Clinical and experimental studies on the action of ethamsylate on haemostasis and on platelet functions

THROMBOSIS RESEARCH 19; 783-791, 1980 0049-3848/80/180783-09!$02.00/0 Printed in the USA. All rights reserved. Copyright (c) 1980 Pergamon Press Ltd

CLINICAL AND EXPERIMENTAL STUDIES ON THE ACTION OF ETHAMSYLATE ON HAEMOSTASIS AND ON PLATELET FUNCTIONS

H. Vinazzer Blood Coagulation Laboratory, Linz, Austria

(Received 19.5.1980; in revised form 7.8.1980. Accepted by Editor A. Henschen)

ABSTRACT In a series of tests,the action of ethamsylate on haemostasis and on plateletfunctionswas examined. Afteroral administrationof the drug,a highly significant diminutionofthe bleeding time and of the bloodloss from a standardwoundwas observed. This effect was demonstrated in healthy individuals and in patients suffering from platelet dysfunctions,andtherewas a distinct dose relation. There wasalso anincreaseofplateletadhesion,of PF 3 availabilityandof PF 4 release. The action of prostacyclin on the epinephrine induced platelet aggregation could be inhibited by ethamsylate. This offers a possible explanation for some oftheeffects on hemostasis. In addition,an action of ethamsylate on the platelet membrane was assumed since an in vitro increase of functions of the platelet membrane like PF 3 availability would not be explainable by a possible inhibition of prostacyclin.

INTRODUCTION Ethamsylate (Diethylamnonium 1.4-dihydroxy-benzenesulfonate)has beeninuseas a hemostaticdrugsince1964 (1). The drug was reported to reduce the blood loss from a standard wound (2), to increase the capillary wall resistence (3,4,5,6,7) andtoincrease the platelet adhesiveness (5,8,9,10).Thecapabil- ityofthedrugto reduce hemorrhage in patients with a bleeding disorder was demonstrated in renal insufficiency (ll), in primary menorrhagia (12) and in secondarymenorrhagia after application of IUD's(l3). A diminution of the blood loss was also found in patients without a bleeding disorder after prostatic resection (14,15) and after tonsillectomy (16,17). The latter was however,not confirmed by a further study (18). Though there seemed to be little doubt on the efficacy of the drug in reducing blood loss in normal individualsand in patients suffering fromvarious bleeding disorders,comparatively little was known about the exact mechanism of the improvement of hemostasis. Attempts were therefore,madeto furtherelucidatethemechanismofactionof this drug.

Key words: Ethamsylate, Bleeding time, Platelet functions, Prostacyclin

783

784 ETHAMSYLATE AND HAEMOSTASIS

MATERIALS AND METHODS -- Ethamsylate (DicynoneR) was obtained by Laboratoires Tablets contained 250 mg of the active substance and in 2 ml solution.

vo1.19, NO.6

OM,Geneva,Switzerland. ampouls contained 500 mg

Thrombin (TopostasinR) was purchased from Hoffmann-La Roche,Basle,Switzer- land.e vial contained 3,000 NIH units lyophilized thrombin. This was dissolved in 30 ml sterile distilled water. The solution was frozen in 1 ml portions and was kept at -360C. Russel viper venom (StypvenR) was from Wellcome,Beckenham, GB. One vial contained 0.1 ma of the lvoohilized substance. This was reconstituted with 1 ml sterile disgilled water'. The stock solution was kept at 4oC. Prior to use, 1 part was diluted with 19 parts isotonic saline solution pH 7.35. Kaolin 500 mg of the powder was suspended in 100 ml sterile isotonic saline man pH 7.35. Calcium chloride solution 0.02 mol/l in isotonic saline pH 7.35. Factor Xa (DiagenK) by Diagnostic Reagents, Thame, Oxon, GB. One vial contained 0 8 units bovine factor Xa. This was reconstituted with 0.8 ml sterile distillid water prior to use. Antithrombin III by AB KABI, Stockholm,Sweden. One vial contained 200 units human AT III The lyophilized substance was reconstituted with 200 ml sterile $i;-illled water. The solution was frozen in 1 ml portions and was kept at

.

2TkT-5 He arin (LiqueminR) by Hoffmann-La Roche,Basle,Switzerland. One ampoul cont- ,000 I.U. per ml. This solution was diluted with distilled water to

get a final concentration of 0.3 I.U. per ml. The heparin dilution was used immediately. Collagen Kollagenreagens by Hormonchemie, Munich, FRG. The stock suspension contained 1 mg horse tendon collagen per ml. This was diluted with SKF Horm buffer pH 2.8 to get a concentration of 10/g per ml. The dilution was used within 1 h at room temperature. Epinephrin (SuprareninR) by Hoechst, FRG. One ampoul contained 1 mg per ml. Prior to use,this solution was diluted with isotonic saline to get a concentration of 4Opg per ml. This dilution was used within 1 h at room temperature. Chromogenic substrate S-2222 by AB KABI, Stockholm,Sweden. The contents of one vial were reconstituted with 17 ml sterile distilled water to obtain a concentration of 2 mmol/l. This solution was stable for 3 months at 4oC. Buffer for tests with chromoqenic substrate Tris-HCl, pH 8.4, M = 0.15. Prostacyclin This was a generous gift by Wellcome,Beckenham, GB. 20 mg of the substance was dissolved in 20 ml 0.01 mol/l sodium hydroxyde. The stock solution was sealed under nitrogen and was kept at 4oC. Dilutions of this stock solution were made with Tris-HCl buffer pH 8.4, M = 0.15. All dilutions were kept in an ice bath and were used within 1 h. Coagulometer by Depex, De Bilt, NL. A re ometer 2 channel Elvi 840 by Elvi, ?%&a---

Milan, Italy. The aggregometer was to a two channel linear recorder by Linear Instruments, Texas.

Photometer Labtronic S 100 by Labtronic,Zurich,Switzerland, connected to a Diehl Certatronic calculating and printing unit.

vo1.19, No.6 ETHAMSYLATE AND HAEMOSTASIS 785

-----+ Microsco e Reichert Zetopan by Reichert,Vienna,Austria. The microscope was equippe with phase contrast and interference contrast and was connected to a Hitachi color video monitor and screen. Bleeding time Ivy (19) Blood loss per minute This method was a modification of Sutor's (20) hemor- rhagiometry. A blood pressure cuff was placed on a forearm and was inflated to 50 mm Hg. The tip of the middle finger was punctured with a 3 mm blood lancet. A stop watch was started and the finger was immediately irmnersed into a beaker containing 50 ml 8.33 mmol/l sodium hydrocarbonate solution warmed to 37oC. This solution was changed every 60 set until bleeding ceased. Hemo- globin was determined in all samples and was compared to the patient's hemo- globin in capillary blood. Thereby,the blood loss irip1 per min and the total blood loss could be calculated. Platelet count Brecher and Cronkite (21) Fibrinogen Clauss (22) Platelet aggregation Born (23). To 200,ul platelet rich plasma (PRP) 50,ul of the aggregating substance was added. The platelet count of all samples was adjusted to 300,000 per,ul by addition of an adequate volume of platelet poor plasma (PPP) to PRP. The final concentration of collagen was 2,ug per ml and the final concentration of epinephrine was 4O,umol/l. For aggregation experiments with prostacyclin,the total volume in the aggregation mixture was 350~1. 100~1 isotonic saline pH 7.35 was therefore,added to blanks in these series of aggregation tests in order to compensate for dilution and volume. The stirrer speed was 1,500 RPM and the recording speed was 2 cm per min. Parameters of aggregation are given according to the suggestion of Yamazaki et al. (24). Platelet adhesion in vitro method of Wright (25), in vivo method of Didis- helm and Bunting (26). Platelet factor 3 (PF 3) and PF 3 availability Sixma and Nijessen (27). Platelet factor 4 (PF 4) and PF 4 release Vinazzer (28) Platelet spreading Breddin (29)

PATIENTS In a first series,the action of ethamsylate was investigated in 15 cases suffering from Werlhoff's disease or from known platelet dysfunctions. Diagnosis and sex of these patients are listed below.

Diagnosis male female Chronic Werlhoff's disease Glanzmann's disease : : van Crefeld's disease Storage pool deficiency :

1

"aspirin-like" type 1 :

Two basic examinations were carried out at an interval of one week. Special care was taken to avoid administration of any drugs which would interfere with platelet functions. After the second examination all patients received four oral doses of 250 mg ethamsylate per day for one week. Tests were repeated on the 7th day of therapy and one week after withdrawal of the drug. In a second series,a dose relation study was carried out. 10 cases suffering from known platelet dysfunctions and 10 healthy individuals were included. Details are listed below.

786 El?lAMSYLATE AN0 HAEMOSTASIS Vo1.19, No.6

Diagnosis male female Storage pool deficiency 2 0 Glantmann's disease 0 "aspirin-like" type : healthy 3 :

Two basic examinations were carried out at an interval of one week. There- after,adailydoseof250mg ethamsylate was given orally for four days followed by 500 mg and 1,000 mg respectively,each for four days. Blood samples were taken on the 4th, 8th and 12th day and 2 weeksafterwithdrawal of the drug.

STATISTICAL ANALYSIS The results obtained in normal cases and in patients were grouped according to the dose of ethamsylate. Significances between groups were calculated by t-tests.

RESULTS The influence of an oral dose of 1,000 mg ethamsylate per day on a series of tests in patients with platelet dysfunctions (or Werlhoff's disease) is shown in table I. Since there was nosignificant difference between the two basic examinations and between basic tests and controls 1 week after therapy,the results of the first basic examinations were omitted in this table. Significances are given between basic tests and results of tests during therapy. -

TABLE I Influence of 1,000 mg Ethamsylate per Dayon Tests OftheHemostatic Mechanism

test basic ethamsylate after 1 week P

bleeding time,sec platelets x 103 fibrinogen, mg/dl PF 3, % PF 3availability,% PF4,% PF 4 release,% Aggregation Collagen lag,sec bm:a,% slope,0 Epinephrine bp:a,% bm:a,% slope,O

M 671.5 234.0 310.6 110.1

9i.i 10:5

102.2 59.7 81.0 74.2 90.4 63.7 30.7 16.2 51.4 16.5 36.9 12.8 55.6 20.3 71.0 13.2 60.8 13.6

10.1 12.7 28.2

1 SD 173.9 87.8 40.9 32.2

2.3 34.5 12.6

1E 13:3

A highly significant reduction of the bleeding time and of the blood loss _ could be demonstrated. There was also an increase of PF 3 availability, of PF 4 release and of some parameters of platelet aggregation. However,the in- homogenous group of patients caused considerable scattering around the mean values. Nevertheless,the increase of most of these parameters was significant. Since there was no variation in the dose of ethamsylate,a dose relation was tried to establish in the following study. Furthermore,the action of the drug

M

300.0 238.4 316.3 107.0

8;*: 31:7

14.6 41.5 41.2

1 SD 71.7 91.9 52.7 25.3 4.5

26.4 17.9

3:.i 18:2

M

716.4 225.2 302.0 111.5

9z 13:s

11.2 22.4 33.4

1 SD 351.2

80.9 35.2 26.7

2.9 33.0 16.1

l?‘: 15:7

i0.0001 n.s. n.s.

$&l

c;;:;oo1

(3% I (0.02

0.02 (0.005 0.03 *

Vo1.19, No.6 ETHAMSYLATE AND HAEMOSTASIS 787

was also examined in healthy persons. In this series,some additional tests such as blood loss per minute and the total blood loss from a standard wound, the in vivo adhesion of platelets and the platelet spreading testwereinclud- ed. Some of the results are shown in figures 1 to 3.

IL00 -)

1200 -

1000 -

600-

600-

LOO -

200 -

% 60 -

LO -

20 -

I

before 250 500 loo0 1 &k Ethamsylatc, mg per d8y after

OJ , , t

bcfow 250 500 tOM l&k Ethtmsyl8tq mg per day ??n8r

FIG. 1 FIG. 2 Dose-related Influence of Ethamsylate on Parameters of Haemostasis

Bleeding time,sec and total bloodloss,~l Platelet Adhesion, % n=lO in each group,average and 1 SD n=lO in each group,average and 1 SD 1 bleeding time,sec 1 Wright test. A: volunteers, B: patients A: volunteers, B: patients

2 total blood loss,,ul 2 Didisheim test. Patients only A: volunteers, B: patients

Significances: + below 5% level, ++ below 1% level

No significant differences were found in platelet count,in total PF 3 and PF 4, and in platelet spreading. The results are therefore,not listed in detail. Platelet aggregation in normal cases showed a significant (p=O.O2) diminution of the lag time at the highest dose of ethamsylate. In patients, there was a trend to an increase of maximum aggregation with collagen and with epinephrine respectively. The difference was however,not significant because of considerable scattering between individual cases. The influence of ethamsylate on PF 3 availability and on PF 4 release is shown in figure 3. A further series of experiments was carried out in vitro. PRP was obtained from 5 healthy individuals, 50 /ul prostacyclin was added to 200,4~1 PRP and platelet aggregation was carried out with collagen and with epinephrine respectively. The lowest concentration of prostacyclin which caused an over 90% inhibition of collagen induced aggregation and a complete inhibition of the second wave of epinephrine induced aggregation was found to be 5 ng per ml plasma. 50,ul ethamsylate in different concentrations was added to PRP with and without prostacyclin and the aggregation experiments were repeated. The results are shown in table II.

788 ETHAMSYLATE AND HAEMOSTASIS Vd.19, No.6

PFIV. 60

I____l___--A----------k___, 2A 40

1 PF 3 availability

8' - P-P-p 'K IA

A: volunteers, B: patients 2 PF 4 release

P A: volunteers, B: patients 6. n=lO in each group, Average and 1 SD le. */*

I\

significances:

fr + below 5% level, ++ below 1% level

:- t- t 18 I I I

before 250 500 Moo IvLk Ethamrylatr,m6 per day attar

FIG. 3

Dose-related Influence of Ethamsylate on PF 3 availability and PF 4 release

TABLE II

Influence of Ethamsylate on Prostacyclin - inhibited Platelet Aggregation Addition to PRP of Prostacyclin, ng/ml 0 Ethamsylate,,ug/ml 0 ; 10: Collagen lag,sec bm:a,% slope,0 Epinephrine bp:a,% bm:a,% slope,0 Significances Collagen: Epinephrine: Significances (row 3): Collagen: Epinephrine:

M 1SD M 1SD M 1 SD 52.8 6.9 not measurable 54.2 4.9 56 79.0 1.6 ;:; 42:; 9:8 62:;

20.2 2.9 2.6 17.8 2.9

53.8 2.6

i-8"

58.8 4.1 17:2 ::: 38.2 45.8 ;::

10: M 1 SD

40.6 2.7 58.8 7.8 79.6 1.1

21.6 3.1 56.4 6.7 60.4 8.5

between blank (row 2 ;nd ethamsylate (row 4): lag p= 0.01 * p= 0.025 slope n.s. bp:a n.s. bmIa n.s. slope n.s. between prostacyclin (row 2) and prostacyclin plus ethamsylate

lag notmeasurable 2:~ nps.O(;OC);) slope p = 0.03 bp:a p= 0.001 : = . slope p = 0.0001

When the concentration of prostacyclin was increased to 10 ng per ml,there was neither an effect of 1OOpg ethamsylate per ml plasma nor was there any effect when the dose of ethamsylate was increased to 1 mg per ml. On the oth- er hand,there was no effect when 5Opg ethamsylate per ml plasma was tested on PRP containing 5 ng prostacyclin per ml.

vo1.19, NO.6 ETHAMSYLATE AND HAEMOSTASIS 789

DISCUSSION

In healthy individuals as well as in patients suffering from different platelet dysfunctions and from Werlhoff's disease respectively, a highly significant diminution of the Ivy bleeding time and of the blood loss from a standard wound was observed when ethamsylate was administered. This effect was dose related and was linear up to an oral dose of 500 mg per day. When the dose was further increased,the additional increase of the effect was less distinct. A similar effect of ethamsylate was found when platelet adhesion was examined. This was however,more distinct in patients than in healthy individuals. The increase in platelet adhesion was also more pronounced in vivo (Didisheim test) than in vitro (Wright test). There was also an increase of PF 3 availability and PF 4 release which too,was more distinct in patients than in healthy persons. Some parameters of platelet aggregation significantly increased but this result was not persistent in all series. This phenomenon can probably be explained by the rather heterogenous group of patients suffering from different platelet dysfunctions. No influence was observed on the platelet count, on platelet spreading and on the fibrinogen level. Attempts of explanation of the hemostyptic mechanism of ethamsylate are rather vague. An influence on capillary fragility and permeability was assumed (6,30) and an alteration of platelets was also suggested (8). The diminution of the bleeding time and of the blood loss would be sufficiently explainable by an exclusive influence on the vessel wall though the exact nature of such an influence remained undefined. The influence on different platelet functions which are measured in vitro requires a different or an additional explanation. In the last test series of this study,a direct inhibition of prostacyclin by ethamsylate could be demonstrated within a limited range of mutual concentrations of both substances. If we can assume that the in vivo reactions are corresponding to the in vitro results,an inhibition of prostacyclin by ethamsylate would be a possible explanation for the increase of platelet adhesion in vivo,and therefore also for the diminution of the bleeding time and of the total blood loss. The effect on platelet functions in vitro is however, not explainable by an inhibition of prostacyclin. An additional effect on the platelet membrane can therefore,be assumed. When an inhibition by ethamsylate of the activity of prostacyclinis suspected, the question arises whether there is a possibility of undesired side effects of this drug. Though an increased adhesion of platelets on the vessel wall could be debated,clinical experience of numerous authors ( 1 - 18) who did not report any thrombotic or microangiopathic complications speaks against such an assumption.

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DAUBRESSE,J.C.: Etamsylate et fragiliti! capillaire chez le diabetique. R’ev. med. Liege 26, 511-514, 1971 CAJOZZO,A., CITA&LLA,P. and MALLEO,C.: Ulteriori osservazioni sulla attivita capillaro-trofica dell'etanisilato in soggetti con turbe dell'emostasi di varia natura. Gazz. med. Ital. 132, 200-207, 1973 MIGNE,J.,DEBRADIER,P. and RABY,C.: Methode d'etude de 1 'adhesivite plaqu- ettaire in vitro par thromb&lastographie,ses modifications sous l'influence d'agents therapeutiques. Proc. 1Oeme Congr. Europ. Soc.Haemat.Strasbourg 1965, part 1, p. 1134, Karger, Base1 CAGIANELLI,M.A.,VOLLANI,C. and MIRENDA,P.: Su alcuni aspetti della sindrome emorrhagica de1 cirrotico.(Modificazioni dell'adesivita piastrinica da ciclonamina. Boll.Soc.med.chir. Pisa 2, 271-289, 1968 BIZZI,B., DONAT1,M.B. and DE GAETANO,C.: Ricerche sull'effetto emostatico della ciclonamina. Gazz. Int. Med. Chir. 75, 501-510, 1970 - COCCONI,G.: Dihydroxybenzenesulfonate de diethylammonium ou cyclonamine (Dicynone). In: Compte-Rendu de la SocietC Italienne de Medecine Interne Rome, 1968, p. 5X JAFFE,Gnd WICKHAM,A.: A double blind pilot study of Dicynene in the control of menorrhagia. J.int.med.Res. 1, 127-129, 1973 CAMPBELL,S. and HARRISON,R.: A double blind trial of ethamsylate (Dicynene) in the treatment of primary and intrauterine-device menorrhagia. Lancet II, 283-285, 1976 -- SYMES,J.M.,OFFEN,D.N.,LYTTLE,J.A.,BLANDY,J.P. and CHAPUTDE SAINTONGE,D.M.: The effect of Dicynene on blood loss during and after transurethral resection of the prostate. Brit. J. Urol. 47, 203-207, 1975 - MACKENNEY,R.P.: Post-prostatectomy bleeding and the use of haemostatic drugs. J.roy.coll.Surg. Edinb. in press DE REYNIER,J.P.: L'influence de la pr'emedication au Dicynone sur la volume de la perte de sang durant l'amygdalectomie. Praxis 52, 1594-1596, 1965 -- PAPATHEODOSSLOU,N.: Essay de la Dicynone en double aveugle dans ces cas d'amygdalectomies. Med. et Hyg.(Gengve) 31, 1079-1088, 1973 VERSTRAETE,M.,TYBERGHEIN,J.,DE GREEF,Y.,GEMS,L. and VAN HOOF,A.: Double-blind trials with ethamsylate,batroxobin and tranexamic acid on blood loss after adenotonsillectomy. Acta clin. Belg. 32,136-141, 1977 - IVY,A.C.,NELSON,D. and BUCHER,G.: The standardization of certain factors in the cutaneous hemostasis bleeding time technique. J. Lab.Clin.Med. 26, 1812-1814, 1941 - SUTOR,A.H.,BOWIE,E.J.W.,MOMPSON,J.H.,DIDISHEIM,P.,~lERTENS,P.F. and OWEN,C.A.JR.: Bleeding from standardized skin pubctures: automated technique for recording time,intensity and pattern of bleeding. Amer.J.Clin.Path. 5, 541-550, 1971 BRECHER,G. and CRONKITE,E.P.: Morphology and enumeration of human blood platelets. J.appl.Physiol. 3, 365-377, 1950 CLAUSS,A.: Gerinnungsphysiologische Schnellmethode zur Bestimmung des Fibrinogens. Acta haemat. 17, 237-246, 1957 - BORN,G.V.R.: Aggregation of platelets by adenosine diphosphate and its reversal. Nature (Lond.) 194, 237-246, 1962

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Clinical and experimental studies on the action of ethamsylate on haemostasis and on platelet functions