1, 17, AND IN SEMINARS THROMBOSIS HEMOSTASIS-VOLUME SUPPLEMENT I99I

Drugs and of Mode Action Heparin Related of

P BE Ph . HE H .C O E N R A A D M K E R , . D a,n dS U Z E T T E G U I N ,h . D .

There are three main classesof antithrombotic and drugs:Plateletinhibitors,oral anticoagulants, drugs such as heparinthat inhibit activatedclotting factors. and Oral anticoagulants heparinsare roughly equally effective in preventingvenousthrombosis.However, different in thesetwo families of drugs are essentially propertheir modeof action.They eachhavenumerous action, but for ties in addition to their anticoagulant that neitherof the two has it been demonstrated it is for actionthat is responsible the indeed anticoagulant the actionis the effect.Yet, the anticoagulant antithrombotic only action that they have in common. Thereforewe considerit more likely than not that the anticoagulant to property bearssomedirectrelationship the therapeutic on effect. Despiteactive research plateletaggregation acknowlinhibitorsfor over25 years,it is now generally make less effective anedged that these substances do.1 thananticoagulants It musteven agents tithrombotic drugsact via a be considered possibilitythat antiplatelet thrombinformation.' an activationon platelet-induced We will thereforediscussthe mode of action of in heparinsand other thrombin scavengers terms of process. inhibitionof the coagulation nor Neither oral anticoagulants heparinsare ideal requireslaboratory treatment drugs.Oral anticoagulant Heparinneeds expertsupervision. controland constant by frequent administration injectionand sideeffectsare common. Bothdrugshavea relativelynarrowtherapeutic the window, that is, the gapbetween effectivedoseand bleeding,is not very wide. This the dosethat will cause is explainswhy at this momentmuch active research spent findingmoreefficientantithrombotics. in thosedrugs,suchas In this article we will discuss active clotting heparin, that are known to scavenge

factors.Within this group, there are drugs with quite for dissimilaractions.Pentasaccharide, instance,only III antithrombin (AT Ill)-mediatedFactorXa enhances inactivation, whereashirudin is a specific thrombin inhibitor.Yet this groupof drugsalsohasmanyfeatures them thatwe mayconsider so in common, many,indeed, call "scavto belongto one "superfamily"that we will engins" (Table l). As a typical examplewe will first the discuss mode of action of heparin.This requiresa of short introductionon the reactionmechanism blood coagulation.

OF MECHANISM THROMBIN THE FORMATION

governthe generation processes Threefundamental actiplasma:(1) sequential of thrombinin platelet-poor (2) ofthe efficiencyof vationofproteases, enhancement by a protease its complexingto a protein cofactorat a phospholipid surface,and (3) activationof the protein by cofactors thrombin,the end product.In plateletrich shouldbe takeninto account: plasmaone more process flip-floP. the plateletmembrane differs accordactivations ofprotease The sequence is ing to the way in which coagulation triggered.When tissuethromboplastin, with excess is the reaction started FactorVII* this is donethroughtheextrinsicpathway: IL In the intrinsic pathway the Factor X-Factor as: reactionscan be summarized Contact Factors* FactorIX-Factor X*Factor II. In that casethe by contactfactorsare activated kaolin, ellagic acid, or other negatively charged "foreign" surfaces. Under conditions,the triggeringof coagulapathophysiologic tion by small'amountsof tissuefactor is probably of the In important. thatcase indirectactivation FactorX via the actionof FactorVII on FactorIX hasto be taken into account.This pathwaywe called the Jossoloop: VII*Factoi Ix*Factor X+Factor II.3'4 Factor Neither activatedFactor VII nor Factor [Xa nor 29

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

Copyright @ 1991 by Thieme Medical Publishers,Inc., 381 Park Avenue South, New York, NY 10016 All rights reserved

30

AND MMOSTASIS_VOLUME 17, SUPPLEMENT1' 1991 SEMINARSIN THROMBOSIS TABLE 1. Scavengins: Drugs That Act Directly or Indirectly by lnactivating Clotting Proteases
Abbreviation heparin Unfractionated Low MW heparin Pentasaccharide Pentosanpolysulfate mucopolysaccharide Japonicus Stichopus Dermatansulfate Lactobionicacid Hirudin UFH LMWH Penta PS SJAMP DS LBA Hir Cofacar

AT III ATM ATM

ATm+x{ctr ATItr+HCtr HCtr HCtr

None

on FactorXa are very efficientenzymes their own' All lipid surface on threeneedto be adsorbed a procoagulant

review). FactorVa and FactorVIIIa do not occur as suchin

c of platelets(processes and d) requiresa,moreor less lig pronounced time. During this lag tirneslowthrombin a causes by generation an as yet unknownmechanism that imall amouniof thrombinto arise'Onecan imagine minute amountsof thrombin can be formed without the cofactorspresentor that occasionalmoleculesof the pathways'Anycofactorsare activatedvia alternative of how, thepresence a smallamountof thrombinduring for a certainiime is necessary the full activation of the systemand for explosivefurther thrombin formation' that Of the threefeedbackmechanisms, of FactorV with the smallestlag time, that of Factor is associated VIII takes more time, and that of platelets still rnore' are the Therefore lag timesin the extrinsicsystem short, andin platelet-rich theyarelonger, in theintrinsicsystem plasma they are the longest. ' determiClotting times areessentiallyexperimental invariablywhen of nations this lag time, clottingensuing about20 nM of thrombinhasfornred.The classicclotting the testsdo not measure amountof thrombin formed' In very probablyboth the lag situations pathophysiologic

FVII-TF
change review). At the sametime, they undergoshape microvesicles' procoagulant PC andproduce containing by mechanism a ThiJconstitutes third positivefeedback thrombin.ll governingthrombinformareactions The essential in Figure 1. tion are summarized

I
I
I

PL
FVa-F

v FVIIIA-FIX

FVIII

i.

mbin
Platelets

CURVE GENERATION THROMBIN THE

In Figure 2 we show the featuresof a thrombin thrombin-dependent curve. The compulsory generation maybe, V of activation Factors andVIII and,asthecase

FlG. 1. A schemeof blood coagulation Solid arrows indidashedarrows show.chemical activations, cate sequential PL: activations' phosdottedarrows,feedback conuersions, pholipid;TF: tissuefactor.

BEGUIN MODE OF ACTION OF HEPARIN-HEMKER,

200

31 is that the intricatekineticsof clotting factor activation betweenthe are not the result of a simplerelationship present duringthe lag time and the amountof tkombin (see formationof prothrombinase later). Heparinhas beenshownto have, in the extrinsic system,only an insignificanteffect on prothrombinase its despite (thatis, Factor Xa-Va-phospholipid).activity, This may be exXa definiteanti-Factor activity.z'rz''r of plainedby the relativeexcess FactorXa in clotting plasma.The maximal velocity of thromtin generation is thatwe observed about300nM' min-'. Theturnover moiety (Factor numberof a completeprothrombinase is on the order of 2000 Xa, Factor Va, phospholipid) min-r.l4 This allows us to estimatethe maximum as plasmaconcentration 0.15 nM. The prothrombinase both Factor V (25 nM) and of plasmaconcentrations X Factor (200nM) aremuchhigherthanthis. This means thatonly a smallfractionof FactorV hasto be activated the to stimulate system.This may explainthe short lag times in the extrinsic system. It also explains why generationis virtually insensitive to prothrombinase ^Factor that et Xa inhibition.Indeed,Pieters alls showed so inhibit FactorXa generation asto have to it is possible (<0.5 nM) and yet to FactorXa present no measurable that activity, pro-vided suffiprothrombinase obtainfull Va (l nM) wasavailable.t) cientFactor In the intrinsic systemwe cannotfor the moment necesguess the amountof tenase of makean educated to trigger explosivethrombin formation, but the sary qualitativedata are clear. The rate-limitingstep is the activationof Factor VIII' Heparin thrombin-dependent it inhibits because diminishesthe thrombin level that FactorVIII duringthelag time' In any casethe activates main actionof heparinis via the inhibition of thrombin 2'12'13'16 mechanisms. feedback andthrombin-rn"diated

I 0

time FlG. 2. The thrombin generationcurve, The ordinate is nonlinear that the rangeof 0 to 1 (grayzone)has been in of the generation small extendedin order to visualize curve; a: during lag phase. normal the of amounts thrombin t: of b: curvein the presence a scavengin; lagtime; p: peak underthe curve. amount; surface s:

time andtheamountof thrombinformedin situ at the site or the injury determine thrombotic hemostatic of a vessel why thereare so often This may be the reason reaction. of the between prolongation a cloftingtime discrepancies action.Often, no and by a scavengin its antithrombotic influence on clotting time tests is interpretedas no From this enor to the on influence thrombingeneration. that that thereexist scavengins do not influconclusion is bloodcoagulation a small,andoftenencountered, ence that but illogical step.In fact, everyscavengin we tested in thosementioned Table 1) markthus far (essentially the edly influenced amountof thrombinformed.That is to a why we are developing test that responds the area curveratherthanto its lag underthe thrombingeneration time.

HEPARINS WEIGHT LOWMOLECULAR

and Apart from pentasaccharide relatedultra low (ULMWH), all heparinsact molecularweight heparins The likJ unfrictionatedheparin(UFH).2'12 essentially low molecularweight heparins(LMWH) that are in medicaluseat the momentdo not belongto the ULMWH plasma actionon thebiochemthe group.In platelet-poor ical mechanismof thrombin formation is essentially groupthem for identical LMWH andUFH. We therefore (theybehave standard heparin). as as together S heparins The ULMWH group we called P heparin(for pentasaccharide).2 Eventhoughthe anti-Xaactionof LMW S heparins is higherthantheir anti-Ilaaction(takingnormalheparin not thisdoes resultin their material),17-22 asthereference inhibiting at the level of protkombinase(whereFactor

OF MODE ACTION HEPARIN OF

Heparin influencesthrombin generationin two ways, both of which are due to its enhancingthe rate. thrombin-inactivation Thereis theeffecton thebulk present anymomentandthe at thrombin of amount active by lag time thatis caused the diminutionof effecton the for of amounts thrombinnecessary feedback the minute Although both effects are causedby the activation. of scavenging thrombin,theyarenot invariablycoupled. hardly influencethe lag time Heparinwill, for instance, (thromboplastin time), but it will of theextrinsicsystem exerta markedinfluenceof the lag time of the intrinsic time) andon the (activated partialthromboplastin system plasma.The reason this for clottingtime of platelet-rich

1' 17, AND IN SEMINARS THROMBOSIS HEMOSTASIS-VOLUME SUPPI.EMENT 1991 of because the large FactorX Xa is the activeenzyme) in reserve plasma,as alreadyexplained.This is why a in expressed anti-Xaunitsdoesnot concentration heparin power' give a realisticpictureof its anticoagulative pose a and Pentasaccharide other P-type heparins they do not problemto somepeoplebecause conceptual This.is acton thrombinandthey still areantithrombotic. with the conceptof thrombin contradiction in apparent inhibitionasbeingcrucialto the antithomboticaction.It however,that anyanti-Xaaction be should remembered, eventuallyresultsin less thrombin being formed. We acts that pentasaccharide on free have demonstrated FactorXa.22In this case, where no anti-Ila action is present, inhibition of FactorXa can be so important the that does rate ihat it becomes limiting. With any heparin havean anti-Ila action,this actionalwaysovemrlesthe anti-Xa action. At the moment,we are trying to find, with the aid of preciselydefinedheparinfractions,at what molecularweight the P-S transitiontakesplace. does Obiter dictum: The fact that pentasaccharide not influence the anti-Ila action of AT III does pose a inducesa conproblem. If pentasaccharide theoretical in change AT III that makesit more active formational why it should towardFactorXa, it is difficult to conceive not also have a certain action on the AT Ill-thrombin is Evidently, the mechanism more compliinteraction. thancommonlythought.That theanti-Xaactionof cated its does pentasaccharide not per seconstitute antithromfrom the fact that oneneeds 6otic potencyis easilyseen to give about 10 times more anti-Xa units of pentasaccharidecomparedwith UFH to obtain a comparable c antithromboti effect.23'24

PLASMA PLATELET.RICH

they Oncethey areactivated, activated. become platelets the heparinpresentand thrombin generation neutralize

less they thanUFH, maybebecause possess PF4binding they containmuch' otheror because sitesper molecule they are wise inert, PF4 binding material or because

SCAVENGINS NONHEPARIN
is scavengins The modeof actionof the nonheparin again principally the same as that for S heparins: feedback' of intriUitibn thrombin andthrombin-mediated Also, the This is withoutdoubtthe casefor hirudin.2s'26 such scavengins as II cofactor (HC Il)-dependent heparin acidactin this way (see sulfateandlactobionic dermatan
100

It thus seemsthat the global mode of action of plasmacan be summarized in scavengins platelet-poor feedas:inhibitionof thrombinand thrombin-dependent this is an noting that for the P heparins back reactions by indirecteffect mediated their direct anti-Xaaction.

10

15

in on of FlG. Theinfluence heparins thrombingeneration 3. heparin a o: plasma. control; : unfractionated platelet-rich 0'2 heparin U/ml r b.t Ul.t (-0.5 rr.g/ml); : unfractionated (e ( - 1 . 0 p r . g / mo):;F r a x i p a r i n 5 p g / m l ) . l

BBCUN.I MODEOFACTION HEPARIN-HBIr,TrNN, OF may explain, at least partly, why in pharmacokinetic betweenanti-Xa and studieson LMWH a discrepancy anti-Ila actionis often observed. in Onemay askwhy heparin vivo hasan antithrombotic actionif this actionis linked to thrombininhibition in and if the amount of thrombin is not decreased plasma, situation platelet-rich a obviouslycloserto thein plasmais. A possible vivo situationthan platelet-poor for is suggestion an answer to be found in the fact that, in vivo, heparinactsin flowing blood. Due to the flow, will automatically a retardation thrombingeneration of meanmoremixing anddilution and hencelower thromevenif the net amountof thrombin bin concentrations, generated not diminished. is

33
platelets the activationof humanfactor VIII by thrombinand in 2:l'l factorXa, andin the activation factorX. Blood'1 6l-l'17 0, of l 988. thrombinis Pieters HC Hemker,T Lindhout:In situ generated J, factorVIII: C andfactor that theonly enzyme effectivelyactivates V in plasma. Pieters Thesis,Universityof Limburg, 1989. lnr J, In: and 1 0 .Bevers EM, J Rosing,RFA Zwaal. Platelets coagulation. III. in Maclntyre,Gordon(Eds):Platelets Biologyand Pathology B.V. New York, 1987,pp 12'7-159. ElsevierScience Publishers of BdguinS, T Lindhout,HC Hemker: Theeffectoftraceamounts in tissuefactor on thrombingeneration plateletrich plasma,its inhibitionby heparin.ThrombHaemost 6l:25-29,1989. Thesis, University of Limburg, t2. Bdguin S: Thrombinoscopy. Maastricht, 1987. lJ. BdguinS, T Lindhout,HC Hemker: Theinodeof actionof heparin in plasma.ThrombHaemost60:457462, 1988. RAF Zwaal, J Rosing: 14. van Rijn JLML, JWP Govers-Riemslag, Effect of factorVa and Kinetic studies prothrombin of activation: phospholipids the formation of the enzyme. Biochemistry on 23:45574563, 1984. 1 5 .PietersJ, T Lindhout: The limited importanceof factor Xa property heparin thromboplasin of inhibitionto the anticoagulant '12:2048-2052, plasma.Blood 1988. tin activated MA MR r6 OfosuFA, P Sie, GJ Modi, F Femandez, Buchanan, Blajchman, B Boneu, J Hirsh: The inhibition of thrombinis positive-feedback reactions critical to the expresston dependent effect of heparin.BiochemJ 243:579-588, of the anticoagulant 1987. GEC t 7 Andersson TW Banowcliffe, E Holmer, EA Johnson, LO, by properties heparin fractionated affinity of Sims:Anticoagulant chromatographyof matrix-bound antithrombin III and by gel 1976. filtration. ThrombRes9:575-583, 1 8 .Andersson LO, TW Barrowcliffe, E Holmer, EA Johnson,G of Molecularweight dependency the heparin-potenS<iderstr<im: tiated inhibition of thrombin and activatedfactor X. Effect of Res15:531-541, 1970. in Thromb heparin neutralization plasma. t 9 CerskusAL, KJ Birchall, FA Ofosu, J Hirsh, MA Blajchman: Effects of heparinfractions of different affinities to antithrombin III and thrombin on the inactivationof thrombin and factor Xa by antithrombin CanJ BiochemCell Biol 62:9'75-983,1984. III. X 20. Fareed JM Walenga,D Hoppensteadt, Huan, A Racanelli: J, Comparative studyon the in vitro and in vivo activitiesof seven weightheparins. Haemostasis l8:719-723, 1988. low molecular D z l . HolmerE, K Sriderberg, Bergqvist,U Lindahl:Heparinandits weightderivates: Anticoagulant antithrombotic and low molecular properties. 16 Haemostasis (Suppl2):l-7, 1986. 22. B6guin S, J Choay, HC Hemker: The action of a synthetic Thromb pentasaccharide thrombingeneration wholeplasma. on in 401, 1989. Haemost 6l:39'7 J z J . Walenga JM, M Petitou,JC Lormeau,M Samama, Fareed,J activity of a syntheticheparinpentasacChoay:Antithrombotic modelusingdifferentthromin thrombosis charide a rabbitstasis 1987. ThrombRes46:187-198, bogenic challenges. J 24. Walenga JM, M Petitou,JC Lormeau,M Samama, Fareed,J antithrombotic activity of syntheticheparin Choay:Intravenous pentasaccharide a human serum induced stasis thrombosis in model.TtuombRes 43:243-248,1986. 1989. communication, 25. LindhoutT: Personal G 26. Markwardt F, G Nowak, J Stiirzebecher, Vogel: Clinicopharmacological hirudin. Thromb Res studieswith recombinant 1988. 52:393-400, J J 2'1.OfosuFA, MA Blajchman, Modi, LM Smit, MR Buchanan, of of Hirsh:Theimportance thrombininhibitionfor theexpression activities of heparin, dermatansulfate, low the anticoagulant

CONCLUSIONS
is 1. The main modeof actionof scavengins to the of decrease amount activethrombinandto impair the positivefeedback thrombingenon thrombin-dependent eration. 2. Anti-FactorXa action is of no importancein either UFH or LMWH now availableto the clinician (S heparins).In dermatansulfate and other HC IIscavengins, plays no role at all and obviit dependent ously not in hirudin either. It comes into play in pentasaccharide some other very low molecular and (P weightheparins heparins). 3. A realistic estimate thepotency a scavengin of of its canonly bemadeif we takeinto account susceptibility platelets. from activated by to beingneutralized products

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34

I, 17, AND IN SEMINARS THROMBOSIS TMMOSTASIS_VOLUME SUPPLEMENT I99I

protein. Adv Exp heparinneutralizing 32. Lane D: Platelet-derived Med Biol 192:427438,1985. MA 33. LevineSP,RR Sorenson, Harris,LK Knieriem:Theeffectof heparin'Br J Haematol of platelet factor4 (PF4)on assays plasma 1984. 57:585-596, 34. van PuttenJ, V.D.M Ruit, M Beunis, HC Hemker. Heparin of neutralizationduring collection and processing blood inhibited 14.,253261,1984. Haemostasis by pyridoxal5'-phosphate. T 35. BdguinS, J Mardiguian, Lindhout,HC Hemker:The modeof (PK actionof low molecularweightheparinpreparation I 0 I 69) and in on two of its major components thrombingeneration plasma. 61:30-34, 1989. ThrombHaemost

Br polysulphate. J Haeweight heparinand pentosan molecular 1985. matol60:695-704, 1989. communication, 28. Dol F: Personal of R, 29. Wagenvoord H Hendrix,C Soria,HC Hemker:Localization in polysulphate blood coagulathe inhibitory site(s)of pentosan 60:220-225,1988. tion. ThrombHaemost cofactorII asthe of 30. ScullyMF, W Kakkar:Identification heparin principal plasmacofactorfor the antithrombinactivity of pentosan 1984. Res36:187-194, polysulphate. Thromb polysulphate activation Pentosan 31. ScullyMF, V Ellis, W Kakl<ar: of heparincofactor II or antithrombinIII accordingto molecular ThrombRes41:489-499,1986' weisht fractionation.