Hepatic uptake of a modified low molecular weight heparin in rats

Hepatic uptake of a modified low molecular

weight heparin in rats.

G Stehle, … , W Maier-Borst, D L Heene

J Clin Invest.

1992;

90(5)

:2110-2116.

https://doi.org/10.1172/JCI116095

.

Fractionated and unfractionated heparins are widely used as antithrombotic agents.

Because of their heterogeneous composition, it is difficult to study the pharmacokinetics of

these drugs. We now report on a new method for labeling low molecular weight heparins

with 131I by binding tyramine to the anhydromannose end of the molecules. We examined

the pharmacokinetics of the compound by intravenous injection of 131I-tyramine-heparin

into Wistar rats. About 18% of the activity was found in the liver, whereas 33% was detected

in urine. Biological activity in terms of Factor Xa inhibition was measurable. Since evidence

from cell culture experiments implies that reticuloendothelial cell system receptors might be

involved in heparin metabolism, maleylated BSA, a substance known to block scavenger

receptors, was injected before the radiolabeled heparin compound. The liver uptake was

reduced from 17.4 to 4.8%. Injection of unfractionated heparin before tracer application

caused a considerable increase in urine excretion of the tracer substance. To our

knowledge, this is the first report that liver uptake of heparins is linked to scavenger receptor

mediated mechanisms in vivo. This interaction of heparins with scavenger receptors might

play an important role in the biology of the vessel wall.

Research Article

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Hepatic Uptake of

a

Modified Low Molecular Weight Heparin

in

Rats

GerdStehle,*Eckhard A.Friedrich, Hannsj6rg Sinn,t Andreas Wunder, Job Harenberg,*

Carl ErikDempfle,*WolfgangMaier-Borst,$ and DieterLudwig Heene*

*FirstDepartmentof Medicine, Faculty for Clinical Medicine Mannheim, UniversityofHeidelberg, W-6800 Mannheim 1, Germany;

andtDepartmentofRadiochemistry and Radiopharmacology, German CancerResearchCenter, W-6900Heidelberg, Germany

Abstract

Fractionated and unfractionated heparins arewidely used as

antithromboticagents. Because of theirheterogeneous

compo-sition, it is difficult to study the pharmacokinetics of these

drugs.We nowreporton anewmethod forlabeling low molecu-larweightheparins with

"31I

by binding tyraminetothe

anhy-dromannose endofthe molecules. We examined the pharmaco-kinetics ofthecompound byintravenous

injection

of

'31I-tyra-mine-heparin into Wistar rats. About 18% of theactivitywas

foundin theliver,whereas 33%wasdetected in urine.

Biologi-cal activityin terms of Factor Xa inhibition wasmeasurable.

Since evidence fromcell cultureexperiments impliesthat

reti-culoendothelialcell system receptorsmightbeinvolved in

hepa-rinmetabolism, maleylated BSA,asubstance knowntoblock scavengerreceptors, wasinjectedbefore the radiolabeled

hepa-rincompound.The liveruptakewasreduced from 17.4to4.8%. Injection of unfractionated heparin before tracer

application

caused aconsiderable increasein urine excretion of thetracer

substance. To ourknowledge,this is the first report that liver

uptake of

heparins

is linked to scavenger receptor mediated

mechanismsinvivo. This interaction ofheparinswith

scaven-ger receptorsmight playanimportantrole in thebiologyof the vesselwall.(J.Clin.Invest. 1992.90:2110-2116.) Keywords:

heparin-pharmacokinetics

* scavenger-receptor *

tyramine-hep-arin*radioiodination

Introduction

Heparinsarewidelyused asantithromboticagents. However, becauseoftheir heterogeneous

composition

andtheirchemical properties, pharmacokinetic studiesaredifficult.Various

stud-ies have tried to clarify metabolic pathways of heparin and othersulfated glucosaminoglycans,using35Sas a tracing agent

Addresscorrespondence to Dr. Gerd Stehle, First Department of Medi-cine, Faculty for Clinical Medicine Mannheim, University of

Heidel-berg,Theodor KutzerUfer,W-6800Mannheim 1, Germany. Receivedfor publication 18 May 1992 and in revisedform 20 Au-gust1992.

( 1-3). Apartfromaroleofthekidneysinheparin pharmacoki-netics, evidencewasfound foran uptake by the reticuloendo-thelial systemof the liver, although resultsgathered with this technique haveto beinterpreted cautiouslybecauseoflow

spe-cific activity andrapid desulfationof the tracer substance.The

useofradioactive iodine coupledtoheparin leadto an

improve-mentintracerquality (4). Considerableamountsof thistracer

substance were detected in the liver of rabbits. To assessthe

roleoftheendothelialcells and thecellsofthe

reticuloendothe-lial system (RES)' in vitro, cell culture experiments were

carried out, andthey revealedanuptake and degradation of heparin by endothelialcells(5, 6). Bleibergetal. (7) described uptakeandinternalization of heparinsby mouse macrophages,

mediated by an unknown receptor. Recently, Falcone

de-scribed that heparin binding to the macrophage cell line

RAW264.7cells wasmediatedby scavenger receptors (8). We nowpresentinvivo evidencethat liver uptake of a modified lowmolecularweight derivative of heparin is mediated by a scavengerreceptor-dependent mechanism.

Methods

Preparation ofthe tracersubstance. Unfractionated porcinemucosa heparin(HeparinNa; Medac GmbH, Hamburg,Germany)was hydro-lyzed usingnitrous acid (9)and atyramineresidue coupledto the anhydromannose end of thepolysaccharidesby reductive amination

(10). This tyramine-heparin preparation was further purified by HPLC ( 11). Carbon and proton nuclearmagneticresonancespectra of thetracercompound had been analyzed by Prof.Casu,(Istituto Scien-tifico diChimicaeBiochimica "G. Ronzoni," Milan). Itwasshown that85% of the substancewasN-sulfated,and therest wasN-acetylated

heparin. N-desulfated heparinwasnotdetected, andmightbe present in<2%. Thetyraminemoleculeswereboundtothe substanceasthe

tyramine peaks shifted, comparedwith the control. Proton spectra

re-vealed that one molecule oftyramine was present for every 20 disaccha-ride units.The meanmolecularmassoftyramine-heparinwas5,800D, witha90% range between3,000 and 8,000D.Assumingameanmol

mass - 600Dforadisaccharideunit,itcanbeestimatedthatupto

50% ofthemolecules presentweretyramine-derivatized.The

anti-Fac-torXaactivityofthesamplewas 110 U/mgusingtheFirst

Interna-tional Standard forLowMolecularWeight Heparinsasreference. The material contained 30% ofhigh-affinitymaterial for antithrombin

1.Abbreviations usedinthispaper:mal-BSA, maleylated BSA; RES,

reticuloendothelial system; ROI, region of interest; SHPP, 3-(4-hy-droxyphenyl) propionicacidN-hydroxysuccinimideester.

2110 Stehleetal.

J.Clin. Invest.

© The American Society for Clinical Investigation, Inc.

0021-9738/92/11/2110/07 $2.00

III.Radioactivelabelingofheparinviathe attached tyramineresidue offers advantagescomparedwiththe conventional labeling procedures

using 3-(4-hydroxyphenyl) propionicacidN-hydroxysuccinimide

es-ter (SHPP), since tyramine residues are firmly linked by H2C-N bridges at a defined binding siteatthe anhydromannose end of about

50% of the heparinmolecules, whereas with the SHPPprocedure,the

peptide bonds ofthe substituents branch off from various undefined

amino groups presenton - 8% ofthe molecules. The H2C-Nbridges

are not cleaved by endogenousproteases,whilethepeptidebondsof

the SHPP technique are (4, 10, 12, 13). Forradioiodination of the

modified heparin, we used atechnique employing

N-bromosuccini-mide as an oxidizingagent. The detailedprocedures for

radioiodina-tion have beendescribed by Sinnetal.(13 ). An additional advantage

of the new technique isthe lower oxidation potentialcomparedwith the conventionallyusedchloramine T procedure (14), which reduces

the risk of unwanted oxidation ofthecompounds ( 13 ). Tracer

chroma-tography afterthe labelingprocedure showedonepeakcontaining

95-98% of the radioactivityboundtotyramine-heparin, 2-5%were

identi-fied as free iodine,andasiodineboundto trace amountsoftyramine.

The introductionof iodine didnotalterthe anticoagulant propertiesof

the substance.Approximately half of the available tyramineresidues

was radiolabeled.MaleylatedBSAserum(mal-BSA)waspreparedby

maleylatingBSAwith maleic acid anhydride.About 50outof 59lysine residuesof the albuminweremaleylated(15).

Animals. 15 healthyfemale Wistarratsweighing250-300g were

used. Theywere obtained from Zentrale Tierversuchsanstalt

(Han-nover, Germany).Theratswerekept understandardliving conditions,

fed a standarddiet, and hadfreeaccessto water.The animal

experi-ments wereapprovedby the German FederalGovernment.

Gamma camera and data evaluation.Allanimalswereplacedona

multihole collimator (420 keV)ofa 10-in. gammacamera. Forthe

on-line evaluationofthe data,acomputersystem(Gaede Medworker,

Gaede GmbH, Freiburg, Germany) was especially adapted to the

gamma camera.To study the distributionofthetracersubstancein the

animals, kineticimageswereregisteredat2-min intervalsfor 60min. After 180min,afinal5-min imagewasrecorded. Theregionsof

inter-est(ROI)weremarked,andthecontentof radioactivity inthethyroid

gland, heart, liver, kidneys, and urinary bladderwasevaluated. The

percentageofactivitypresentin each region of interestwascalculated

from the countsofthatareainrelationtothe whole body count. A

color scale fromred followed by yellow,green,and blue,with decreas-ing amountofradioactivity, wasusedtovisualize the data.

Animal experiments. Throughout theexperiments, the ratswere anaesthetized byamixture ofhalothane, N20, and02(1.2%/60%/

38%).The animalsreceived 3.7MBq(100MCi)

of1'I-tyramine-hepa-rin containing100Mg(340-400Mgoftyramine-heparin/kgbodywt)of

the substance. Thetracerwasadministered byanintravenousinjection into a lateral tail vein. 5, 10, 20, 40, 60, and 180 min aftertracer

application, bloodsamplesweredrawn by cuttingthe tailtip,andafter discarding the firstdropofblood, 90 Mlofwhole bloodwascollected

and mixed with 10

_Al

of sodium citrate inasiliconizedglasscapillary.

Anti-FactorXa activitywas measured byacoagulometric method

(Heparimat0 Test;BioMerieux,Paris,France)usingaKC4

coagulo-meter (KC4; Amelung Lemgo, Germany), as described previously (16). The normalrangeof thistestin the untreatedratwasdetermined

to be between 12and 15s.Anadditional 10Mlofbloodwascollected for radioactivitymeasurement. Areferencecurve waspreparedusing serial dilutionsofthetracersubstance.Theequationbloodvol=0.06

body wt+0.77 (17)wasusedtoestimatethe blood volumeof the

animal. From thesedata,thepercentage oftheappliedradioactivity

present in thebloodatdifferenttimeswascalculated.The blood lossof

the animalswas< 2 mlor<10%of the respective total blood volume.

A catheter wasplacedin theurinary bladderforanalysisofthetracer

composition inurine.Theamountof urineexcretedwascontinuously monitored.Theradioactivitywasdeterminedinall samplesat 15-min

intervals for 60 min.Fractionsofthese sampleswereassayedbythin

layerchromatography_{to assess tracer stability. Afterthe final}

measure-mentand bloodsampling (180

min),

theanimalswerekilled and their organswereremoved.Aliquotsofthe organsweretaken andafter deter-mination of theweight,theradioactivityof the samples was measured inagamma counter. Thespecific activity per gram of tissue was

calcu-lated, and multiplied with the weight of therespective organs. The resultswereexpressedaspercent ofradioactivity appliedtotheanimal

initially.Thefollowingorganswereexamined: liver, kidneys, spleen, thymus, lungs, stomach, intestines, colon, muscle, and the thyroid

gland.

Three groups were formed for the study, and all animals were ran-domly distributed. Group 1 (mal-BSA) received 2 mg of mal-BSA 2

min

before tracer application to block the scavenger receptor of the RES cells.Group 2

(heparin

competition) received 2mg of unfraction-ated

heparin

tocompete with the tracer substance for binding sites. Group 3 (control) received only the tracer substance.

Results

Five female Wistarratsreceived 100

_,g

of

'1I-tyramine-hepa-rin (3.7 MBq = ₁₀₀

/Ci,

11 anti-Xa units) by_{anintravenous} injection. Anotherfive animals received 2 mg ofmal-BSAto

blockscavenger receptorsofcells 2

min

before thetracer

sub-stance. To

provide further

information onthebehavior of the

tracersubstance, fiverats werepreinjected with2mgof unfrac-tionated heparintocompetefor binding sites.Blood samplesof

allanimals wereassayed foranti-Factor Xa activity

(Hepari-mat' test system;normal range 12-15 sforuntreated rats). A

considerable prolongation of the clotting timewas seenfor the mal-BSAgroupand the control confirming the anticoagulant propertiesofthetracersubstancein vivo (Fig.1 ).Theclotting time of thecontrolgroupafter5

min

wasprolongedto a mean

of53s,whereas the mal-BSApretreatedanimalshada consider-ably higher value (80s). Thesedifferences prevailed

through-outthe studyperiodof 180

min.

After3 h, clotting timeswere

still prolonged with 24.5 sfor thecontrolgroupand 32.7sfor the mal-BSA group, respectively. The clotting times for the heparin competitiongroup wereabove the measuring rangeof

120 *100 * 80 o ₆₀ C 2 40-U 20, 0I

.1

r U control

TJ

T

mal-BSA

T

T

I

5 1 0 20 40 60 1 80 time after injection

(min)

Figure 1. Anti-FactorXaactivity (inseconds)afterinjection of 100

Mg

3I-tyramine-heparin

(3.7 MBq = _{100MCi).The controlgroup}

received'3I-tyramine-heparin alone, whereasthe mal-BSAgroup

re-ceived the tracer2 min aftertheapplication of2mgof mal-BSA.

The mean valuesandSDofthe control, and themal-BSAgroupare

shown (n= 5).

---F

r

_T

o control

0

40

-a10

X

20

C

5 1 0 20 40 6 0 18 0 time after

injection

(min)

Figure2.Percentageofradioactivitypresentin bloodafterthe

injec-tion of100 gg

"3'I-tyramine-heparin

(3.7 MBq= 100y4i).The

per-centage is calculatedonbasis of theappliedamountofradioactivity.

Thecontrol groupreceived131_{1-tyramine-heparinalone,whereasthe}

mal-BSAgroupreceived thetracer 2 minafter theapplicationof2 mg

of mal-BSA. Thehepanincompetitiongroupwaspreinjected2 mg

ofunfractionatedheparinfollowedbytracerapplication2 minlater.

Themeanvaluesand SDareshown(n=₅₎.

the

coagulation

test

_(>

180

s)

throughout

the

experiment

and

arethereforenotshown.

An

analysis

ofthepercentage ofthetracer presentin the

bloodofthe controlanimalsrevealedaninitial

peak

of 53.4%

of the

applied

radioactivity

after5

_min,

afastdeclineto25.4%

after20

_min,

to 7.5% after60

min,

and to 1%after 180min

(Fig. 2).

The data forthe mal-BSA treatedanimals diffiered

from these

findings.

After5

min,

46%ofthetracerwas

measur-able in the blood. After20

_min,

30% of the

activity

wasstill

present in the

circulation,

andafter 60

_min,

the values'were

twiceas

high

asin the control group.Five timesmore

activity

waspresentin the bloodoftheanimals withblocked scavenger

TableI. Specific Activity oftheTracer(xI,000cpm/gtissue)in Various Organs180 min

after

Application

of

100,ug

of

131I-Tyramine-Heparin (3.7MBq = 100,uCi),

Showing

the

Avidity

Per Gram TissuetowardstheTracer

Mal-BSA group Heparincomp. Control

Organ Mean SD Mean SD Mean SD

Liver 120 8 71 16 419 58 Kidney 912 116 810 180 779 167

Spleen 489 25 35 4 252 26 Intestine 344 52 78 8 297 86

Colon 215 49 103 25 190 18 Stomach 170 14 75 20 114 32

Lung 95 20 133 42 82 23

Thymus 78 14 33 4 49 2

Muscle 16 5 18 2 16 4

Before thetracerapplication,theanimals of the mal-BSA group

re-ceivedaninjection of2 mgmal-BSA.Theanimalsin theheparin competitiongroupreceived2 mgunfractionatedheparinbeforetracer

applicationinstead of mal-BSA (n=_{5animals,mean values, andSD).}

receptors

(5.5%

vs

1%)

after 180min.In the

_heparin

competi-tiongroup, 70% of thetracersubstancewasremoved from

circulationwithin 5min.After 3h,3.2% oftheinitially

applied

activity

was

measured, giving

a value betweenthe mal-BSA

group and the control. The SDwas < 10% of the

respective

meanvalues, supportingthereliabilityof the data and the dif-ferences recorded.

After180 mintheanimalswerekilled and theirorganswere

removed. Table I displaysthe specific activity of the organs;

i.e.,theamountofcounts per gramoftissue. The data show the

capacityoftissuestoaccumulate thetracersubstance,butdo notreflect theimportanceof the wholeorgansinmetabolizing

thetracer. Thehighest specific activitywasfound in the

kid-neyswithmeanvalues of 779,000 cpm/g in the controlgroup

and 810,000 cpm/g in the heparin competition group vs

912,000cpm/g inthe mal-BSAgroup.Rankingsecondin the controlgroupwastheliver with419,000 cpm/g.Because ofthe

blockingof thescavenger receptors,this valuewasreducedto

120,000 cpm/g in the mal-BSAgroup.In the competition

ex-perimentwithunfractionated heparin,theuptake of the radio-active material by the liver was further diminished to only 71,000 cpm/g, implying additional non-mal-BSA blockable binding. Thespleen, rankingfourth in the controlgroupwith

252,000 cpm/g, rankedsecondafterblockingwith mal-BSA with 489,000 cpm/g, suggesting alternative binding sites that

are not blocked by mal-BSA, but by unfractionated heparin

(35,000 cpm/g). The tissues of the gastrointestinal tract

showed nosignificant differences after mal-BSAapplication,

but preinjected unfractionated heparin significantly lowered thetracerbinding of stomach, small intestines, and colon. The contentofradioactivityof the other organs seemedtobenot

decisively altered. The muscle samples taken showed the lowest specific activities measured.

To assessthe share of the organs on the metabolism of the

substance,itwasnecessarytocheck the totalamountof activ-ity per organ (Fig. 3, Table II). In Fig. 3, the percentage of

radioactivityatthetime of organ removal and the percentage of activity in blood and excreted into urine are shown to

indi-Table I. Amount ofRadioactivity in Various Organs 180min

afterApplication of100,ugof3I-Tyramine-Heparin

(3.7MBq= 100uCi)

Mal-BSA group Hepann comp. Control

Organ Mean SD Mean SD Mean SD

Liver 4.77 0.23 2.31 0.21 17.36 1.09

Kidney 8.51 0.69 6.12 0.86 6.82 0.50

Stomach 1.05 0.08 0.43 0.11 0.73 0.22 Spleen 0.96 0.32 0.10 0.03 0.44 0.03 Lung 0.59 0.13 0.84 0.27 0.43 0.13 Thyroid 0.15 0.07 0.11 0.02 0.24 0.14

Thymus 0.05 0.01 0.04 0.01 0.04 0.01

Percentages calculatedfromtheoriginally administeredamount. The

controlgroupreceived1311-tyramine-heparin.The mal-BSAgroup

received2 mgmal-BSA2 minbeforetracerapplication. The animals in the heparin competitiongroupreceived2 mgunfractionated hepa-rinbeforetracerapplication instead of mal-BSA(n=5 rats, mean

thekidneysisconsiderablyenhanced(Fig. 5B).From these

scintigraphic data,itwaspossibletocalculate that within 3h,

55±5.6% of the initially administered dosagewasexcreted in urine among the animals of theheparin competition group, whereasurinary excretionin the mal-BSA group and the

con-trol groupwas - 20% lower(Fig. 3).

To receive additional information on tracer stability in vivo, urine was collected from animals of the control group by

acatheter. About 25%ofthe amountofradioactivity injected intravenously was recovered within 30 min. An additional

2-6% wasfound duringthe next30 min. Tracer chromatogra-phy revealed that themajoramountofthe tracer compound waschemically intact in urineandconsisted of93-98% 131

-ty-liver kidneys blood urine Figure 3. Percentage of radioactivity found in theliver, kidneys, blood, and urine 3 h after the injection of 100_Ag '31I-tyramine-hepa-rin(3.7 MBq= 100 MCi). Thepercentageis calculatedonbasis of

theappliedamountof radioactivity. The controlgroupreceived

'311-tyramine-heparin alone, whereas the mal-BSAgroupreceived the tracer2min after the application of 2mgof mal-BSA.The heparin competitiongroup waspreinjected 2mgofunfractionated heparin followedbytracerapplication 2 min later. Themeanvalues and SD

areshown(n=5).

catethetracerbalance ofthe examinedorgansystems. In the

control animalsthe liver accounted for 17.4%ofthe radioactiv-ity, ranking well above the kidneys withatotal of 6.8% of the activity. Aftertreatment with mal-BSA only 4.8% of the ap-plied activitywaspresentinthe liver after 180min. Thetracer

uptakeof the liverwasdiminishedto2.3% intheheparin com-petitiongroup. These data disclose theimportanceof thecells

of the liver RES in the uptake of the heparin tracer. The

amountofradioactivity foundinthekidneyswas8.5%, illus-tratingtheincreasingshare ofthekidneysinheparin pharmaco-kinetics after blocking the hepatic uptake. In spite of the high specific activity,thespleencontributedto<0.5%tothe radio-activitymeasured in the controlgroup vs 1% inthe mal-BSA

group. Like in other organsthetracer substance wasalmost completely displacedfrom thespleen (0. %) byunfractionated heparin. Only 0. 1,0.15, and 0.24% ofthe applied activitywas

foundinthethyroid gland, denotingthe in vivostabilityofthe

tracersubstance.

Togain furtherknowledge on the fate ofthetracer

sub-stance, the different kinetics of accumulationordisappearance

in the organs were monitored by whole body scintigraphy.

ROlsweredefined for the liver,thekidneys,and the urinary

bladder. The percentages statedinthescintigraphic figuresare

not fully comparable numerically to the percentages, calcu-lated from the removedorgans on abasis ofabsolute values,

since thescintigraphicdata reflectareasandnotwholeorgans.

As expected, similar effects were visible on a relative basis.

With thedecreasingrole of the liverROIaftermal-BSA

block-ingorcompetition by unfractionated heparin, the increasing

role of thekidneysand of the urine excretion in the

pharmaco-kinetics ofthe substancewasobvious(Figs. 3 and4,AandB).

The liverregion is clearlyvisibleonthescintigramsrecorded after180minfor thecontrolgroup(Fig.5A).Inthe animals of

the mal-BSAgroup,theradioactivity registered overthe liver

region isdepressed, whereas the radioactivity measured over

A

₂₀

0 40 8 0 120 160 200 time after injection (min)

B _{20 T}

0 4 0 8 0 120 160 200

time after injection (min)

Figure4.The kineticsof influx and efflux of 100 Mg

31I-tyramine-heparintracer(3.7 MBq = 100 MCi)overtheROIsof

the liver(A)and thekidneys (B),asregistrated byscintigraphy.The

controlgroupreceived'3'I-tyramine-heparinalone,whereas the

mal-BSAgroupreceived thetracer 2minafter theapplicationof 2mgof

mal-BSA. Theheparin competitiongroupwaspreinjected2_mgof

unfractionatedheparinfollowedbytracerapplication2minlater. The

meanvalues and SDareshown(n=_5).

*0 0

a.

._

0

0

0

0

I-- I--I--I--I--I--I--I--I--I--I--I--I--I--I--I--I--I--I--I--I--I--I--

---lo

_---I

I

A

B

Kidneys

Figure 5.Scintigraphicimagesofa ratfrom the control group (A) and ofa ratfrom the mal-BSA group(B)180 minafterapplicationof

100l

g

31I-tyramine-heparin

tracer(3.7 MBq= 100

MCi).

The control groupreceived

'31I-tyramine-heparin

alone.The mal-BSA group received the

tracer2 minaftertheapplicationof 2 mg of mal-BSA.

ramine-heparin.Coagulationtests were

performed

withan

ali-quotof pooled

urine,

containing

'31I-tyramine-heparin,

show-ing prolonged

clotting

times.

Only

trace amountsof free

131I

andof

131I

coupled with

tyramine

weredetected inurine.

Discussion

Radiopharmacokinetic studies withlow molecularweight hepa-rinsshowed that aconsiderable amountofthematerial is re-movedfromthecirculationwithin 15 min and can befoundin urine. The hepatic uptake mechanism was blocked by mal-BSA,leadingto anincreasedbloodlevel ofthe tracer, whereas the rate ofurinary excretion increased only slightly. In the

competition experiment

with unfractionated

heparin,

the

he-patic

uptakewasfoundtobereduced

(liver 2.3%),

whereas the

amountoftracersubstance inurineincreased

by

20%

com-pared with the control and mal-BSA groups

(Fig. 3).

Thesedatasupportthe

hypothesis

thatthehepatic uptake

of

heparin depends

on a

receptor-mediated

mechanism.

Gold-stein et al. (18) found a receptor for acetylated low-density

lipoproteins on cultured murine macrophages, which was

named "scavenger receptor."Subsequent researchonthis re-ceptorresultedindescribingamajorroleofthisreceptor and themacrophagesinthe formation ofthefoamcell, thelatter

beinga majorconstituentoftheearly atherosclerotic lesions (19). Type I and type II bovinescavenger receptors have

re-centlybeencharacterizedintheir tertiarystructures(20,21).

Atleast threedifferent kinds ofscavenger receptors have been

postulated,andextensive

investigations

have beencarriedout

byourgroupthroughoutrecentyears(15, 22-24). Mal-BSA

was foundto be areliableagent to detect and to characterize thedifferentscavenger receptorsin vivoand invitro (15,25).

Hiebert (26)describedtheuptake ofheparin byliver sinu-soidal cells in rabbits. Bleiberg et al. (7) postulated heparin receptors on mouse macrophages in culture. Evidence fora scavengermediated heparin uptakeincultured macrophages

waspublished byFalcone(8). However, likeall results from

cell culture experiments, these findings cannot easily be

adoptedforexplanationof in vivofindings.Our data now sug-gest a role of scavenger receptors in heparin metabolism in vivo. Bypreinjecting2 mgofmal-BSA, it was possible to block theuptake of theheparintracerbyscavengerreceptor-bearing cellsalmostcompletelyfor aperiodof 3 h. The liveruptakeof radioactivity was reduced from an average of 17.4 to 4.8% of theactivity, paralleled by an increase of blood levels. Injection

of unfractionated heparinas acompetingagentfortracer bind-ingpreventedaccumulation of thetracer substanceintheliver

with a mere 2.3% of activity left. Tracer binding was also re-ducedbycompetition of unfractionatedheparin in thetissues

of thegastrointestinaltractand in thespleen (TableI). Thus, a scavengerreceptor-mediateduptakeofheparin by

livermacrophages andsinusoidal endothelialcellsmightplay animportantrole fortheliver metabolismofboth the tracer compoundoflow molecular weightandofunfractionated

hepa-rin. Additional non-scavenger receptor binding sites can be

occupied by nonlabeled unfractionated heparin causing uri-nary excretion of thetracer substance to increase. It can be

suspectedthattheseunsaturatedbinding sitesarelocalizedon theendothelial cellsofthe vascularsystem that are under physi-ologic conditions devoidof scavenger receptors.Thiswould be

supported bydatapublishedfromHiebertet al.(5) andBarzu etal.(6)ontheroleofendothelial cells in heparin metabolism. Thus,ourpharmacokineticdata suggest thatin heparin

me-tabolism there are two saturable uptake mechanisms. One mechanism is mediatedby scavengerreceptor-mediated

hepa-rinuptake by the macrophage cell system, whereas the otheris formed by the endothelialcell system.Saturation ofboth bind-ingsitesbyadministration ofunfractionatedheparinbeforethe

heparintracercompound results inanincrease intheexcretion ofthesubstance by thekidneysto urine.These results arein

agreementwith the resultspublished byBoneu et al.(27, 28),

whodescribedthese twodistinctmechanisms ofelimination in rabbits by studying the disappearance of '25I-SHPP-heparin fromblood.

The interaction withscavenger receptors of macrophages might be

responsible

for several

biological properties

of hepa-rin.Stimulation ofthemacrophagetumorcell line RAW264.7 cells

by

adding

heparin

hadbeen foundtoincreasethe

secre-tion of urokinase type

plasminogen

activator (8). Studies carriedout

by

Markwardtand

Klocking (29)

andVinazzeret

al.(30) in

perfused

organ systemsshowedanincreasein fibri-nolytic activity after

heparin

stimulation. Studies

using

arabbit

jugular

vein clot

lysis

model

suggested

that low molecular

weight heparins exhibiteddose-dependent in vivo

fibrinolytic

activity comparable

to

clinically

effective dosesof urokinase

(31 ).Arecentclinical trialon treatmentof

deep

venous throm-bosis with either unfractionated

heparin intravenously

orlow

molecular weight heparin

subcutaneously

revealed consider-able recanalizationratesof the occluded vesselsas

judged by

phlebography (32),also

supporting

a

profibrinolytic potential

of

heparin preparations.

An effect of

heparin

onthe

metabo-lism oflowdensitylipoprotein metabolism hasrecentlybeen

presented. A high affinity

heparin

subfraction (8%) forlow

density

lipoprotein

was found to cause an accumulation of

cholesterylester inmouse

macrophages (33).

Soluable

heparin

proteoglycansreleasedfrommast cells

might

inducethe

uptake

of low

density lipoproteins by macrophages

via a scavenger

receptor-mediated

phagocytosis(

34).The

regulation

ofmacro-phagelipoprotein lipase secretion

might

also beinfluenced by

scavenger receptor-mediated uptake of glycosaminoglycans

(35).Thefindingscouldprovide an additional explanation for theproperties of heparins to stimulate lipoprotein lipase activ-ity invivo, apartfromthehypothesized competitionofheparin and lipoprotein lipase for binding sites on endothelial cells(36).

Theinteraction of heparins with scavenger receptors thus appear toplay a complex and important role in the biology of the vesselwallwith implications for physiologicaland

patholog-ical mechanisms, in whichcellsoftheliverRES areinvolved.

Acknowledgments

For technical assistance, the authors thank H. H. Schrenk, R.

Kuhnlein,andA.Tietz. For the nuclearmagneticresonanceanalysisof thetyramine-heparincompound,weexpressourgratitudetoProf.B.

Casu, G.

Toni,

and M.Guerrini from Istituto Scientifico di Chimicae

Biochimica "G. Ronzoni," Milan.

This workwassupported by grant Ste647/1-1 from Deutsche

For-schungsgemeinschaft.

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