IN I ERNA . FIONAI. JOUKtill OF LEPROSY Volume 49, Number IPrinted in the U.S.A.

Low Serotonin Uptake by Platelets in Leprosy and aNew Approach to Prevent It'

Lenke Szabados, Jean-Marie Launay, Madeleine Mester, FrancoisCottenot, Jean Pennec, and LAszlo Mester'

Looking at the long history of leprosy inhuman civilization, it is surprising that onlyduring the last two decades has the bloodof leprosy patients become a subject of in-tensive investigation. Some major prob-lems related to the clotting system, how-ever, still remain open.

The study of hemostasis in leprosy is sig-nificant not only for the treatment of lep-rosy patients showing coagulation abnor-malities and platelet defects ( 3 ) but also fora better understanding of the mechanismsthrough which thromboembolic phenomenaare less frequent in leprosy ( 15 ).

Today it is generally accepted that a greatmany of the coagulation abnormalities inleprosy are due to the presence of a higherlevel of immunoglobulins, especially ofIgM, in the plasma of leprosy patients(8 . 1 "). Hypergammaglobulinemia (4 ) can in-terfere both with the action of coagulationfactors as well as with the functions ofplatelets. However, the fact that not onlya quantitative but also a qualitative changeoccurs in immunoglobulins in leprosy hasbeen neglected for a long time.

Modifications in serum sialic acid levels( 2 ) and in platelet membrane sialic acidlevels have been observed in leprosy andare almost certainly involved in the abnor-mal responses of blood constituents in thedisease. Sialic acid levels affect a variety of

' Received for publication on 11 September 1979;accepted for publication in revised form on 26 June 1980.

2 L. Szabados, D. Sci., Charge de Recherches; J.-M. Launay, D. Pharm.; M. Mester, D. Univ., Institutde Chimie des Substances Naturelles, Centre Nation-ale de Ia Recherche Scientifique; F. Cottenot, NI.D.,Director; J. Pennec, M.D., Service de Dermatologie,Pavillon de Malte, HOpital St. Louis, 75010 Paris,France; L. Mester, Ph.D., Directeur de Recherches,Institut de Chimie des Substances Naturelles, CentreNationale de Ia Recherche Scientifique, 91190 Gif-sur-Yvette, France. Dr. Launay's present address is La-boratoire de Biochimie, HOpital St. Louis, 75010Paris, France.

platelet functions such as adhesiveness andaggregation of platelets, release and uptakeof serotonin, etc. Serotonin uptake is a par-ticularly relevant parameter not only forblood but also for the nervous system. In-deed, abnormal serotonin uptake observedwith platelets can be directly correlatedwith abnormal functions of tryptaminergicneurons ( 7 ).

For these reasons, we have investigatedchanges in mucoproteins in plasma and inplatelet homogenates from leprosy pa-tients, considering the sialic acid level to bea measure of structural modifications. Wethen found a decrease in serotonin uptakeby platelets from leprosy patients. Finally,we tried to prevent this decreased serotoninuptake by: a) using normal human plasmainstead of plasma from leprosy patients andb) using desoxyfructo-serotonin, a newdrug, which has been found to be effectivein other cases showing low serotonin up-take. In these experiments desoxyfructo-serotonin was added to the platelet prepa-ration of leprosy patients in vitro. It shouldbe noted that the drug has been found to beeffective against leprosy in mice after oraladministration (").

MATERIALS AND METHODSClinical material. This study was con-

ducted on a group of 29 patients with Han-sen's disease, hospitalized in the Pavillonde Malte, HOpital Saint-Louis, Paris, Thegroup was heterogenous in relation to age,sex, and type of leprosy. Diagnosis wasbased on clinical, bacteriological, immu-nological, and histological examinations(smears stained by the Ziehl-Neelsen meth-od, whole lepromin and skin biopsies of theMitsuda test). At the beginning of our ex-periments, out of these 29 patients, 18 hadlepromatous leprosy (LL), five tuberculoid(TT), four borderline leprosy (BL or BT)and two erythema nodosum leprosum

42

49, 1^.S'zabados, et al.: Lou Serotonin Uptake by Platelets in Leprosy^43

(ENL), according to the International Clas-sification.

Although these patients had receivedclassical chemotherapy (dapsone, Disu-lone, Sultirene, rifampin, and Solupred),they were in different clinical states, cor-responding to different phases of the dis-ease at the time of the collection of bloodfor analysis. Some patients were examinedrepeatedly, in different phases of the evo-lution of the disease. For this reason thenumber of examined "cases" (45) is higherthan the number of "patients" (29). In allexperiments the number of parallel mea-surements of the same sample was three(Ns = 3). In France the use of thalidomidebeing not allowed: only one patient had re-ceived such treatment before his arrival inFrance.

We have categorized the patients as fol-lows:

I. Active phase (leprosy in evolution)Clinical reactional episodes in anytype, BL (2), BT (10), LL (7); total 19cases.

II. Reactive phase (lepra reaction)Lepromatous patients with fever,etc., due to incipient ENL but beforeskin eruptions appear, LL (7), ENL(4): total 11 cases. (sic.)

Ill. Stabilized phase (subsided lepra re-action)All types of cases, but after stabili-zation; 15 cases.

During the third phase, we were partic-ularly interested in bacteriologically andimmunologically unstabilized severe cases,in which very little, if any, hope exists forrecovery. Case I (age 36, male) and case2 (age 20, male) both are bacteriologicallypositive, untreated lepromatous cases withfever.

The average of cases in Phase I, II, andIII, as to age, sex, race, and therapy wasnearly identical and very similar to the av-erage of normal cases.

Statistical significance of the results wastested using the Student-Fisher "t" distri-bution ("). The particular methods and de-nominations used in Table 3 were takenfrom Graf and Pletscher ( 7 ).

Biochemical analysis of blood plasma andwashed platelets. Blood samples were col-lected from each patient two or three times

during the development of the disease.Twenty-seven healthy donors served ascontrols. The 27 healthy donors werematched as much as possible to the pa-tients' age, sex, and race. Only 11% of thehealthy donors were hospital personnel.

Platelet rich plasma (5) (PRP) was ob-tained by centrifuging citrated blood (1 vol.3.8% citrate solution plus 9 vol blood) sam-ples at 180 x g for 10 min. The plateletcount was made using a Mclassez chamberwith phase interference optics.

Washed platelets (') were isolated by cen-trifugation of PRP at 2500 x g at 4°C for 10min. The sediment was suspended andwashed 3 times with 50 vol saline (0.3 M).Washed platelets were homogenized andsonicated.

Protein concentration was determined byLowry's method, modified by Markwell, etal. ("), using crystallized bovine albumin(Sigma Chemical Co., St. Louis, Missouri,U.S.A.) as a standard.

The mucoprotein fraction of the plasmaproteins was precipitated by 5% phospho-tungstic acid by the method of Winzler ('s).

Sialic acid was determined in samples ofPRP (0.05 ml), platelet suspension (0.5 ml),and precipitated mucoprotein (0.5 ml) byadding sulfuric acid to a total volume of Iml in order to carry out the hydrolysis at afinal concentration of 0.05 M HoSO„ at80°C for 30, 45, and 60 min, respectively.Sialic acid was determined with thiobarbi-tunic acid by the method of Warren ( 17 ), us-ing N-acetyl neuraminic acid (Sigma Chem-ical Co., St. Louis, Missouri, U.S.A) as aninternal standard.

Uptake of 5-hydroxytryptamine-( 3 H) byplatelet rich plasma and by isolated plateletsfrom leprosy patients. Platelet rich plasma(PRP) and the isolated platelet suspensionwere diluted to a concentration of 1()) plate-lets (pis)/mm;. After 10 min preincubationwith shaking at 37°C, 100 Al of PRP orplatelet suspension was incubated for 1min with shaking at 37°C with different con-centrations of "H-labeled 5-hydroxytrypt-amine (5HT) binoxalate in 0.9% w/v NaClsolution to give final concentrations of5 x^10-8, 5 x 10 -8 , and 10 -7 M, re-spectively. Two ml of 0.9% NaCI-0.4%EDTA was added to stop the reaction, theplatelets were then filtered on nitrated cel-lulose filter (Millipore, Type GS 0.22 Am),

44^ International .low-nal of /,eprosy^ 1981

washed six times with 5 ml of 0.9% NaC1—0.4% EDTA, and the radioactivity on thefilter was counted in a liquid scintillationcounter (Intertechnic, Plaisir, France) ateach concentration. Eight healthy donorsserved as controls. The percent uptakeshown in Fig. I represents the average ofvalues obtained at different concentrationsand with patients in the three phases of thedisease. The same method was used tomeasure serotonin uptake by normal humanplatelets in plasma from leprosy patientsand vice versa.

In order to study the effect of desoxy-fructo-serotonin on the uptake of "H-la-beled 5-hydroxytryptamine by PRP of lep-rosy patients, 25 ,LL1 of 0.9% NaCl (control)and 25 id of 0.9% NaCI containing desoxy-fructo-serotonin, respectively, were addedto a constant volume (75/11) of "H-labeled5-hydroxytryptamine (5HT) to provide finalconcentrations of 1-1-5HT varying from10- " to 2.5 x 10 7 M. The final concentra-tion of desoxyfructo-serotonin was 2.15 x10-7 M in each case. The observed increasein the uptake of 3 H-5HT by leprosy PRP isrepresented in Fig. 3 by the shaded areas(N = 3).

Desoxyfructo-serotonin (") was pre-pared from the oxalate salt by adding anaqueous solution of Ca(OH), and removingthe precipitated calcium oxalate by suction.The effect of the compound on plateletshape-change and on the uptake and releaseof serotonin by normal platelets were mea-sured by the methods reported earlier(7.9. 12, 1:3) .

RESULTS AND DISCUSSIONIt has been reported that coagulation ab-

normalities and platelet defects are due toa high level of mucoproteins, especiallyIgM (8 ' '") in the plasma of leprosy patients.The present investigations support the as-sumption that a structural change related tothe sialic acid content both in the plasmaand in the platelets is involved in these phe-nomena.

Mucoprotein level and sialic acid contentin the plasma of leprosy patients (Table 1).In addition to a small increase in the totalplasma protein level (up to 7%), leprosypatients had marked elevations in plasmamucoprotein concentrations; these in-

FIG. I. Uptake of 5HT by leprous PRP, by normalplatelets in leprous plasma and by isolated leprousplatelets."

Leprous PRP

Normal plateletsLi^in leprous plasma

„.,.,_,^Isolated leprousLj^platelets

NORMAL 100%

CONTROL

r.

*

Ph is 1

* One asterisk indicates statistically significantchange (p < 0.05) from the control.

** rwo asterisks indicate statistically highly signif-icant change (p < 0.01) from the control.

Normal control (N = 8), Phase I (N = II), Phase II(N = II), Phase Ill (N = 9), Severe cases (N = 2).

" Average of the uptake of 5HT by leprous PRP, bynormal platelets in leprous plasma and by isolated lep-rous platelets in buffer or in normal human plasmaduring the three phases of the disease and in severecases (uptake by normal PRP = 100%).

creases ranged between 30 and 113% abovenormal during the evolution of the disease.In spite of a significant increase in the sialicacid concentrations and a significant in-crease in the percent of sialic acid in thetotal serum proteins (150% of normal in thethird phase of the disease), the sialic acidcontent of isolated plasma mucoproteins isclearly diminished in the second and thirdphases of the disease (to 87 and 73% re-spectively of the normal value). Thus thereare definite structural changes in the sugarmoiety of the mucoproteins in these leprosypatients.

The sialic acid content of the platelet mem-brane in leprosy (Table 2). As with the sialicacid content of plasma mucoproteins, thesialic acid content of isolated platelets cal-culated on platelet protein also diminished.

200%

100%

* *

P ase Ill^Severecases

49, 1^Szahados, et al.: Lott' Serotonin Uptake by Platelets in Leprosy^45

TABLE 1. Protein, mucoprotein, and sialic acid concentrations in the plasma of leprosypatients. (Mean ± S.D.).

Plasma No. Proteinsmg/m1

Sialic acidmg/mI

% Sialic acidon protein

Mucoproteinmg/m1

% Sialic acidin mucoprotein

Control 27 72.8 ± 5 0.64 ± 0.1 0.88 ± 0.10 0.68 ± 0.10 9.48 ± 0.7(100%) (100%) (100%) (100%) (100%)

Phase I 19 76.4 ± 2.4 0.82 ± 0.2" 1.06 ± 0.04'' 0.90 ± 0.04" calculated(Active) ( 105%) (127%) (120%) (130%) (95%)

Phase 11 11 77.2 ± 4.2" 0.78 ± 0.7" 1.09 ± 0.05" 0.92 ± 0.05" 9.20 ± 0.47"(Reactive) (106%) (120%) (110%) (134%) (87%)

Phase Ill 15 78.0-±22'2.2" 1.03 ± 0.2" 1.32 ± 0.04" 1.50 ± 0.10" 0.34"6.90±0.34"(Stabilized) (107%) (161%) (150%) (213%) (73%)

" Statistically significant change compared to normal controls, p < 0.05.'' Statistically significant change compared to normal controls, p < 0.01.

The rate of this progressive dimunition is17%, 19%, and 22% respectively, suggestinga structural change in the carbohydratemoiety of the platelet membrane.

It should be noted that the increase insialic acid poor protein on the platelet mem-brane is much higher, 19%, in the thirdphase than in the two precedent phaseswhere the increase is only 7% and 9%, re-spectively (Table 2). The change in abnor-mal protein on the platelet membrane in thethird phase is statistically significant (p <0.01). This is probably the main reason thatthe abnormal serotonin uptake by plateletscannot be corrected by leprosy plasma inthe third phase.

As a consequence of the low sialic acid

TABLE 2. Sialic acid and protein contentin platelet membrane of leprosy patients.(Mean ± S.D.).

Platelets No.Proteinsnig/1(1"

platelets

Sialic acidµg/mgprotein

Control 27 2.51 ± 0.57 8.4 ± 0.96(100%) (100%)

Phase I 19 2.70 ± 0.38 7.0 ± 0.38"(Active) (107%) (83%)

Phase 11 11 2.74 ± 0.46' 6.9 ± 1.00"(Reactive) (109%) (81%)

Phase III 15 3.00 ± 0.43" 6.6 ± 0.74"(Stabilized) (119%) (78%)

" Statistically significant change compared to nor-mal controls, p < 0.05.

'' Statistically significant change compared to nor-mal controls, p < 0.01.

level in the platelets of leprosy patients, adecrease of 20-30% in the platelet aggre-gation induced by collagen has been ob-served. This decrease parallels the earlierreported decrease in adhesiveness and ag-gregability of leprosy platelets ( 8 ).

Uptake of serotonin by leprosy PRP, bynormal human platelets in leprosy plasmaand by isolated leprosy platelets in buffer ornormal human plasma. Isolated platelets ofleprosy patients show a significantly dimin-ished uptake of serotonin all through thethree phases of the evolution of the disease.The existence of defective platelets is dem-onstrated by the fact that the low serotoninuptake of leprosy platelets could not be cor-rected with normal human plasma. In con-trast, the sialic acid rich plasma of leprosypatients can prevent the reduced serotoninuptake of the defective leprosy platelets inthe first and second phases of the diseaseand increase by nearly 100% the serotoninuptake in normal human platelets (Fig. 1).In the third phase, the corrective effect ofthe leprosy plasma is significantly dimin-ished. In this phase the number of severecases in which defective platelet functionsare no longer corrected by their abnormalplasma become more and more frequent. Insevere cases, normal human platelets alsoshow a decreased serotonin uptake in theabnormal leprosy plasma. From these ex-periments the existence in leprosy of bothabnormal plasma and defective plateletsbecomes evident.

Attempt to prevent the low uptake of se-rotonin by platelets in leprosy. The failure

Released

( 3 H)5 -HT

20^pmoles/lOgpls15,6-di0H-T+dF-5HT

/1^(Theoretical^values )

15^

5,6- di0H-T

5,6-60H-TrrIF-5HT( Observed^values )

dF- 5HT

0

10 ^Cone^in Indolamine

46 International .lournal of I,eprosy^ 1981

FR). 2. Inhibition by desoxyfructo-serotonin of therelease of serotonin by human platelets (pis) inducedby 5.6-dihydroxylrvptaminc.

-.^Release of 5-HT by 5,6-dihydroxytiypta-

mine.

—•— Release of 5-HT by desoxyfructo-serotonin.

— — — Theoretical values of the release of 5-HT by

a mixture of equimolecular amounts of the

two agents. Observed values of the release of 5-HT by

a mixture of equimolecular amounts of the

two agents, showing inhibition by desoxy-

fructo-serotonin of the release induced by

5,6-dihydroxylryptamine. Normal human

platelets (N = 8).

to prevent the reduced serotonin uptake byplatelets in the third phase of the disease insevere cases, using either normal humanplasma or leprosy plasma, incited us to lookfor new approaches to the problem. Wehave recently reported that some 2-desoxy-2-keto-sugar derivatives of serotonin areactivators of the uptake and inhibitors ofthe release of serotonin by platelets ("•': 3 ).We reasoned that these properties couldhelp to correct the reduced serotonin up-take by platelets in leprosy. This is the casefor desoxyfructo-serotonin, which exhibitsthe expected properties. Even at low con-centrations (<10 -" M), this compound is apowerful inhibitor of the release of seroton-

TABLE 3. Concentration required to pro-duce 50% o/ the maximal shape-chan,:x ofrabbit platelets.

5HTDesoxyribulo-5HT

(oxalate)^

8.4 x 10 " (4.7-15.0)^74%^Desoxyfructo-5HT 1.7 x 10 ' (1.5-2.1)^93%

Desoxyfructo-5HT(oxalate)^

7.2 x 10 5 (4.2-12.3)^33%

" EC,,„ indicates the concentration inducing halfmaximal shape-change.

" 95% confidence limits.

in, showing an inhibition of 43.8 ± 6.48%(Mean ± S.D., N = 8) at a concentrationof 10 -4 M. The compound stimulates trip-taminergic receptors on the platelet mem-brane, as demonstrated by the induction ofa nearly total (93%) shape-change and ofthe uptake of serotonin (Table 3).

The release of serotonin induced by 5,6-dihydroxytryptamine is inhibited by des-oxyfructo-serotonin, as shown in Fig. 2.

In the case of lepromatous leprosy in thethird phase of the evolution of the disease,in severe cases, showing markedly reduceduptake of serotonin in PRP, addition of des-oxyfructo-serotonin at a concentration of2.15 x 10 -7 M raised the serotonin uptakesignificantly (Fig. 3, shaded area).

FIG. 3. Uptake of serotonin by the platelets of lep-rosy patients before and after addition of desoxyfruc-

to-serotonin."

icr 9^

10^

25110"'MN

10

..."

[s1 = Concentration of serotonin.* The asterisk indicates statistically significant

change (p < 0.05)." Uptake of serotonin by leprosy platelets at con-

centrations of serotonin varying from 2.5 x 10 7 M to10- 9 M with (upper line) and without (lower line) ad-dition of desoxyfructo-serotonin at concentration of

2.15 x 10 M.

Maxi-^EC 5„"^SE 95%5^mal

effect

^

1.9 x 10^(1.8-2.0)^100%

49, 1^Szabados, et al.: Lou' Serotonin Uptake by Platelets in Leprosy^47

The concentration of desoxyfructo-sero-tonin (2.15 x 10 -7 M) was chosen to be halfthe concentration producing 50% inhibitionof the uptake of serotonin by normal plate-lets in order to avoid an eventual inhibitoryeffect of the compound on the uptake ofserotonin by the leprosy platelets.

CONCLUSIONMeasurements of the sialic acid content

in isolated plasma mucoproteins and plate-lets of leprosy patients show a decrease inthis sugar component. However, due to theincreased mucoprotein concentration, thetotal sialic acid content of leprosy plasmais increased when compared to normal hu-man plasma. A low sialic acid level wasalso found with isolated leprosy platelets.Both sialic acid deficient platelets and ab-normal plasma seem to be responsible forthe abnormal clotting and low incidence ofthrombotic phenomena in leprosy patients.

]'he low content of sialic acid is correlat-ed with a decreased uptake of serotonin byisolated platelets. The change in serotoninuptake by platelets is of particular interestin leprosy because this parameter reflectsnot only a change in platelet functions butcan be directly correlated with defectivefunction of tryptaminergic neurons in thenervous system.

The failure to correct low serotonin up-take in isolated leprosy platelets with nor-mal human plasma during the third phaseof the disease is an argument in favor of adefinite structural change in leprosy plate-lets, related to their low sialic acid content.

In the first and second phases of the dis-ease, leprosy plasma can prevent the lowuptake of serotonin observed with the iso-lated platelets. The reactifying effect of lep-rosy plasma was also demonstrated by itsenhancing the serotonin uptake of normalhuman platelets. In the third phase of thedisease, however, leprosy plasma is muchless effective in stimulating serotonin up-take in platelets from leprosy patients. Thereason for this failure is probably due to thefact that in the third phase, especially insevere cases, not only is the sialic acid levelof the protein on the platelet membrane de-creased by 22% but also the proportion ofthis defective protein is increased by 19%(while only 9% in the second phase).

A new approach to prevent the low up-

take of serotonin was possible in vitro usingdesoxyfructo-serotonin. The success of thismethod in severe cases may be of practicalinterest.

SUMMARYThe plasma of leprosy patients contains

high levels of mucoproteins which are de-ficient in sialic acid. However, due to theincreased mucoprotein level, the total sialicacid content of leprous plasma, calculatedon protein, is increased when comparedwith normal human plasma. The low sero-tonin uptake observed with isolated plate-lets is probably due to their low sialic acidcontent. The inability of normal humanplasma to correct the diminished serotoninuptake by isolated leprous platelets is in fa-vor of a definite structural change in lep-rous platelets, related to their low sialicacid content.

In patients with active disease and inthose with lepra reactions, leprous plasmaitself can correct the diminished uptake ofserotonin by the isolated platelets. In pa-tients with subsided lepra reactions, theleprous plasma is much less effective. Insevere cases, where serotonin uptake is de-creased even in platelet rich plasma, des-oxyfructo-scrotonin increased the uptakeof serotonin.

RESUMENEl plasma de los pacientes con lepra conticne altos

niveles de mucoproteinas qUe son deficientes en acido

sialico. Sin embargo, debido al increment ado nivel demucoproteina, el contenido total de acido sialico en el

plasma lepromatoso resulta increment ado cuando secompara con el plasma ha IMMO normal. El bajo con-sumo de serotonina observado con plaquetas aisladas

se debe probablemente a su hajo contenido en acidosialico. La incapacidad del plasma humano normal

para corregir el consumo disminuido de scrotonina por

las plaquetas provinientes de pacientes con lepra estaen favor de on camhio estructural definido en las pla-quetas de los pacientes relacionado con su bajo con-

tenido en acido sialico.

En los pacientes con la enfermedad activa y enaquellos con reacciOn leprosa, el plasma leproso si

puede corregir el bajo consume) de serotonina por lasplaquetas aisladas. En los pacientes con reacciein lep-rosy en regresiOn el plasma es mucho menus efectivo.

En los casos severos donde el consume) dc scrotonina

esta disminuido aim en plasmas ricos en plaquetas, la

desoxifructo-serotonina es capaz de incrcmcntar elconsumo de serotonina.

48^ International Journal of Leprosy^ 1981

RESUMELe plasma des malades de la lepre contient de

grandes quantites de mucoproteines qui sont

cientes en acide sialique. Neanmoins, par suite de('augmentation du taux de mucoproteines, le contenutotal en acide sialique du plasma lepreux, lorsqu'il est

calcule en quantites de proteines, est accru Iorsqu'onle compare an plasma humain normal. La faible in-

corporation de serotonine que I' on observe dans les

plaquettes isolees est probablement dire a few - content'

faible en :wide sialique. Le fait que le plasma humainnormal ne petit retablir l'incorporation &ewe de sero-

tonine par les plaquettes lepreuses isolees, est en fa-veur de ('existence de modifications structurales ca-

racteristiques dans les plaquettes lepreuses,

modifications qui doivent etre en relation avec cur

contenu faible en acide sialique.

Chez les malades presentant une affection en evo-lution, de meme que chez ceux qui souffrent de re-actions lepreuses, le plasma lui-meme petit retablir

l'incorporation diminuce de serotonine par les pla-

quettes isolees. Chex les malades qui sont en conva-lescence de reactions lepreuses, cette activite du plas-

ma est beaucoup moins prononcee. Dans les casgraves, lorsque l'incorporation de serotonine est di-

minuee en presence d'un plasma riche en plaquettes,la desoxyfruto-serotonine augmente l'incorporation deserotonine.

Acknowledgements. This work was executed in theframework of the Indo-French Scientific and Techni-

cal Cooperation Program and supported, respectively,

by the Fondation pour la Recherche Medicale Fran-

caise and by the Oeuvres Hospitaliers de l'Ordre Sou-verain et Militaire de Malte.

The authors are grateful to Prof. A. Pletscher and

Dr. M. Graf (Basel, Switzerland) for measurement ofplatelet shape-change, to Drs. C. Labrid and G. Du-

reng (Riom, France) for investigation of tryptaminer-

gic receptor affinity, to Prof. K. P. Bhargava (Luck-now, India) for useful advice, and to Miss D. Pesques

(Htipital Saint-Louis, Paris) for technical assistance.

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