Fibrinogen degradation products generation is the major determinant of platelet inhibition induced by plasminogen activators in platelet-rich plasma

fihrino/ysrs(lY93)7, 379-385 ~19Y3LongmanGroup UK Ltd

Fibrinogen Degradation Products Generation is the Major Determinant of Platelet Inhibition Induced by Plasminogen Activators in Platelet-rich Plasma

P. Parke,

J. Hauert,

A. Iorio,

P. Callegari,

G. G. Nenci

SUMMARY. The platelet function defect induced by thrombolytic agents has been referred either to the degradation of platelet surface receptors or to the anti-aggregatory effect of fibrinogen degradation products (FgDPs). In the present study we have evaluated platelet aggregation induced by ADP, collagen and ristocetin after incubation of washed platelets or platelet-rich plasma (PRP) with plasmin (l.l-3.4IU/ml), plasminogen activators (PAS) (streptokinase 250-1000 IU/ml; urokinase, 10-1000 IU/ml; t-PA OS-lOpg/ml) or FgDPs (0.062-2mg/ml). In parallel the surface levels of platelet GP Ib and IIb/IIIa complex were determined by fluorescence flow cytometry using specific monoclonal antibody. Washed platelets treated with plasmin (l.lIU/ml) for 10 to 90 min showed a progressive reduction of ristocetin-induced platelet agglutination and a progressive reduction of surface GP Ib. Surface expression of GP IIb/IIIa complex was significantly increased after plasmin exposure. The addition of PAS to PRP resulted in a marked reduction of ADP-induced platelet aggregation. Collagen-induced platelet aggregation was only slightly affected. Similar changes were observed when PRP was preincubated with high concentrations of FgDPs. In PRP treated with PAS platelet surface levels of GP Ib and GP IIb/IIIa complex did not show any significant changes. In conclusion our results show that in plasma no proteolysis of platelet adhesive receptors occurs after plasminogen activation. The platelet inhibition observed after incubation of PRP with PAS is likely to be caused by FgDPs generation.

Modifications of platelet function induced by the activation of the fibrinolytic system may have an important role in the pathogenesis of some shortcomings of thrombolytic therapy, including failure to achieve reperfusion, reocclusion after initially successful thrombolysis and hemorrhages.‘,2 Both activation and inhibition of platelets have been observed after their exposure to plasmin or plasminogen activators.’ Recently, it has been shown that both these phenomena occur after exposure of platelets to PAS, platelet activation appearing firstly whereas platelet inhibition being a late consequence.-3-6

P. Parke, A. Iorio, G. G. Nenci, Institute of Internal Medicine and Vascular Medicine, University of Perugia, Perugia, Italy, J. Hauert, P. Callegari, Division of Hematology, Department of Medicine, University Hospital Center, CHUV, Lausanne, Switzerland.

The activation of platelets during thrombolysis may preclude recanalisation or predispose to early reocclusion due to the adhesion and aggregation of platelets onto a ruptured atherosclerotic plaque or onto a residual thrombus.7 This is confirmed by a number of experimental 8,9 and clinical”’ observations showing that a more rapid and sustained restoration of vascular patency is achieved when antiplatelet drugs are used conjunctively with thrombolytic agents. On the other side, the bleeding time is prolonged in experimental animals” as well as in patientsI who have received thrombolytic agents. This prolongation appears to be correlated with the incidence of spontaneous hemorragic events12 suggesting that platelet dysfunction can account, at least in part, for the hemorrhagic tendency observed during thrombolysis. Platelet inhibition during thrombolysis has been referred to several mechanisms including:

380 Plasminogen Activators and Platelets

1. the inhibition of thrombin-induced thromboxane AZ (TXAz) generation through the block of arachidonic acid mobilization from membrane phospholipid@ 2. the cleavage of surface Gp Ib13 and/or Gp IIb/IIIa complex’4 receptors 3. the dispersion of platelet aggregates through the lysis of coadhesive fibrinogen. l5 Furthermore, platelet inhibition in aggregometry studies’6,‘7 may be the consequence of plasminof fibrinogen. Plasma mediated degradation fibrinogen is an essential cofactor for platelet aggregation and fibrinogen degradation products (FgDPs) can interfere with platelet to platelet interactions by competitive binding with activated GP IIb/IIIa complex.” The present study was designed to determine the mechanism of platelet inhibition induced by plasmin and PAS. Platelet aggregometry was performed after exposure of platelets to plasmin, streptokinase (SK), urokinase (UK), tissue-plasminogen activator (t-PA) and FgDPs. In parallel with platelet aggregometry platelet surface levels of GP Ib and IIb/IIIa complex were measured by fluorescence flow cytometry.

MATERIAL AND METHODS Human fibrinogen (grade L), plasminogen, plasmin, H-D-valyl-L-leucyl-L-lysine-p-nitroanilide dihydrochloride (S-2251) and streptokinase were purchased from KabiVitrum (Stockholm, Sweden). Plasmin and plasminogen were dissolved in Tris-saline buffer (10mM Tris, 150mM NaCl, pH 7.4) and stored in aliquots at -70°C. Recombinant t-PA was obtained from Boehringer Ingelheim (Ingelheim am Rhein, West Germany) and urokinase (Ukidan) from Serono Laboratories SA (Aubonne, Switzerland). Apyrase, prostaglandin El, aprotinin and a*-antiplasmin were supplied by Sigma Chemical Co. (St. Louis, MO, USA) and ristocetin by Lundbeck A/S (Copenhagen, Denmark).

Collection

and Preparation

of Human Platelets

After having obtained informed consent, blood was collected by gentle venipuncture from an antecubital vein of healthy adult donors who had not ingested aspirin or other medications known to interfere with platelet function for at least 10 days before donation. Washed Platelets

The blood was drawn directly into a tube containing l/7 volume of ACD (64mM citric acid, 85 mM trisodium citrate, 1lOmM dextrose, pH 4.5). After mixing, the sample was centrifuged at 15Og for 15 min at room temperature, and only the upper two thirds of the platelet-rich plasma (PRP) were collected to minimize leukocyte and red cell contamination. PRP was kept at 22°C for up to 1 h before use. For the preparation of washed platelets, PRP was centrifuged once more at 150g for 4 min and the occasional pellet of erythrocytes discarded. The pH of the supernatant PRP was adjusted to 6.5 by drop-wise addition of ACD. Platelets were then washed twice by centrifugation at 1200g for 15 min at room temperature and resuspension in a modified Tyrode’s buffer (138mM NaCl, 50mM KCl, 11.9mM NaHC03, 8mM NaH2PO4,5.5 mM dextrose, 5 mM HEPES, 0.35% bovine serum albumin, pH 6.5) containing 0.025 mg/ml apyrase and 50ng/ml PGEi. The final platelet resuspension was made in Tyrode’s buffer, pH 7.4, containing 2 mM calcium chloride and 1 mM magnesium chloride. Platelet counts were evaluated in a cell counter (Coulter counter model S-Plus STKR, Coulter Electronics, Inc., Hialeah, FL, USA). Platelet-rich Plasma

The blood was collected in polystyrene tubes containing 3.8% trisodium citrate (1:lO v/v) and centrifuged at 2OOxg for 15 min. The PRP was removed and the platelet count adjusted to 4.0~ lO*/ml by dilution with platelet-poor plasma (PPP) obtained from the same donor blood by centrifugation for an additional 15 min at 1700g.

Antibodies

MoAbs AN51, which reacts with GP Ib, and MoAb P2, which recognizes the GP IIb/IIIa complex, were purchased respectively from Dakopatts (Glostrup, Denmark) and from Immunotech SA (Marseille, France). An affinity purified goat anti-mouse IgG F(ab’)2 fragment conjugated with dichlorotriazinyl amino fluorescein, at a concentration of 1.4mg/ml, was obtained from Immunotech SA. ELISA kit for FgDP determination was from Organon Technica (Oss, The Netherlands). All substances added to platelet suspensions are expressed as final concentration after all additions. Appropriate solutions were used in control experiments.

Pretreatment FgDP

of Platelets with Plasmin,

PAS or

Plasmin, PAS or Tris-saline were added to platelet suspensions and incubated at 37°C in a water bath. The mixture was swirled only once just before incubation because constant mixing may promote formation of large platelet aggregates which interfere with flow cytometric assays. After various incubation periods, an aliquot was removed for plasmin assays and the reaction stopped by adding an excess of aprotinin (500U/ml) to ensure total inhibition of plasmin activity. Plasmin activity was determined with the chromogenic substrate S-2251 following the manufacturer’s method (Kabi Diagnostic Data Sheet

Fibrinolysis

for S-2251) and expressed in IU; 1 IU corresponds to about 0.40nmol of plasmin. A standard curve was established daily using known amounts of plasmin. In order to define the effect of FgDPs on platelet aggregation, different dilution of FgDPs were added to PRP 10 min before aggregation studies. FgDPs were prepared by incubating human purified fibrinogen (lOmg/ml) with plasminogen (lOO~g/ml) and UK (5000 IU/ml) for 24 h at 37°C. Aprotinin was then added in excess to block residual plasmin and UK activity. Platelet Aggregation

Studies

Platelet aggregometry was performed in a Payton dual channel aggregometer (Payton Scientific Inc., Buffalo, NY, USA) at 37°C with a stirring rate of 1100 rpm. Aliquots of washed platelets suspensions, preincubated with plasmin, were mixed with l/4 volume of PPP prepared from the original donor. After 2 min 360 111of this mixture was placed in an aggregometer cuvette and platelet agglutination initiated by adding 40~~1 of a 12.5mg!ml solution of ristocetin. The remaining was further diluted with Tyrode’s buffer for flow cytometric assay of GPs levels. Platelet aggregation was evaluated in PRP by adding ADP (0.6, 2 and 4pM), collagen (2 and 4kg/ml) or ristocetin (1.25, 1.5 and 2mg/ml). Preparation

of Samples for Flow Cytometry

In experiments performed with washed platelets, 0.2 volumes of PPP from the original donor was always added prior to the incubation with antibody to saturate the platelet surface Fc receptor and thus to avoid a non-specific binding of MoAbs. Washed platelets or PRP diluted to a final platelet count of 0.3x108/ml were incubated for 30 min at room temperature with MoAbs directed against human platelet glycoprotein Ib and IIb/IIIa complex at saturating concentrations. Platelets were then washed twice as described above and resuspended in the same volume. Fluorescein isothiocyanate-conjugated goat antimouse IgG were used as second antibody and incubated for 20 min at room temperature. After two further washings platelets were resuspended at a final concentration of 0.3 x lOs/ ml and fixed by addition of an equal volume of 2% formaldehyde. Samples treated by this way and stored at 4°C in the dark had a stable fluorescence for at least 3 days but were always analysed within 15 h. Flow Cytometric

Assay

A flow cytometer (Profile Epics, Coulter Electronics Inc., Hialeah, FL, USA) equipped with an air-cooled argon ion laser adjusted to deliver 150 milliwatts at 488nm was used to analyse platelets. For the measurement of green fluorescence emission, a 515-

381

530 short-pass filter was placed in front of the photomultiplier. Logarithmic amplification was used for forward-angle (l-19°C) light scatter (to analyse the size of cells), side-angle light scatter (to examine the density of cells) and for the green fluorescence signal. Standard calibrated fluorescent beads (Immunocheck and DNA-check, Coulter Electronics Inc.) were used before each series of experiments to ensure orthogonal alignment and day-to-day stability of the instrument. Samples of washed platelets or PRP were analysed in the flow cytometer to determine a platelet cytogram using forward and right angle scatter signals generated by the argon laser. Erythrocytes and leucocytes as well as debris and ‘machine noise’ demonstrated a light scatter profile different from that of platelets and were excluded from the analysis by setting a gate around the region of maximal platelet concentration. Platelet samples were passed through the laser beam at a flow rate of 1000 cells/s: at least 50000 cells were analysed in each sample. The mean relative fluorescence of a given platelet population, which is directly related to the surface binding of fluorochrome, was determined by analysis of singleparameter, 256-channel, gated, log integral green fluorescent histograms. Non-specific binding of the antibodies was evaluated by adding to control platelets either the FITC-conjugated goat antimouse antibody alone or an equal amount of an irrelevant first antibody. Results were expressed as changes in fluorescence intensity, i.e. the ratio of the mean relative fluorescence intensity of treated platelets over that of controls. Statistical

Analysis

The normality of the distributions of the data was tested with the Kolmogorov-Smirnov test. Normally distributed data were indicated as mean values+1 standard deviation and analysed by one-way analysis of variance for repeated measures. When a variance was found, further analysis was performed with the Fisher’s Protected Least Significant Difference (PLSD) procedure to detect the existence of significant differences for each pairs of possible comparisons (a p-value<0.05 was considered to represent a significant difference). Non parametric data were reported as median and range and analysed by means of the Friedman’s test. l9 Analyses were performed by the Statview SESprogram (Abacus Concepts Inc., Berkeley, CA, USA) with the aid of a Macintosh LC personal computer. RESULTS Effects of Plasmin and Plasminogen Washed Platelets

Activators

on

Plasmin treatment of washed platelets resuspended in Tyrode’s buffer induced a dose- and time-dependent

382

Plasminogen Activators and Platelets

% .

-

GPlb

--+-

GP /lb/l//a

160-

120-

80 -

unless platelets were preincubated with plasminogen (data not shown). Preincubation of washed platelets with 1.2 PM plasminogen and the subsequent addition of t-PA or UK resulted in a degradation of GP Ib, which was correlated to the amount of plasmin generated in the medium (Table 1). Surface expression of GP IIb/IIIa complex shows a significant increase only after addition of UK at concentrations higher than 50U/ml.

40* o-

1 Basal

10

20

30

60

90

minutes Fig. 1 GP Ib and GP IIb/IIIa complex surface levels in washed platelets treated with plasmin (1.1 W/ml). The binding of the type specific MoAbs AN51 and P2 is expressed as per cent of mean fluorescence intensity of treated versus control platelets. Values represent meanfl SD of 6 experiments. *=p
reduction in the binding of the MoAb AN 51, directed against GP Ib (Fig. 1). The maximal reduction of the binding was seen after incubation with 1.1 IU/ml for 90 min, which resulted in GP Ib levels of 9..5+3.5%. A similar reduction of surface GP Ib was seen after exposure of washed platelets at higher plasmin concentrations (2.3-2.8IU/ml) for shorter incubation time (40-50 min). In contrast, the binding of the MoAb P2, directed against the GP IIb/IIIa complex was significantly increased by plasmin treatment (Fig. 1). 20 min after exposure to 1.1 IU/ml plasmin the level of surface GP Ib/IIIa complex rose up to 160% of controls. An increase of surface expression of GP IIb/IIIa complex was also observed after addition of several platelet agonists, including low doses thrombin (data not shown). To evaluate the functional consequence of GP Ib reduction, we performed a series of experiments in which GP Ib levels were assayed in parallel with ristocetin-induced platelet agglutination. Figure 2 shows the behaviour of ristocetin-induced platelet agglutination (slope and maximal amplitude) after treatment of washed platelets with 1.1 IU/ml plasmin for 90 min. As expected, plasmin treatment resulted in a progressive impairment of ristocetin-induced platelet agglutination. The maximal velocity of aggregation (slope) was markedly reduced after 20 min incubation with plasmin, when surface GP Ib level was about the 50% of its basal value (Fig. l), whereas the maximal amplitude of the agglutination curves was reduced only when more than 75% of GP Ib had been cleaved. The direct addition to washed platelets of t-PA or urokinase in concentrations up to 20 &ml and 10000 U/ml, respectively, did not induce any change in the surface expression of GP I and IIb/IIIa complex,

Effects of Plasminogen Plasma

Activators

on Platelet-rich

The addition of UK (250 and lOOOU/ml), SK (250 and lOOOU/ml) or t-PA (2 and 5 l.rg/ml) to PRP resulted in a marked inhibition of ADP-induced platelet aggregation (Table 2). A significant but mild impairment of collagen-induced platelet aggregation was seen with UK and with the highest concentration of t-PA. Agglutination curves induced by ristocetin were not affected by all plasminogen activators. The parallel evaluation of GP Ib and IIb/IIIa complex levels did not show any significant modification in comparison with controls (Table 3). Table 4 and Figure 3 show the results of platelet aggregation induced by ADP and collagen after incubation of PRP with various concentration of FgDPs. ADP-induced platelet aggregation was inhibited by preincubation of platelet-rich plasma with FgDPs at concentration ranging from 0.25 to 2mg/ml, whereas collagen-induced platelet aggregation was inhibited only in the presence of the highest FgDPs concentration. Ristocetin-induced platelet agglutination was not modified by FgDPs (data not shown).

10

60

90

minutes

Fig. 2 Inhibition

of ristocetin-induced platelet agglutination (slope and maximal amplitude) after treatment of washed platelets with plasmin (1.1 IU/ml). Values represent meanfl of 6 experiments. *=p
SD

Fibrinolysis

Table 1 Plasmin generation plasminogen activators

and platelet

surface

levels of GP Ib and IIbiIIIa

10 50 250 t-PA 0.5

IU/ml IUlml IU/ml &ml

5 6 6 5

1 wdml

6

5 pg/ml 10 pg/ml

6 4

Washed platelets were incubated represent mean+SD. *=p
SK

1000 250 1000 250

t-PA

IU/ml IU/ml IU/ml IU/ml

5 &ml 2 pg/ml

78* 51* 63* 55* 86* 82*

(49; (32; (47; (43; (59; (61;

98) 66) 87) 71) 100) 95)

19* (3; 10* (2; 8 (0; 3 (-4; 23* (2; 6 (-5;

31) 21) 20) 11) 40) 12)

58i6* 21?5* J+2* 93f9 50+6* 18+6* 13+5*

9s+9 121+9** 125fll** 96f8 91f6 108fll 109f9

-7 (-11; 2 (-3;6) 5 (0; 4 (0; -2 (-12; -5 (-18;

3) 7) 8) 8) 2)

UK SK

1000 250 1000 250

t-PA

IU/ml IU/ml IU/ml IU/ml

5 &ml 2 &ml

levels (‘X of

GP Ib (% of controls)

GP IIb/IIIa (% of controls)

85k9 10027 88fll 89+10 81f12 99f5

116f5 104f7 127+12 119+9 109+6 98f5

Platelet-rich plasma was incubated for 1 h at 37°C with UK, SK or t-PA at concentrations indicated. Values represent mean+SD of 6 experiments. Table 4 Platelet

aggregation

after addition

ADP (0.6pM)

of FgDPs Collagen

(1 .~JLM) and UK or t-PA at concentrations

DISCUSSION

Platelet-rich plasma was incubated for I h at 37°C with UK, SK or t-PA at concentrations indicated. Values represent median and range of 6 experiments. *=p
with

0.36+0.05 1.81f0.22 2.16zkO.21 0.11~0.02 0.26kO.07 1.59+0.13 2.08kO.19

Ristocetin (1,25 mg/ml)

(4 @ml)

(2 wM) UK

platelets

GP IIb/IIIa (% of controls)

for 1 h at 37°C with plasminogen

Collagen

of washed

GP Ib (“Y0of controls)

Table 2 Inhibition (% of controls) of platelet aggregation (maximal amplitude) induced by addition of PAS to PRP ADP

after treatment

Plasmind (IU/ml)

No. exp. UK

complex

383

to PRP (2 &ml)

FgDP (mg/ml)

Slope

Max. amplitude

Lag time

Max. amplitude

2 1 0.5 0.25 0.125 0.062 Control

6 58 76 97 108 100 112

0 0 24 75 88 90 90

1’30” 51” 40” 30” 29” 30” 29”

74 85 94 96 95 96 95

Slope is expressed as a rate (arbitrary units min-I). It was calculated by fitting the line tangent to the steepest portion of the aggregation curve. Maximal amplitude is the percentage of light transmission at 4 min. Lag time represents the time intercurring from addition of collagen and the initial slope of the aggregation. Values represent the mean of 3 experiments.

indicated.

Values

AND CONCLUSIONS

Platelet changes observed after exposure to plasmin or PAS have been claimed to play a role in the pathogenesis of both thrombotic and hemorrhagic complications of thrombolytic therapy.‘,*,’ Plasmin addition in vitro or the infusion of PAS in vivo activate platelets leading to the release reaction and fibrinogen binding. 4~s,2(b22On the other hand, an inhibition of platelet function occurs after prolonged exposure to plasmin. The responsible mechanism for plasmin-mediated platelet inhibition is not yet fully elucidated. A direct effect of plasmin on platelets, particularly the cleavage of membrane receptors for plasma adhesive proteins, e.g. fibrinogen and von Willebrand factor, has been suggested by in vitro studies.‘“,14 In addition, thrombolytic agents can indirectly inhibit platelet aggregation, through the lysis of plasma fibrinogen and the generation of FgDPs, which interfere with platelet aggregation.‘s In order to better characterize the platelet defect induced by plasmin or PAS we performed studies both with washed platelets and with PRP. To evaluate the expression of platelet surface GP Ib and IIb/IIIa complex we took advantage of the sensitivity of fluorescence flow cytometry and of the specificity of well defined MoAbs. Flow cytometric analysis offer several advantages over conventional radioligand binding and gel electrophoretic methods. First, platelets may be studied in PRP or in whole blood2”.24 with little or no manipulations. This allows a more severe and accurate comparison of the expression of specific epitopes in resting versus activated platelets. Second, since flow cytometry permits to individually examine every cell in a definite sample, small populations displaying distinct characteristics can be readily identified,25 which is not possible with other methods. Third, very small numbers of cells are required for the analysis. The evaluation of surface GPs in washed platelets incubated with plasmin, or with UK or t-PA in presence of plasminogen, confirmed previous datai of a dose- and time-dependent reduction of GP Ib surface levels. We demonstrated that GP Ib degra-

384 Plasminogen Activators and Platelets

Collagen

(2

uglml)

Contrd 0.125

0.5

2

minutes

minutes

Fig. 3 Example of the dose-dependent platelet inhibition induced by FgDPs addition to PRP. Values reported on the right side of the figures indicate FgDPs final corwzentration (mg/ml).

dation was accompanied by an impairment of ristocetin-induced platelet agglutination. An important observation was that the effects of PAS on GP Ib surface levels were entirely due to plasmin generation, because (1) no modification was observed in the absence of plasminogen, even when very high concentrations of t-PA or UK were used; (2) the reduction of GP Ib expression was always well correlated with the amount of plasmin generated in the mixture and (3) it was prevented by pretreatment with cw2-antiplasmin (data not shown). We also found that platelet surface GP IIb/IIIa complex does not undergo significant plasminmediated proteolysis. It is now well established that the concentration of calcium ions in the suspending medium determines the sensitivity of the GP IIb/IIIa complex to lysis.‘*26 Only at a very low calcium concentration (
The different sensitivity of platelet GP Ib to plasmin added directly to suspension of washed platelets or generated in PRP by PAS is not easy to explain. It can be taken into account that the maximal plasmin concentration theoretically achievable in vitro in PRP is about 2km. More than */3 of this amount can be blocked by physiologic plasmin inhibitors, e.g. a*-antiplasmin and cc*-macroglobulin. Thus, an insufficient, or a too slower, generation of plasmin could explain the difference observed. Another possible explanation is that in plasma platelets retain their metabolic activity, the GP Ib receptors degraded by plasmin are replaced by intact receptors from an internal ~001.~”Preliminary results of ex vivo studies are in agreement with this hypothesis. Administration of rt-PA, SK, or UK in patients with acute myocardial infarction resulted in no change or in a small increase of platelet surface GP Ib while a concomitant 19% decrease in total platelet GP Ib occurs.31.32 The resistance of platelet adhesive receptors to plasmin-mediated degradation in plasma suggests that the inhibition of platelet aggregation in PRP is most likely due to other mechanisms. The generation of FgDPs, which compete with fibrinogen for binding to the activated GP IIb/IIIa complex but do not support platelet aggregation, is likely to be involved.3~‘8*33In fact, we found that when PRP was supplemented with high concentrations of FgDPs, aggregation induced with either ADP or collagen was inhibited. In conclusion, our data confirm and extend previous observations on the mechanisms of platelet dysfunction induced by PAS. The platelet surface receptor GP Ib is readily hydrolysed on washed platelet by plasmin or PAS whereas was not modified by the addition of PAS to PRP. The main mechanism of platelet inhibition induced by PAS in plasma appears to be the generation of FgDPs. Thus, taking into account the role of platelet inhibition in the hemorrhagic tendency during thrombolysis, it is

Fibrinolysis

possible that really selective thrombolytic agents can significantly lower the hemorrhagic risk by preventing or lowering FgDPs generation. ACKNOWLEDGEMENTS The authors thank Dr Anna Maria Mezzasoma for her excellent assistance in performing platelet aggregation studies.

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Received: 11 November 1992 Accepted after revision: 19 March 1993 Offprint orders to: Pasquale Parise, Istituto di Medicina Interna e di Medicina Vascolare, Universitl degli Studi di Perugia, via Enrico dal Pozzo, 06100 - Perugia, Italy.

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