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Thrombomodulin is present in human plasma

and urine.

H Ishii, P W Majerus

J Clin Invest.

1985;

76(6)

:2178-2181.

https://doi.org/10.1172/JCI112225

.

Thrombomodulin is an endothelial cell membrane protein that is a cofactor required for the

rapid activation of plasma protein C. We now report that plasma and urine of normal

subjects contains a modified form of thrombomodulin that is soluble. The levels measured

by radioimmunoassay were 292 +/- 60 ng thrombomodulin/ml plasma and 102 +/- 38 ng

thrombomodulin/ml urine. Thrombomodulin was isolated from both plasma and urine by

immunoaffinity chromatography using a polyclonal anti-human thrombomodulin IgG column.

The apparent molecular weight of soluble thrombomodulin was estimated by immunoblot

analysis using 125I-monoclonal anti-thrombomodulin IgG. When run without

2-mercaptoethanol, soluble thrombomodulin appeared as two polypeptides, Mr = 63,000 and

54,000, while samples run with 2-mercaptoethanol migrated mainly at Mr = 85,000. These

results imply that the soluble form of thrombomodulin is smaller than the cellular form,

presumably because of a lack of the membrane-binding domain. Soluble thrombomodulin is

similar to cellular thrombomodulin in its intrinsic protein C-activating cofactor activity as

measured by antibody neutralization. The apparent Km for protein C was the same for

cellular and soluble thrombomodulin, while the soluble form requires a higher concentration

of thrombin (three- to fivefold) for one-half maximal activity than the cellular form.

Thrombomodulin functional activity cannot be directly measured in plasma because of

some inhibitory substance(s). The physiological significance of circulating and urinary

thrombomodulin is presently […]

Research Article

Find the latest version:

(2)

Thrombomodulin

Is Present in Human Plasma and Urine

Hidemi Ishii and Philip W.Majerus

DivisionofHematology-Oncology, Departments ofInternal Medicine and BiologicalChemistry,

Washington UniversitySchool ofMedicine, St. Louis, Missouri 63110

Abstract

Thrombomodulin

isan endothelial cell membrane

protein

that isacofactor

required

forthe

rapid

activation of

plasma

protein

C.Wenowreportthat

plasma

andurine of normal

subjects

con-tainsa modified form ofthrombomodulin that issoluble. The

levels measured

by

radioimmunoassay

were292±60ng

throm-bomodulin/ml

plasma

and102±38ng

thrombomodulin/ml

urine.

Thrombomodulin was isolated from both

plasma

and urine by

immunoaffinity chromatography

using

a

polyclonal

anti-human

thrombomodulin

IgG

column. The apparent molecular

weight

of

soluble thrombomodulinwasestimated

by

immunoblot analysis

using

1251-monoclonal

anti-thrombomodulin IgG. When run

without

2-mercaptoethanol,

soluble thrombomodulin appeared

as two

polypeptides,

Mr =

63,000

and

54,000,

while samples

runwith

2-mercaptoethanol migrated mainly

at

Mr

= 85,000.

These results

imply

that the soluble form of

thrombomodulin

is smaller than thecellular

form, presumably

because ofalack of

the

membrane-binding

domain. Solublethrombomoduiinis

sim-ilar to cellularthrombomodulin in its intrinsic

protein

C-acti-vating

cofactor

activity

asmeasured

by

antibody

neutralization. The apparent Kmfor

protein

Cwas the same for cellular and

soluble

thrombomodulin,

while the soluble form

requires

ahigher

concentration of thrombin

(three-

to

fivefold)

for one-half

max-imal

activity

than the cellular form. Thrombomodulinfunctional

activity

cannot be

directly

measuredin

plasma

because ofsome

inhibitory

substance(s).

The

physiological significance

of

cir-culating

and

urinary

thrombomodulinis

presently

obscure.

Introduction

Protein C isavitamin

K-dependent glycoprotein

of62,000mol

wt,whichcirculatesin humanplasmaataconcentration of -4 ,ug/ml(1).Protein C is converted to a protease withanticoagulant

function

by

thrombin. Theconversion requiresan endothelial

cell membrane

protein cofactor, thrombomodulin,

which was

firstisolated from rabbit

lung

(2).Wehaveisolatedthe human form ofthrombomodulin from placenta (3) and lung(4) and have

prepared

both

polyclonal

(3)andmonoclonal (5)antibodies

directed

against

the

protein.

We have shown that thrombo-modulin is

widely

distributed on the endothelium of human

arteries,

veins, capillaries,

and lymphatics in allorgansexcept

AddresscorrespondencetoDr.Majerus, Division of

Hematology-On-cology, Washington UniversitySchoolofMedicine,660South Euclid, St.Louis,MO63110.

Receivedfor publication22July1985.

brain(6,

7).

Thegreatestcontentof thrombomodulin is inlung

andplacentaasmeasuredby radioimmunoassay of extracts of these tissues(7).Wenowreportthat humanplasmaand urine contain a soluble form of thrombomodulin that has been de-tectedbyboth

radioimmunoassay

andbyafunctionalassay.

Methods

Exceptwhereindicated,allreagentswerefromSigmaChemicalCo.,St. Louis,MO.ProteinC(8),thrombin(9),andantithrombinIII(10)were

isolatedfrom humanplasma.Humanthrombomodulinwasisolated from

placentaaspreviously reported (3). Polyclonal anti-human thrombo-modulinantibody (3)andmousemonoclonalanti-thrombomodulin an-tibody (5)wereprepared using placentalthrombomodulinastheantigen (3). The iodination ofthrombomodulin wasperformedaspreviously

described (7).TheiodinationofproteinAwasperformed accordingto Mellmanand Unkeless(11).Free1251wasremovedbychromatography

on a I-mlcolumn ofSephadexG-25. Protein concentrationwasmeasured usingthe Bio-Rad assay (Bio-Rad Laboratories, Richmond, CA)with humanIgGas astandard.

Immunoaffinity chromatography.Anti-humanthrombomodulin

IgG

agarosewaspreparedbycoupling 10mgof rabbitanti-human throm-bomodulin IgG (10 mg/ml)in0.1Mmorpholino-ethane-sulfonicacid,

pH 6.5,to 1 ml ofAffi-gel 10(Bio-Rad Laboratories) overnightat4VC.

Residualactive esterswereblocked bythe addition of0.1 ml of 1 M ethanolamine, pH 8.0, for 6 h. Thegelwaswashed with 20 vol of 20 mMTris, pH 7.4, containing0.15 M NaCl and 0.2% Nonidet-40,and 10 vol of 20 mMTris,pH 7.4, containing2 MNaSCN,0.15 MNaCl,

and0.2%Nonidet-40.Thegelwasfinallyequilibratedwith 20mMTris,

pH 7.4, containing0.15 MNaCl,0.2%Nonidet-40,and 0.02% sodium azide.60-ml samplesof urine andplasmaweredialyzed against20 mM Tris, pH 7.4, containing 10 mMbenzamidine,0.15 MNaCl,and0.2% Nonidet-40.The dialyzed urine andplasmawereappliedtoseparate 0.5-ml columns of theanti-thrombomodulin IgGat3ml/hat4°C.After washingwith 15 ml of theequilibrating buffer, thrombomodulinwas

eluted with 20 mMTris, pH 7.4, containing2.0M NaSCN and 0.2% Nonidet-40. Fractions (0.5 ml)werecollected, dialyzed against 100vol of 0. 15 MNaCl,0.2%Nonidet-40,and20 mMTris, pH7.4,andassayed

forproteinC-activatingcofactoractivityand forthrombomodulinby

radioimmunoassay.

Radioimmunoassay.Theradioimmunoassayofthrombomodulinwas

performedaspreviously reported(7).Reaction mixturescontainedthe following:testsample,0.25,gofanti-thrombomodulin IgG,and0.5 rd of nonimmune rabbit serum in 20 mMTris, pH 7.4, containing0.15 M NaCIand0.2% Nonidet-40,in0.2 ml.After incubationat4°Cin 1.5 mlEppendorftubes(Brinkman Instruments Co., Westbury, NY) for2 h,2ngof'25I-thrombomoduliii (16,000 cpm,8000cpm/ng)was added and theincubationwascontinued for anadditional 16 h. A 10%

sus-pension of Staphylococcus aureus (Pansorbin,Calbiochemical-Behring

Corp.,SanDiego, CA)in0.1 ml,wasthen added to each reactionmixture. Incubationwascontinuedfor anadditional 1 h at roomtemperature. TheS. aureusorganismswerecollected by centrifugationat 14,000g for 10min,the supernatant wasremoved,and the1251I-thrombomodulin

bound was measured in agamma scintillation spectrometer.

Cofactor activity of thrombomodulin. Reaction mixtures contained thefollowing:0.5gM protein C,40 nMthrombin, andasampleina

total volof30 pAcontaining 20 mM Tris, pH 7.4, 0.15 M NaCI, 1 mM J.Clin. Invest.

© The American

Society

for Clinical

Investigation,

Inc.

(3)

CaC12, and 5 mgbovine serum albumin/ml. ProteinCaformed was measured asdescribed previously (3).

Immunoblot ofthrombomodulin. Sodium dodecyl sulfate-polyacryl-amidegelelectrophoresiswasperformedby the method of Laemmli(12) using4%acrylamide in the stacking and 10% acrylamide intherunning gel. Samples of thrombomodulin from urine,andplasmawere electro-phoresed with and without2-mercaptoethanol. Immunoblotting was performed bythemethod ofBurnette(13). The thrombomodulin was detectedbytwomethods. In onemethod,polyclonal anti-thrombom-odulinserum wasusedat a1:250dilutionanddetectionwasmadeusing

'251I-proteinAandautoradiography. Theothermethod used

'251-mono-clonalanti-thrombomodulinIgGfor direct detectionofthrombomodulin.

Results

Properties ofplasma and urine thrombomodulin. In preliminary studies we assayed samples of human urine and plasma for thrombomodulin antigen as described below. In these experi-mentsweconsistently detected thrombomodulin antigen in both fluids eventhough we could not detect thrombomodulin activity byproteinC-activating cofactor assay ofthe fluids. To definitively establish that thrombomodulin wasactually contained in both plasma and urine, we performed immunoaffinity chromatog-raphy as described in methods. The eluates of both plasma and urine were found to have protein C-activating cofactor activity that waslinear with time and completely dependent on both protein C and thrombin. The protein C dependence of urine

thrombomodulin isshown inFig. 1.Similarresults wereobtained

using thrombomodulin isolated from plasma. The apparent Mi-chaelis constant

(Km)

for protein C was 2 uM for both cellular andsoluble thrombomodulin. The effect of varying thrombin

concentrationonthrombomodulin functionalactivity is shown in Fig. 2. Plasma thrombomodulin had an apparent

K.

for thrombin of 1.1nMcompared with 0.33nMfor cellular

throm-E

-E

z

"I-0~ ll

To

0

PROTEIN C ( pM)

Figure1.Activation ofproteinCby thrombin-thrombomodulin

com-plex.Reaction mixtures(30

Ml)

contained 1.25 nMtissue thrombo-modulinstandard(- *-),or10Mlof fraction of urine thrombomodu-linfromanti-thrombomodulin IgGagarose column(-o-),40nM

thrombin,and theindicatedconcentrations ofproteinC. Insetshows double-reciprocal plot forthesamedata. TheKm forproteinCwas2 MM.

6

5

4

3

2

B

0.2

0 It

0 2 3 4 5 -3 -2 -I 0 2

THROMBIN(nM) [THROMBINnM]

Figure 2. Thrombin dependence of protein Cactivation.Ashows sat-urationcurve.Reaction mixtures (30ul)contained 1.25 nMtissue thrombomodulin standard (- * -),or10Mlof fraction of plasma thrombomodulin from anti-thrombomodulin IgGagarosecolumn (-0-),0.5 AMprotein C, andtheindicatedconcentrations of throm-bin.Bshowsdouble-reciprocalplotof thesame data. TheKmof

stan-dardandurinethrombomodulinwas0.33and 1.1 nM,respectively.

bomodulin. Urine thrombomodulin had an apparent Km for thrombin of 1.6 nM (data not shown). The protein C-activating cofactor activity isolated from plasma and urine reflected thrombomodulin asevidenced by the finding that theactivity

in both cases was completely inhibited by an excess of rabbit anti-thrombomodulin IgG. The relative catalyticactivityof urine

thrombomodulin vs. cellular thrombomodulin was estimated by theimmunotitrationexperimentshown inFig.3. Weestimate

that urine thrombomodulin has -v3 the activity ofcellular thrombomodulin based onthe findingthat the tangent tothe inactivation curveofeach thrombomodulin extrapolatestothe

same point using -2/3 as much urine as cellular

thrombo-modulin.Asimilar result was obtained usingplasma thrombo-modulin as shown in Fig. 4. In this experiment plasma

throm-bomodulinhad

½1/2

theintrinsicactivityofthatobservedusing

cellularthrombomodulin. We next compared plasma

throm-bomodulintocellularthrombomodulinby immunoblotanalysis

using

1251-monoclonal

anti-thrombomodulin IgGfor detection

asshown inFig. 5. This gelwas runin the presenceof

2-mer-captoethanolwherecellularthrombomodulin hasanapparent molecular weight of 105,000. Themajor polypeptidedetected in plasmathrombomodulin has an apparent molecularweight

of85,000. Thebands detectedatlowermolecularweightsmost

2.0 Figure 3.Inhibition of

tis-sueorurine

thrombomodu-E lin by

anti-human-throm-E 6 bomodulinrabbit IgG.

Tis-sue(0.94ng,-* -) and

E 2 urine(aneluatefraction

Gas fromanti-thrombomodulin,

10

Ml,

-o-)

thrombomodu-° 08 linwereincubated with

0-U

Ad

150

'a

ngpolyclonal

anti-hu-z man-thrombomodulin

rab-H0.4 ' bitIgGfor 15 min at

370C.

ai Thecofactoractivityof

thrombomodulin inprotein

0 50 100 150 Cactivationwasassayedas

ANTITHROMBOMODULIN IgG (ng) described in Methods. In

controlexperimentsitwasshown that theanti-thrombomodulin

IgG

(4)

04

I

\A

\\A

0 50 100 150

ANTITHROMBOMODULIN IgG(ng)

Figure 4. Inhibition of tissueorplasma thrombomodulin by anti-hu-man-thrombomodulin rabbit IgG. Tissue (0.63ng,-o -) and plasma

thrombomodulin (an eluate fraction from anti-thrombomodulin IgG column, 10gl,-o-)wereincubated with 0-150ngpolyclonal

anti-thrombomodulin rabbit IgG for 15 minat37°C. The cofactor activity ofthrombomodulin in protein C activationwasassayedasdescribed in Methods.

likely reflect further degradation products of thrombomodulin. Since one of the lower molecular weight peptides detected in

lane 2 migrated similarlytothe heavy chain of IgG,we

consid-eredthe possibility thatthesepeptides might reflect,inpart,low

affinitybindingof'25I-anti-thrombomodulintoIgGthateluted fromtheaffinitycolumn. Weran 5,ugof human IgG in lane 3

which is slightly detected by the monoclonal anti-thrombo-modulin. Theamountofthrombomodulin appliedtothe gelin lane2was7ngbasedonfunctional activityandtheproteinin thissamplewas<2,gby Bio-Rad proteinassay.Thus the bands

detectedinlane 2reflectamuchsmalleramountof protein than

the IgGin lane3. We therefore conclude that the material de-tectedin lane 2reflects peptides derived from thrombomodulin. We also performed immunoblots on samples electrophoresed

intheabsence of 2-mercaptoethanol.Underthis conditionboth soluble and cellular thrombomodulin contain high molecular weight peptides thatdonotrunassharpbands.Thereisadiscrete

protein migrating withapparentmolecularweightof75,000 in cellularthrombomodulin.Inthe plasma thrombomodulin there were two discrete bands detected with apparent molecular

weights of 63,000and54,000.

E E c

E,

0

2 3

-92500

-66200 Figure 5.Immunoblotanalysis of

thrombomodulin. Immunoblot for

-45000 plasmathrombomodulin. The immu-noblotwasperformedasdescribedin

_31

000 Methods. '25I-monoclonal

anti-throm-bomodulin IgGwasused for detec-tion. Lane1,isolatedtissue

throm-14400 bomodulin(1.0jyg);lane2,

immunoaf-finity-isolated plasma

thrombo-modulin(100,l);lane 3, human

IgG (5ptg).

a

CL.

cI

a.

0

U-0C

H-THROMBOMODULIN (ng)

2 5 10 20 50 100

PLASMA, URINE OR FRACTION

FROM IMMUNOAFFINITY COLUMN(jil)

Figure6.Radioimmunoassayofthrombomodulin.

Radioimmunoas-sayof thrombomodulinwascarriedoutasdescribed in Methods.(-) Standardplacenta thrombomodulin, (A)urineconcentrated 10times, (n) plasma, (A) affinity purified urine thrombomodulin, (o) affinity

pu-rified plasma thrombomodulin.

Radioimmunoassayofplasma and urinethrombomodulin.

Astandardcurveofthe radioimmunoassay of thrombomodulin

using isolated placental thrombomodulin is shown in Fig. 6. Assays ofurine, plasma, and affinity-purified materials from

bothsourcesare alsoshown and allappeartohave similarslopes. Wealso added known amounts of placental thrombomodulin

toboth plasma and urine, and showed that therewasgood re-coveryof the added material, further indicatingthat thrombo-modulin is accurately measured. We determined thecontentof thrombomodulin in the urine and plasma ofnormalsubjectsas

shown in TableI. Wehave alsomeasuredthrombomodulin

an-tigen in serum andfindlevels identicalto thoseinplasma. In

order toexclude the possibility that soluble thrombomodulin wasnottrulysoluble butwascontained in fragments of mem-brane,we subjected serumto ultracentrifugation at 150,000 g

for I hand carriedoutaradioimmunoassayofthesupernatant

fraction. All ofthe thrombomodulin antigen remained in the

supernatant,indicatingthat the material isactually soluble.

Table I. Thrombomodulin Content in Urine andPlasma of Normal Human Subjects

Thrombomodulin

ng/ml

Urine (n=9) 102±39

Plasma(n=6) 292±60

Thecontentof thrombomodulinwasmeasuredbyradioimmunoassay

asdescribed in Methods. Sampleswereobtained from normalsubjects aged25-48. Sixmenand threewomenprovidedurinesamples.All of theplasma sampleswerefrommen.

E

E

CL.

0 w

0 U-0 z w

0

(5)

Discussion

We havefound that human plasma and urine contain soluble forms of thrombomodulin that appear to be smaller than cellular thrombomodulin. The origin ofthis material is presently unclear. We haveconsidered two major possibilities to explain the find-ings: (a) The soluble circulating forms of thrombomodulin are produced insome cell specifically for secretion into plasma and the molecule functions to promote protein C activation in the circulation. According to this hypothesis soluble thrombo-modulin might be synthesized without the appropriate mem-brane binding domain to allow its insertion into membranes. (b) The second possibility is that the circulating forms of thrombomodulin reflect proteolysis of endothelial membrane thrombomodulin that is produced either as a result of the normal turnoverof thrombomodulin or as a result ofendothelial damage in areasof endothelial injury or inflammation. We have noticed that elastase treatment of isolated placental thrombomodulin generatespeptides thatmigrate on sodium dodecyl sulfate gels similarly to circulating thrombomodulin (unpublished obser-vations). It is estimated that adult man has approximately

6 X 1013endothelial cells (14). If the average content of throm-bomodulin is 40,000molecules/cell as wefindoncultured

um-bilical vein endothelial cells (5), then the totalcontentof throm-bomodulin in endothelium is -300 mg. Circulating throm-bomodulin would total -0.8 mg (0.3

,g/ml

X2600 ml plasma). Thus theproteolysis ofasmall fraction of cellular

thrombom-odulin couldconceivablyaccountfor thecirculatingform. We

cannotestimate thisfraction since the plasma circulation time of solublethrombomodulinis unknown. We also donotknow the source ofurinary thrombomodulin. It appears essentially identicaltoplasma thrombomodulin byimmunoblotting, sug-gesting that it is probably derived from plasma. We also donot

knowwhyweareunable to measuredirectlythrombomodulin

activity byprotein Cactivation assay in plasma. The material isolated from plasma by immunoaffinity chromatography is nearlyfullyactive; however, theyieldof materialpurified from

plasma is only 2-5%. The lowyieldcould resultfrom the very small amount ofprotein involved, which may be lost during chromatography orthe subsequent dialysis after elution from

theaffinitycolumnto removeNaSCN. Alternatively,it may be thatmostof theimmunoassayedthrombomodulin inplasmais

degraded toinactiveforms that donotbind

sufficiently

tightly

totheaffinity columntoberecovered upon purification.

Fur-thermore, it is difficultto measureproteinCactivationdirectly

in plasma since antithrombin III inhibits thrombin relatively rapidly.

The finding that thrombomodulin exists ina form that is both soluble and functionally active haspotential therapeutic implications. Thus, if thestructureof soluble thrombomodulin

canbe

determined,

it may bepossibletoproduce largeamounts

ofthis moietyby molecularcloningtechniques. Such aprotein could be used as a natural anticoagulant that could inhibit thrombus formation in pathological states.

We thank Dr. Ikuro Maruyama, Dr. Thomas M. Connolly, and Dr. DavidB.Wilsonfor helpful discussions.

References

1.Kisiel,W. 1979. Human plasmaprotein C. Isolation, character-ization,andmechanismofactivation bya-thrombin.J.Clin. Invest.64: 761-769.

2. Esmon, N. L., W.G. Owen,andC.T. Esmon. 1982. Isolation of membrane-boundcofactor for thrombin-catalyzed activation of protein C.J.Biol.Chem. 257:859-864.

3.Salem,H. H., I. Maruyama, H.Ishii, andP. W. Majerus. 1984. Isolationandcharacterization of thrombomodulin fromhumanplacenta. J. Biol. Chem. 259:12246-12251.

4. Maruyama, I., H. H.Salem, H.Ishii, andP. W.Majerus. 1985. Humanthrombomodulin isnot anefficient inhibitor of procoagulant activityof thrombin. J. Clin. Invest. 75:987-991.

5.Maruyama, I., and P. W.Majerus. 1985. Theturnover ofthrombin-thrombomodulin complex in cultured human umbilical vein endothelial cellsandA549lungcancercells:endocytosis anddegradationofthrombin. J. Biol. Chem.Inpress.

6. Maruyama, I.,C. E.Bell,and P. W. Majerus. 1985. Thrombo-modulin is foundon endothelium ofarteries, veins, capillaries,

lym-phatics, and onsyncytiotrophoblast of human placenta. J Cell Biol. 101:363-371.

7. Ishii, H., H. H. Salem, C. E. Bell,E. B. Laposata, and P. W. Majerus. 1986. Thrombomodulin,anendothelialanticoagulantprotein,

isabsentfromhumanbrain.Blood. Inpress.

8. Suzuki, K., J. Stenflo, B. Dahlback, and A.Toedorsson. 1983. Inactivationof human coagulation factorVby activatedprotein C. J. Biol.Chem. 258:1914-1920.

9.Miletich,J. P.,C.M.Jackson, andP. W.Majerus. 1978.Properties ofthefactorXabinding siteon human platelets.J. Biol.Chem. 253: 6908-6916.

10.Owen,W.G. 1975.Evidence for theformation ofan esterbetween thrombin andheparin cofactor. Biochim.Biophys.Acta.405:380-387. 11.Mellman, I.S.,and J.C. Unkeless. 1980.Purification ofa func-tionalmouseFc receptorthrough theuseof monoclonalantibody.J. Exp. Med. 152:1048-1069.

12.Laemmli,V. K. 1970.Cleavage of structuralproteins duringthe assembly oftheheadof bacteriophageT4. Nature(Lond.).227:680-685. 13. Burnette,W.N.1981. "Westernblotting": electrophoretic transfer ofproteins from sodium dodecyl sulfate-polyacrylamide gelsto un-modified nitrocellulose and radiographic detection with antibodyand radioiodinatedproteinA.Anal. Biochem. 112:195-203.

14.Wolinsky, H. 1980.Aproposallinkingclearanceofcirculating

References

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