mode of action of heparin and related drugs

7/29/2019 Mode of Action of Heparin and Related Drugs

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Mode fAction f HeparinndRelated rugsH.COENRAADEMKER,h.D.,ndSUZETTEEGUIN,h.D.

There are three main classesof antithromboticdrugs:Platelet nhibitors,oralanticoagulants,nddrugssuchas heparin hat inhibit activated lotting factors.Oral anticoagulantsnd heparinsare roughly equallyeffective n preventingvenous hrombosis.However,these wo families of drugsare essentially ifferent ntheirmodeof action.They eachhavenumerous roper-ties in addition to their anticoagulant ction, but forneitherof the two has t beendemonstratedhat it isindeed he anticoagulantction hat s responsibleor theantithromboticffect.Yet, theanticoagulantctions theonly action that they have in common.Thereforeweconsidert more ikely than not that the anticoagulantproperty ears ome irect elationshipo the herapeuticeffect. Despiteactiveresearch n plateletaggregationinhibitorsor over25years, t is nowgenerally cknowl-edged hat thesesubstancesmake less effective an-tithrombotic gentshananticoagulantso.1 t mustevenbe considered possibility hatantiplatelet rugsactviaan activation n platelet-inducedhrombin ormation.'

We will thereforediscuss he mode of action ofheparinsand other thrombin scavengersn terms ofinhibitionof the coagulation rocess.Neitheroral anticoagulantsor heparinsare dealdrugs.Oral anticoagulantreatmentequiresaboratorycontroland constant xpertsupervision.Heparinneedsfrequent dministrationy injectionand sideeffectsarecommon.Bothdrugshavearelativelynarrow herapeuticwindow, hat s, thegapbetweenhe effectivedoseandthe dose hat will cause leeding,s not verywide. Thisexplainswhy at this momentmuch activeresearchsspentn findingmoreefficientantithrombotics.In this article we will discusshosedrugs, suchasheparin, that are known to scavenge ctive clotting

From the Universityof Limburg, Department f Biochemistry,Maastricht,TheNetherlands.Reprint requests:Dr. Hemker, Departmentof Biochemistry,Universityof Limburg, P.O. Box 616, 6200MD Maastricht,TheNetherlands.

SEMINARSN THROMBOSISND HEMOSTASIS-VOLUME7,SUPPLEMENT, I99I

factors.Within this group, thereare drugswith quitedissimilaractions.Pentasaccharide,or instance,onlyenhancesntithrombinII (AT Ill)-mediatedFactorXainactivation,whereashirudin is a specific thrombininhibitor.Yet thisgroupof drugsalsohasmany eaturesin common, omany, ndeed,hatwe mayconsiderhemto belong o one"superfamily" hatwe will call "scav-engins"(Table l). As a typical examplewe will firstdiscusshe mode of actionof heparin.This requiresashort ntroductionon the reactionmechanism f bloodcoagulation.

THEMECHANISMFTHROMBINFORMATIONThree undamentalrocessesovern hegenerationof thrombinn platelet-poor lasma: 1) sequential cti-vationofproteases,2) enhancementfthe efficiencyof

a protease y its complexingo a protein cofactorat aphospholipid urface,and (3) activationof the proteincofactors y thrombin, he endproduct. n plateletrichplasma nemoreprocess houldbe taken nto account:theplateletmembranelip-floP.Thesequencefprotease ctivations iffersaccord-ing to theway in which coagulations triggered.Whenthereactions startedwith excessissuehromboplastin,this s done hroughheextrinsicpathway:FactorVII*FactorX-Factor IL In the intrinsic pathway thereactionscan be summarized s: ContactFactors*Factor X-Factor X*Factor II . In that case hecontact actorsare activated y kaolin, ellagicacid, orother negativelycharged"foreign" surfaces.Underpathophysiologiconditions, he triggeringof coagula-tion by small'amounts f tissue actor is probablyimportant.n thatcasehe ndirectactivation f FactorXvia theactionof FactorVII on Factor X has o be akeninto account.This pathwaywe called the Josso oop:FactorVII*Factoi Ix*Factor X+Factor II.3'4NeitheractivatedFactorVII nor Factor [Xa nor

Copyright @ 1991by Thieme Medical Publishers, nc., 381 ParkAvenue South,New York, NY 10016 All rights reserved 29


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30 SEMINARS N THROMBOSISAND MMOSTASIS_VOLUME 17, SUPPLEMENT1' 1991

TABLE 1. Scavengins: Drugs That Act Directly orIndirectly by lnactivating Clotting ProteasesAbbreviation Cofacar

Unfractionated eparinLow MW heparinPentasaccharidePentosanpolysulfateStichopus aponicusmucopolysaccharideDermatansulfateLactobionicacidHirudin

UFHLMWHPentaPSSJAMPDSLBAHir

AT IIIA T MA T MA T m + x { c t rA T I t r + H C t rHCtrHCtrNone

FactorXa arevery efficientenzymes n their own' Allthreeneedo beadsorbednaprocoagulantipid surface

review).FactorVa andFactorVIIIa do not occuras such n

review).At the same ime, theyundergo hape hangeandproducePC containing rocoagulant icrovesicles'ThiJconstitutes thirdpositive eedbackmechanismythrombin.llThe essentialeactions overninghrombin orma-tion aresummarizedn Figure1.

THETHROMBINENERATIONURVEIn Figure 2 we show the featuresof a thrombingenerationurve.The compulsoryhrombin-dependentactivation f FactorsV andVIII and,as hecasemaybe,

of platelets processes and d) requiresa,moreor lesspronouncedig time.During his ag irneslow hrombingeneration y an as yet unknownmechanism ausesimall amouniof thrombin o arise'Onecan maginehatminuteamountsof thrombincanbe formed without thecofactorspresentor that occasionalmoleculesof thecofactorsare activated ia alternative athways'Any-how, thepresencef a smallamount f thrombinduringa certain ime is necessaryor the full activationof thesystemandfor explosive urther thrombin formation'Of thethree eedbackmechanisms,hatof FactorVis associated ith the smallestag time, that of FactorVIII takesmore time, and that of plateletsstill rnore'Thereforehe ag times n theextrinsicsystem reshort,in the ntrinsicsystemheyare onger,and n platelet-richplasma heyare he ongest.'

Clotting timesareessentiallyexperimental etermi-nations f this ag ime,clottingensuingnvariablywhenabout20 nM of thrombinhas ornred.Theclassicclottingtestsdo not measurehe amountof thrombinformed' Inpathophysiologicituations ery probablyboth the lag

IIIIIvFVI I IA-F IX+i .FVI I I

FV I I -TFPL

mbinPlatelets

FlG.1. A schemeof blood coagulation Solidarrows ndi-cate sequential ctivations, ashedarrowsshow.chemicalconuersions,ottedarrows,eedback ctivations' L:phos-pholipid;TF: issue actor.

FVa-F


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I

MODE OF ACTION OF HEPARIN-HEMKER,BEGUIN

20 0

t imeFlG. 2. The thrombin generation urve,The ordinate snonlinearn that he range f 0 to 1 (gray one) as beenextended n order to visualizehe generation f smallamounts f hrombin uring he agphase. : normal urve;b: curve n hepresencef a scavengin;: lag ime;p: peakamount; : surface nder he curve.

time and heamountof thrombin ormed n situat thesiteof avesselnjurydeterminehe hrombotic r hemostaticreaction.Thismay be the reasonwhy therearesooftendiscrepanciesetweenheprolongation f a clofting imeby a scavengin nd ts antithrombotic ction.Often,noinfluenceon clotting time tests is interpretedas noinfluence n thrombingeneration. rom his enor to theconclusionhat hereexist scavenginshat do not influ-ence loodcoagulations asmall,andoftenencountered,but llogical step. n fact, everyscavenginhatwe testedthus ar (essentiallyhosementionedn Table1) mark-edly nfluencedhe amountof thrombin ormed.That swhy we aredeveloping test hat respondso the areaunder he thrombingeneration urverather han o its lagtime.

MODEOFACTION FHEPARINHeparin influences hrombin generation n twoways, both of which are due to its enhancing hethrombin-inactivationate.Theres theeffecton hebulk

amount f active hrombin resent t anymomentand heeffecton he ag time hat s caused y thediminutionofthe minuteamounts f thrombinnecessaryor feedbackactivation.Although both effects are causedby thescavengingf thrombin, heyarenot nvariablycoupled.Heparinwill, for instance, ardly nfluencehe ag timeof theextrinsicsystemthromboplastinime), but it willexerta markednfluenceof the ag time of the ntrinsicsystemactivated artial hromboplastinime) andon theclotting imeof platelet-rich lasma.The reason or this

3 1is that he intricatekineticsof clottingfactoractivationare not the resultof a simple elationship etween heamount f tkombin present uring he ag time and heformationof prothrombinasesee ater).Heparinhasbeenshown o have, n the extrinsicsystem,only an insignificanteffecton prothrombinase(thats, FactorXa-Va-phospholipid).activity,espitetsdefiniteanti-Factor a activity.z'rz''r his may be ex-plainedby the relativeexcess f FactorXa in clottingplasma.The maximalvelocityof thromtin generationthatwe observeds about 00nM' min-'. The urnovernumberof a completeprothrombinase oiety (FactorXa, FactorVa, phospholipid)s on the order of 2000min-r.l4 This allows us to estimate he maximumprothrombinaselasmaconcentrations0.15 nM. Theplasmaconcentrationsf both FactorV (25 nM) andFactorX (200nM) aremuchhigher han his.Thismeansthatonly a small ractionof FactorV has o beactivatedto stimulatehe system.This mayexplain he short agtimes in the extrinsic system. t also explainswhyprothrombinase eneration s virtually insensitive to^FactorXa inhibition. ndeed,Pieters t alls showedhatit ispossibleo nhibitFactorXa generationoas o haveno measurableactorXa present


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SEMINARSN THROMBOSISNDHEMOSTASIS-VOLUME7,SUPPI.EMENT' 1991

Xa is the activeenzyme) ecause f the argeFactorXreserven plasma,as alreadyexplained.This is why aheparin oncentrationxpressedn anti-Xaunitsdoes otgivea realisticpictureof its anticoagulativeower'Pentasaccharidend otherP-typeheparins ose aconceptualroblem o somepeoplebecauseheydo notacton thrombinand heystill areantithrombotic. his.isin apparent ontradictionwith the conceptof thrombininhibitionasbeingcrucial o theantithomboticaction. tshould eremembered,owever,hatanyanti-Xaactioneventually esults n less thrombinbeingformed.Wehave demonstratedhat pentasaccharidects on freeFactorXa.22 n this case,whereno anti-Ila action spresent,he nhibitionof FactorXa canbe so mportantihat t becomesate imiting. With anyheparinhat doeshavean anti-Ilaaction, his actionalwaysovemrlesheanti-Xaaction.At the moment,we are trying to find,with the aid of preciselydefinedheparin ractions,atwhat molecularweight the P-S transition akesplace.Obiterdictum:The fact that pentasaccharideoesnotinfluence he anti-Ila action of AT III does pose atheoretical roblem. f pentasaccharidenducesa con-formational hangen AT III thatmakest moreactivetowardFactorXa, it is difficult to conceivewhy t shouldnot alsohave a certainactionon the AT Ill-thrombininteraction.Evidently, he mechanisms morecompli-catedhancommonlyhought.That heanti-Xaactionofpentasaccharideoesnot per seconstitutets antithrom-6oticpotency s easilyseenrom the act thatoneneedsto give about10 timesmoreanti-Xaunitsof pentasac-charidecomparedwith UFH to obtain a comparableantithromboti effect.23'24

NONHEPARINCAVENGINSThemodeof actionof thenonheparincavenginssagain principally the same as that for S heparins:intriUitibn f thrombinand hrombin-mediatedeedback'This s withoutdoubt hecaseor hirudin.2s'26lso, theheparin ofactorI (HC Il)-dependentcavenginsuchasdermatanulfateand actobionic cidact n thisway (see

It thus seems hat the global modeof actionofscavenginsn platelet-poor lasmacan be summarizedas: nhibitionof thrombinand hrombin-dependenteed-back reactions oting hat for the P heparinshis is anindirecteffectmediated y theirdirectanti-Xaaction.

PLATELET.RICHLASMA

platelets ecome ctivated.Once heyareactivated,heyneutralizehe heparinpresentand hrombingeneration

thanUFH, maybe ecauseheypossessessPF4bindingsitesper molecule r becauseheycontainmuch'other-wise inert, PF4 binding materialor becausehey are

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0 o s 1 0 1 5FlG. . The nfluence f heparins n hrombingenerationnplatelet-richlasma. : control; : unfractionatedeparinb.t Ul.t (-0.5 rr.g/ml); : unfractionatedeparin '2 U/ml(-1.0pr.g/ml) ;: Fraxipar ine5pg/ml) .

I


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MODEOFACTIONOF HEPARIN-HBIr,TrNN,BCUN.Imay explain, at leastpartly, why in pharmacokineticstudieson LMWH a discrepancy etweenanti-Xa andanti-Ila action s often observed.Onemayaskwhy heparinn vivo hasanantithrom-botic action f thisaction s linked o thrombinnhibitionand if the amountof thrombin is not decreasednplatelet-rich lasma, situation bviously loser o the nvivo situation han platelet-poor lasma s. A possiblesuggestionor an answers to be found n the fact that,in vivo, heparin cts n flowing blood.Due to theflow,a retardation f thrombingenerationwill automaticallymeanmoremixing anddilutionandhence ower throm-bin concentrations,ven f the net amountof thrombingenerateds not diminished.

CONCLUSIONS1. The main modeof actionof scavenginss to

decreaseheamount f active hrombinand o impair hethrombin-dependentositive eedback n thrombingen-eration.2. Anti-FactorXa action s of no importance neitherUFH or LMWH now available o the clinician(S heparins). n dermatansulfate and other HC II-dependentcavengins,t playsno role at all and obvi-ously not in hirudin either. It comes into play inpentasaccharidend some other very low molecularweightheparinsP heparins).3. A realistic stimatef thepotency f a scavengincanonly bemade f we ake nto accountts susceptibilityto beingneutralized yproductsrom activated latelets.

REFERENCESI . LoeligerEA: Oralanticoagulationn patientsurvivingmyocardial

infarction.A new approacho old data.Eur J Clin Pharmacol26:137-139,1984.2. Hemker HC: The mode of action of heparin n plasma. n:Thrombosis nl Haemostasis. erstraeteM, J Vermylen,HRLijnen,J Arnout(Eds): nternational ocietyon Thrombosis ndHaemostasis,euvenUniversityPress, euven,1987.3. HemkerHC: In memoryof Frangois osso.Why do hemophiliacsbleed? cand Haematol 0:11-19, 984.4. Ma Xi, S B6guin,HC Hemker: mportancef factor X dependentprothrombinaseformation-The Josso Pathway-in clottingplasma. aemostasis9:301-308, 989.5. TansG, J Rosing:Multicomponent nzyme omplexes f bloodcoagulation.n: ZwaalRFA, HC Hemker Eds):BloodCoagul.a-rion. ElsevierScience ublishers .V.6. Biggs R, RG Macfarlane, WE Denson,BJ Ash: Thrombinandthe nteraction f factorsVIII and X. Br J Haematol l:276-295,1965.7. SuzukiK, B Dahlbiick, Stenflo: hrombin atalyzed ctivation fhuman oagulationactorV. J Biol Chem257:655G{565,1982.8. Neuenschwander, J Jesty:A comparison f phospholipid nd

33plateletsn the activation f human actor VIII by thrombinandfactorXa, and n the activation f factorX. Blood'1 :l'l 6l-l'17 0,l 988.Pieters , HC Hemker,T Lindhout: n situgeneratedhrombinstheonly enzymehateffectively ctivatesactorVIII: C and actorV in plasma.nr Pieters , Thesis,Universityof Limburg,1989.BeversEM, J Rosing,RFA Zwaal. Platelets ndcoagulation.n:Maclntyre,Gordon Eds):Platelets n Biologyand PathologyII.ElsevierScience ublishers .V. New York, 1987,pp 12'7-159.BdguinS,T Lindhout,HC Hemker:Theeffectoftraceamounts ftissue actor on thrombingenerationn platelet ich plasma, tsinhibitionby heparin.ThrombHaemost l:25-29,1989.Bdguin S: Thrombinoscopy. hesis, University of Limburg,Maastricht, 987.BdguinS,T Lindhout,HC Hemker:The nodeof action f heparinin plasma.ThrombHaemost 0:457462, 1988.van Rijn JLML, JWP Govers-Riemslag,AF Zwaal, J Rosing:Kinetic studies f prothrombin ctivation: ffect of factorVa andphospholipids n the formationof the enzyme.Biochemistry23:45574563, 1984.PietersJ, T Lindhout: The limited importanceof factor Xainhibition o the anticoagulantroperty f heparinn thromboplas-tin activated lasma.Blood 12:2048-2052,1988.OfosuFA, P Sie, GJ Modi, F Femandez,MR Buchanan,MABlajchman, B Boneu, J Hirsh: The inhibition of thrombin-dependentositive-feedbackeactionss critical o the expresstonof the anticoagulantffect of heparin.BiochemJ 243:579-588,1987.Andersson O, TW Banowcliffe,E Holmer, EA Johnson,GECSims:Anticoagulantroperties f heparinractionatedy affinitychromatographyof matrix-bound antithrombin III and by gelfiltration. hrombRes9:575-583, 976.Andersson O, TW Barrowcliffe,E Holmer, EA Johnson,GS


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34 SEMINARSN THROMBOSISNDTMMOSTASIS_VOLUME7,SUPPLEMENT, I99I32. LaneD: Platelet-derivedeparinneutralizing rotein. Adv ExpMed Biol 192:427438,1985.33. LevineSP,RR Sorenson, A Harris,LK Knieriem:Theeffectofplatelet actor4 (PF4)on assays f plasma eparin'Br J Haematol

57:585-596, 984.34. van PuttenJ, V.D.M Ruit, M Beunis,HC Hemker.Heparinneutralizationduringcollectionandprocessing f blood inhibitedby pyridoxal5'-phosphate.aemostasis4.,253261,1984.35. BdguinS, J Mardiguian,T Lindhout,HC Hemker:The modeofactionof low molecularweight heparinpreparationPK 0 69) andtwo of its major componentsn thrombingenerationn plasma.ThrombHaemost 1:30-34,1989.

molecularweightheparinandpentosan olysulphate. r J Hae-matol60:695-704, 985.28. Dol F: Personalommunication, 989.29. Wagenvoord, H Hendrix,C Soria,HC Hemker: ocalization fthe inhibitorysite(s)of pentosan olysulphaten bloodcoagula-tion. ThrombHaemost 0:220-225, 988.30. ScullyMF, W Kakkar: dentificationf heparin ofactorI as heprincipalplasma ofactor or the antithrombinactivity of pentosanpolysulphate.hrombRes36:187-194, 984.31. ScullyMF, V Ellis, W Kakl

mode of action of heparin and related drugs

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