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Partially N-desulfated heparin as a non-anticoagulant heparin: Some physico-chemical and biological properties
THROMBOSIS RESEARCH 55; 247-258, 1989 0049-3848/89 $3.00 t .OO Printed in the USA. Copyright (c) 1989 Maxwell Pergamon Macmillan plc.
All rights reserved.
PARTIALLY N-DESULFATED HEPARIN AS A NON-ANTICOAGULANTHEPARIN : SOME PHYSICO-CHEMICAL AND BIOLOGICAL PROPERTIES
E. Sache, Sanofi-Recherche,
M. Maillard,
P. Malazzi,
and H. Bertrand
Institut Choay, 46 Av. Theophile 75782 Paris-Cedex 16, France
Gautier
(Received 12.12.1988; accepted in revised form 11.5.1989 by Editor H. Vinazzer)
ABSTRACT (ITFH) obtained by A partially N-desulfated preparation of hepar-in iO‘C (TJH.4) thermally inactivating heparinic acid for 24 hours at was examined for its physico-chemical and biological proper-ties in 14 ,TOO and 27% vitro and in vivo.TIH.4 has a molecular weight of remaining N-sulfate (UFH = 17,500 ; 100% Ii-sulfate g1-oups measurable lJ\. groups) .TIH.4 has no anticoagulant acti\‘ity i( r-al)hi t conventional amidol>? ic 0 r clotting tests.Howe\.er-, in stasis-induced thrombosis mode 1 and two different using thrombogenic stimuli PCC(Kon>ne)/RV1‘), TIHA ‘iffor-ded a dose-dependent (1 0-’ iFeiba and protection impa i I mu/kg) sufficient to thrombosis (UFH *: ‘fully0 effective at 0.13 mg/l;g) .TIHA did not produce any bleeding a r-at at supramasimal antithrombotic dosage in tail bleeding and a r-abbit ear blood loss model and it did not augment ADP-induced aggregation cont1-ast, a Of platelets.ln c:ompletely N-desulfated deriv.ative of lJFH (Inoue and Nagasawa, \-itr-0 Carbohydr- .Res. 46, 67-95, 1976) also lacking measur-able in activity was stud>completelyinactive in \,ivo.The I-esul ts in this suggest that TIH.4 ma!- be consider-ed as a norl-anticoagul,int hel~a:-i ri 1OKCI still retaining antitill-ombot ii rtc t i 1.i t >Cln d ill s 0 1 ittr haemorr-hagic effect than I.FH. INTRODUCTJB Heparin i.s a major- anticoagulant widely used in the pr-eventiorr o! post* f1~01~1 s sllo\:irly, operative thrombosis .However , there has been a number of and biceriing that complications mainl! associated wi t h ttil-omboc?-topt,rlia t h<~l c-1]1! ( f 01. events could occur in patients receiving hel’ai-in ant.icoagulant a review, see r-ef I-3). consider-able inter-est was r-aised 1)). the 1~0ssib1 I it! last decade, During the low molrcul axof obtaining heparin-der-ived fractions or frmagnrents, i.e. the weight (LMW) heparins.When compar-ed to unfractionated heparin (IQV), LML Keywords
partially non-anticoagulant
: Hepar in,
N-desulfated, hepar-in.
247
antithrombotic,
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hep.~r ins show lower anticoagul‘xnt xtivi t.y wit-h J. mot-c or less altered ratio of antiFNa : nntithrombin (antiFIIa) activity i& gredter than 1 .O).They rrt,.lin the capacity to act as antithrombotic agents and .llso are devoid, at least partially, of unwanted side-effects..4 number of such LNW heparins are now in clinical use. Other sulfated polysaccharides (heparinoids) either with a glycosaminoglycan (GAG) structure (a Org 10172; SSHA ,\73025; Sulodeside) or a different carbohydrate polysaccharide backbone (u Pentosan polysulfate) also show antithrombotic properties with low, or even negligible, effect on clotting tests.Recently, a synthetic pentasaccharide with high antiFXa activity the critical binding representing sequence of heparin to antithrombin III (,\TIII) (4) was shown to possess antithrombotic properties in a rabbit stasis thrombosis model (5), in the absence of any antiFIIa activity. Over the past years a variety of chemically modified heparins have been extensively s t uclied, mos t 1y
AND NETHODS
Thrombin (bovine ; 50 NIH u/mg) was obtained from HoEfman-Laroche (Basel, Swit.zerland) .Bovine Factor Xa (FXa), the chromogenic substrates S-2238 (H-DPhe-Pip-Arg-pNA) and S-2222 (Bz-Ileu-Glu-Gly-Xrg-pNA) were from Kabi-Vitrum assay kits (Haemachem, St.Louis, USA) Sweden).Heptest heparin (Stockholm, were purchased from Diamed (Paris, France). Adenos ine 5’ phosphate (ADP) was France).Plasma source was outdated from Boehringer-Mannheim France (Paris, transfusion blood collected in citrate-acid glucose, centrifuged (2000 g, 20 stored at -2O”C.Blood for platelet aggregation studies minutes at 4°C) and was taken from forearm veins of healthy adult donors.Fresh blood was mixed citrate (3.8 % solution of sodium citrate, 5.5 HzO) in a immediately with ratio of 9 parts blood to 1 part citrate.Platelet-rich plasma (PRP) was the supernatant after whole blood sedimentation (1 hour ; separated as 20°C) .The tube was then further centrifuged (20 min ; 2000 g) to obtain platelet-poor plasma (PPP).Michaelis buffer (veronal-acetate, pH 7.3) was from Stago (Asnisres, France).A human FactorIX complex (prothrombin complex concentrate, PCC, containing Factors II, VII, IX and X ; Konyne) was from Cutter Laboratories (Berkeley, USA).Russell’s viper venom (RVV) was from Sigma Chemicals Co. (St. Louis, USA).A human activated prothrombin complex concentrate (Feiba) was from Immuno AG (Vienna, Austria).Porcine mucosal heparin, sodium salt (UFH ; 170 U/m::) was pharmaceutical grade from Choay (Paris, France).
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Prepnr,~tion of heparin fraction-s depleted of anticoagulant activity : (JFH transformecl into its heparinic acid form hy passage over a Dower %i-X8 (H’ ) column, in wnter.The solution was kept 2!, h at 5O”C, then neutralized to pH 7.0 by NaOH, dialyzed against distilled water and lyophilized (thermally inactived heparinic acid, TIHA). Affinity chromatography of TIHA (32 mg) was performed on an ATIII-Sepharose column (2.5x16cm) with elution conditions as previously reported (7). Completely N-desulfated heparin (NJ-DSH) was prepared by sol.volytic Ndesulfation of the pyridinium salt of heparin using dimethylsulfoxide in the presence of water, as described by Inoue and Nagasawa (8). The contents of N-sulfate in UFH and modified heparins were determined by turhidimetry of the inorganic sulfate 1 iberated after treatment of the samples with nitrous acid (Nethod A) (9). Pet- ioc1at.e oxidation : UFH ~;as subjected to periodate oxidation in water at pH 5. 3, followec! by NaBH.i reduction (RO-Hep) in the conditions outlined by Casu et ,ll. (IO), in the absence of any subsequent acid hytlrolysis. Xoleculalweight estimations r;ere cat-ried out using d Pharmac i;i (i’ppsala, Sxeclen) Liquid Chromatography System (FPLC) equipped vith a UV detector at 2 14 nm.High performance gel fi.ltration experiments were performed on a Superose IL HR IO/30 column wi.th O.OlY Tris-HCl-IX YaCl, pH7.0 buffer as eluent.The chromatographic data wet-e processed through a Yerck-Brukec LCltl series Software-Epson QX-16 microcomputer sys tern. The column was c,\l ibrated with heparin fractions standards of known molecular weights. I3 C-N.Y.R. Spectra were recorded with a Bruker AC-100 spectrometer at a sample concentration of 200 mg/ml in DzO.Chemical shifts ate-e r-eported to internal methanol (S 51.75 ppm in DzO with reference to internal TSPtrimethyl silyl-3 propionic acid D4-2,2,3,3 ; sodium salt). .4ssav procedures for biological activities.The activities of heparin and ; smodified heparins were measured with chromogenic substrates (S-2238 “‘2) clotting described (7). In the chromogenic and by tests as determination of antiFXa activity using S-2222 4.53 substrate, bovine FXa (0.43 nkat) was used, however, in place of endogenous plasma FSa activation by- RVV..jctivated partial thromboplastin time (.APTT) and thrombin clotting time (TT) were as previousl>described (7) .Heptest determin,ltions were c
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One vial PCC/Nonyne was reconstituted to 20 ml with saline (i.e. 25 units/ml).RVV was also reconstituted with saline (0.1 unit/ml).Each material administered separately to animal in was the immediate succession. Circulating concentrations of 25.0 u/kg PCC and 0.01 u/kg RVV were used. Each vial of lyophilized Feiba was reconstituted to 25.0 Feiba u/ml with sa1ine.A circulating concentration of 5.0 u/kg was used. Blood samples (5 ml) were collected from each animal on citrate immediately after anaesthetizing and 5 minutes after procoagulant challenge. Thromboelastography (TEG) tests were performed on whole blood and APTT, TT and Heptest determinations were on PPP after centrifuging blood samples (2000 g, 20 minutes at 4°C). PLat.elet aggregation studies were performed using a Icare aggregometer (Marseille, France) coupled to a Speedomax XL model 680 recorder (Meci ; Paris, France).Aggregation was induced by the addition of 50 ul ADP (1.38 nmol/L) in 0.15 M veronal-acetate, pH 7.3 buffer (Michaelis buffer) to PRP sample (200 ~1) containing TIHA or UFH. RESULTS The physicochemical and in vitro anticoagulant properties of TIHA are given in Table I, relatively to UFH and N-DSH.In the amidolytic and anticoagulant assays, essentially identical values were obtained for TIHA and N-DSH when outdated and freshly collected human plasmas were used.The values for UFH potency were however slightly, higher in the presence of fresh plasma.
TABLE I The Physicochemical
and Biological
Properties
Weight average molecular weight (Mw) a D (polydispersity) Contents of N-sulfate groups b (as percentage of UFH) AntiFIIa activity with S-2238 (U/mg) AntiFXa activity with S-2222 (U/mg) USP XXI assay (U/mg) Heptest (U/mg) c a. With reference to heparin samples sulfonate markers (Pressure Chemical the following values (calculated as 0.01 (N-DSH). C. Standard calibration
Properties TIHA
UFH
14 700 1.83 27
17 500 1.46 100
co.1 CO.1 (2.0
218 176 170 190
of
TIAA N-DSH
Ii
400 1.83 (I.0 CO.1 CO.1 (2.0 Cl.0
standardized with sodium polystyrene Co., Pittsburg, USA). b. Based on umol/mg) : 0.31(TIHA),1.15 (UFH), curve established with UFH.
These data show that, similarly to N-DSH, TIHA has lost its in vitro anticoagulant properties.However TIHA and N-DSH differ in their sulfate contents since only partial N-desulfation is achieved under the conditions used for TIHA preparation.Regarding the amidolytic and anticoagulant values it is noteworthy that TIHA and N-DSH were employed at dosages at least 300fold higher than UFH.TIHA was also characterized by 13C-NMR spectroscopy. Fig. 1 shows the partial 25-MHz 13C-NMR spectra of TIHA and UFH.The TIHA spectrum shows reduced signals at 699.5 and 60.6 ppm corresponding,
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PARTIALLY N-DESULFATED HEPARIN
(b)
251
Fig. 1 : 25-MHz llC-NHR spectrum of TI HA (a) . The spectrum of the corresponding starting heparin (b) is given for purpose of comparison. Assignments are taken from (15).
(a) 110.0
90.0
70.0
50.0
PPM ~-92pt~c‘ ir.-i:l:,., t t0
cilrIm11s Cl ant1 C2 of N-\ulfated glucosamine r-esidues in i t ,tlso shows the appear,lnce of ner; signals (69>.8 and 57 ppm) c orrt~spon~l in:: tc carbons Cl ,intl C:! of glucosamine residues lacking the N,ulEatr ::roup..1 signal at 565.6 ppm attributed to glucosamine devoid C? of of 4-sulLrte group is also obser-ved.It could also be seen that a slight Loss by the relative ol 0-sull-ate group might have occurred in T1H.A as indicated intensities of at 669.1 ppm and 62.5 ppm that correspond to C6 the signals with or group, respectively.The of glucosamine, without a 6-O-sulfate uranic acids has possibility that some hydrolysis of O-sulfate groups in occurred is also suggested by the difference in proportions of signals ohserved it S101.8 and 104 ppm which correspond to Cl of sulfated and nonbrrlE,jted I,-iduronic acid residues, respect.ic-ely. From the results presented above it may he concluded that TIH.1 has undergone with complete loss of measrrrable in vitro biological p<11t i,ll Li-desulfat ion, (ictivitk. a Sepharose-AT111 chromatography on Lihen TLH.1 IGAS subjected to affinity as estimated IIy a cnrbazole method, was i?SS than 1% by weight, column, activity (S-2238) less retained css high-affinity material with an antiFIIa than 1 .O u/m::. at pH 5.3 followed by NaBH-r reduction was independently Periodate oxidation the antithrombogenicity of a nonper-for-meI1 on UFH with the aim of comparing structural characteristics different anticoagulant heparin (10) possessing from TIHA, although with a molecular weight comparable to TIHA.The in vitro of periodate-oxidized (pH 5.3) and reduced UFH are anticoagulant properties given in Table II. The results indicate that periodate oxidation at pH 5.3 is associated with not complete, loss of anticoagulant activity thus confirming marked, though the findings of Casu et al. (10). effects of TIHA were examined in a rabbit stasis vivo biological The in administration of two different the intravenous in which thrombosis model PCC/RVV and Feiba) were studied. thrombogenic challenges (i.e. The dose-related antithrombotic effects of TIHA using Feiba as thrombogenic response occurred in vivo challenme0 (I?) indicate that a dose-dependent (Table III).At 1.0 mg/kg only minimal clotting is observed.The antithrombotic properties of TIHA were further studied (n-10 animals) using this trtapc+t-irt.
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TABLE II Molecular
Weight
and Biological Properties Borohydride-Reduced
Weight average molecular weight (Mw) a D (polydispersity) AntiFIIa activity, S-2238 (U/mg) AntiFXa activity, S-2222 (U/mg) CSP XXI assay (U/mg) Heptest (U/mg) b See footnote
Periodate-Osidized UFH Periodate-oxidized and reduced
Properties
a.
of
a in Table
I.
b.
(pH 5.3)
(pH 5.3) UFH
13 000 1.67 3.8 4.0 30 13
See footnote
c in Table
I.
TABLE III A Comparison of at Different Dose (mg/kg/IV Route)
2.0 1.0 0.5 0.25 0.12 0.05 0
the Antithrombotic Doses in a Rabbit
Activities of TIHA and UFH Stasis Thrombosis Model
TIHA (mean + s)
0.21 0.73 1.58 3.85 4.89 6.46
+ + + + +
LJFH (mean + s)
0.32 0.92 0.83 1.15 1.00
0 0 0 0 0.38 0.88
+ 0.8 + 1.1
+ 0.94
The thrombogenic stimulus is Feiba (5.0 u/kg).Clot grading represent a mean of right and left jugular vein clots from stasis time of 10 minutes.All other experimental conditions
scores (0 to IO) 6 animals after a are as in (12).
dose.A mean clot rating value of 1.17 + 0.94 (s ; S.D.) was obtained, thus confirming the antithrombogenic properties of TIHA in this model. When the thrombogenic challenge was PCC/RVV (IV route) with stasis time also of 10 minutes, TIHA at 1.0 mg/kg (n=lO animals) gave a mean clot rating 3.87 + 0.92.At 2.5 mg/kg (n=lO animals) the rating was 1.05 + 0.78 value of (saline control value : 6.62 + 0.84 ; n=lO animals).Thus, with both Feiba and PCC/RVV challenges, TIHA produces a dose-dependent response.The response was, however, weaker when the challenge was PCC/RVV. The antithrombotic properties of TIHA were examined in another series of venous stasis experiments where PCC/RVV was again the thrombogenic with a 20 minutes’ stasis time (Table IV).Rabbits were divided challenge, into three groups (n=6 per group) : a control saline the heparin group, (UFH) control (0.13 mg/kg) and the TIHA-receiving (1 mg/kg).Blood group samples (control pre-injection samples) were drawn from the left ear median vein of each animal exactly 5 minutes prior to injection of either saline,
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253
T.\BLEIV Thr
In
I.V.
\i\.o
Biological
Route
Saline 5 min
pre-inject.
5 min
po5I--inject.
Effects TT (set
Dose (mg/kg)
UFH 5 min p~.~‘-illject. ; m i n p 0 ~1t - i n j e c F
0.13
TIHA 5 mi n p r t - i n .j e c t . J min post-inject.
1.0
of
TIH,\
in
cl Rdhbit
;\PTT :I
Stasis
Heptes (set)
(set)
t
‘Thrombosis
TEG Jugular Vein (r+k, Stasis Thrombosis in mm)
L&.3+1.7 ‘5.0+O.S
35. 5+':.h i,.;+/+.i,
'2.O+l.i _ ‘:._,cl.i
2’1. ?+I .‘i
: \.o+>. ‘~l).(ltl1.~l
itI. ;+I .8
lH.lfl1.7
‘tO.I,+l,.;
‘x.o+!;.i
‘&.!.5+11.0
3
.St0
2’c.Stl.?
0
;
Yodel
19.8+10..! 1’).8+11).rl
f,
Cl’& +J 2
1.,i+i.:
';l.h+yL 20. 3%>. 0
C;FH or TIH.1 in the right ear nralzindl vein. Clotting tests were performed on each control sample. Five minlll es dfter PCC/(?_, u/k::)/RL-l’(l1.01 u/kg) saline or antithrombotic agents administration, vrin..\ftetexact- ly i minrltas the were injected in the left ear marginal veins were tied.Five minutcas post-injection of thrombo::rnic exposed jugular agents another blood sample wds rlrawn (post-inject.ion sdmplr) ,111ri (‘lots wer-e est.imdtec1 after 20 minutes’stdsis time. The (latL1 in Table TV indicate that IV administration I)[ TIfl.\ .~t 1.0 nrp,/k:: in 11~ .intico,tglll.lnt f:ff(xr:t I‘his is In measurdb rabbits does not produce any c 0 n t r ‘1s t t 0 LIFH which morlif ies blood p.lr<+metPl-s <1t Imuctl IOW~~l IlOSt? (_1,:2 II. 13 mg/ks) .TIIl;\, however, shows ,+nti thl-ombot il: propeL-ti(:5, ~1 I hou,Jtr less /> in the experiments ~erc Feiba bias the challen::e. l:llrt hernror-e, pot-entl>than occur red between these results also indic,lte th,xt no signifi~:ant diffcl-tanc:t, the scores of TIHA (1 mg/kg) whtxn st,isis time xere either IO or 20 minlltes. Further studies would evidently be necessary to support this conclusion. The antithrombotic effects of TIHA were also recently confirmed in a laserat the .J-ti. Goethe induced thrombosis model in Pr. K. Breddin’s laboratory Crniversity Hospital in Frankfurt/Main (FRG) (personal communication). Since N-DSH and TIHA both totally lack anticoagulant properties (T
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prolongation of the bleeding time at the relatively lower dosages of 0.5 and 1.0 mg/kg.In this model, TIHA at IO-time weight (10 mg/kg) produced a lower increase in comparison to UFH at 1.0 mg/kg.In the rabbit ear blood loss model (Table VI) UFH produced a dose-dependent increase in the red-cell loss at 1.25 and 2.5 mg/kg.In contrast, TIHA at relatively higher concentrations of up to 10 mg/kg did not produce comparable blood loss effects.It is indeed apparent that the amount of UFH needed to produce prolongation of bleeding times is much higher than the dosage needed for producing the antithrombotic effect.The same, however, applies for TIHA where higher amounts are needed to produce extensive prolongation of bleeding times. TABLE V Effects of TIHA and UFH on Rat Tail Bleeding Times (Template) Template Bleeding Time (set) 120.8 + 4.3 620.8 + 10.3 648.0 + 48.9 150.5 + 8.3 250.0 + 18.9 363.5 + 18.4 456.0 + 14.8
Treatment Saline (control) UFH (0.5 mg/kg) UFH (1.O mg/kg) TIHA (1.0 mg/kg) TIHA (2.5 mg/kg) TIHA (5.0 mg/kg) TIHA (10.0 mg/kg)
(S.E.).All Results for each test group (n=lO rats) are given as mean +sm tests were performed 15 minutes after animals received treatment.
TABLE VI Effects of TIHA and UFH on Rabbit's Ear Blood Loss Treatment Saline (control) UFH (1.25 mg/kg) UFH (2.5 mg/kg) TIHA (2.5 mg/kg) TIHA (5.0 mg/kg) TIHA (10.0 mg/kg)
Blood Loss (IO9 red blood cells/L) 0.095 + 0.005 0.620 t 0.022 1.450 + 0.089 0.143 + 0.009 0.156 + 0.010 0.330 + 0.019
Results for each test group (n=lO rabbits) are given as mean +sm (S.E.).All tests were performed 15 minutes after animals received treatment. The effects of UFH and TIHA on ADP-induced platelet aggregation are shown in Fig.Z.In the presence of UFH an important enhancement of platelet aggregation (tracing B) is observed relatively to the saline control (tracing A).In contrast, TIHA, in the presence of an equal amount of ADP gives an aggregation (tracing C) very comparable to the saline control.Since the sensitivity of platelet aggregation widely varies from one blood donor to another, the experiment was repeated on 10 different blood samples, with identical results.These experiments indicate that TIHA does not produce any enhancement of platelet aggregation even in 80-fold weight excess over UFH.
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PARTIALLY N-DESULFATED HEPARIN
! Time
255
F ier ,.2 : Influence of ‘fIH;1 on ADPinduced platelet aggregation.0. I ml saline, tiFH or TIHA were added to 0.3 ml PRP. Platelet aggregation was induced by the atlrli tion of 5Ocll ADP solution (1 . ‘38 I.lmol/L jn >fichdelis bufft:1- ( pH 72) to 100 1-11 of each mizture..1:cont rol (sdl ine + ADP) . C:LFil, 5 units/ml + ADP. C: TTHA, L.’ 5 mg/mt + .\I)P.
DISCUSS ION The primary aim study of this was t0 investigate the antithrombotic properties of a partially N-desulfated heparin (TIHA) obtained by thermally inactivating heparinic acid.hlthough completely lacking in vitro ant.iFIIa and antiFNa activities, TIHA showed antithrombotic action in an in vivo model. rabbit stasis-induced thrombosis The effect of X-desulfation on the properties of heparin has been largely addressed in recent years (16-Zj).Despite differences in the methods used to achieve N-desulfation, have all shown that an increase in the these studies degree of N-desulfation is xcompanied b) a decrease of anticoagulant properties .However, and to the our know ledge best of there has been, to date, no N-desulfated partially’ N-desulfated) studies using (or nonanticoagulant heparin vith relevance to it_; in viva antithrombotic action. The present studies indicate that TIHA, when compared to UFH on a weight is at least 300 times less active in amidolytic and clotting tests. basis, Khen N-DSH, a completely N-desulfated heparin preparation (8) also lacking anticlotting properties (Table I) was compared to TIHA in anticoagulant and in vivc: experiments for possible antithrombotic action, it was found totally inactive. noteworthy that TIHA produces a dose-dependent antithrombotic effect It is the antithrombotic dosage, no in the stasis thrombosis model.However, at was noted.In contrast UFH produced a modif ication of the clotting times tests.Indeed signif icant modif ication the various the antithrombotic of the with than one observed relatively weaker effects of TIHA were significant antithrombotic effects UFH.However, the fact that TIHA produces that its effect could be anticoagulation suggests without. producing any u involving endothelium. mediated through presumably other mechanisms, effect on the bleeding TIHA dj.d not produce any significant models at the mg/kg .There was a dose-dependent increase in antithrombotic dosage of 1.0 the rat tail bleeding and the rabbit ear blood loss models with TIHA.However was relatively minor in contrast to the observed effects with this increase UFH.In both animal models TIHA at 10.0 mg/kg produced lesser effect than the
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one observed by one-t.enth the (losage of [:FH (1 .O nrg/kg) .This data clearly suggests that liFH produces a mot-e pronounced haemorrhagic effect and its s~lfety - efficacy index is narrower than for TIHA. possibility could esist that anticoagulant
: We are grateful for his expert advice on bleeding aggregation tests.
to Dr. J. Choay for many useful comments ; assistance in NMR experiments ; to Miss A. experiments, and to Mrs S.Roussange for
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activit:- of heparin.
&
zidv. Carbohycl.
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15.
observations AYO’ITE, L., anll PERLIN, A.S. NMR spectroscopic the function of sulfate groups in heprrin. C.1Lcium binding biological activity. Carbohyrl. Res. m, 267-277, 1386.
16.
LEVY, L., and PETRACEK, F. J. Chemical N-resulfated heparin. Proc. Sot. Exp.
17.
HELBERT, J.R., I. Hydrolytic
and pharmacological Biol.
Med.
109,
related vs.
studies
to
on
901-905,
1962.
and M.ZRINI, M.A. Structural studies of heparin. cleavage of sulfates. Biochemistrv 2, 1101-1106,
1963.
18.
LASKER, S.E., and STIVALA, S. S. Physicochemical studies of f r-act ionated bovine heparin. 3. Some dilute solution properties. ;\rch. Biochem. Ilj, 360-372, 1966. Biophys.
19.
YOSIZA-\E;_-\, Z. , KOTOKU, T. , P.UlAUCI~I, F. , and !l.:TSUNO, M. Stability the biological activ-ities of hep
20.
SPMTK, G.B., N-desulfated
21.
EIKA, C. Anticoagulant ant-i plnt,elet Thromb. Res. 2, 349-360, 1973.
3 ‘I
CTFONELLI, J.A. The relationship of molecular rfeight, content and distribution to anticoagulant activity of preparations. Carbohvd. Res. 37, 145-154, 1974.
23.
CIFONELLI, J.A. Relation of chemical structure of heparin to its anticoagulant activity. In : Heparin-Structure, Function and Clinical Implications. R.A. Bradshaw and S. Kessler (Eds) New York : Plenum Press, 1975, pp.95-103.
24.
NAGASAWA , K. , TOKUYASU, T. and INOGE, Y. Studies on the N-substitution in heparin on its anticoagulant activity. 8l, 989-993, 1977.
25.
DANISHEFSKY, T. modification of
26.
HEUCK, C.C., SCHIELE, E., HORN, D., FROND,\, D., and RITZ, E. The role OF surface charge on the accelerating action of heparin on the antithrombin III-inhibited activity of a-thrombin. J. Biol. Chem. 260,
“_.
4598-4603, 27.
and YOCPM, C.F. Gel Eiltration behavior of heparin. J. Chromatogr; 35, 101-103, 1968.
Studies on the its structure.
aggr-egating
hcparin
activities
mechanism of heparin Fed. Proc. 16, 33-35,
of
of
and
heparin.
and sulfate heparin
influence of J. Biochem.
action 1977.
by
1983.
BARZU, T., VAN RIJN, J.L.M.L., PETITOU, M., MOLHO, P., TOBELEM, G., and CAEN, J. Endothelial binding sites for heparin ; specificity and role in heparin neutralization. Biochem. J. 238, 847-854, 1986.
All rights reserved.
PARTIALLY N-DESULFATED HEPARIN AS A NON-ANTICOAGULANTHEPARIN : SOME PHYSICO-CHEMICAL AND BIOLOGICAL PROPERTIES
E. Sache, Sanofi-Recherche,
M. Maillard,
P. Malazzi,
and H. Bertrand
Institut Choay, 46 Av. Theophile 75782 Paris-Cedex 16, France
Gautier
(Received 12.12.1988; accepted in revised form 11.5.1989 by Editor H. Vinazzer)
ABSTRACT (ITFH) obtained by A partially N-desulfated preparation of hepar-in iO‘C (TJH.4) thermally inactivating heparinic acid for 24 hours at was examined for its physico-chemical and biological proper-ties in 14 ,TOO and 27% vitro and in vivo.TIH.4 has a molecular weight of remaining N-sulfate (UFH = 17,500 ; 100% Ii-sulfate g1-oups measurable lJ\. groups) .TIH.4 has no anticoagulant acti\‘ity i( r-al)hi t conventional amidol>? ic 0 r clotting tests.Howe\.er-, in stasis-induced thrombosis mode 1 and two different using thrombogenic stimuli PCC(Kon>ne)/RV1‘), TIHA ‘iffor-ded a dose-dependent (1 0-’ iFeiba and protection impa i I mu/kg) sufficient to thrombosis (UFH *: ‘fully0 effective at 0.13 mg/l;g) .TIHA did not produce any bleeding a r-at at supramasimal antithrombotic dosage in tail bleeding and a r-abbit ear blood loss model and it did not augment ADP-induced aggregation cont1-ast, a Of platelets.ln c:ompletely N-desulfated deriv.ative of lJFH (Inoue and Nagasawa, \-itr-0 Carbohydr- .Res. 46, 67-95, 1976) also lacking measur-able in activity was stud>completelyinactive in \,ivo.The I-esul ts in this suggest that TIH.4 ma!- be consider-ed as a norl-anticoagul,int hel~a:-i ri 1OKCI still retaining antitill-ombot ii rtc t i 1.i t >Cln d ill s 0 1 ittr haemorr-hagic effect than I.FH. INTRODUCTJB Heparin i.s a major- anticoagulant widely used in the pr-eventiorr o! post* f1~01~1 s sllo\:irly, operative thrombosis .However , there has been a number of and biceriing that complications mainl! associated wi t h ttil-omboc?-topt,rlia t h<~l c-1]1! ( f 01. events could occur in patients receiving hel’ai-in ant.icoagulant a review, see r-ef I-3). consider-able inter-est was r-aised 1)). the 1~0ssib1 I it! last decade, During the low molrcul axof obtaining heparin-der-ived fractions or frmagnrents, i.e. the weight (LMW) heparins.When compar-ed to unfractionated heparin (IQV), LML Keywords
partially non-anticoagulant
: Hepar in,
N-desulfated, hepar-in.
247
antithrombotic,
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PARTIALLY N-DESULFATED HEPARIN
Vol. 55, No. 2
hep.~r ins show lower anticoagul‘xnt xtivi t.y wit-h J. mot-c or less altered ratio of antiFNa : nntithrombin (antiFIIa) activity i& gredter than 1 .O).They rrt,.lin the capacity to act as antithrombotic agents and .llso are devoid, at least partially, of unwanted side-effects..4 number of such LNW heparins are now in clinical use. Other sulfated polysaccharides (heparinoids) either with a glycosaminoglycan (GAG) structure (a Org 10172; SSHA ,\73025; Sulodeside) or a different carbohydrate polysaccharide backbone (u Pentosan polysulfate) also show antithrombotic properties with low, or even negligible, effect on clotting tests.Recently, a synthetic pentasaccharide with high antiFXa activity the critical binding representing sequence of heparin to antithrombin III (,\TIII) (4) was shown to possess antithrombotic properties in a rabbit stasis thrombosis model (5), in the absence of any antiFIIa activity. Over the past years a variety of chemically modified heparins have been extensively s t uclied, mos t 1y
AND NETHODS
Thrombin (bovine ; 50 NIH u/mg) was obtained from HoEfman-Laroche (Basel, Swit.zerland) .Bovine Factor Xa (FXa), the chromogenic substrates S-2238 (H-DPhe-Pip-Arg-pNA) and S-2222 (Bz-Ileu-Glu-Gly-Xrg-pNA) were from Kabi-Vitrum assay kits (Haemachem, St.Louis, USA) Sweden).Heptest heparin (Stockholm, were purchased from Diamed (Paris, France). Adenos ine 5’ phosphate (ADP) was France).Plasma source was outdated from Boehringer-Mannheim France (Paris, transfusion blood collected in citrate-acid glucose, centrifuged (2000 g, 20 stored at -2O”C.Blood for platelet aggregation studies minutes at 4°C) and was taken from forearm veins of healthy adult donors.Fresh blood was mixed citrate (3.8 % solution of sodium citrate, 5.5 HzO) in a immediately with ratio of 9 parts blood to 1 part citrate.Platelet-rich plasma (PRP) was the supernatant after whole blood sedimentation (1 hour ; separated as 20°C) .The tube was then further centrifuged (20 min ; 2000 g) to obtain platelet-poor plasma (PPP).Michaelis buffer (veronal-acetate, pH 7.3) was from Stago (Asnisres, France).A human FactorIX complex (prothrombin complex concentrate, PCC, containing Factors II, VII, IX and X ; Konyne) was from Cutter Laboratories (Berkeley, USA).Russell’s viper venom (RVV) was from Sigma Chemicals Co. (St. Louis, USA).A human activated prothrombin complex concentrate (Feiba) was from Immuno AG (Vienna, Austria).Porcine mucosal heparin, sodium salt (UFH ; 170 U/m::) was pharmaceutical grade from Choay (Paris, France).
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PARTIALLY N-DESULFATED HEPARIN
249
Prepnr,~tion of heparin fraction-s depleted of anticoagulant activity : (JFH transformecl into its heparinic acid form hy passage over a Dower %i-X8 (H’ ) column, in wnter.The solution was kept 2!, h at 5O”C, then neutralized to pH 7.0 by NaOH, dialyzed against distilled water and lyophilized (thermally inactived heparinic acid, TIHA). Affinity chromatography of TIHA (32 mg) was performed on an ATIII-Sepharose column (2.5x16cm) with elution conditions as previously reported (7). Completely N-desulfated heparin (NJ-DSH) was prepared by sol.volytic Ndesulfation of the pyridinium salt of heparin using dimethylsulfoxide in the presence of water, as described by Inoue and Nagasawa (8). The contents of N-sulfate in UFH and modified heparins were determined by turhidimetry of the inorganic sulfate 1 iberated after treatment of the samples with nitrous acid (Nethod A) (9). Pet- ioc1at.e oxidation : UFH ~;as subjected to periodate oxidation in water at pH 5. 3, followec! by NaBH.i reduction (RO-Hep) in the conditions outlined by Casu et ,ll. (IO), in the absence of any subsequent acid hytlrolysis. Xoleculalweight estimations r;ere cat-ried out using d Pharmac i;i (i’ppsala, Sxeclen) Liquid Chromatography System (FPLC) equipped vith a UV detector at 2 14 nm.High performance gel fi.ltration experiments were performed on a Superose IL HR IO/30 column wi.th O.OlY Tris-HCl-IX YaCl, pH7.0 buffer as eluent.The chromatographic data wet-e processed through a Yerck-Brukec LCltl series Software-Epson QX-16 microcomputer sys tern. The column was c,\l ibrated with heparin fractions standards of known molecular weights. I3 C-N.Y.R. Spectra were recorded with a Bruker AC-100 spectrometer at a sample concentration of 200 mg/ml in DzO.Chemical shifts ate-e r-eported to internal methanol (S 51.75 ppm in DzO with reference to internal TSPtrimethyl silyl-3 propionic acid D4-2,2,3,3 ; sodium salt). .4ssav procedures for biological activities.The activities of heparin and ; smodified heparins were measured with chromogenic substrates (S-2238 “‘2) clotting described (7). In the chromogenic and by tests as determination of antiFXa activity using S-2222 4.53 substrate, bovine FXa (0.43 nkat) was used, however, in place of endogenous plasma FSa activation by- RVV..jctivated partial thromboplastin time (.APTT) and thrombin clotting time (TT) were as previousl>described (7) .Heptest determin,ltions were c
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PARTIALLY N-DESULFATED HEPARIN
Vol. 55, No. 2
One vial PCC/Nonyne was reconstituted to 20 ml with saline (i.e. 25 units/ml).RVV was also reconstituted with saline (0.1 unit/ml).Each material administered separately to animal in was the immediate succession. Circulating concentrations of 25.0 u/kg PCC and 0.01 u/kg RVV were used. Each vial of lyophilized Feiba was reconstituted to 25.0 Feiba u/ml with sa1ine.A circulating concentration of 5.0 u/kg was used. Blood samples (5 ml) were collected from each animal on citrate immediately after anaesthetizing and 5 minutes after procoagulant challenge. Thromboelastography (TEG) tests were performed on whole blood and APTT, TT and Heptest determinations were on PPP after centrifuging blood samples (2000 g, 20 minutes at 4°C). PLat.elet aggregation studies were performed using a Icare aggregometer (Marseille, France) coupled to a Speedomax XL model 680 recorder (Meci ; Paris, France).Aggregation was induced by the addition of 50 ul ADP (1.38 nmol/L) in 0.15 M veronal-acetate, pH 7.3 buffer (Michaelis buffer) to PRP sample (200 ~1) containing TIHA or UFH. RESULTS The physicochemical and in vitro anticoagulant properties of TIHA are given in Table I, relatively to UFH and N-DSH.In the amidolytic and anticoagulant assays, essentially identical values were obtained for TIHA and N-DSH when outdated and freshly collected human plasmas were used.The values for UFH potency were however slightly, higher in the presence of fresh plasma.
TABLE I The Physicochemical
and Biological
Properties
Weight average molecular weight (Mw) a D (polydispersity) Contents of N-sulfate groups b (as percentage of UFH) AntiFIIa activity with S-2238 (U/mg) AntiFXa activity with S-2222 (U/mg) USP XXI assay (U/mg) Heptest (U/mg) c a. With reference to heparin samples sulfonate markers (Pressure Chemical the following values (calculated as 0.01 (N-DSH). C. Standard calibration
Properties TIHA
UFH
14 700 1.83 27
17 500 1.46 100
co.1 CO.1 (2.0
218 176 170 190
of
TIAA N-DSH
Ii
400 1.83 (I.0 CO.1 CO.1 (2.0 Cl.0
standardized with sodium polystyrene Co., Pittsburg, USA). b. Based on umol/mg) : 0.31(TIHA),1.15 (UFH), curve established with UFH.
These data show that, similarly to N-DSH, TIHA has lost its in vitro anticoagulant properties.However TIHA and N-DSH differ in their sulfate contents since only partial N-desulfation is achieved under the conditions used for TIHA preparation.Regarding the amidolytic and anticoagulant values it is noteworthy that TIHA and N-DSH were employed at dosages at least 300fold higher than UFH.TIHA was also characterized by 13C-NMR spectroscopy. Fig. 1 shows the partial 25-MHz 13C-NMR spectra of TIHA and UFH.The TIHA spectrum shows reduced signals at 699.5 and 60.6 ppm corresponding,
Vol. 55, No. 2
PARTIALLY N-DESULFATED HEPARIN
(b)
251
Fig. 1 : 25-MHz llC-NHR spectrum of TI HA (a) . The spectrum of the corresponding starting heparin (b) is given for purpose of comparison. Assignments are taken from (15).
(a) 110.0
90.0
70.0
50.0
PPM ~-92pt~c‘ ir.-i:l:,., t t0
cilrIm11s Cl ant1 C2 of N-\ulfated glucosamine r-esidues in i t ,tlso shows the appear,lnce of ner; signals (69>.8 and 57 ppm) c orrt~spon~l in:: tc carbons Cl ,intl C:! of glucosamine residues lacking the N,ulEatr ::roup..1 signal at 565.6 ppm attributed to glucosamine devoid C? of of 4-sulLrte group is also obser-ved.It could also be seen that a slight Loss by the relative ol 0-sull-ate group might have occurred in T1H.A as indicated intensities of at 669.1 ppm and 62.5 ppm that correspond to C6 the signals with or group, respectively.The of glucosamine, without a 6-O-sulfate uranic acids has possibility that some hydrolysis of O-sulfate groups in occurred is also suggested by the difference in proportions of signals ohserved it S101.8 and 104 ppm which correspond to Cl of sulfated and nonbrrlE,jted I,-iduronic acid residues, respect.ic-ely. From the results presented above it may he concluded that TIH.1 has undergone with complete loss of measrrrable in vitro biological p<11t i,ll Li-desulfat ion, (ictivitk. a Sepharose-AT111 chromatography on Lihen TLH.1 IGAS subjected to affinity as estimated IIy a cnrbazole method, was i?SS than 1% by weight, column, activity (S-2238) less retained css high-affinity material with an antiFIIa than 1 .O u/m::. at pH 5.3 followed by NaBH-r reduction was independently Periodate oxidation the antithrombogenicity of a nonper-for-meI1 on UFH with the aim of comparing structural characteristics different anticoagulant heparin (10) possessing from TIHA, although with a molecular weight comparable to TIHA.The in vitro of periodate-oxidized (pH 5.3) and reduced UFH are anticoagulant properties given in Table II. The results indicate that periodate oxidation at pH 5.3 is associated with not complete, loss of anticoagulant activity thus confirming marked, though the findings of Casu et al. (10). effects of TIHA were examined in a rabbit stasis vivo biological The in administration of two different the intravenous in which thrombosis model PCC/RVV and Feiba) were studied. thrombogenic challenges (i.e. The dose-related antithrombotic effects of TIHA using Feiba as thrombogenic response occurred in vivo challenme0 (I?) indicate that a dose-dependent (Table III).At 1.0 mg/kg only minimal clotting is observed.The antithrombotic properties of TIHA were further studied (n-10 animals) using this trtapc+t-irt.
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PARTIALLY N-DESULFATED HEPARIN
Vol. 55, No. 2
TABLE II Molecular
Weight
and Biological Properties Borohydride-Reduced
Weight average molecular weight (Mw) a D (polydispersity) AntiFIIa activity, S-2238 (U/mg) AntiFXa activity, S-2222 (U/mg) CSP XXI assay (U/mg) Heptest (U/mg) b See footnote
Periodate-Osidized UFH Periodate-oxidized and reduced
Properties
a.
of
a in Table
I.
b.
(pH 5.3)
(pH 5.3) UFH
13 000 1.67 3.8 4.0 30 13
See footnote
c in Table
I.
TABLE III A Comparison of at Different Dose (mg/kg/IV Route)
2.0 1.0 0.5 0.25 0.12 0.05 0
the Antithrombotic Doses in a Rabbit
Activities of TIHA and UFH Stasis Thrombosis Model
TIHA (mean + s)
0.21 0.73 1.58 3.85 4.89 6.46
+ + + + +
LJFH (mean + s)
0.32 0.92 0.83 1.15 1.00
0 0 0 0 0.38 0.88
+ 0.8 + 1.1
+ 0.94
The thrombogenic stimulus is Feiba (5.0 u/kg).Clot grading represent a mean of right and left jugular vein clots from stasis time of 10 minutes.All other experimental conditions
scores (0 to IO) 6 animals after a are as in (12).
dose.A mean clot rating value of 1.17 + 0.94 (s ; S.D.) was obtained, thus confirming the antithrombogenic properties of TIHA in this model. When the thrombogenic challenge was PCC/RVV (IV route) with stasis time also of 10 minutes, TIHA at 1.0 mg/kg (n=lO animals) gave a mean clot rating 3.87 + 0.92.At 2.5 mg/kg (n=lO animals) the rating was 1.05 + 0.78 value of (saline control value : 6.62 + 0.84 ; n=lO animals).Thus, with both Feiba and PCC/RVV challenges, TIHA produces a dose-dependent response.The response was, however, weaker when the challenge was PCC/RVV. The antithrombotic properties of TIHA were examined in another series of venous stasis experiments where PCC/RVV was again the thrombogenic with a 20 minutes’ stasis time (Table IV).Rabbits were divided challenge, into three groups (n=6 per group) : a control saline the heparin group, (UFH) control (0.13 mg/kg) and the TIHA-receiving (1 mg/kg).Blood group samples (control pre-injection samples) were drawn from the left ear median vein of each animal exactly 5 minutes prior to injection of either saline,
Vol. 55, No. 2
PARTIALLY N-DESULFATED HEPARIN
253
T.\BLEIV Thr
In
I.V.
\i\.o
Biological
Route
Saline 5 min
pre-inject.
5 min
po5I--inject.
Effects TT (set
Dose (mg/kg)
UFH 5 min p~.~‘-illject. ; m i n p 0 ~1t - i n j e c F
0.13
TIHA 5 mi n p r t - i n .j e c t . J min post-inject.
1.0
of
TIH,\
in
cl Rdhbit
;\PTT :I
Stasis
Heptes (set)
(set)
t
‘Thrombosis
TEG Jugular Vein (r+k, Stasis Thrombosis in mm)
L&.3+1.7 ‘5.0+O.S
35. 5+':.h i,.;+/+.i,
'2.O+l.i _ ‘:._,cl.i
2’1. ?+I .‘i
: \.o+>. ‘~l).(ltl1.~l
itI. ;+I .8
lH.lfl1.7
‘tO.I,+l,.;
‘x.o+!;.i
‘&.!.5+11.0
3
.St0
2’c.Stl.?
0
;
Yodel
19.8+10..! 1’).8+11).rl
f,
Cl’& +J 2
1.,i+i.:
';l.h+yL 20. 3%>. 0
C;FH or TIH.1 in the right ear nralzindl vein. Clotting tests were performed on each control sample. Five minlll es dfter PCC/(?_, u/k::)/RL-l’(l1.01 u/kg) saline or antithrombotic agents administration, vrin..\ftetexact- ly i minrltas the were injected in the left ear marginal veins were tied.Five minutcas post-injection of thrombo::rnic exposed jugular agents another blood sample wds rlrawn (post-inject.ion sdmplr) ,111ri (‘lots wer-e est.imdtec1 after 20 minutes’stdsis time. The (latL1 in Table TV indicate that IV administration I)[ TIfl.\ .~t 1.0 nrp,/k:: in 11~ .intico,tglll.lnt f:ff(xr:t I‘his is In measurdb rabbits does not produce any c 0 n t r ‘1s t t 0 LIFH which morlif ies blood p.lr<+metPl-s <1t Imuctl IOW~~l IlOSt? (_1,:2 II. 13 mg/ks) .TIIl;\, however, shows ,+nti thl-ombot il: propeL-ti(:5, ~1 I hou,Jtr less /> in the experiments ~erc Feiba bias the challen::e. l:llrt hernror-e, pot-entl>than occur red between these results also indic,lte th,xt no signifi~:ant diffcl-tanc:t, the scores of TIHA (1 mg/kg) whtxn st,isis time xere either IO or 20 minlltes. Further studies would evidently be necessary to support this conclusion. The antithrombotic effects of TIHA were also recently confirmed in a laserat the .J-ti. Goethe induced thrombosis model in Pr. K. Breddin’s laboratory Crniversity Hospital in Frankfurt/Main (FRG) (personal communication). Since N-DSH and TIHA both totally lack anticoagulant properties (T
254
PARTIALLY N-DESULFATED HEPARIN
Vol. 55, No. 2
prolongation of the bleeding time at the relatively lower dosages of 0.5 and 1.0 mg/kg.In this model, TIHA at IO-time weight (10 mg/kg) produced a lower increase in comparison to UFH at 1.0 mg/kg.In the rabbit ear blood loss model (Table VI) UFH produced a dose-dependent increase in the red-cell loss at 1.25 and 2.5 mg/kg.In contrast, TIHA at relatively higher concentrations of up to 10 mg/kg did not produce comparable blood loss effects.It is indeed apparent that the amount of UFH needed to produce prolongation of bleeding times is much higher than the dosage needed for producing the antithrombotic effect.The same, however, applies for TIHA where higher amounts are needed to produce extensive prolongation of bleeding times. TABLE V Effects of TIHA and UFH on Rat Tail Bleeding Times (Template) Template Bleeding Time (set) 120.8 + 4.3 620.8 + 10.3 648.0 + 48.9 150.5 + 8.3 250.0 + 18.9 363.5 + 18.4 456.0 + 14.8
Treatment Saline (control) UFH (0.5 mg/kg) UFH (1.O mg/kg) TIHA (1.0 mg/kg) TIHA (2.5 mg/kg) TIHA (5.0 mg/kg) TIHA (10.0 mg/kg)
(S.E.).All Results for each test group (n=lO rats) are given as mean +sm tests were performed 15 minutes after animals received treatment.
TABLE VI Effects of TIHA and UFH on Rabbit's Ear Blood Loss Treatment Saline (control) UFH (1.25 mg/kg) UFH (2.5 mg/kg) TIHA (2.5 mg/kg) TIHA (5.0 mg/kg) TIHA (10.0 mg/kg)
Blood Loss (IO9 red blood cells/L) 0.095 + 0.005 0.620 t 0.022 1.450 + 0.089 0.143 + 0.009 0.156 + 0.010 0.330 + 0.019
Results for each test group (n=lO rabbits) are given as mean +sm (S.E.).All tests were performed 15 minutes after animals received treatment. The effects of UFH and TIHA on ADP-induced platelet aggregation are shown in Fig.Z.In the presence of UFH an important enhancement of platelet aggregation (tracing B) is observed relatively to the saline control (tracing A).In contrast, TIHA, in the presence of an equal amount of ADP gives an aggregation (tracing C) very comparable to the saline control.Since the sensitivity of platelet aggregation widely varies from one blood donor to another, the experiment was repeated on 10 different blood samples, with identical results.These experiments indicate that TIHA does not produce any enhancement of platelet aggregation even in 80-fold weight excess over UFH.
Vol. 55, No. 2
PARTIALLY N-DESULFATED HEPARIN
! Time
255
F ier ,.2 : Influence of ‘fIH;1 on ADPinduced platelet aggregation.0. I ml saline, tiFH or TIHA were added to 0.3 ml PRP. Platelet aggregation was induced by the atlrli tion of 5Ocll ADP solution (1 . ‘38 I.lmol/L jn >fichdelis bufft:1- ( pH 72) to 100 1-11 of each mizture..1:cont rol (sdl ine + ADP) . C:LFil, 5 units/ml + ADP. C: TTHA, L.’ 5 mg/mt + .\I)P.
DISCUSS ION The primary aim study of this was t0 investigate the antithrombotic properties of a partially N-desulfated heparin (TIHA) obtained by thermally inactivating heparinic acid.hlthough completely lacking in vitro ant.iFIIa and antiFNa activities, TIHA showed antithrombotic action in an in vivo model. rabbit stasis-induced thrombosis The effect of X-desulfation on the properties of heparin has been largely addressed in recent years (16-Zj).Despite differences in the methods used to achieve N-desulfation, have all shown that an increase in the these studies degree of N-desulfation is xcompanied b) a decrease of anticoagulant properties .However, and to the our know ledge best of there has been, to date, no N-desulfated partially’ N-desulfated) studies using (or nonanticoagulant heparin vith relevance to it_; in viva antithrombotic action. The present studies indicate that TIHA, when compared to UFH on a weight is at least 300 times less active in amidolytic and clotting tests. basis, Khen N-DSH, a completely N-desulfated heparin preparation (8) also lacking anticlotting properties (Table I) was compared to TIHA in anticoagulant and in vivc: experiments for possible antithrombotic action, it was found totally inactive. noteworthy that TIHA produces a dose-dependent antithrombotic effect It is the antithrombotic dosage, no in the stasis thrombosis model.However, at was noted.In contrast UFH produced a modif ication of the clotting times tests.Indeed signif icant modif ication the various the antithrombotic of the with than one observed relatively weaker effects of TIHA were significant antithrombotic effects UFH.However, the fact that TIHA produces that its effect could be anticoagulation suggests without. producing any u involving endothelium. mediated through presumably other mechanisms, effect on the bleeding TIHA dj.d not produce any significant models at the mg/kg .There was a dose-dependent increase in antithrombotic dosage of 1.0 the rat tail bleeding and the rabbit ear blood loss models with TIHA.However was relatively minor in contrast to the observed effects with this increase UFH.In both animal models TIHA at 10.0 mg/kg produced lesser effect than the
256
PARTIALLY N-DESULFATED HEPARIN
Vol. 55, No. 2
one observed by one-t.enth the (losage of [:FH (1 .O nrg/kg) .This data clearly suggests that liFH produces a mot-e pronounced haemorrhagic effect and its s~lfety - efficacy index is narrower than for TIHA. possibility could esist that anticoagulant
: We are grateful for his expert advice on bleeding aggregation tests.
to Dr. J. Choay for many useful comments ; assistance in NMR experiments ; to Miss A. experiments, and to Mrs S.Roussange for
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J.P.,
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The heparin induced : a review. PatholoPv
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KING, D.J., and KELTON, J.G. Heparin-associated Int !lecl IO0 535-540, 1984 2d-..--9
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KELTON, J.C., and HIRSH, J. Bleeding associated therapy. Semin. Hematol. l7, 259-291, 1980.
with antithrombotic
4.
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