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Low-affinity heparin potentiates the action of high-affinity heparin oligosaccharides
THROMBOSISRESEARCH34; 125-133,1984 0049-3848/84$3.00 + .OO Printed in the USA. Copyright (c) 1984 Pergamon Press Ltd.
All rights reserved.
KW-AFFINITYHEPARINKYl'EWMAT~'IHEACTI0NOF HIM-AFFINITY HEPARINOLIGOS~IDES T.W. Barrowcliffe,R.E. Me&on, S.J. Havercroft, L. Thunberg*,U. Lindahl* and D.P. Thanas National Institute for BiologicalStandardsand Control, Holly Hill, Hampstead,London lW3 6RB, U.K. and * SwedishUniversityof AgriculturalSciences,College of VeterinaryMedicine,S-751 23, Uppsala,Sweden.
(Received 17.11.1983; Accepted in revised form 6.2.1984 by Editor P. Olsson)
ABSTRACT Previous studies have shown that high-affinity(HA) heparinoligosaccharides,with molecularweights of 3,000-5,000,were less effectivethan uufractionatedheparin in preventingseminduced vemus thrombosisin rabbits,using a Wessler stasismodel. In the present study, a largerhigh-affinityfragment (M.Wt.6,000-6,500) was also found to be less effectivethan unfractionatedheparin as an antithrcmtxkicagent. Hmever, additionof 80 &kg low affinity (IA) heparin to 80 &kg of this HA fragmentsignificantlypotentiated its antithranboticactivity,and the antithretic action of the mixture was equivalentto that of unfractionatedheparin. Significantpotentiationof antithrcmlmticactivitywas also observed on the additionof IA heparin to a HA decasaccharide(M.Wt.3,0003,500)with anticoagulantactivityonly againstFactor 2Ia. The LA heparin contentof law rmlecularweight heparin fractions appears to be an importantdeterminantof their antithranbotic activity.
The anticoagulantactivityof heparin dependsupon binding to antithranbin III (At III) and it is well establishedthat only about one-thirdof the moleculesin commercialheparin binds with high affinityto At III (1,2). Structuralanalysisof high-affinityoligosaccharides has revealeda unique pentasaccharide moiety which constitutesthe actual binding sequence (3,4). Nonetheless,high affinity (HA) oligosaccharides which containedthis binding sequencewere shown in our previousstudies to be less effectiveantithrm botic agents than heparin in a rabbitmodel (5) and the poor antithranbotic activityof very low molecularweight heparin fractionswas reportedalso Keywords:Heparin;Law affinity;High attrnity;Oligosaccharide; Antithrcmbotic. 125
126
LOW-AFFINITY HEPARIN ACTION
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fran two other laboratories(6,7). Althoughhigh-affinityheparinrrplecules are responsiblefor most of the anticoagulantactivityof heparin in vitro, anticoagulanteffects of heparin have recentlybeen describedwhich are independentof At III (8,9) and it is passible that mlemles with law affinity to At III could contributeto the overallbiologicalactivityof heparin. We have thereforetested the effect of additionof km-affinity heparin (LA heparin)on the antithranboticactivities of heparinoligosaccharides, using a rabbit model. Anticoagulant activitiesof the oligosaccharides, with and without LA heparin, have also been measured,both in vitro and in ex viva blood samples,by several assay methods.
Heparin oligosaccharides with high affinity for At III were prepared by nitrousacid degradation,affinitychramtography on insolubilisedAt III, and gel chranatography, as describedpreviously(5). The two fractionsused in the present study containedlo-12 monosaccharideunits (M.Wt. 'L3,0003,500)and > 18 (predominantly 20-22) units (M.Wt.G 6,000-6,500). LA heparinwas preparedfran unfractionatedheparin by affinity chrmatography on insolubilisedAt III, as describedpreviously(1). Only material which did not bind to the column was used in the present study and its mlecular weight range was similarto that of the unfractionatedmaterial, i.e. 5,000-30,000. In vitro anticoagulantactivitieswere measuredby AFlT, anti-Ha (clotting and chromogenicmethods)and calcium thranbintime assays, as describedpreviously (5). l'heanti-Xaassays were carried out by the method of Denson and Bonnar (101, modifiedby anissionof the defibrinationstep and shorteningof the incubationtime, as describedby Bggletonet al. (11). The same methods were used to measure heparinactivitiesin ex vim blood saqles by ccqarison with standardcurves preparedin rabbit pm-injection plasma. The 3rd InternatiohalStandardfor heparinwas used as a standard in all assays. The generationof Factor Xa in plasma after additionof purifiedFactor IXa (kindlyprovidedby Dr. D.L. Aronson,Office of Biologics,Bethesda, Maryland,UsA) was measured in vitro by the followingmethod. 3003 of Factor IX deficientplasm, after defibrinationwith ax-mod, were mixed at 37“C with 3OOl.1.l phospholipid(HIBSC79/508) and 50~1 of either heparin or buffer (0.05M Tris, 0.15 M HaCl, pH 7.4). 501-11 purifiedhuman IXa (5&ml) were added and, after exactly1 minute, the mixture was recalcifiedwith 300 ul of 30 nM Cac12, 100 r<&Cl, 50 nM Tris, pH 7.4. The Factor Xa generated was measured at mtervals by subsampling1001~1into lOO@ of a ltinogenic substrate,S-2613 (HabiVitrum), 0.005 x 10e4 M in Tris, pH 7.4 at 37'C. After exactly 1 l/4 minutes' incubation,the enzyme substratereactionwas guenchedby additionof 20~1 acetic acid. The liberatedisolumiholwas measured in a luminamter after additionof 100~1 henin and H202 as &scribed by Lindahl et al. (12) except that, becauseof the presence of plasm, the concentrationof hemin was increased20 times to 125N and that of H202 decreased10 times to 8uM. Antithranboticactivitywas assessedafter 10 minutes and 20 minutes stasis in a rabbit nxidel,as previouslydescribed (5).
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127
RSULTS
The in vitro anticoagulantactivitiesof the tw9 HA oligosaccharidepreparations aud of the LA hsparin are shown in Table 1. All assays are against the TABLE1 In Vitro AnticoagulantActivitiesof Heparin Fractions (iu/mgl Material
Calcium thranbin 144
Anti-Xa Clotting 500
20-22 Oligosaccharide
42
10-12 Oligosaccharide
2.5
LA heparin
8 9.8 42* * Non-parallelassays, range15-71
1,097
S-2222 660 1,382
3rd InterhatiohalStandard for Heparin,which has ah activityof 173 i&q (by all assay methods). The HA lo-12 saccharide,like the HA decasaccharide Eragmsntpreviouslystudied (5) had high activityonly by anti-Xa assays, whereas the HA 20-22 preparationhad in addition sub&antial activity against thrcmbinand mxderateAPTI!activity. TheLAheparin hadlowanticcagulant activityby all assays (the calciumthranbinactivitywas difficultto estimtebecauseof non-parallelism). Effects of LA heoarin on in vitro activities Table 2 shows the effect of additionof LA heparin to the 20-22 oligosaccharide, on a 1:l weight basis, on its in vitro anticoagulantactivities. Comparisonof the activitiesfound in the mixturewith those expectedfran assays of the separateaqonents showed ho significantdifferencesfor APTIkalcium thranbinor anti-Xa S-2222 assays, kt the anti+& clottingactivityof the mixturewas higher than expected. mJ3LE2 Effect of LA Heparin on In Vitro AnticoagulantActivities of the High Affinity20-22 Oligosaccharide Potency of 1:l mixture, iu/mg Found Expected 27.4 26 Calcium thrcmbin Anti-Xa: Clotting S-2222
93
100
254 330
421 304
Generationof Factor Xa 'Iheeffect of the HA 20-22 fragmenton the generationof Factor Xa in plasm is shown in Figure 1. At a concentration of 0.5 pg/ml, the oligosaccharidewas only partiallyeffective,whereas unfractionatedheparin completelyinhibitedthe generationof Factor Xa. Hmever, additionof 1 1.19 of LA heparin to 0.5 pg of oligosaccharidemarkedly reduced the amount of Factor Xa generated (Fig.1). The resultsof four such experimentsare shcnm in Table 3; in each case the mixture of oligosaccharide
Vo1.34, No.2
LOW-AFFINITY HEPARIN ACTION
128 and LA heparin gave mre
inhibitionof Xa generationthan did the oligosaccharide alone. LA heparinalone had m inhibitoryactivity in this systan.
n
12c
100
/Buffer
80
2
4
6
8
10
12
Min. Incubation
FIG.
1
The effectsof heparin,20-22 oligosaccharideand a 1:2 mixture of 20-22 oligosaccharide and IA heparin on the generationof Factor Xa in plasm !lYBJ_&3 Effect of LA Heparin on Inhibitionof Xa Generationby 20-22 Oligosaccharide Jxxpt. No. 1
Corm. of heparin w/d LA 20-22 1 1
PeakXa,% 20-22alone 28
of buffer value 20-22 + LA 15
129
LOW-AFFINITY HEPARIN ACTION
Vo1.34, No.2
Antithrrn&oticactivities The antithranboticactivityof the HA 20-22 oligosaccharide,ccmparedwith that of unfractionatedheparin,is shown in Table 4, togetherwith the ex vivo blood levels. At a c&e of 80 ug/kg, heparin preventedthranbosisin all 10 rabbitstested,whereas preventionoccurred in only 5 out of 10 rabbitsgiven oligosaccharide, in spite of the fact that blood levelswere higher by all assay methods tested. This differencewas statisticallysignificant(P < 0.05) as was the differencein antithranbotic activitiesafter 20 minutes stasis. On additionof 80 pg/kg LA heparin to 80 &kg of the 20-22 oligosaccharide, the antithranboticactivityof the latterwas clearlypotentiated,as shown in Table 4. The activityof the mixturewas significantlygreater than that of the oligosaccharidealone (P < 0.05) and not significantlydifferentfrom that of heparin. Blood levels were slightlyhigher after injectionof the mixture than after oligosaccharide alone, but the differencewas statistically significantonly for the calciumthranbinand anti-Xaclottingassays (P < 0.05). The effect of LA heparin on the antithranboticactivityof the lo-12 oligosaccharidewas also tested. In this case, becauseof shortageof material, resultscould only be comparedwith those obtainedwith a previousdecasaccharide fragnvznt (51, which was found to be largely ineffectiveas an antithranand 80 &kg LA botic agent. The mixture of 80 &kg lo-12 oligosaccharide heparinpreventedthranbosisin 5 of 6 rabbitsafter 10 minutes'stasis, oznpared with 1 out of 6 for 80 &kg of the previousdecasaccharide; this differencewas statisticallysignificant(P < 0.05). However,after 20 minutes' stasis,thranbosisoccurred in all six rabbitsgiven the mixture, indicating that the axnbinationof lo-12 oligosaccharide and IA heparinwas less effective than that of 20-22 oligosaccharide and IA heparin. LA heparin had virtuallyno antithranboticactivitywhen testedalone; at a dose of 160 &kg, thrcnbosisoccurredin 5 out of 6 rabbitsafter 10 minutes' stasis and in all 6 after 20 minutes stasis (Table4). TABLB4 AntithranboticActivitiesand Bx Vivo Blood Levels Material injected (per kg)
No. of rabbitswith thranbosis
Heparinactivityex vivo, iu/ml Anti-Xa Clotting S-2222
APIT
Calcium thranbin
4/10
0.15
0.12
0.12
0.16
s/10
8/10
0.16
0.43
0.29
0.82
o/9
4/9
0.19
0.55
0.34
0.86
10' stasis
20' stasis
80 ng heparin
o/10
80 ug 20-22 80 1.19 20-22 +8OngLA heparin
160 m LA S/6 6/6 0.09 0.08 0 0 heparin All ex vivo blood sarqleswere taken 2 minutes after injectionof heparin. DISCDSSION In our previous studies (51, it was shown that, whereas a decasaccharidewas virtuallyinactiveas an antithranboticagent, a fragmentwith 16-18 saccha-
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LOW-AFFINITY HEPARIN ACTION
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ride units was partiallyeffective. These resultssuggestedthat full antithranboticactivitymight be attainedby a sanewhatlarger fragnent. However,the 20-22 fragmantused in the present study was still less effectiveas an antithrcmboticagent than heparin,despite the fact that, unlike the 16-18 fragmentused previously,it had inhibitoryaction against thranbinaswellas Factor Xa. These resultsmay be contrastedwith those of Holmer et al. (6) and Ockelford et al. (71, who found that fractionswith Mr valuesof about 4,000 (12-14 monosaccharideunits) and 4,600 (14-16monosaccharideunits), respectively, were as effectiveas unfractionated heparinwhen given on an qual unitage basis, as measured by anti-Xaassays. These fractions,in spite of their ~CYNX Mr, thus appear to have higher antithranboticactivitythan the HA 2022 oligosaccharideused by ourselves. Amajor differenceas regardsaxnposition is that our fragments,isolatedby affinitychrmtography on insolubilised At III, containonly moleculeswith high affinityfor At III, whereas those of Holmer et al. (6) and Ockelfordet al. (7) containedboth hi&- and low-affinityccmponents. The resultsof the present study show that the presenceor absence of low-affinityheparinmoleculesis a major determinant of the antithranboticactivityof these oligosaccharides.The antithranbotic activityof the HA 20-22 oligosaccharide was clearlypotentiatedon addition of IA heparin (Table4) and the mixturewas as effectiveas unfractionated heparin,albeit at twice the total dose. Potentiationof the activityof the HAdecasaccharidewas also evidentand,although thismixturewasless effectivethan that of the 20-22 oligosaccharide and LA heparin,these results show that a decasaccharidecontaininglittlemore than the AT III binding region can have antithranboticactivity,providedthat low-affinity moleculesare also present. Since the decasaccharidepotentiatesthe inhibitionof Factor Xa but is inactiveagainst thrcxnbin, it is concluded that thranbin inhibitionis not an absolutereguirsmentfor antithranbotic activity. There are a number of possiblereasonsfor the potentiatingeffect of LA heparin. The injectionof LA heparin,togetherwith the HA 20-22 fragment, gave slightlyhigher ex vivc blood levels than the HA oligosaccharidealone, possiblydue to bindingto antiheparinproteins in plasma (Table41. Hover, since after injectionof the HA-oligosaccharide alone, blood levels by all four assay methodswere higher than those after unfractionated heparin,it seems unlikelythat a further slight increasein blood levels could account for the extra antithranboticactivityon addition of LA heparin. There was little evidenceof extra activityin anticoagulantassays in vitro, with the possibleexceptionof anti-Xa clottingassays (Table2). HokFever, Ofosu et al. (8) have describedan anticoagulantaction of heparin which is independentof At III and is thereforeshown by both LA and HA heparin (13). This actionwas ascribedto disruptionby heparin of the binding of Factor Xa to phospholipid(PL),Ca* and Factor V. Since Factor Xa is protectedfrcm the action of At III/heparinwhen bound to PL, Ca* and Factor V, the additionof LA heparinmid lead to less protecticn,and hence increasethe inhibitionof Factor Xa by the HA oligosaccharide.Thismight explain the extra effect of LA heparin in the Xa generationsystem (Fig. 1, Table 31, in which Factor Xa is generatedin the presenceof Ca*, phospholipid and Factor V. However,part of the activityof heparin in this assay system is due to inhibitionof Factor IXa, and the effect of LA heparin could be due to enhancedIXa inhibition. In any case, themixture of oligosaccharideand LA heparinwas still less effectivethan unfractionated heparin in inhibitingthe generationof Factor Xa and it seems that the effectsof ILAheparinon thrcmbusfomtion must be at least partly due to
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131
interactionstaking place in viva. An importantaspect of the action of heparin in vivo is its interactionwith vascularendothelialcells. These cells contain specificbinding sites for heparin (14) and binding of w heparin to such sites my conceivablyaffect the thranbogenicpropertiesof the endothelialsurface. Moreover, endogenous heparin-relatedpolysaccharideat this surface,presumablyheparan sulphate, apparentlybinds the platelet-derived protein,plateletfactor 4 (15) and may thus becane inaccessibleto other conponentsof the circulatingblood. Since HA and LA heparin have qual affinityfor plateletfactor 4 (161, IA heparin may displacethe protein fran the endothelim and expose the endogenouspolysaccharide(17). Since heparan sulphatehas been shown tohave anti-Ka activity in vitro (18) and antithrcmboticactivityin animals (191, its exposure may increasethe antithranboticpotentialof the vascularsurface. Yet anotherpossible in vivo effect could be related to the action of a recenly discoveredendoglycosidasein platelets(20). This enzyme cleaves 8-glmuronidic linkagesin heparin and, since one such linkageoccurs in the antithranbinbinding region,the enzyme has been shown to degrade this site (21) and hence destroy anticoagulantactivity (22). It is possiblethat addition of IA heparin,which muld act as an alternativesubstratefor this enzyme, muld lead to less degradationof the HA oligosaccharida, therebypotentiating its activity. Theeffectwouldnotbedetectedin exvivoassays, which are carriedout on platelet-poorplasma taken before activationof the rabbit'scoagulationsystem by injectionof serum. Whatever the reasons for the effect of LA heparin,these resultsenphasise that, whilst HA heparinmoleculesare responsiblefor most of the anticoagulant activity,IA heparinmoleculesplay an iqortant additionalrole in the overall antithrcmboticaction of low-molecular weight heparin fractions.
1.
HaK, M., BJtjRK,I., HO-, J. and LIMXHL, U. Anticoagulantactivity of heparin:separationof hi@-activity and lmactivity heparin species by affinitychraaatographyon immbilised antithranbin. m Letts. 66, 90-93, 1976.
2.
L?M, L.H., SILBERT,J.E. and RGSENBERG,R.D. The separationof active and inactiveforms of heparin. Biochem.Biophys.Res.Carm. 69_,570-577, 1976.
3.
m, I. aud LINDAHL,U. Mechanismof the anticoagulantaction of heparin. Molec.Cellul.Biochem. 48, 161-182,1982.
4.
IHUNBERG,L., I#CKSTR&& G. and LItX)AHL, U. Further characterisation of the antithranbinbinding sequencein heparin. CarbohydrateRes. 100, 393-410,1982.
5.
H-XXIAS, D.P., MERTCN, R.E., HARRCXLIFFE, T.W., lHUNBERG,L. and with high affinity for LINDAHL,u. Effects of heparinoligosaccharides antithrmbin III in experimentalvenous thranbosis. mraTlbos.Haezms. 47, 244-248,1982.
6. HOIMER, E., MXMSON, C. and NILSSON,S. Anticoagulantand antithranbotic effects of heparinand low mlecular weight heparin fragmentsin rabbits. lhranbosisRes. 25, 475-485,1982.
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Vol.34,
No.2
7.
OCKELFORD,P.A., CAEtTER, C.J., MITCHELL,L. and HIRSH, J.
8.
OEUSU, F., BLAJC!&IAN, M.A. and HIRSH, J. The inhibitionby heparin of the intrinsicpathway activation of factor Xa in the presenceof anti-
Discordance between the anti-Xa activityand the antithrcmboticactivityof an ultra low mlecular weight heparin fraction. lhrcskosisRes. 28, 401-409, 1982.
thranbinIII. ThranbosisRes. 20, 391-403,1980. 9. OE0SU, F., BLAJCHMAN,M.A., MODI, G., CERSKUS,A.L. and HIRSH, J. Activation of factorXa and prothranbinin antithranbinIII depletedplasma: the effects of heparin. ThranbosisRes. -23. 331-345,1981. 10. DENSON, K.W.E. and BONNAR, J. The measurementof heparin. A method based on the potentiationof anti-factorXa. Thranb.Diath.Haemrrh.?o, 471-475,1973.
\
11. ESGLEION,C.A., BARRCWCLIFFE,T.W., MERlDN, R.E. and 'IHCMAS,D.P. In vitro and in viva studiesof the anti-Xa activityof heparin. ThranbosisRes. 24, 319-328,1981. 12. LINDAHL,u., KoImr, S.O., BU@lALD,J., BTERUD, B. and SELJELID,R. Studies on blood coagulationfactor X/Xa producedby muse peritoneal macrophages,using a luminogenicpeptide substrate. Bide&J. 206, 231-237,1982. 13. OEOSU, F.A., MDDI, G., mKU.9, A.L., HIRSH, J. and BLAJCHMAN,M.A. Heparin with law affinityto antithrunbinIII inhibitsthe activationof prothrcmbinin normal plasm. ThrcmbosisRes. 28, 487-497,1982. 14. GLIMELIUS,B., BUSCH, C. and Hm, M. Binding of heparin on the surfaceof culturedhumn endothelialcells. 'Ihranbosis Res.l.2, 773-782,1978. 15. BUSCH, C., DAWES, J., PEPPm, D.S. and WASTESON,A. Binding of platelet factor 4 to culturedhuman umbilicalvein endothelialcells. Thrantmsis Res. 19, 129-137,1980. -S., RUCINSKI,B., JAMES, P. and LINDAHL,U. Plateletanti16. NIEWWSKI, heparin proteins and antithranbinIII interactwith differentbinding sites on the heparinmlecule. E'EBSLett. 102, 75-78, 1979. 17. DAWES, J., PUMPHREY,C.W., MC-, K.M., PROWSE,C.V. and PEPPER,D.S. The in viva releaseof human platelet factor 4 by heparin. Thranbosis Res. 27, 65-76, 1982. -18. 'IIEOMAS, D.P., MER'I0N,R.E., BARRC%LIFFE, T.W., MULLOY, B. and JOHNSON, E.A. Anti-FactorXa activityof heparan sulphate. Thra&osis Res. 2, 501-506,1976. 19. 'IHCMAS,D.P., JDHNSDN,E.A. and DIFFE, T.W. The antithrankkic action of heparan sulphate. In: VascularOcclusion:Epideniological, Pathophysiological and TherapeuticAgents, Ed. Teri, M. and Dommdy, J ., AcademicPress, London, pp 377-382,1981. 20. WASl%ON, A., Ha,
M. and WESTEE@%RK, B. Dermstration of a platelet
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enzyme, degradingheparan sulphate. FE8s Lett. 64, 218-221,1976. L., B&X3X&l, G., WAS!CESON, A., ROBINSON,H.C., &W3N, S. and 21. Tf-IuNBmG, of heparin-related LINDAHL,u. Enzymaticdepolymerization polysaccharides.J.Biol.Chem.257, 10278-10282,1982. 22. OLDBERG,A., HELDIN, C.-H., WASlWON, A., BUSCH, C. and HWK, M. Chara* terizationof a plateletendoglycosidasedegradingheparin-like polysaccharides.Biochemistry19, 5755-5762,1980.
All rights reserved.
KW-AFFINITYHEPARINKYl'EWMAT~'IHEACTI0NOF HIM-AFFINITY HEPARINOLIGOS~IDES T.W. Barrowcliffe,R.E. Me&on, S.J. Havercroft, L. Thunberg*,U. Lindahl* and D.P. Thanas National Institute for BiologicalStandardsand Control, Holly Hill, Hampstead,London lW3 6RB, U.K. and * SwedishUniversityof AgriculturalSciences,College of VeterinaryMedicine,S-751 23, Uppsala,Sweden.
(Received 17.11.1983; Accepted in revised form 6.2.1984 by Editor P. Olsson)
ABSTRACT Previous studies have shown that high-affinity(HA) heparinoligosaccharides,with molecularweights of 3,000-5,000,were less effectivethan uufractionatedheparin in preventingseminduced vemus thrombosisin rabbits,using a Wessler stasismodel. In the present study, a largerhigh-affinityfragment (M.Wt.6,000-6,500) was also found to be less effectivethan unfractionatedheparin as an antithrcmtxkicagent. Hmever, additionof 80 &kg low affinity (IA) heparin to 80 &kg of this HA fragmentsignificantlypotentiated its antithranboticactivity,and the antithretic action of the mixture was equivalentto that of unfractionatedheparin. Significantpotentiationof antithrcmlmticactivitywas also observed on the additionof IA heparin to a HA decasaccharide(M.Wt.3,0003,500)with anticoagulantactivityonly againstFactor 2Ia. The LA heparin contentof law rmlecularweight heparin fractions appears to be an importantdeterminantof their antithranbotic activity.
The anticoagulantactivityof heparin dependsupon binding to antithranbin III (At III) and it is well establishedthat only about one-thirdof the moleculesin commercialheparin binds with high affinityto At III (1,2). Structuralanalysisof high-affinityoligosaccharides has revealeda unique pentasaccharide moiety which constitutesthe actual binding sequence (3,4). Nonetheless,high affinity (HA) oligosaccharides which containedthis binding sequencewere shown in our previousstudies to be less effectiveantithrm botic agents than heparin in a rabbitmodel (5) and the poor antithranbotic activityof very low molecularweight heparin fractionswas reportedalso Keywords:Heparin;Law affinity;High attrnity;Oligosaccharide; Antithrcmbotic. 125
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LOW-AFFINITY HEPARIN ACTION
Vo1.34, No.2
fran two other laboratories(6,7). Althoughhigh-affinityheparinrrplecules are responsiblefor most of the anticoagulantactivityof heparin in vitro, anticoagulanteffects of heparin have recentlybeen describedwhich are independentof At III (8,9) and it is passible that mlemles with law affinity to At III could contributeto the overallbiologicalactivityof heparin. We have thereforetested the effect of additionof km-affinity heparin (LA heparin)on the antithranboticactivities of heparinoligosaccharides, using a rabbit model. Anticoagulant activitiesof the oligosaccharides, with and without LA heparin, have also been measured,both in vitro and in ex viva blood samples,by several assay methods.
Heparin oligosaccharides with high affinity for At III were prepared by nitrousacid degradation,affinitychramtography on insolubilisedAt III, and gel chranatography, as describedpreviously(5). The two fractionsused in the present study containedlo-12 monosaccharideunits (M.Wt. 'L3,0003,500)and > 18 (predominantly 20-22) units (M.Wt.G 6,000-6,500). LA heparinwas preparedfran unfractionatedheparin by affinity chrmatography on insolubilisedAt III, as describedpreviously(1). Only material which did not bind to the column was used in the present study and its mlecular weight range was similarto that of the unfractionatedmaterial, i.e. 5,000-30,000. In vitro anticoagulantactivitieswere measuredby AFlT, anti-Ha (clotting and chromogenicmethods)and calcium thranbintime assays, as describedpreviously (5). l'heanti-Xaassays were carried out by the method of Denson and Bonnar (101, modifiedby anissionof the defibrinationstep and shorteningof the incubationtime, as describedby Bggletonet al. (11). The same methods were used to measure heparinactivitiesin ex vim blood saqles by ccqarison with standardcurves preparedin rabbit pm-injection plasma. The 3rd InternatiohalStandardfor heparinwas used as a standard in all assays. The generationof Factor Xa in plasma after additionof purifiedFactor IXa (kindlyprovidedby Dr. D.L. Aronson,Office of Biologics,Bethesda, Maryland,UsA) was measured in vitro by the followingmethod. 3003 of Factor IX deficientplasm, after defibrinationwith ax-mod, were mixed at 37“C with 3OOl.1.l phospholipid(HIBSC79/508) and 50~1 of either heparin or buffer (0.05M Tris, 0.15 M HaCl, pH 7.4). 501-11 purifiedhuman IXa (5&ml) were added and, after exactly1 minute, the mixture was recalcifiedwith 300 ul of 30 nM Cac12, 100 r<&Cl, 50 nM Tris, pH 7.4. The Factor Xa generated was measured at mtervals by subsampling1001~1into lOO@ of a ltinogenic substrate,S-2613 (HabiVitrum), 0.005 x 10e4 M in Tris, pH 7.4 at 37'C. After exactly 1 l/4 minutes' incubation,the enzyme substratereactionwas guenchedby additionof 20~1 acetic acid. The liberatedisolumiholwas measured in a luminamter after additionof 100~1 henin and H202 as &scribed by Lindahl et al. (12) except that, becauseof the presence of plasm, the concentrationof hemin was increased20 times to 125N and that of H202 decreased10 times to 8uM. Antithranboticactivitywas assessedafter 10 minutes and 20 minutes stasis in a rabbit nxidel,as previouslydescribed (5).
Vo1.34, No.2
LOW-AFFINITY HEPARIN ACTION
127
RSULTS
The in vitro anticoagulantactivitiesof the tw9 HA oligosaccharidepreparations aud of the LA hsparin are shown in Table 1. All assays are against the TABLE1 In Vitro AnticoagulantActivitiesof Heparin Fractions (iu/mgl Material
Calcium thranbin 144
Anti-Xa Clotting 500
20-22 Oligosaccharide
42
10-12 Oligosaccharide
2.5
LA heparin
8 9.8 42* * Non-parallelassays, range15-71
1,097
S-2222 660 1,382
3rd InterhatiohalStandard for Heparin,which has ah activityof 173 i&q (by all assay methods). The HA lo-12 saccharide,like the HA decasaccharide Eragmsntpreviouslystudied (5) had high activityonly by anti-Xa assays, whereas the HA 20-22 preparationhad in addition sub&antial activity against thrcmbinand mxderateAPTI!activity. TheLAheparin hadlowanticcagulant activityby all assays (the calciumthranbinactivitywas difficultto estimtebecauseof non-parallelism). Effects of LA heoarin on in vitro activities Table 2 shows the effect of additionof LA heparin to the 20-22 oligosaccharide, on a 1:l weight basis, on its in vitro anticoagulantactivities. Comparisonof the activitiesfound in the mixturewith those expectedfran assays of the separateaqonents showed ho significantdifferencesfor APTIkalcium thranbinor anti-Xa S-2222 assays, kt the anti+& clottingactivityof the mixturewas higher than expected. mJ3LE2 Effect of LA Heparin on In Vitro AnticoagulantActivities of the High Affinity20-22 Oligosaccharide Potency of 1:l mixture, iu/mg Found Expected 27.4 26 Calcium thrcmbin Anti-Xa: Clotting S-2222
93
100
254 330
421 304
Generationof Factor Xa 'Iheeffect of the HA 20-22 fragmenton the generationof Factor Xa in plasm is shown in Figure 1. At a concentration of 0.5 pg/ml, the oligosaccharidewas only partiallyeffective,whereas unfractionatedheparin completelyinhibitedthe generationof Factor Xa. Hmever, additionof 1 1.19 of LA heparin to 0.5 pg of oligosaccharidemarkedly reduced the amount of Factor Xa generated (Fig.1). The resultsof four such experimentsare shcnm in Table 3; in each case the mixture of oligosaccharide
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128 and LA heparin gave mre
inhibitionof Xa generationthan did the oligosaccharide alone. LA heparinalone had m inhibitoryactivity in this systan.
n
12c
100
/Buffer
80
2
4
6
8
10
12
Min. Incubation
FIG.
1
The effectsof heparin,20-22 oligosaccharideand a 1:2 mixture of 20-22 oligosaccharide and IA heparin on the generationof Factor Xa in plasm !lYBJ_&3 Effect of LA Heparin on Inhibitionof Xa Generationby 20-22 Oligosaccharide Jxxpt. No. 1
Corm. of heparin w/d LA 20-22 1 1
PeakXa,% 20-22alone 28
of buffer value 20-22 + LA 15
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LOW-AFFINITY HEPARIN ACTION
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Antithrrn&oticactivities The antithranboticactivityof the HA 20-22 oligosaccharide,ccmparedwith that of unfractionatedheparin,is shown in Table 4, togetherwith the ex vivo blood levels. At a c&e of 80 ug/kg, heparin preventedthranbosisin all 10 rabbitstested,whereas preventionoccurred in only 5 out of 10 rabbitsgiven oligosaccharide, in spite of the fact that blood levelswere higher by all assay methods tested. This differencewas statisticallysignificant(P < 0.05) as was the differencein antithranbotic activitiesafter 20 minutes stasis. On additionof 80 pg/kg LA heparin to 80 &kg of the 20-22 oligosaccharide, the antithranboticactivityof the latterwas clearlypotentiated,as shown in Table 4. The activityof the mixturewas significantlygreater than that of the oligosaccharidealone (P < 0.05) and not significantlydifferentfrom that of heparin. Blood levels were slightlyhigher after injectionof the mixture than after oligosaccharide alone, but the differencewas statistically significantonly for the calciumthranbinand anti-Xaclottingassays (P < 0.05). The effect of LA heparin on the antithranboticactivityof the lo-12 oligosaccharidewas also tested. In this case, becauseof shortageof material, resultscould only be comparedwith those obtainedwith a previousdecasaccharide fragnvznt (51, which was found to be largely ineffectiveas an antithranand 80 &kg LA botic agent. The mixture of 80 &kg lo-12 oligosaccharide heparinpreventedthranbosisin 5 of 6 rabbitsafter 10 minutes'stasis, oznpared with 1 out of 6 for 80 &kg of the previousdecasaccharide; this differencewas statisticallysignificant(P < 0.05). However,after 20 minutes' stasis,thranbosisoccurred in all six rabbitsgiven the mixture, indicating that the axnbinationof lo-12 oligosaccharide and IA heparinwas less effective than that of 20-22 oligosaccharide and IA heparin. LA heparin had virtuallyno antithranboticactivitywhen testedalone; at a dose of 160 &kg, thrcnbosisoccurredin 5 out of 6 rabbitsafter 10 minutes' stasis and in all 6 after 20 minutes stasis (Table4). TABLB4 AntithranboticActivitiesand Bx Vivo Blood Levels Material injected (per kg)
No. of rabbitswith thranbosis
Heparinactivityex vivo, iu/ml Anti-Xa Clotting S-2222
APIT
Calcium thranbin
4/10
0.15
0.12
0.12
0.16
s/10
8/10
0.16
0.43
0.29
0.82
o/9
4/9
0.19
0.55
0.34
0.86
10' stasis
20' stasis
80 ng heparin
o/10
80 ug 20-22 80 1.19 20-22 +8OngLA heparin
160 m LA S/6 6/6 0.09 0.08 0 0 heparin All ex vivo blood sarqleswere taken 2 minutes after injectionof heparin. DISCDSSION In our previous studies (51, it was shown that, whereas a decasaccharidewas virtuallyinactiveas an antithranboticagent, a fragmentwith 16-18 saccha-
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LOW-AFFINITY HEPARIN ACTION
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ride units was partiallyeffective. These resultssuggestedthat full antithranboticactivitymight be attainedby a sanewhatlarger fragnent. However,the 20-22 fragmantused in the present study was still less effectiveas an antithrcmboticagent than heparin,despite the fact that, unlike the 16-18 fragmentused previously,it had inhibitoryaction against thranbinaswellas Factor Xa. These resultsmay be contrastedwith those of Holmer et al. (6) and Ockelford et al. (71, who found that fractionswith Mr valuesof about 4,000 (12-14 monosaccharideunits) and 4,600 (14-16monosaccharideunits), respectively, were as effectiveas unfractionated heparinwhen given on an qual unitage basis, as measured by anti-Xaassays. These fractions,in spite of their ~CYNX Mr, thus appear to have higher antithranboticactivitythan the HA 2022 oligosaccharideused by ourselves. Amajor differenceas regardsaxnposition is that our fragments,isolatedby affinitychrmtography on insolubilised At III, containonly moleculeswith high affinityfor At III, whereas those of Holmer et al. (6) and Ockelfordet al. (7) containedboth hi&- and low-affinityccmponents. The resultsof the present study show that the presenceor absence of low-affinityheparinmoleculesis a major determinant of the antithranboticactivityof these oligosaccharides.The antithranbotic activityof the HA 20-22 oligosaccharide was clearlypotentiatedon addition of IA heparin (Table4) and the mixturewas as effectiveas unfractionated heparin,albeit at twice the total dose. Potentiationof the activityof the HAdecasaccharidewas also evidentand,although thismixturewasless effectivethan that of the 20-22 oligosaccharide and LA heparin,these results show that a decasaccharidecontaininglittlemore than the AT III binding region can have antithranboticactivity,providedthat low-affinity moleculesare also present. Since the decasaccharidepotentiatesthe inhibitionof Factor Xa but is inactiveagainst thrcxnbin, it is concluded that thranbin inhibitionis not an absolutereguirsmentfor antithranbotic activity. There are a number of possiblereasonsfor the potentiatingeffect of LA heparin. The injectionof LA heparin,togetherwith the HA 20-22 fragment, gave slightlyhigher ex vivc blood levels than the HA oligosaccharidealone, possiblydue to bindingto antiheparinproteins in plasma (Table41. Hover, since after injectionof the HA-oligosaccharide alone, blood levels by all four assay methodswere higher than those after unfractionated heparin,it seems unlikelythat a further slight increasein blood levels could account for the extra antithranboticactivityon addition of LA heparin. There was little evidenceof extra activityin anticoagulantassays in vitro, with the possibleexceptionof anti-Xa clottingassays (Table2). HokFever, Ofosu et al. (8) have describedan anticoagulantaction of heparin which is independentof At III and is thereforeshown by both LA and HA heparin (13). This actionwas ascribedto disruptionby heparin of the binding of Factor Xa to phospholipid(PL),Ca* and Factor V. Since Factor Xa is protectedfrcm the action of At III/heparinwhen bound to PL, Ca* and Factor V, the additionof LA heparinmid lead to less protecticn,and hence increasethe inhibitionof Factor Xa by the HA oligosaccharide.Thismight explain the extra effect of LA heparin in the Xa generationsystem (Fig. 1, Table 31, in which Factor Xa is generatedin the presenceof Ca*, phospholipid and Factor V. However,part of the activityof heparin in this assay system is due to inhibitionof Factor IXa, and the effect of LA heparin could be due to enhancedIXa inhibition. In any case, themixture of oligosaccharideand LA heparinwas still less effectivethan unfractionated heparin in inhibitingthe generationof Factor Xa and it seems that the effectsof ILAheparinon thrcmbusfomtion must be at least partly due to
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interactionstaking place in viva. An importantaspect of the action of heparin in vivo is its interactionwith vascularendothelialcells. These cells contain specificbinding sites for heparin (14) and binding of w heparin to such sites my conceivablyaffect the thranbogenicpropertiesof the endothelialsurface. Moreover, endogenous heparin-relatedpolysaccharideat this surface,presumablyheparan sulphate, apparentlybinds the platelet-derived protein,plateletfactor 4 (15) and may thus becane inaccessibleto other conponentsof the circulatingblood. Since HA and LA heparin have qual affinityfor plateletfactor 4 (161, IA heparin may displacethe protein fran the endothelim and expose the endogenouspolysaccharide(17). Since heparan sulphatehas been shown tohave anti-Ka activity in vitro (18) and antithrcmboticactivityin animals (191, its exposure may increasethe antithranboticpotentialof the vascularsurface. Yet anotherpossible in vivo effect could be related to the action of a recenly discoveredendoglycosidasein platelets(20). This enzyme cleaves 8-glmuronidic linkagesin heparin and, since one such linkageoccurs in the antithranbinbinding region,the enzyme has been shown to degrade this site (21) and hence destroy anticoagulantactivity (22). It is possiblethat addition of IA heparin,which muld act as an alternativesubstratefor this enzyme, muld lead to less degradationof the HA oligosaccharida, therebypotentiating its activity. Theeffectwouldnotbedetectedin exvivoassays, which are carriedout on platelet-poorplasma taken before activationof the rabbit'scoagulationsystem by injectionof serum. Whatever the reasons for the effect of LA heparin,these resultsenphasise that, whilst HA heparinmoleculesare responsiblefor most of the anticoagulant activity,IA heparinmoleculesplay an iqortant additionalrole in the overall antithrcmboticaction of low-molecular weight heparin fractions.
1.
HaK, M., BJtjRK,I., HO-, J. and LIMXHL, U. Anticoagulantactivity of heparin:separationof hi@-activity and lmactivity heparin species by affinitychraaatographyon immbilised antithranbin. m Letts. 66, 90-93, 1976.
2.
L?M, L.H., SILBERT,J.E. and RGSENBERG,R.D. The separationof active and inactiveforms of heparin. Biochem.Biophys.Res.Carm. 69_,570-577, 1976.
3.
m, I. aud LINDAHL,U. Mechanismof the anticoagulantaction of heparin. Molec.Cellul.Biochem. 48, 161-182,1982.
4.
IHUNBERG,L., I#CKSTR&& G. and LItX)AHL, U. Further characterisation of the antithranbinbinding sequencein heparin. CarbohydrateRes. 100, 393-410,1982.
5.
H-XXIAS, D.P., MERTCN, R.E., HARRCXLIFFE, T.W., lHUNBERG,L. and with high affinity for LINDAHL,u. Effects of heparinoligosaccharides antithrmbin III in experimentalvenous thranbosis. mraTlbos.Haezms. 47, 244-248,1982.
6. HOIMER, E., MXMSON, C. and NILSSON,S. Anticoagulantand antithranbotic effects of heparinand low mlecular weight heparin fragmentsin rabbits. lhranbosisRes. 25, 475-485,1982.
132
LOW-AFFINITYHEPARINACTION
Vol.34,
No.2
7.
OCKELFORD,P.A., CAEtTER, C.J., MITCHELL,L. and HIRSH, J.
8.
OEUSU, F., BLAJC!&IAN, M.A. and HIRSH, J. The inhibitionby heparin of the intrinsicpathway activation of factor Xa in the presenceof anti-
Discordance between the anti-Xa activityand the antithrcmboticactivityof an ultra low mlecular weight heparin fraction. lhrcskosisRes. 28, 401-409, 1982.
thranbinIII. ThranbosisRes. 20, 391-403,1980. 9. OE0SU, F., BLAJCHMAN,M.A., MODI, G., CERSKUS,A.L. and HIRSH, J. Activation of factorXa and prothranbinin antithranbinIII depletedplasma: the effects of heparin. ThranbosisRes. -23. 331-345,1981. 10. DENSON, K.W.E. and BONNAR, J. The measurementof heparin. A method based on the potentiationof anti-factorXa. Thranb.Diath.Haemrrh.?o, 471-475,1973.
\
11. ESGLEION,C.A., BARRCWCLIFFE,T.W., MERlDN, R.E. and 'IHCMAS,D.P. In vitro and in viva studiesof the anti-Xa activityof heparin. ThranbosisRes. 24, 319-328,1981. 12. LINDAHL,u., KoImr, S.O., BU@lALD,J., BTERUD, B. and SELJELID,R. Studies on blood coagulationfactor X/Xa producedby muse peritoneal macrophages,using a luminogenicpeptide substrate. Bide&J. 206, 231-237,1982. 13. OEOSU, F.A., MDDI, G., mKU.9, A.L., HIRSH, J. and BLAJCHMAN,M.A. Heparin with law affinityto antithrunbinIII inhibitsthe activationof prothrcmbinin normal plasm. ThrcmbosisRes. 28, 487-497,1982. 14. GLIMELIUS,B., BUSCH, C. and Hm, M. Binding of heparin on the surfaceof culturedhumn endothelialcells. 'Ihranbosis Res.l.2, 773-782,1978. 15. BUSCH, C., DAWES, J., PEPPm, D.S. and WASTESON,A. Binding of platelet factor 4 to culturedhuman umbilicalvein endothelialcells. Thrantmsis Res. 19, 129-137,1980. -S., RUCINSKI,B., JAMES, P. and LINDAHL,U. Plateletanti16. NIEWWSKI, heparin proteins and antithranbinIII interactwith differentbinding sites on the heparinmlecule. E'EBSLett. 102, 75-78, 1979. 17. DAWES, J., PUMPHREY,C.W., MC-, K.M., PROWSE,C.V. and PEPPER,D.S. The in viva releaseof human platelet factor 4 by heparin. Thranbosis Res. 27, 65-76, 1982. -18. 'IIEOMAS, D.P., MER'I0N,R.E., BARRC%LIFFE, T.W., MULLOY, B. and JOHNSON, E.A. Anti-FactorXa activityof heparan sulphate. Thra&osis Res. 2, 501-506,1976. 19. 'IHCMAS,D.P., JDHNSDN,E.A. and DIFFE, T.W. The antithrankkic action of heparan sulphate. In: VascularOcclusion:Epideniological, Pathophysiological and TherapeuticAgents, Ed. Teri, M. and Dommdy, J ., AcademicPress, London, pp 377-382,1981. 20. WASl%ON, A., Ha,
M. and WESTEE@%RK, B. Dermstration of a platelet
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133
enzyme, degradingheparan sulphate. FE8s Lett. 64, 218-221,1976. L., B&X3X&l, G., WAS!CESON, A., ROBINSON,H.C., &W3N, S. and 21. Tf-IuNBmG, of heparin-related LINDAHL,u. Enzymaticdepolymerization polysaccharides.J.Biol.Chem.257, 10278-10282,1982. 22. OLDBERG,A., HELDIN, C.-H., WASlWON, A., BUSCH, C. and HWK, M. Chara* terizationof a plateletendoglycosidasedegradingheparin-like polysaccharides.Biochemistry19, 5755-5762,1980.