Molecular Biology of Plasminogen Activators: What Are the Clinical Implications of Drug Design?

Molecular Biology of Plasminogen Activators: What Are the Clinical Implications of Drug Design?

M o Biology l of Plasminogen e c Activators: What Am the Clinical Implications of Drug Design? u Richard W. Smalling, MD, PhD Theinitialworkan thram...

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M o Biology l of Plasminogen e c Activators: What Am the Clinical Implications of Drug Design?

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Richard W. Smalling, MD, PhD Theinitialworkan thrambolytie thempy& fUU@qq+Otiti reSU~hOW beenobluinedin Iofge, Anothernewagentisthe eardialinfaretian (AM) focused on infracz odin’w mrukwed trialsof reteplase. research hasgiven TNKmutantof t-PA.whichalsohasa waknaedhalf-life istmtianof slreptokinase. Continuing marempid and comb thromriseb thedeW@men t of bothsecond-and third=gen- and seemsto &e bleedingthan in thrambo- bolysis,aswellas lessriskof intraemnial _ and eansequent refinements inanimalmodels. Although large,mndamM W@ns. Intravenous recombinant tissueplasmin- withakspiase encouraging n3r (t-PA,oralteplase) provedsuperiarto both izedtrialshavenotyetbeencandueted, ~~ ntmomnaryandintmveno dase-mnging trials usStrept&nase withregard suhshaveemergedfrompreliminary ta mpeffusian effkaeyand impacton suwiwd.An accel+ withTNK.A thirdnewagmt, n-PA,hasan mm Iongw half4feandhasshovmimpmvedIytkwtivityin animal +.mgti was hr Md ti cflkwk under a&m&okm of &PAavera shah pm%daf$mq,IJn- madsk A dosemmg” M of n~PAis currently of thethir$generation hwwiely, t-PAfailedtolessen theriskafb&ding cam- WW, Despitethefact ‘%@ .,4 * ~~ Pkl=d ~ u~ ~ -;~ wdd~ t-PAmoiede ks sinceh ~ atkmpt~.achieveimproved~ characteristics+ -*. AmOflgthesethitd b @pA); -d wih OF _ half-lifeandseems to3$%:;%?!E*G

~ @ 1996 by Excwpto@dica, Inc. AmJCardial 1996;78(suppl12A):2-7

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arly attemptsat achieving successful reperfusion of the infarct-related artery in patients with F acute myocardial infarction (AMI) utilized direct

that in the treatment arm of the LATE trial.2 Linderer et a14also showed that patients treated within 1.5 hours of pain onset experienced a 21-day morinfusicm of streptokinase (Streptase; Astra USA, tality rate of l%, compared with 7% in patients Inc., Westboro, MA) into the coronary artery dur- treated beyond this point. ing an acute catheterization. Improvement in perIt is equally important to maintain patency once fusion, as demonstrated by thallium imaging after thrombolysis is successful. Many investigators have successful thromb,olysis, seemed to be associated shown that the 4-6% risk of mortality during the with imprmwl hfi ventricular ftinc$$~qeven in pa- acute hospital phase increases to 12–14% when a tients presm$iag aa late as 18 -llotl* @er onset of reperfused artery undergoes reclosure.5’c Furtherpain.l Similiwly; tk@ate Assess~wt of Throm- more, the risk of mortality continues to increase at bolytic Efficacy (LATE) TriaI, a@&~ others, also an accelerated rate in patients whose arteries remain showed a benefi$of iWravenQustMg@$olytic ther- closed over the following year.5’7 apy in Patien$$ ~~~ 6-12 hours#kiar symptom Not only is restoration of blood flow in the inonset.z For @&e time, there was WqcWs on push- faret-relatd artery important, but the degree of reing the treatmemt window later aftq &set of pain stored flow is a major factor in predicting outcomes. in an attempt to gain a better ugekwstanding of It is now clear that achieving reperfusion without which patitwitsshotdd be treated ~~ $wombolyt- restoration of normal flow (Thrombolysis in Myoics. However, Wavqr and the Is@@#dial Infarc- cardial Infarction trial grade 3 ITI.MI grade 3]) gention Triage and Iakwventicm P@@% (MITI) in- erally does not improve outcome in terms of either vestigators demonstrated convhmiN#y that “the survival or left ventricular function.8-10In the first emphasis should be on treating @tie$tts very early Global Utilization of Streptokinase and Tissue Plasin the course of an AMI. The I@w @al showed minogen Activator for Occluded Coronary Arteries that treatmtmt within 70 minutes ‘@f’ onset of pain (GUSTO-I) angiographic trial, TIMI flow grade corresulted in a mortality rate as low as 1.270,whereas related closely with mortality.l’ The correlation cotreatment beyond 70 minutes resulted in a mortal- efficient between observed and predicted mortality, ity rate of 8.7~03—a number strikingly similar to TIMI grade 3 flow status as a variable, was r >0.9. Thus, it is now clear that the goals of thrombolytic therapy should include rapid restoration of normal From the Division of Cardiology, Universi~ of Texas Medical School at Houston, and Hermann Heart Center, Hermann Hospital, Houston, flow (TIMI grade 3) in the infarct-related artery, Texas. avoidance of reocclusion of the infarct-related artery, Address for reprints: Richard W. Smallin MD, PhD, Division of and avoidance of bleeding complications, particuCardiology, University of Texas Medical Scf’001 at Houston, 6431 larly intracranial hemorrhage. Fannin, MSB 1.246, Houston, Texas 77030. a

@1996

by Excerpta Medic.,

All rights reserved.

Inc.

0002-9149/96/$15.00 Pll S0002-9149(96]

00736-9

EVOLUTIONOF SECONDGENERATIONTHROMBOLYTIC. AGENTS

[email protected]

I Problems

With

Current

Thrombolytic

Agents

Infusion protacols are complex (especially with Front-looded t-PA]

and require an Inbion pumpand 2 separate intravenous lines As stated previously, the initial efforts in the IMld Optimal reperfuaion. (TIMI grade 3 flow) takes a relatively long of coronary thrombolysis utilized intracoronmy adtime [often >90 minutes] ministration of streptokinase. A so-called secondOptimal reperfusion is not achieved by 90 minutes in generation thrombolytic recombinant tissue plasmirtcrpproximotely 50% of patients _ Risk of intracranial ~morrhage ogen activator (t-PA, or alteplase; Activas~; GenenteCh Inc., San Francisco, CA) was subseTIMI = Thrombolysis in Myocardial Infarction; t-PA = tissue plasminogen acquently developed. The initial hope was @at ahe!ivoror, w“al&plaw plase would target coronary thro.mbi.pre$nx%tially, therefore reducing the @k of intracranial bleeding. b ( When administered intravenously over 3’hdurs; alTABLE II Desirable Features of New Thromb+tic Agents teplase achieved thrcxnbolysismore q~ckly @m did More ropid restoration of Tlfvll,grade 3 (normal) flow in the intracoronary streptokinase ,but was associatqil with inFarct@crted artew similar bleeding complications and ilbrinogeii de@eRestoration of TIMI grade 3 flow in the malority of patients tion.12Subsequently, an angiographic trial demonLonger halFJife, permitting bdus crdministration less risk of intracranial hemorrhage strated that the reperfusion efficacj of intravenous alteplase was superior to that of intravenous strep- * Reosarwble cost tokinase.13The alteplase dose was later mmlifiod @ TIMI = Tlt~mboly$is in Myocardial Infarction. optimize reperfusion by giving gore Iytic qver a shorter period of time. The “accelerated’” dosage i regimen consists of an initial 100 mg bolus dose, ~ TABKE It} Modification of Wild-Type t-PA followed by a 50 mg infusion over 30 minutes, and Function then 35 mg infused over the next 60 minutes,147i5 Region of Molecule High-affinity fibrin binding After the initial positive results showing apparent suFibroriectin’ finger periority of alteplase in achieving successful throm~ Epidermal, growth factor Receptor binding (liver) Receptor binding Kringle-1 bolysis, the GUSTO-I trial demonstrated that a.lte- Krin$k-2 Lowdfinity fibrin binding [stimulation) plase achieved more complete throml?olysis,which Protwme &main Plasminogen-specific, PAI-1 binding site was, in turn, associated with improtied survivai.lG Carbahy+ates Mediators of plasma clearance Administration of akeplase remained cumbersome, PAI-1 = pkminogem activator inhibitor 1; t-PA = tissue plasminogencrctivotor, requiring 2 intravetibus access sites (for heparin mid or altsystrwe. alteplase, respectively) as well as a fhirly complicated administration regimen. Although, initial results with a double-bohs dose of 50 mg of rdteplttse would. ,permit bolus administration. The drug seemed promising, subsequent larger; randomized would also act more quickly and achieve normal series suggested that the accelerated alteplase re$i- TZMI grade 3 flow in a greater percentage of pamen might be safer as well as more efficacious than tients. A$,the same time, bleeding complications the easier double-bolwi regimen.17’1* I-h&mtunaiely, should be”no worse than those associated with curthe initial hope that alteplase would cause fewer rently available agents. Finally, it would be most bleeding complications, partictdarly intiacmnial desirabl~.if su~h an agent ‘wereless expensive than hemorrhage, has not @en borne out. It therefore currently available thrombolytics (Table 11). Stratseemed that a plateaubf efficacy had been reached egies for the gerwtic modification of thromboly tic with alteplase. In a@ition, early reports suggested agents have be~n beautifully described by Losthat ‘intravenous thrombolysis rbay be les$ effica- calzo21 AIMColleti and colleagues.22 The wild-type t-PA (cm alteplase) molecule cious and associated with more complications, as well as higher mortality, than direct coronaij angio- shown on the top of Figure 1 consists of several morphologically identi~able regions that have various plasty.19’20 functi~ (Table III): the fibronectin finger-like doEVOLUTIONOF THIRD-GE$4HRATION main, which is associated with high-affinity fibrin binding; the epidermal growth factordomain, which THROMBOLYTICS Using the wild-type t-PA molecule as a “gold is responsible =forreceptor binding in the liver and standard,” many investigators sought tomodify al- acceleratwl clearance; the tingle-l domain, which tep~ase in an attempt to achieve better Iytic charac- is associated with receptor binding; the kringle-2 doteristics and less bleeding risk. Because the half-life main, which is associated with low-affinity fibrin of alteplase is only 3 minutes and its ability to binding (and, in part, accelerates conversion of plasachieve TEvlI grade 3 flow in GUSTO-I was only rninogen to plasmin); and the protease domain, 54% at 90 minutes, it was desirable to develop an which is a plasrninogen-specific protease and also improved, thrombolytic using genetic engineering contains,the binding-site region for plasminogen ac/. tivator inhibitor 1 (PAI-1 ). The zig-zag symbols techniques (Table I). One desirable feature of such a new thrornbo- demonstrate glycosylation sites. The glycosylation Iytic agent would include a longer half-life that site on kringle-1 is a so-called high mannose-type ●





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A SYMPOSIUM:

CONTROVERSIES

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side chain at asparagine 117; this is associated with rapid clearance of the molecule from plasma. The remaining complex-type carbohydrate side chains enlarge the physical size of the molecule and tictually slow clearance. By deleting portions of the molecule or rnalchig substitutions within various regions, the 4

THE

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biologic properties of the molecule maybe substantially changed. _ Re@plase (r~PA; Rqtavase; Boehringer Mannheim, Gaithersburg, MD) is a deletion mutant plasrninQgenactivator in which the finger, epidermal growth factor, and kringle-1 regions of the wild-type

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t-PA molecule have been deleted (Figure ”l; Table IV). Because r-PA is produced in Escheridia coli cells, it is unglymrsylated. In other words, by production in E. coli, the molecule does not contain carbohydrate side chains. By virtue of retaining the kringle-2 region, the molecule retains its fibrin s@cificity (orpreferential conversion of clot-bound plasminogen). However, beeause it does not have the finger domain, it does not have high fibrin affinity. It has a prolonged half-life of 18 minutes {approximately 6 times longer than that of alteplase). Because the molecule is produced in E. coli rather than. mamm~ian cells, it does require refolding, a process that is somewhat complex although effedive in restoring its biologic activity.23Compared with t-PA, reteplase (both in vitro and in vivo) has demonstrated a prolonged half-life and markedly reduced

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fibrin binding, and it appears to lyse coronary thrombi more rapidly (Figure 2) and more frequently.u-2G After these promising data were obtained in animals; prdimirmry dose-ranging trials appeared to uphold the a ,parent .superkwity of reteplase in”humans.27;28& ‘ e results of larger, randomized trials with this agent are also promising (for ,details, see the articles by Bode and Wilcox in this supplement). TNK:Another approach to modification of the alteplase molecule involves multiple point mutations. In the tWA mutant, T103N,N117Q,KHRR(296– 299}AAAA (TNK-t-PA; Table IV), threonine 103 has been changed to aspamgine 103, which creates a new glycosylation site for a complex-type side chain. This effectively enlarges the molecule and results in a longer half-life. The asparagine 117 has

A SYMpOSIUM: cONTROvERsIES IN fHROMBOLWIc THERAPY 5

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half-life (30-45

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AMA.

been changed to glutamine 117, eliminating the high mannose-type side chain, which also reduces clearance and extends the half-life of tlie”molecide. The sequence within the protease of Lys 296-His 297Arg 298-Arg 299 has been replaced by 4 alanines, which reduce inhibition by I?AI-1. The molecule is produced in Chinese hamster ovqy cells, and the remaining carbohydrate side chairis we tierefore conserved. The, net result is a molecule with a pr~ longed half-life (approximately 18 minutes) that is resistant to PAI-1 inactivation, has high fibrin specificity and binding, and is a more active Iytic agent than’wild-type t-PA.29Ip animal models, TNICseems to produce more rapid and complete thrombolysis with less risk of intracranial bleeding.30’31 Large, randomized trials have not yet been conducted with this agent; however, preliminary dose-ranging trials (TIMI 10A) are encouraging.” n-PA:The thrombolytic agent n-PA @ a deletion and point mutant of wild-type t-PA (Table’If!). The finger and epidermal growth factor regicms have been deleted, and a point mutation at tie Am 117 site in kringle-1 has been mutated to Gln 117.33This eliminates the high mannose side chain in kringle-1, but the complex carbohydrate side chains in l@ngle2 and the protease domain are preserved @ause n-PA is produced in Chinese hamster ovary cells. The consequences of this mutation group are,a more extended half-life (30-45 minutes) with improved lytic activity (in anim~ models) and reduced fibrin affinity. A dose-ranging trial (InTIME) has recently commenced, but preliminary data are not yet av@lable.

reteplase in the ongoing Global Utilization of Strategies to open Occluded Coronary Arteries (GUSTO-3) trial and previous Reteplase Angiegraphic ,Phase II International Dose Finding Trial (RAPID)’’’” and International Joint Efficacy Compariso.QQf Thrombolytics (INJECT)36 trials. Since TNK-WM has a similar half-life, it is likely that it will re@@ a double-bolus regimen as well. On the other &@, n-PA, with a significantly prolonged half-lif~’;~ll probably require a single bolus for adequate ‘I@i@olysis. Fil@iaaffinity is also an area where the drugs seem iOdiffer. Altho&gh data are somewhat difficult td~btain on rI-PA, its structure suggests it has less fibrin affinity than TNK. If that is the case, n-p+ s~ould be less fibrin- and fibrinogen-sparing than TNIL CMthe other hand, reduced fibrin binding has been shown to improve clot penetration and lysis of established clots.37’38As shown in Figure 3, increasing concentrations of alteplase >1 ~g/rnL in the dynamic plasm? model appear to inhibit thrombolysis, probably secondary to plasminog,tmconsumption. In contrast, however, a deIetion mutant plasminogen activator produced in E. cdi and consisting only of the protease domain gives a progressive reduction in clot lysis time with increasing concentration. This protease domain plasminog~nactivator has very little fibrin-binding activity and therefore is able to penetrate the clot more teadilyi thus preventing fibrinogen and plasminogen consumption in the supematant fluid. Alteplase binds tightly to the surface of the clot; therefore, in increasing concentrations it depletes the plasminogen above the clot and is unable to activate deep, clot-bound plasminogen.

Ccma.usms TMsis an exciting

time in the evolution of thrornbcdytic therapy for AMI. We now have at leaat 3 new plasminogen activators under clinical investigation that promise more rapid thrombolysis and significantly improved ease of administration. Risk of intracranial bleeding remains a significant issue, however, and will have to be addressed by large, randomized trials. In theory, the production of lytic agents in E. coli should be less expensive than in cultured mammalian cells. Whether this will lead to less costly agents is yet to be determined. Ackwwkdgnwnh The author would like to thank Dr. StepiwmFischer for his patience in trying to educate an interventional cardiologist in the subtleties of molea.k biology as it applies to thrombolytic drug design.

Dlscusmohl

All the new genetically modified .thmmbolytic agents under investigation seem to Iyse clots better than wild-type t-PA. Chief differences include their half-lives and fibrin affinity. An agrht with a halflife of 18 minutes will most likely need to be given in a double-bolus regimen, as is being done with 6

l. SmaUiWRW,FuerstesF, FreundGC,RedutoLA,Warrta-MWhews M, Gaeta JM,WrdkerW, Ster@rgR, GouldXL.Beneficialeffectsofintmcoronarythrombolysisup to eighteenhoursafteronsetof pain in evolvingmyecsrdisJinfarction.AmHeart J 1982;104:912–920. 2. LATEStudyGroup.Late assessmentof the thrombelytkefficacy(LATE) study with akeplsse 6-24 hours after onset of acute myomrdiatinfarction. Larcet 1993;342:759-766.

THE AMERICAN JOURNAL OF CARDIO1OGY” VOL. 78 [12A)

DECEMBER 19, 1996

3. WeaverWD.C$rqueiraM, H~W.rnmAP,LitwinP5Martin JS, Kudeochuk ~C ~:’ ~hlJ@f&&i~ PJ, @ae&#qj,,~ ~h+,~&@J aPY.-fW93;270:1211-1M6. 4. Lin&rer T,,Scbr6dexR, Arntz IL HeirtekingM, W~fierli@iW; KUtl K,, ForyckiF, HenzgenR, WagnerJ. ~apital%trqka601yaia: (5&@&d@@4tS of very earlytreatmenton infarctsize and left ventricularfunction.JAm Coil Cardiol 1993;22:13W-131O. 5. Dalen JE, Gore JM, BrmmwaldE, Borer J, GoldbergRJ, Paaaamani ER, ForrrumS, KmttterudG, and tie TIMI Investigators.Six- and’twelve-month follow-upof the Phase1‘fhrnmbelysisin MyecardialInfarction(’JTMI)trial. AmJ Cmdiol 1988;62:179 -185. 6. CXVnanRNf,CaliffRM, TopoIJ3J, CmadcrlaR,Ab*~C,E.JJi8 S, Si@non RN, KereiakesD,.GeorgeB, Stack R, and theTAMI S*Y Gruup.CoWqwencesof reocoltiafonafteisrrccmzfalreperfusiontherapyin a6utemyumrdird -791. ittfarcrion.Cirwr%sth 1~82:781 7. l.amaa (i& F@ker w MitchellG, ($rnithSC, Oer@BJ,WurJC, MOW L, RouleauJL, Rut@ford JD,PfefferMA, BraunwaldE. Eff~t of infarctartery pawney on pm@tosis atler acute myoear@l irrfarztion. Cireufation 199592:1101-1109. 8. AnderaoaJ~ wunis L& FIwkerLC, SomrssenSG, kferdow RL.TJl+4J perfusiongrade 3’butnot grade 2 resultsin improvedoutcomeafter @ornblyaisfor myuodr&aJinfarction.Cirmdurkm1993;S7:1S29-1839. 9. Vogt& vor@saemR,TebbeU, FeuererW, AP@ KF,NeuhauaKL.Imp@ of c+arlypcrfuaionstatus of the infarct-rekwdartery on @mrt.tcrrpmorplity offorrr aftartlminbolysisfnracrrte myncardialinfarction: rewospwtive’anidy.+is Germanmoltieenterstudies.7Am COZJ C#rdiol’1993;21:1391-1395. 10, GUSTOAngiographicInvestigators.Theeffectsof tiaaWphwmhtogenactivator, s**aae, or botb on wpxrary-arrmy patency,ventricular function, and survivalafieracutemyocardidinfarction.N Engl.JMed 1993;329:1615– 1622. 11. Simes RJ, TopolEJ, HolmesDR Jr, white HD, RutmhWR, Vahauiam A, SirnnunsML, Morris D, ~etriu A, Cafiff RM, Ross AM, for tbe GUSTO-I Invearigatnrs.Linkbetweenthe engiographicsubstodyand mortalityoutcnmes in a largerandomfmdtriidof mybmrdirdscperfuaion;impwkm@of earlyand’ completeinfamtarteryreperfwion.Ciwdrition 1995;91:1923-1928. 12. TopcdEl, MorrisDC, SmallingRW, Schumacher,W, TaylorCR, Niahikawa A, LibermanHA, COllenD, Tufta ME, tiSSbUd EB,‘0’Neill WW. A “X, ptacebn-controlledtrialof a new ford ofiritravemnrs multicmtwr,omdomr recumbinaattiaaue-~ pJaaminogenactivator(Activase)in wute myocardird infarction.JAm Call Cardiol 1987;9:1205–1213. 13. CheaebrnJH, KnattrmrdG, RobertaR, BorerJ, CohenLS, DrdesrJ, Dodge HT. FrancisCK, H@ D, LodbmokP, MarkisJE, MuellerH, PasaamardER, PowessER, J@ ~ RobertaonT, ROESA, RyanTJ, S@sl BE, WiffemonJ, WilfiamsDO, ZaretBL, BramrwaldE. Tbmmbolysisin MyocardialInfarction (TTMI)Trial, phase I: a compadsenbetweenintravenous‘tissueplaarainogen 76:142-154. activatorand intravenousslrept&inaae.Circrdat&n 1987; 14. NeubausKJ..,FeuererW, Jqr-Tebbe S, NiedemrW, Vogt A, TebbeLJ. Improvedtbrmqbolysiswith a muditieddose regimenof rwombiwmrttissue@ PISaOrinOgen activator.JAm CoZfCardiol 1989;14:1566-1569. 15.Srmdlitig RW, SchumacherR, MorrisD, HarderK, FrrenMsF, Valentine RP, B@fteyLL Jr, MerbigeJ, PittaDa LiebermanHA, NishiJ@wa.&A@ySUthayaA, HopfdnaA, GrossbardE. h~vditi=t-mktimrid.~~ncy @er bhzhdose.weight-adiusted.remidintirsionof tissue-winulaaridrromrr activator in-myocar&ali%arc&m:= of amrdticenterramd~-tih triafo~twodosage requirements.JAM COll c.ardiol1990;15:915-921. 16, GUSTOInvestigatura.An interna.tionafrandomisedtrial Gomptig four tbmmbnlytic strategies for acute myneardial infarction. N Engl J Med 1993;329:673-682. 17. Pervis JA. McNeiUAJ, Siddiqui RA, Roberts MJ, McClmnentsBM, McBnearteyD,C2urrpbellNPS, KhanMM,WebbSW,WilsonCM,Adge~AAJ. Bt%cacyuf lCQmg of dnuble-bufusafteplaae’inachievingcompletepetfnsirm in the tremrnetttofacmemywerdialinfarction.YAmCOUCanfki 1994;23:6– 10. 18. B]eichS. An angiogmp~cassessmentof the efficacyand Safetyof fTUntloadedand bultwregimensof Activase(alteplaaerecombinant).The DoubleBoluaLyricEf6cawyTrial (The DouJ3LETrird). Pmaentkdat the 68tb Annual ScientificSessionsof the AmericanHaartAwciation, Anaheim CA,November 1995. 19. Grines CL,’BrowneKF, Marco J, RothbaumD, Stone GW, O’KeefeJ, OverlieP, DoamboeB, ClmffiahN, TimmisGC, VlielatraBE, StrseleckiM, Puchrowicz.oehuckiS, O’NeillWW. A compariaunof immedktcSUgiOP@y

therapy,for kute m~ardial ihfaretion. N Engl J Med W93;338:623+67!?. 20. ZijMraF, de BeerJM,HoomtjeJCA,ReifferaS, ReiberJHC,Suryapranata H. A com@Wq of irnrqediatecurmraryangioplastywitlrintravenousstreptoW @ac!d&luY&arf@di# @vIL N@gl JMed 1993;328:680-684. 21. LoscalzoJ. Molecularbiologicmodificationof plaaminogenactivators,an artfulscience.Circulation1990;82:1062 -1063. 22. CohenD, LijnenHR,GoldHK.Towardsbcttcrtfrmmbolytictherapy,Prog Cardiovusc Dis 1991;34:101-112. 23. KohnertU, RudolphR, VerheijenJH, Weening-VerhucffEJD, Stem A, OpirzU, MartinU, Lill H, PrirrzH, LecfmerM, ~esse GB,Bu.kel P, Fischer S. Biecherr@lpropcrdes,ofthe khtgle 2Wdproreasedomairrsaremaintained in the refoldedt-PAdeletionvariantf3M,06.022.P’rarein.Eng1992;5’:93 -100. 24. MartinU, I%cherS; KohrrertU, IQdph R, SpenerG, SternA, StminK. tfxombolytieppertim ofanmd recombittamplaaminugenactivator c~ (BM 06.022)in a caninemodel.J Gwdioya.sc P/wvnucol 1991;18:111-119. 25. Martio U, lhder R, Bohm E, KohncrtY, vmt Mollendm-fE, Fischer S, Sperm G. BMti,022: a nevelrwurnblrmntplaaminogenactivator.Cardiovasc Drug Jfev 1993;11:299-311. 26. MardrrY, SpunerG, $trein K. Eh@uationof t@ombnlyticand systcrnic effects’ofthe novelrecombbramplasmhrogenaztivatr?rBM 06.022compered wjtb sJw@~I,wJistrepIw atrepmkirraae midomk@aaein a +nc modelof corooaryat’teryrbrorrdwk. {Am Cdl Ca@iol1992;19:433-440. 27. TebbeU, vonEaaenR, SmolarzA, LirhbourgP, RoxJ, RustigeJ, VogtA, NeuhmrsK-L.Open,nmrdcmtralicd deac-tinding WagnerJ, Meyer-SabeRbk’W, Stwdywith 0 novelr@mbaet Pk+smhl~ SOtivatm (3M 06.022) @V~ as a double bolus in patients with acute myocardialinfarction.Am J Cardiol 1993;72:518-524. 28. NeuhanaK-L, %rt EsacriR, Vugt A, Tebbe ~, RustigeJ, WagnerH-J, AppelK-R St@mrrU, K6nigIL [email protected] novel recombinantplaaminogenactivatur(BM 06.022)in patientswith acutemyocardiafinfarction:results of the Germanrccombinamtplasmirrogenactivator -60. study.JAm Coil Cam301 1994;24:55 29. KeytBA,.PanniNF, RefineCJ,BcrleauL, NguyenH, ChowA, IA J, Pena L, PatcrC, ClgezJ, JStcheverryT, J30tsteinD, BennettWF. A faarer-acrhrgand mom potent fdnmof tissue plaaminngcnactivater. Proc Natl Acad Sci USA 1994;91:M70-3674. 30. ThomasOR, l%ibndeauxH, Errett CL BadilloJM, Keyt BA, RefimrCJ. ZivinJA, BennettWF. A long-half-lifeand fibrin-specificformof tissneplasminngenaativ@erin rabLiitmodelsof ernbolicstrokeand peripheralbleeding. Stroke1994;25:~2 -X379. 31. BenedictCR, RefinoCJ, KeytBA, PakrdaF, PamriNF, ThomasGR, Bennett WF. Newvariantof bumarrtisimeplaarrrinogmr activator(TPA) with enhaneedeffieacyand lowerincidencerd bleedingcomparedwith recombinant humanTPA. Circulation 1995;92:3032-3040. 32. Braunwald,E. TNKEPA. Proc ??srornbolysisCollegiurn 1996.(In press). 33. LarsenGR,TimonyGA,HorgrmPG, BaroneKM,HensonKS,AngusLB, StOu&mireJB. Proteinemrineerirw of novel nlaaminoeenactivatorswith in. creased thmmbcdyticpntancy in ~bbits rel~tiveto &tivaae. ./ Biol C/tern 1991:266:8156-8161. 34. Sr@irrg RW,BrideC, KalbfleischJ, SenS, LimbmrrgP, ForyckiF, Habib G, FeldmanR, HohnloserS, SealsA, andthe RAPJDfrrvestigators.Mumrapid, complete,and stablecororrmythrumbolysiswith bulus administrationof reteplaaecomparedwith alteplawintirsionin acutemyoeardirdinfarction.Circr4with tkombolytif

Iation 1995;91 :2725-2732. 35. BadeC, SmaflingRW,BergG, BurnettC, @h G, KalbfleischJM, ChernoffR, ChristieLG. FeldmanRL, SealsAA, WeaverWD, md the RAPID11 Investigators.Randomizedcompariaunof cornnarythrombulysisachievedwith doublebuhrareteplaae(r-PA) andfmnt-luaded“accelerated”alteplaae(rt-PA) iII@emS with aCUtGmyUCSrdiSl infarction.Circrdarion1996;94:891-898. 36. InternationalJoint EfficacyCompa@onof llrrombolytics.Randomised, dorrbfe-blindcomparismrof reteplaaeduubie-bdrs administrationwith strepOJfdIMSS in acqtGmyecardiaJinfarction(INJECT): triafto investigateequivalence.Luncet 1995;346:320-336. 37. Kohnert U, HeIlerbrahdK, Mardn U, Stem A, Popp F, Fischer S. ‘Ilre recombinamEschericlriaaoi&&rivedprutease-domainof tissue-tyy plaarninogen activatesis a patent @ fibrin speciiicfibrinolyticagent. Fibrirralysis 1996;10:93-102. 38. MartinLJ,KohnertU, HellerbrandK, StemA, PnppF, DucrgeL, Stegmeier K, Mtiller-Be&raanB, FiaoherS. Effectiverhmmbnlysisby a rexmmbinant Esc/cerichia coli–pmduced prutcaaedrmrainnf tissue-typeplaaminogenactivatorin the rabbitmedelofjugularveinthrombosis.Fibrbrdysis 1996;10:8792.

A SYMPOSIUM: CONTROVERSIES IN THROMBOIWIC THERAPY 7