Institute of Nephrology, St. Orsola University Hospital, Bologna, Italy
Correspondence and offprint requests to: Dr Giuseppe Cianciolo, Servizio di Nefrologia e Dialisi, Policlinico S. Orsola, Via Massarenti 9, I-40138 Bologna, Italy.
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Abstract |
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Methods. Using the ELISA method, PDGF-AB, platelet factor-4 (PF4) and ß-thromboglobulin (ß-TG) levels were determined in peripheral blood, as well as in arterial and venous haemodialyser lines, in 10 patients each of whom underwent five consecutive dialysis sessions with a CU membrane. Blood samples were taken at 0, 15, 30, 60, 120, 180 and 240 min during dialysis and at 1, 4 and 20 h after the end of the session. In the same group of patients the levels of the same molecules were also determined after a heparin bolus injection of 4500 IU, blood samples were taken at 0, 15 and 30 min after injection of the bolus.
Results. PDGF-AB serum levels increased, remained consistently high during the haemodialysis session (in particular +134±20% after 30 min, P<0.001, and +140±5% after 240 min, P<0.001) and returned to basal values only after 20 h following the end of the session. PF4 and ß-TG showed a similar trend to PDGF. The heparin bolus injection caused only a small increase (+15±5% at 30 min) in PDGF-AB serum levels.
Conclusions. PDGF-AB is released during dialysis mainly as consequence of the bloodmembrane contact and it returns only slowly to basal values.
Keywords: accelerated atherosclerosis; dialysis; growth factors; platelet activation; platelet-derived growth factor
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Introduction |
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In haemodialysis (HD) patients, in addition to the well-known risk factors that can predispose or trigger the atherosclerotic process (old age, smoking, hypertension and hypercholesterolaemia) other factors seem to accelerate the process, namely, hyperparathyrodism, hyperhomocysteinaemia and prothrombotic factors [25].
In recent years, some authors have suggested a possible role in the atherosclerosis process for platelet activation, as well as the release of growth factors. Among these, platelet-derived growth factor (PDGF) seems to have special importance since many experimental observations have recognized its specific role in the progression of vascular lesions [68].
PDGF is the most important growth factor acting on all cells of mesenchymal derivation. Its molecular mass is in the range 28 00035 000 Da, with a half-life of less than 2 min. It is synthesized by fibroblasts, endothelial cells, macrophages and platelets. PDGF is composed of two different glycoprotein chains (A and B), connected by covalent linkages. The two chains are codified by two different genes, which are expressed independently and enable cells to synthesize three isomorphic molecules: PDGF-AA, PDGF-AB and PDGF-BB. The AB isoform is stored mainly inside -platelet granules [9]. The serum level for PDGF-AB in the normal population is 29.7±14 ng/ml.
PDGF (and mainly the AB isoform) causes the proliferation of fibroblasts, smooth muscle cells and any type of mesenchymal cells, all of which, in turn, can synthesize PDGF itself. PDGF stimulates extracellular matrix synthesis, promotes smooth muscle cell migration and acts as a powerful trigger to their contraction [10]. It is well known that all these changes characterize the evolution of an atherosclerotic plaque [11].
Nevertheless, all reports about the role of PDGF in the atherosclerotic process have hitherto referred to non-renal patients. The pathogenesis of accelerated atherosclerosis in HD patients is multifactorial but it is probable that, in addition to the well-known risk factors, other mechanisms, triggered or accelerated by dialysis therapy, play an important role.
Intradialytic platelet activation, with the potential release of several growth factors that act as mediators, may represent an interesting and underexplored area.
As is well known, during HD sessions, bloodartificial material contact causes the release of the content of both platelet dense granules (adenosine diphosphate (ADP) and serotonin) and -granules, which contain, in addition to PDGF, platelet factor-4 (PF4, 358 000 Da) and ß-thromboglobulin (ß-TG, 35 800 Da). With regard to the release reaction, there is a general agreement that PF4 and ß-TG are parameters of platelet activation [12]. Also intradialytic administration of heparin into the extracorporeal circuit has been proven to stimulate platelet aggregation. The pathway for this aggregating effect is not well known and could be mediated by: (i) the inhibition of platelet adenylate cyclase, regardless of any alteration of ADP action; (ii) changes in prostaglandin metabolism [13,14].
Considering the important biological effects of PDGF and its role in atherosclerotic vascular changes, it should be interesting to know whether its serum levels change during and after a HD session, reflecting the platelet activation caused by bloodartificial material contact.
Therefore, in the present study we evaluated the intra- and post-dialytic changes in PDGF-AB serum levels, together with ß-TG and PF4 plasma levels, during HD sessions performed using a cellulosic membrane.
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Subjects and methods |
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In all procedures the same heparinization modalities were used: in the washing phase, 2 l of saline solution containing 20 000 IU of standard heparin were used (Sodic Heparin, Vister by Parke-Davis), thus during the connection procedure 500 IU was administered to the patients, and then during dialysis 4 000 IU in continuous infusion followed.
Plasma heparin activity, measured as anti-factor Xa activity, was maintained between 0.7 and 0.95 IU/ml (after 2 h), its levels were determined using an anti-factor Xa chromogenic substrate.
Sample collection
To evaluate the intra- and post-dialytic changes in the target molecules studied, PDGF-AB was determined in serum, whereas PF4 and ß-TG were determined in plasma.
Blood samples were taken at 0, 15, 30, 60, 120, 180 and 240 min during the dialysis sessions both in the peripheral blood and, to evaluate the arterious-venous (AV) variation, in the arterial and venous extracorporeal blood lines. Blood samples were taken at the arterial and venous injection sites of the arterial and venous blood lines. Peripheral blood was obtained from the contralateral arm. During HD sessions, arterial, venous and peripheral blood samples were taken at the same times from three different operators. The blood pump was maintained close to 100 ml/min and the ultrafiltration rate close to 0 1 min before the blood sampling collection phase. For the post-dialytic evaluation samples were taken 1, 4 and 20 h after the end of the session. The platelet count was determined both for the intra- and post-dialytic period at the same time intervals from the peripheral blood. All data obtained were corrected for haemoconcentration (evaluated as variation in total serum protein concentration measured from the peripheral blood) to avoid overestimation of the molecules released. The blood volume per cent reduction was calculated at 0, 30, 60, 120, 180 and 240 min, and this per cent reduction was applied to the PDGF-AB, ß-TG and PF4 values obtained from the study.
In order to distinguish the effect of HD on these target molecules from the effect due to the aggregating effect of heparin, the same patients received a heparin bolus injection alone (4500 UI) on three separate occasions. The blood samples were taken at 0, 15 and 30 min after any bolus, and the patients then underwent a planned dialysis using a lower heparin dose. All tests were performed in duplicate.
To obtain the serum sample necessary for the PDGF-AB determination we used serum separator tubes and allowed the sample to clot for at least 30 min at room temperature. We centrifuged the samples for 10 min at ~1000 g. This procedure was repeated until we obtained a clear serum, without corpuscular elements. Serum was collected in a second tube and stored at 20°C until the test was performed.
For PF4 and ß-TG plasma values, blood was collected in DiaTubes, containing citrate, adenosine, theophylline and dipyridamole. After venepuncture the first 1 ml was discarded and 4.5 ml was collected and added to 0.5 ml of an anticoagulant solution. Tubes were placed in melting ice and centrifuged 15 min later at 2000 g for 30 min. One-third of the plasma was collected in the middle region of the supernatant, aliquoted and immediately frozen at -80°C. Quantitative tests were performed within 2 weeks.
PDGF was determined by enzyme-linked immunosorbant assay (ELISA), using commercial kits (Quantikine, R&D System). This assay employs the quantitative `sandwich' enzyme immunoassay technique. A monoclonal antibody specific for PDGF-AA is first coated onto the microtitre plate provided in the kit. Standards and samples are pipetted into the wells and any PDGF-AB present is bound by the immobilized antibody. After any unbound sample proteins are washed away, an enzyme-linked polyclonal antibody specific for PDGF-BB conjugated to peroxidase is added to the wells to `sandwich' any PDGF-AB immobilized during the first incubation. Following washing to remove any unbound antibodyenzyme reagent, enzymatic activity was revealed with tetramethylbenzidine and hydrogen peroxide as the substrate solution, the colour developing in proportion to the amount of PDGF-AB bound in the initial step. The colour development was stopped with 2 N sulphuric acid and the intensity of the colour is measured at 450 nm in a spectrophotometer microplate reader (Kinetic Analyzer Milenia).
ß-TG and PF4 were again determined by ELISA, using commercial kits (Stago, Boehringer Mannheim). Microtitre plates were coated with Fab'2 fragments of IgG specific for ß-TG and PF4. Anti-ß-TG or anti-PF4 peroxidase coupled antibodies react with the specific epitopes. Enzymatic activity was revealed with O-phenylendiamine and hydrogen peroxide as substrate. Colour development was stopped with 3 M sulphuric acid and absorbency measured at 492 nm in a spectrophotometer microplate reader (Kinetic Analyzer Milenia).
Data analysis
Considering the high individual variability of PDGF, PF4 and ß-TG basal values, we decided to assess percentage variation of serum levels against the T0 value, both for intra- and post-dialytic evaluation. We also determined the per cent variation of the venous concentration compared with the arterial concentration (AV variation) at each time interval for the three target molecules studied.
The data are presented as mean±SD. Statistical evaluation was performed by means of the statistical program Statview 4.01 for Macintosh. One-way ANOVA was used to determine statistical significance; the paired Student's t-test was used to confirm the data obtained with the one-way ANOVA.
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Results |
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The data showed that the decrease was maximal 1 h after the end of the session (from 140±5% to 77±12%). After 4 h from the end of the session PDGF-AB changed from 77±12% to 45±7%, and returned to basal values only following 20 h after the end of the session. After the heparin bolus, the PDGF-AB peripheral serum levels showed an increase of 15±5% (P<0.001) after 30 min (Table 3).
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Post-dialytic evaluation showed a decrease in ß-TG values, which changed from 81±22% to 51±14% 4 h after the session end and returned to basal values after 20 h.
After the heparin bolus the ß-TG peripheral plasma levels, showed an increase of 14±5% (P<0.001) after 30 min (Table 3)
PF4
PF4 peripheral levels remained higher than basal values throughout the session (Figure 3) The results showed a significant increase in peripheral levels after 15 min (+43±14%); a second peak in PF4 peripheral levels occurs 240 min after the beginning of the session (+49±16%).
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Post-dialytic evaluation showed a progressive decrease in PF4 venous levels, which returned to the basal value 20 h after the end of the session.
After the heparin bolus the PF4 peripheral plasma levels, showed a peak of 21±5%, (P<0.001) after 15 min with lower values at 30 min (15±6%; Table 3).
Platelet count
In the intradialytic phase, the platelet count showed a transient fall of ~15% after 30 min, with stable values during the session and in the post-dialytic period.
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Discussion |
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After the protein layer has been adsorbed onto the membrane surface, the platelets adhere, lose their discoid shape, become irregularly spherical with a reduction of their mean platelet volume, spread out and begin the `release reaction' [15]. The platelet `release reaction' is the secretory process following primary platelet aggregation whereby the contents of the platelet granules are released into the blood. The intradialytic `release reaction' is induced either by surface factors (micro-macroscopic characteristics and the physicochemical status of the dialysis membrane) or by circulating factors, such as thrombin, heparin, ADP, thromboxane A2, fibrinogen, Von Willebrand factor and others [1618].
It is widely agreed that platelet activation and the consequent release of active biological molecules, are due mainly to plateletmembrane contact [19]. Our study confirms a sizeable platelet activation; PDGF-AB, ß-TG and PF4 are largely released into the blood from the first minutes of the dialysis session, so that they may be considered, among other things, as suitable platelet activation markers.
PDGF-AB peripheral blood levels increased by ~134% after 30 min, with high levels persisting at all the time intervals tested, to this initial increase presumably contributes, even if to a lesser degree, the aggregating effect of heparin as indirectly assessed by the PDGF increase (+15±5%) found 30 min after the heparin bolus injection. The effective PDGF-AB release could be underestimated, if we consider the short half-life (<2 min ) of this molecule, which results in its fast removal from the blood circulation.
The further increase in PDGF-AB peripheral blood levels observed in the second half of the dialysis session is probably multifactorial and could be related to: (i) the further triggering of platelet activation related to the progressive increase in fibrinogen content within the protein layer on the membrane surface, which causes a more marked platelet activation over the dialysis membrane [20]; (ii) the progressive appearance in the circulation of new platelets that are more reactive to pro-aggregating factors. In fact during extracorporeal circulation platelets are continuously removed from, and added to, the blood so that there is soon a heterogeneous population of new and old platelets [21,22]; (iii) the intradialytic haemoconcentration with a relative increase of activated coagulative factors.
The higher PDGF-AB values found in the venous line compared with the arterial line (measured as AV variation) directly reflect the platelet activation and point to continuous release inside the dialyser.
Post-dialytic evaluation shows a slow and progressive decrease in the PDGF-AB peripheral level, which returns to basal values only after 20 h from the end of the session. This post-dialytic trend seems to reflect a persistent platelet activation (see below) beyond the end of the session. Furthermore, it is possible to hypothesize that PDGF itself causes, by a cascade activation, PDGF-AB production and release from its target cells.
With regard to the other target molecules studied, both ß-TG peripheral levels and ß-TG AV variation present, in the intradialytic period, a trend similar to PDGF, with an increase during the first phase of the dialysis session, and a new and further peak at the end. For PF4, the initial peak observed at 15 min could presumably be related to the heparin-induced release of PF4 from heparan sulphate-binding sites in endothelial cells [23]. This seems to be confirmed by the analogous increase that we pointed out 15 min after the heparin bolus injection.
The lesser intradialytic increase in ß-TG and PF4 values, if compared with PDGF-AB, could be explained by the neutralization of the anticoagulant activities of heparin; in fact, it is well known that these two molecules (and PF4 in particular) have heparin-binding capacity.
The post-dialytic levels of both ß-TG and PF4 decrease during the 20 h following the end of the session. The fact that during this period ß-TG and PF4 plasma values decrease more slowly than PDGF-AB could also be induced by their longer half-life (100 and 13 min, respectively) [24] in addition to the persisting platelet activation (see below). With regard to PF4 it is very interesting that this protein exerts a chemotactic effect on neuthrophils and monocytes with consequent further damage to the vascular wall [25].
This prolonged platelet activation is probably a multifactorial phenomenon that could be caused mainly by: (i) the presence of younger and more reactive platelets (see above), (ii) the progressive exhaustion of the heparin activity that involves a reduced neutralization of the activated coagulative factors.
While the existence of intradialytic PDGF-AB release, mainly as a consequence bloodmaterial interaction, seems established, one is led to speculate on the possible clinical implications.
Among systemic pathologies, accelerated atherosclerosis is of special importance in terms of morbidity and mortality, and it remains the most common cause of death in patients on renal replacement therapy according to several national registries. The importance of cardiovascular disease begins to emerge not only from the statistics but also from morphological and longitudinal evaluations that show the precocity and the impact of artheriosclerotic disease on dialysis patients.
Recently, Burdick et al. [26] reported a significant correlation between carotid atherosclerosis and time on dialysis. Bommer et al. [27] found that aortosclerosis is significantly more common in patients dialysed for more than 5 years.
In dialysis patients several atherosclerosis risk factors, related both to the uraemic syndrome and to dialysis therapy, can accelerate the vascular changes: (i) secondary hyperparathyroidism; (ii) elevated concentrations of prothrombotic factors (Von Willebrand factor and fibrinogen); (iii) oxidant stress; (iv) hyperhomocysteinaemia; (v) ß-lipoprotein-lipase deficit, related to the cyclic use of heparin and uraemic dyslipoproteinaemia; and (vi) lipoprotein (a) [2833]. However, no combination of the above-mentioned risk factors has provided a convincing explanation of why accelerated atherosclerosis sets in among dialysis patients. Therefore, we think that it may be useful, with a view to understanding the pathophysiology of this process, to extend our attention to the biological effects of growth factors.
PDGF, in particular, has been proposed to play a key role in the development of advanced atherosclerotic lesions by stimulating the migration and proliferation of vascular smooth muscle cells [34,35]. Recently Caplice et al. [36] found that PDGF-AB released into the coronary circulation after angioplasty promotes the proliferation of smooth muscle cells in culture. As a further demonstration of its role, inhibition the PDGF receptor by CGP 53716 (a specific PDGF receptor tyrosine kinase inhibitor) prevents smooth muscle cell migration and proliferation in vitro and, to a lesser extent, prevents the proliferation of these cells after balloon injury in vivo [37], this confirms a causal role for PDGF activation in the development of neointimal lesions.
The observation that PDGF-AB is released during dialysis and, above all, its slow return to basal values, seem to qualify it as a possible cardiovascular risk factor in uraemic patients; this intradialytic release could be related, over time and together with other atherosclerosis risk-factors, to the appearance and worsening of atherosclerotic lesions either by direct linkage to the target cells (smooth muscle cells, macrophages, endothelial cells) or by inducing PDGF production in these cells. This hypothesis could be of special interest considering the duration of dialysis therapy and PDGF cyclic release.
Further evaluations of PDGF-AB release are certainly necessary, checking it together with other atherosclerotic risk factors against other artificial membranes, alternative dialysis procedures, different anticoagulation modalities and prospective studies.
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Acknowledgments |
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References |
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