels of TGFß1, compared with patients on tacrolimus. However, there are clearly increased levels of platelet activation as evidenced by increased ßTG levels in the cyclosporine samples; the apparent high plasma TGFß1 levels in patients on cyclosporine merely result from greater platelet degranulation during venepuncture. This is not surprising since cyclosporine augments platelet reactivity [15,16] whereas tacrolimus does not [17,18]. Therefore, expressing the plasma results as TGFß1 per unit of ßTG identifies no difference in the plasma total TGFß1 levels in patients on cyclosporine or tacrolimus. Thus, any study claiming to show a difference in TGFß1 levels between groups or treatments that relies on deliberate activation of the latent TGFß1 must control for the level of platelet activation in the sample as has been previously recommended [3]. Failure to include proper controls for platelet activation as a source of circulating TGFß1 has resulted in a claim that cyclosporine stimulates in vivo TGFß1 expression [5]. Yet this effect is arguably no more than that demonstrated in this paper, i.e. that cyclosporine renders platelets more sensitive to secretion of {alpha} granule proteins such as ßTG and TGFß1 when subjected to the stress of venepuncture. Similarly, a study suggesting that ethnic differences in rates of ESRF are dependent on overexpression of TGFß1 has not controlled for differential platelet activation between the groups, resulting in artefactual secretion of TGFß1. It is established that patients in ESRF on chronic haemodialysis show enhanced platelet activation [19]. Therefore, this apparent ethnic difference in expression of TGFß1 may simply reflect uncontrolled effects on platelet activation caused by different patterns of haemodialysis and prescription of anti-hypertensive drugs [20].

In order to ensure meaningful data on circulating TGFß1 levels, the ideal strategy is the adoption of the rigorous procedures necessary to avoid platelet activation during venepuncture as described by workers in the field of platelet biology [12]. To date, only one study of TGFß1 has adopted these precautions [3] and they claim that there is a low level of circulating TGFß1 from a non-platelet derived pool. These authors advocate strongly the use of a platelet release marker correction factor, which we have demonstrated does control for spurious effects of drugs causing release of TGFß1 into the blood sample. Concentrating on producing ‘platelet poor plasma’ overlooks the variable platelet secretion of TGFß1 that has already happened during venepuncture.

We suggest that the quality and validity of interpretation of most of the clinical data on circulating TGFß1 levels is seriously flawed due to the failure to control for the level of platelet activation in the blood samples. The future for measurement of clinically relevant pools of TGFß1 must be in the development of assays that quantitate the products of in vivo activated TGFß1 which may circulate as TGFß1 complexes. Stable complexes of TGFß1–endoglin have already been described and similar complexes with other natural antagonists may exist. Understanding the role of TGFß1 in disease pathology is an important goal that is not being helped currently by the assay of a spuriously high platelet-derived pool of TGFß1, as if it represented a real in vivo circulating pool of TGFß1.



   Notes
 
Correspondence and offprint requests to: Dr PEC Brenchley, Renal Research Labs, Manchester Institute of Nephrology and Transplantation, The Royal Infirmary, Oxford Road, Manchester M13 9WL, UK. Back



   References
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 

  1. Sanderson N, Factor V, Nagy P et al. Hepatic expression of mature transforming growth factor beta 1 in transgenic mice results in multiple tissue lesions. Proc Natl Acad Sci USA1995; 92: 2572–2576[Abstract]
  2. Border WA, Noble NA, Yamamoto T, Tomooka S, Kagami S. Antagonists of transforming growth factor-beta: a novel approach to treatment of glomerulonephritis and prevention of glomerulosclerosis. Kidney Int1992; 41: 566–570[ISI][Medline]
  3. Wakefield LM, Letterio JJ, Chen T et al. TGF beta 1 circulates in normal human plasma and is unchanged in advanced metastatic breast cancer. Clin Can Res1995; 1: 129–136[Abstract]
  4. Grainger DJ, Mosedale DE, Metcalfe JC, Weissberg PL, Kemp PR. Active and acid-activatable TGF-beta in human sera, platelets and plasma. Clin Chim Acta1995; 235: 11–31[ISI][Medline]
  5. Shin GT, Khanna A, Ding RC et al. In vivo expression of transforming growth factor-beta(1) in humans—Stimulation by cyclosporine. Transplantation1998; 65: 313–318[ISI][Medline]
  6. Suthanthiran M, Khanna A, Cukran D et al. Transforming growth factor-beta 1 hyperexpression in African American end-stage renal disease patients. Kidney Int1998; 53: 639–644[ISI][Medline]
  7. Fredericks S, Holt DW. TGFß quantitation can be tricky. Transplantation1999; 68: 468–469[ISI][Medline]
  8. Assoian RK, Komoriya A, Meyers CA, Miller DM, Sporn MB. Transforming growth factor-beta in human platelets. Identification of a major storage site, purification and characterization. J Biol Chem1983; 258: 7155–7160[Abstract/Free Full Text]
  9. Kropf J, Schurek JO, Wollner A, Gressner AM. Immunological measurement of transforming growth factor-beta I (TGF-beta 1) in blood, assay development and comparison. Clin Chem1997; 43: 1965–1974[Abstract/Free Full Text]
  10. Dawes J, Pratt DA, Dewar MS, Preston FE. Do extra-platelet sources contribute to the plasma level of thrombospondin? Thromb Haemost1988; 59: 273–276[ISI][Medline]
  11. Reinhold D, Bank U, Buhling F et al. A detailed protocol for the measurement of TGF beta 1 in human blood samples. J Immunol Methods1997; 209: 203–206[ISI][Medline]
  12. Pepper DS. Radioimmunoassay of platelet proteins. In: Patrono C, Peskar BA, eds. Handbook of Experimental Pharmacology. Springer-Verlag, Berlin, 1987; Vol. 82: 517–541
  13. Coupes BM, Newstead CG, Short CD, Brenchley PE. Transforming growth factor beta 1 in renal allograft recipients. Transplantation1994; 57: 1727–1731[ISI][Medline]
  14. Krasagakis K, Tholke D, Farthmann B, Eberle J, Mansmann U, Orfanos CE. Elevated plasma levels of transforming growth factor (TGF)-beta 1 and TGF-beta 2 in patients with disseminated malignant melanoma. Br J Cancer1998; 77: 1492–1494[ISI][Medline]
  15. Cohen H, Neild GH, Patel R, Mackie IJ, Machin SJ. Evidence for chronic platelet hyperaggregability and in vivo activation in cyclosporin-treated renal allograft recipients. Thromb Res1988; 49: 91–101[ISI][Medline]
  16. Fishman SJ, Wylonis LJ, Glickman JD et al. Cyclosporin A augments human platelet sensitivity to aggregating agents by increasing fibrinogen receptor availability. J Surg Res1991; 51: 93–98[ISI][Medline]
  17. Pelekanou V, Trezise AE, Moore AL, Kay JE. FK 506 and rapamycin do not affect platelet aggregation or mitochondrial function. Transplant Proc1991; 23: 3200–3201[ISI][Medline]
  18. Mysliwiec J, Azzadin A, Chabielska E, Takada A, Mysliwiec M, Buczko W. The effect of tacrolimus (FK506) and cyclosporin A (CyA) on peripheral serotonergic mechanisms in uremic rats. Thromb Res1996; 83: 175–181[ISI][Medline]
  19. Viener A, Aviram M, Better OS, Brook JG. Enhanced in vitro platelet aggregation in hemodialysis patients. Nephron1986; 43: 139–143[ISI][Medline]
  20. Wilson J, Orchard MA, Spencer AA, Davies JA, Prentice CR. Anti-hypertensive drugs non-specifically reduce ‘spontaneous’ activation of blood platelets. Thromb Haemost1989; 62: 776–780[ISI][Medline]
Received for publication: 29. 3.00
Accepted in revised form: 26. 5.00