DIFFERENT APROTININ APPLICATIONS INFLUENCING HEMOSTATIC CHANGES IN ORTHOTOPIC LIVER

TRANSPLANTATION

G. HIMMELREICH ,I.2 M . MUSER.' P. NEUHAUS,' W. O. BECHSTEIN.3 K-J . SUhlA," M. JOCHUhI,' AND H. Rlus2

lHpcutmrnt. ot J,.urnoJ MediciM, SIJI'IIf'Y. ond Anouthui.ol4Dt. Un.iwrtity Ho.pWl. &rlin; twllHpartnwnl of Phyfiolt4ical CMmi.ltf)', Ußiwrtity of MiWch, M~h, Gtrmany

TIle .ffect 01 dill.reDt .protbUa .ppllcatloDS Oll h.­moetatlc eb ... _ ud blood. product requlreme.t. In ortbotopic liver trauplautatloD w .. mv..tilated lD • proepecUve. open, ud randomized Iludy,

Prom November 1989 to Jtme 1990. 18 patt.DU re. oelved aprotlalD ... bo1u 01 0.0 MW. k.lJIkrelD. iAac­U •• tor uaJ.ts (KIU) OD tiu'ee OOCMloaa lD. the coune 0' aD OLT, .. urea 10 otIMr paU.DU were tr..ted with OODtblUOUII .protbdD lDtuDOD 010.1-0 •• MW. KIU/hr. BeloN -.4 alter repert.loD 01 the p-aft liver. mpa. 01 JonerlIbrlDolyala, m-.ed by Ihn>mhe~.pby, weN lifD.lfleutly lower la tu lDluloD IP'OUP. T ........ type plumlaoa:eD ac:t1vator (t·PA) activity lacreued du:rIJaC lbe aallepatie phue but to • lipiIleudy l ... r e .. teDt lD. lbe batuiOD lP'Oup. Blood. produci requil'e. lIMati du..rbla OLT were teDdeDtlOtWy hleber ba tbe bolllll lJ'Oup but DOt eJca1ftcaaily 10. Bowever, th. UM 01 packed ncl blood ceu. ... QnUleaatly 10wer iD. tu po.&operaUve period. wbereu tlMre w .. llO .tcaHIca.at dlttereDCe in treü trosen pluma reqlllrelDent. bMween tIM two II'OUPI.

AU 38 patieJ:lt. have aurvived, ud oDly ODe WOJDaJl 01 Melh ~p reqa.ired retramplaDtatloD. due to Mvere hoet-venu.paft reactio ....

l'urthermore. we mveet.lp.ted. the pertaaate 01 the araft Uver La both ll'Ou,. ud deteeted ~ 01 a de-o ........ t-PA releuelD the bafu;ioD IJ'OUp.

Our renlt. demoutrate aa advaataae 01 aprotlain liven U coDtmuoa. lDtUaloD over bola. appUcaUoD lD OLT.

In OLT, hyperfibrillolyail h.u been recopized .. an impor­tant ClUM of intra- and poet.operative bleedin& (1-.. 1), intluenc­inI both the ahort- and lon,-term outcome (4, 6). PreviOUI inVflltigationa have indicated that hyperfibrinolyaia ia due to an increue olüuue-type pwminopn activator (t-PA)· activ-

I Addreu COrNIpondence 00: Dr. med. G. Himmelnicb. Gaubiua Laboratory TNO, P.O. BoI 430, 2300 AR Leiden, Tb. NetherWtd..

• Deputment of Interna! MediclM, Univeraity Ha.pital, Berlin. • Depart.ment of Surpry. Univeraity Hotpital, Serlin. • Deputment of ~o~, Univn.ity Hotpital, Berlin. · l)ep&rtment of Pby.ioIocical Cbemimy, Univeraity of Municb,

Munk h. • AbbNViationt: «rAP, «r antiplu.min; AT 111, u titluombin 111; B,

bolue POUP of aprotinin app1ieation; Cl-inb, Cl -iI'Ihibitor; FPP, rn.h frozen plalzu; I, inlueion POUP of IPz'otinin application; KIU, blli· hein inactivltor UDi"; PAI, plalll1inopn activator inhibitor; PC, protein C: RBC, pad,ed red blood ~u.: TAT, thrombin-antithrombin J1J: TEe, thrombelutorraphy: t·PA, tiuue.type pluminopn .ctiva· tor; u·PA, urolr.in ... ·type pluminopn . m vltor; WBLT, wbole·blood­clot Iylil time.

ity (1, 3) and a concomitant increue of urokinue-type plu­minogen aetivator (u-PA) activity (1) .

Aprotinin (Traaylol, Bayer, Leverkueen, Germany), a par­enterally applied proteinue inhibitor from bovine lune (6-8), inhibite plumin, kallikrein, tryp&in, ud to IOme enent human urokinaM (7). Tb. toxicity of aprotinin iI ennmely low (7), ud even hieb. doIOI are well tolerated (6, 9). AI randomiJed et:udi. in cardiac l\lI"Pry bave demonatrated ailnificantly re­ducecl tranafuaion requirementa when aprotinin w ... dmin; •• tered durine lurpry (10, ll ), we teated ita application in patienta undergoing OLT. Boh" application of 0.6 MüI. kaIli· krein inactivator unita (KIU) aprotinin liven three timet dur­lnJ OLT led. to reductiona in blood product requiremant and in the increue olt·PA activity (1 ) .. compared with tbe reporta of other inveetipton (2, 3, 12). Nevertbeleu, thrombeluto­pphy (TEG) ahowed aipu oi hyperfibrinoly.i. in moo of the OLT., in apite ol aprotinin bolua application.

In the prMOnt etudy, we have inveetipted tbe efrett of two different aprotinin reaU:nena on tbe helDOltatic ehanpa ud blood product roquitementa in 23 OLTa.

MATERIALS AND METHODS

In an open, proepective, and rudomized Itudy 23 ~tiente witb terminal liver diIeue (Table 1) underwent their flJ'lt OLT It tbe Univel"lity Hoepital Rudolf Vircbow, Berlin, Germany, betwNn No· vember 1989 and JUDe 1990. Tbe patienu were nmdomDed by aealed envelopee with aeria1 numbal'l contlininf the .Iy of IProtinin admin­iltretioD. Thirteen patien&l wen thereby randomized to racelV'l Lv. boli oe 0.6 Mill KIU aprotin.i.D three tImee duriIi,. OLT, liven .t tbe berinninl of tbe operation, at tbe onMt o( tIM anbepatk pbue, and at tbe berinninr of reperfueion, .bereu 10 patiente were contin\JI:M1y treated with aprotinin mfulion (Tab!. 2). OL T .... cam.d out by Htablilhed 1UJ'lica1 techniquM uainc a vellOWlDOUl bypue (13, 14). P.cI,ed red blood ceJ.a (RBC) ud fnah frosen pla.une. (FFP) wera eubetituted to compenu.te for intra· and poRoperative blood 10M. PoatoperatiV'lIy, RBe concentra .... wert! pnerally liven .hen tbe bemOilobin concentraUon .... lower tban 9 &/dl, while FFP conctn­tratea were admini.ttered wben tbe prottin concentration .... Iower than 6.0 &/dl. For cold .wrqe o( tbe pd liver, Beidr UW·CSS eolution (Dupont, Paria, France) .... UNd.

8lood Mmplee _re taken from tbe arterialline; (1) aftar induction of aneatbeIia; (2) 6 min berore; e.od (3) 10 min alter the berinnina: of the ubepe.tic ltap. Fu.rtber Mmplee _re eoUected; (4) 6 lI1in before repedueiol'l ; (6) .. _U u 6 miI'I: (6) 16 min; (7) 60 miD; e.od (8) 12 br aftarwardl. In addition, a Mmple or the perfuute releued from tbe li"r pd vein durin, the nUlJUn, proc.dun witb uterial. blood .,u taken prior to openin( oe the hepatocaval anutolDOlii (P). Blood aampl .. weN collected in ptuÜc Iyrinpa prefdled with 1/ 10 volume of triaodium citrate. For the determination of t·p A activity uticoqulat.ed blood wu immed.i.etely acidified witb tcetate acid Another blood

TAIILB 1. Chuacteriatice o( 23 patientll who underwent their first orthotopic liver transplantation wben aprotinin boli (B) or continuoUl aprotinin infulion (I) were given

Number Diqnoait

B

Poatnecl'Otic cirrboaie 5 8 Alcohol-toIic cirrhoait , 0 Primary biliuy cirrhoaia 1 1 Secondary biliary cirrhoeie (erythrohepatic proto- 1 0

porphyrle) Budd Chiari Iyndrume I 0 Liver cell carcinoma 1 0 Metutatic Iiver • 1

Total 13 I. TABLE 2. Aprotinin application UNd in 23 patientll who underwent

their firat orthotopic liver tranaplantation

Bolu.IfOVP

0.6 Mill. KIU Induction o( aDeatheaia 0.6 Mill. KIU Anhepatic phaae 0.6 Mill. KIU Raperfuaion

Skin doaure (heparin 260 IU/hr) (heparin 500 IUf hr at&rtini 12 br After operation till the 3rd poatoperative day)

Infuooion ........ P

0.2 Mill. KIU/hr 0.4 Mill. KIU/hr

0.1 Mill. KIU/hr

aample wu anticoagulated with a mature o( triaodium citratll, theo­phyllin, adenoaine, and dipyridamol (or the determination o( plu min­opn activator inhibitor (PAIl activity. Tbe epecimena were gently milled, centrifupd at 3000 Ufmin (or 20 min, and the aupernataot (ro:r.en at -70·C until meuurement.

Tbe (ollowin, parametera wen meuured uaini oommereially avail­.!Ible kib: protA;n C (PC) aetivity (Boehringer, MannlMim, Gnm. ny), antithrombin 111 (AT 111) ACtivity, pluminopn ACtivity, a t-antiplumin (a,-AP) activity, Cl-inhibitor (C1-inh) activity (all: Rehring Werke AG, MarbW'J, Germany), fibrinogen according to Clauaa (Hoffmann­LaRoche, Baael Switierland). 1--PA and P AI activitiea wen determined by chromogenie eubetratll methoda, where .. t-PA antigen w .. me .. -uNd Ulini a solid-phase enzyme immunouaay (alI: Kabi, Stockholm, Sweden). Thrombin-antithrombin III (TAT) compler.ea were deter­mined by enzyme immunouaays (Behrinperke AG, Muburt, Ger­many). Aprotinin w .. eltimated by ELiSA u previoWlly described (16). TEG oe reealcified wbole blood wu performed Uling a thromb­elutolHph (HeUige, Fniburg, Germany). When whole-blood-c1ot lysis time (WBL T), tbe time between tbe maximal amplitude and complete lyait in tbe TEG, wu aborter tban 90 min, it wo denned .. aevere h)perfibrinolysia, and between 90 and 120 min, .. mild hyperfibrino­lyail (8).

StotwticaJ onaly,w. Uaing the M&rtinez and Iglewicz teilt (16), the dietribution oe the parameters Wal round t.o be abnormal. Thererore the nonparametric Mann-Whitney telt WH uaed to teilt the sirnifieance oe differenoea between grou~ . Values ror P<0.05 were oonaidered to be aignificant.

RESULTS Median valuea of blood product requirementa during OLT

were 8 (range: 4- 26) unita for RBC and 10.5 (7-35) unita for FFP in the bolus group and 7 (2- 20) unita for RBC and 8 (2-28) unita for FFP in the infusion group. The difference wo not .ignificant. However, the UIe of RBC waa aignificantly lower (P-O.04Ö) in the infuaion group (median values (range): 3.5 (0-9) unita [bolus groupJ); 1.5 (0--6) unita [infusion groupJ) in the

Female M" ... M~ I_' B B B I

I , , , 50 (43-65) 49 (24~) 3 0 I 0 49 (40--66) 1 1 0 0 " 55 1 0 0 0 52

1 0 0 0 " 1 0 0 0 50

• 1 0 • 55

8 6 5 • 47 (40-65) 53 (24--65)

fint three postoperative daya alter the operation, whereas there waa no aignificant difference in FFP requiremente between tbe two groupa (6.5 (0-17J unita (infusion groupJö 9.5 (4-22) unita (bolus group) at the ume stage. Tbe median time o(intensive care in the bolus group wo 25 days (range: 14-58 days) 88

compared with 23 dayB (11-36 daya) in the infusion group (NS). In TEG, hyperfibrinolysia waa stated in 7 out of 13 OLTs in

the bolus group but ooly in 1 out of 10 OLTs in the infusion group (Fig. 1). Levels oft·PA activity increaaed in both groupa during the anhepatic phase, reaching their peaks immediately be(ore reperfusion. These muima, however, were significantly higher in the bolus group (Fig. 2). Tbe t-PA antigen levels in plaama did not differ between the two groups. In both groupa PAI levels increased alter reperfusion to comparablelevela (Fig. 3). The cour&e of C1-inh did not differ for either group (Fig. 3). The levela of a2-AP were higher in the infusion group, reaching significance only 10 min after the beginning o( the anhepatic phaae and 12 hr alter reperfusion (Fig. 4). Plaaminogen concen­tration ahowed no ditference in either group (Fig. 4). TAT complens showed significantly higher levels in the infusion group prior to the anhepatic phase and at ita oneet end alter reperfusion (Fig. 5), Similarly, fibrinogen levels were higher in the bolus groUp shortly before the anhepatic phaae and alter reperfusion (Fig. 5). The course o(PC and AT III activities did not differ in the two groupa (Fig. 6). Aprotinin levels were significantly higher in the infusion group (Fig. 7).

In the perfusate the comparilOn of different hemostatic pa­rameters betwe~m tbe groups revealed a aignificantly lower level oft-PA antigen and a tendentiously lower level in t-PA &etivity in the infusion group while activity o( the inhibitors PAI and a z-AP waa significantly increased in thia group. All otber pa­rameten in the perfuaate were comperable in both groups (Table 3).

D1SCUSSION

Our previous e:rperience (1) prompted UI to postulate a beneficial effeet of aprotinin bolus application on the course of OLT, but signs of hyperfibrinolysis as meaaured by TEG Were .till preaent in most patienta undergoing OLT. Here, we have investigated the effeet o( intermittent bolus vereus continuous infusion application of aprotinin during the course of 23 OLTa in an open, prospective, and randomized study.

We conrll'tDed previous inveetigations suggeating a beneficial effect of aprotinin bolus application in reducing signs of hyper­fibrinolyeis end bleeding complications in patientl undergoing OLT (1).

BOLUS INFUSION

• ..... t.

• Will. " •• !. : ~. "1,.,U', 'u" .I,

.... ll. "0 .. , •• nO' H .. ,. : .11. """U"'.ol •• I. wILl · " '".~ . ...... ",''',,,.,, •• ,,,,,

FIClUMr. 1. Sijl:na of hyperlibrinol)'si8 during orthotopic liver trans­plantation in thromboelastogram in 13 patitnlll of 8protinin bolu8 applicillion lind 10 pHlientll of Hprotinin infu.ion.

I " c': 01"

I " " ; 0 . " , " .~

,dJ: ...... ... : • : ........ ., ... ' , ,

, ......... , .0 ....... ,-" ...... _, - •

"

" !\\----I :\ 0 0 10

.l \ i' ~ • : + " ,

• •• \;._ •• _.-- 0 , . ""­-~,

•

i i . .

FIGUIH! 2. I· PA flct ivity and Rnligen during orthotopic livet Irlln8-p]an\a\ion in ]3 plilienu of ap rOlinin bolus apllliclltion Ind 10 patientll of aprotinin infusion.

Aprolinin levels were higher in the infusion group during the whole length of the ÜL Ts (Fig. 7). Signs o( hyperfibrinolysis. according to TEG and increased t ·PA aetivity (rigs. 1 and 2) oceurred more often in the bolus group especially before and after reperfusion, whereas PAI and a 2·A P levels (Figs. 3 and 4) were lower during the anhepatie phase and after reperfusion in the infusion group, respe<:tively. These results suggest that higher apfotinin levels maintained by continuou. infusion re· duee plasminogen activator aetivity in plasma and diminish signs of hyperfibrinolysis in the infusion group.

After reperfusion a more pronounced inerease of TAT eorn· plexes and a coneomitant decrease of fibrinogen levels in the infusion group indieate an inereased prothrombin aetivation. Higher aprotinin levels may shift the hernostatic balance to· ward coagulation. As on the other hand aetivities of pe, AT 111 , and CI · inh were nOl seriously decreased in either group no aceelerated eonsumption of eoagulation factor inhibitors eould be suspe<:ted.

". " " "

1 " I " .. " " "

.. .. ..... ........ .. """" ", , • .. - .. -- ......... , - •

. • • "'--" '''-' ' _., --, -- •

" . "

" • ,

I !

FIGUME 3. PA l ltnd CI ·inhibitor IIc tiYit ies du ring orthotopic liyer tTl1ßsphtntlition in 13 plltienUi of aprotinin bolus application and 10 plltienu of IIp rOlinin infUllion.

I

--::::- I 1·· .. ~w< ... 1 " '-"~::::::~::::::::~::::::=------" "

o 40 0 :

" '"

" " . , , . • • •

, _ . . ~- .-,., ........ ,_., -~, ....... _.-- • -- •

I

FIGUR": 4. Plasminogen ooncenwuion lind o" lIntiplalimin IIctiyity during orthotopic liver transplllntalion in 13 plltientl of aprotinin bolus IIpplicalion and 10 plltien13 of IIprotinin infusion.

Dur results showed higher blood product requiremenlS during OLT in the bolus group but not to a significant level. All 23 patienUJ sre still alive. and only one woman of the bolus group and one woman of the infusion group needed retransplantation because of seyere host·versus·graft resetioDs. Neither had he· patie artery thrombosis. Our preyious investigations of the perfu88te showed signifieantly higher TAT eomplexes and sig­niricantly lower aetivities of the protease inhibitors pe, AT 111 , and CI · inh when compared with the systemic cireulation 5 min before reperfusion. This indieates increased prothrombin acti · vslion and a coDsumption of inhibitors in the graft liver (I). Furthermore. parameters of actiYAted phElgC)l:yteli such EIS elal ' tase and cathepsin 8 were found to be signifieantly eleyated in the perfusate (17). In addition, it hall recently Ileen demon· strßted that aprotinin exerts a protf!(:tive effect on ischemic hepatocellular damage oceurring in the rat liver.transplant model (/8). We wondered whether considering these results higher systemie leyels of aprotinin in the reperfusion phose wilJ

• I f •

_. _ ... '" -0 '''

"'­-•

, .. '"

'" ..

FrGUElE 5. Thrombin antithrombin III complnel and fibrioopn concentration during orthotopic Iiver tran.plantation in 13 patienUi of Ilprotinin bolua application and 10 patient. of aprotinin infuJion.

I .,""ft· ... ·.11 -- I I '.p •• l .... ton I -......... .. .. · . . • ., +

" , , " 7 .. : :,. . ..... + : +,,' +~ ....&-' ..... .. • •. ~.+ .. , • . -" .. • .... .;.@' • + ....... : :

l ... . . ..

" ! : : ./ , ! " :: ,/ .. • ' . ," , '. " : )'.0' .... ".. .0 ........ 0. " " · .. 0··. ·· .. ·····,···· .. ····°: : . "

" " , , • • • • , _., ••• ...

-~. ... ... , _.-.... 0- . .• - •

FIGURE 6. Protein C and antithrombin III activities during ortho· topic liver traruplantation in 13 patienu of aprotinin bolus application and 10 patienta of aprotinin infusion.

change hemoatatic parameters of the gratt liver perfuaate. Indeed, t-PA antigen and activity were lower and PAI and a2-AP activitiea ware higher in tbe perfueate ofthe infusion group. In vitro atudies ahowed DO .ffeet ol different amount8 of aproti­nin in t·PA end PA! activity assays (not publiahed). Presuming that hilh 8y1temic aprotinin levels lU'e mucin, endothelial damage in the flushed graft liver, the lower t·PA activity and antipn levela in the perfueate cu be nplained. In addition, it hu to be diecuued whether t-PA liberation is reduced by inhibition or kallikrein, a potent stimulator or t·PA release (19). However, the diminished t-PA releue in tbe graft liver did not resu1t in alower t·PA level in the aystemic circulation after reperfuaion.

Parameten of thrombin formation and protease inhibiton did not differ in the perfueate and the syatemic circuJ.ation.

In summary, aprotinin application by infusion resulte in sienificantly diminished signs of hyperiibrinolyais end a leuer inCnlue in t-PA activity intraoperatively. On the other band,

! I

..

.. . ...

"

: o ' : .... 0·'

.' : ,.----

'"c~-------c,,.-,,------7.' • .-•• ----~------... •

--FlOURE 7. Aprotinin concentrationa during orthotopic liver tran.­

planution in 13 patient. of aprotinin bolu. application and 10 patienta of aprotinin infwlion and P (bolue/infwlion) are demon.trated.

TABLE 3. Comp.nltOn of different hemoetatic parameteR in the perfull8.te of 13 patienta in the bolue group u compared with 10

patientl of the infwlion group who underwent their first orthotopic liver tr8l\.lplantation

M.diaD (B) Ranp(B) P (BIl) median (I) ranp (I)

t-PA activity (IU) I' 0.7-23.6 0.101 , 0.7-17.4 t-PA antigen (nI/mI) 12.3 7.2-26.6 0.060

10.1 1.2-19.3 PAI activity (AU) 6.' 0-22 0.043

17.8 8.8-30.4 QI-AP (%) 63 .... 95 0.010 ., 68-102 Pluminogen (%) " 3-77 0.288

60 30-87 Fibrinocen (I/L) 1., 0-2.2 0.476

1.' 0-2.6 TAT (mI/mi) 93.2 40.5-324 0.288

73.5 32.5-1500 pe(%) 9.' 0-2< 0.063

I' 3-50 AT III (%) 31 3-56 0.476

37 9-55 CI·inh (%) .. &-9' 0.254

73 34- 109

aprotinin given in higher concentrationB may influence the hemoatatic balance toward hypercoagulation. A beneficiaJ ef­fect of aprotinin for the pnlvention of endothelial damage in the graft liver il auggelted.

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11, Fraedrich G, Weber C, Benwd C, Hettwer A, SchlOller V. lWduc­tiOD of blood tranlfuaion requirement in open heut IUrpry by adminietration of hia;h doeea of aprotinin-preliminuy rnulte. Thorac Cardiovuc SUfi 1989; 37: 89.

12. PllIanti G. Oe RoM V, Fortunato G, et al, Control of hemOltuia

during orthotopic liver traruplantation. Fibrinolylis 1988; 2 (Iuppi 3): 61.

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15. Müller-Elter! W, Oettl A, Truacheit E, Fritz H. Monitorin, of aprotinin pluma levell by an enzyme.linkld immunOMln:.nt uaay (ELlSA), Freeenius Z Anal Chem 1984; 317: '118.

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Received 16 February 1991. Accepted 30 April 1991.