Inhibition of the activation of Hageman factor (factor XII) by peripheral blood cells

(1)

Inhibition of the activation of Hageman factor

(factor XII) by peripheral blood cells.

O D Ratnoff, … , M M Emanuelson, N P Ziats

J Clin Invest.

1987;

80(4)

:1180-1189.

https://doi.org/10.1172/JCI113177

.

Suspensions of peripheral blood mononuclear cells (PBMC), monocytes, T or B

lymphocytes, platelets or granulocytes, and cell-depleted supernatant fluids of these

suspensions inhibited activation of Hageman factor (HF, Factor XII) by ellagic acid, a

property not shared by erythrocytes. PBMC also inhibited HF activation by glass or

sulfatides. Contaminating platelets may have contributed to inhibition by PBMC. Elaboration

of agents inhibiting HF activation required metabolically active cells. The inhibitor(s) in

PBMC supernates were not identified with known agents, but had properties of a

nonenzymatic protein. PBMC supernates did not contain fibrinogen, nor alter the thrombin,

prothrombin, or partial thromboplastin times of normal plasma, amidolysis by activated

plasma thromboplastin antecedent (Factor XIa) or activated Stuart factor (Factor Xa) or

esterolysis by C1 (C1 esterase); they inhibited plasmin minimally. These experiments

suggest that peripheral blood cells may impede intravascular coagulation. Whether this

property helps maintain the fluidity of blood is unclear.

Research Article

Find the latest version:

(2)

Inhibition of the Activation of Hageman

Factor (Factor

XII)

by

Peripheral Blood Cells

Oscar D. Ratnoff, Marlene M. Emanuelson, and Nicholas P. Ziats

DepartmentsofMedicine and Pathology, Case Western ReserveUniversitySchoolofMedicine, and University Hospitals ofCleveland, Cleveland, Ohio 44106

Abstract

Suspensions of peripheral

blood mononuclear cells (PBMC), monocytes, T or B

lymphocytes,

platelets or

granulocytes,

and

cell-depleted

supernatant

fluids

of

these

suspensions

inhibited

activation of Hageman factor (HF,

Factor

XII)

byellagic acid, a property not

shared by erythrocytes. PBMC

alsoinhibited

HF

activation

by

glass

or

sulfatides. Contaminating

platelets mayhave

contributed

toinhibition by PBMC.

Elaboration

of

agents

inhibiting

HF

activation

required

metabolically active cells.

The

inhibitor(s) in

PBMC super-nates

were

not

identified

with

known

agents,

but

had

properties

of

a

nonenzymatic

protein.

PBMC

supernates did not contain

fibrinogen,

nor alter

the

thrombin, prothrombin,

or partial

thromboplastin times of

normal

plasma, amidolysis

by acti-vated

plasma

thromboplastin

antecedent

(Factor

XMa)

or acti-vated

Stuart factor (Factor

Xa) or esterolysis by

Ci

(Cl

ester-ase); they

inhibited

plasmin

minimally.

These

experiments

suggestthat

peripheral

bloodcells may

impede

intravascular coagulation. Whether this

property helps

maintain the

fluidity

of blood

is unclear.

Introduction

A

variety

of

devices

appear to

maintain the fluidity of

circu-lating blood.

Among

these

are a

multitude of plasma proteins

that inhibit the

enzymatically active

forms of

clotting factors

or, under suitable

circumstances, digest procoagulant

proteins orthe fibrin clot itself. The

experiments

tobe

described

suggest that another class of substances may deter the

formation

of

intravascular

clots.

Suspensions of peripheral blood

mononu-clear

cells,

granulocytes,

and

platelets

all appear to

inhibit

the activation

of

Hageman

factor

(HF,' Factor XII). The

responsi-Address reprint requests to Dr.

Ratnoff,

Department of

Medicine,

University HospitalsofCleveland,2074

Abington

Road,

Cleveland,

OH44106.

Receivedfor publication27October1986andin

revisedform

25 March 1987.

1.Abbreviations usedinthispaper:BS,barbital-saline

buffer; BS-Alb,

BScontaining0.25%bovine

albumin; Cl,

Clesterase, activated form

ofthefirstcomponentof

complement;

CI-INH,

Clesterase

inhibitor,

Clq,theClq subcomponentof the first component of

complement;

DFP,diisopropylfluorophosphate; DPBS,Dulbecco's

phosphate-buf-feredsaline; DPBS-CaMg,Dulbecco'sphosphate-bufferedsaline

with-outcalcium andmagnesium

ions;

HF, Hageman

factor,

Factor

XII;

HPPAN, H-D-prolyl-L-phenylalanyl-L-arginine p-nitroanilide

hydro-chloride(S2302); LBTI,lima beantrypsininhibitor; PF4, platelet

fac-tor4;p-NA,p-nitroaniline; PTA, plasma

thromboplastin

antecedent (FactorXI); SBTI, soybeantrypsin inhibitor; S2222, benzoyl-isoleu-cyl-glutamyl-glycyl-argininep-nitroanilide; S2238,

H-D-phenylalanyl-pipecolyl-arginine-p-nitroanilide.

bleagent or agents aredetected in cell-depleted supernatant

fluid separated from cell suspensions. The inhibitory

proper-ties are associated with proteins that are synthesized by the cells. Thebiological significance of the inhibitory agent or agents in deterring intravascular coagulation remains to be determined.

Methods

Heparinized bloodwaspreparedby drawing 50 ml blood from the antecubital vein of human subjects into polypropylene syringes

con-taining1 mlheparin (1,000NIHU/ml); the donorswerenormaladults except fora19-yr-oldwomanwithidiopathic aplastic anemia (5,000

platelets/tal).

Citrated bloodwaspreparedbytransferring 10 ml normal venous blood,drawn intoapolypropylene syringe, toapolystyrene

centrifugetubecontaining0.2 mlsodium citrate buffer (0.5 M, pH 5.0). Human ABserum wasseparated fromvenousblood of fasted donors thatwasincubatedovernight in glassat370C. Pooled normal plasma,arbitrarilysaidtocontain 1.00 U HF/ml, was prepared from thevenousblood of 24 normal menaged 21 to40 yr and storedat

-70°C (1). Informedconsent wasobtainedfrom all subjects.

HF(FactorXII)waspurified bytwodifferent methods(2,3), and wasdevoid of otherclottingfactors andplasminogen. Itdisplayeda

singleband upon unreducedorreduced polyacrylamide gel electro-phoresis. Before use,HF wasdiluted, usuallyto0.05 U/ml, in 0.25% bovineserumalbumin in barbital-saline buffer(BS-Alb).

Apparent solutions of 10-4 M ellagic acid (synthesized by

Dr. James Crum in 1966 orDr. Miklos Bodanszky in 1983, Case Western ReserveUniversity)in BSwerepreparedasdescribed earlier

(2)anddilutedtothedesiredconcentration with BS.

The buffersused included BS (2), BS-Alb, tris-imidazole-saline

buffer (0.025 M, pH 8.2) (4), Dulbecco's phosphate-buffered saline

(DPBS)or DPBSwithout calcium andmagnesiumions (DPBS-CaMg) (MABioproducts,Walkersville,MD; KCBiological, Lenexa,KA;or

Gibco,GrandIsland, NY)(5),DPBSwith 1%bovine albumin

(DPBS-Alb), RPMI 1640 with 2mML-glutamine(RPMI)(6),RPMIwith 10% humanABserum(RPMI-HS), and RPMI with 10% fetal bovineserum

(RPMI-FBS) (MABioproducts).

Sheep erythrocytes, suspendedin Alsever'ssolution,were sedi-mentedat 1,800gat 10°C,washed threetimes withRPMIand

sus-pendedat aconcentration of 1%(vol/vol)in RPMI.

FBS(Sterile Systems, Logan, UT)washeatedat56°Cfor 30 min beforeuse.

H-D-prolyl-L-phenylalanyl-L-argininep-nitroanilide

dihydrochlor-ide (HPPAN, S2302), benzoyl-isoleucine-glutamyl-glycyl-arginine

p-nitroanilide (S2222), H-D-valyl-L-leucyl-L-lysine p-nitroanilide (S2251), and H-D-phenylalanyl-pipecolyl-arginine p-nitroanilide (S2238) wereobtained fromHelenaLaboratories (Beaumont, TX),

dissolvedat aconcentration ofI mMindistilled water, and storedat

4°C.Before use, HPPAN and S2238werediluted withanequal

vol-umeof Tris-imidazole-saline buffer. N-Acetyl-L-tyrosine ethyl ester

(Calbiochem,LaJolla, CA)wasdissolvedat aconcentrationof 1Min ethyleneglycolmonomethylester(FisherScientific,FairLawn, NJ).

CrudeIgG fractions ofnormalgoatand rabbitsera

(Pel-Freez

Biologicals, Rogers, AR), rabbit antiserum

against

human

Clq

(Boehringer-Mannheim Biochemicals, Houston, TX)andgoat

anti-serum against

#2-glycoprotein-I (Calbiochem)

were

_separated

as

de-1180 0.D.Ratnoff7M.M.Emanuelson,and N. P. Ziats

J.Clin.Invest.

Society

for Clinical

Investigation,

Inc.

0021-9738/87/10/1180/10

$2.00

(3)

scribed earlier (7, 8). CrudeIgGfractions of normal goat and rabbit sera, goat antiserum againsthuman platelet factor 4 (PF4; Atlantic Antibodies, Scarborough, ME) and rabbit antiserum against human Cl-INH(Cl inactivator, Behring Diagnostics, La Jolla, CA)were pre-pared in the same way and further purified by filtration through DEAE-Affigel Blue (Bio-Rad Laboratories,Richmond,CA) and

con-centrated, first bynegativepressuredialysis againstBS and then ina

Centricon 30 microconcentrator (Amicon Corp., Danvers, MA). Other reagents used included heparin (beef lung, 100U/ml, Up-john Co., Kalamazoo, MI), Ficoll-Paque (Pharmacia Fine Chemicals, Piscataway, NJ), crystallized bovine serum albumin (BSA, Pentex, Miles Laboratories, Naperville, IL), bovine brain sulfatides (monoga-lactosyl-stearate, Supelco, Bellefonte, PA), bovine thrombin (Throm-bostat, U. S. Pharmacopeia, Parke-Davis Co., Detroit, MI), and bovine trypsin (Worthington Biochemical Corp., Freehold, NJ). EDTA, lima beantrypsininhibitor (LBTI), soybeantrypsininhibitor (SBTI),

diiso-propylfluorophosphate(DFP),benzamidinehydrochloride, cyclohexi-mide andsodiumazide were from Sigma Chemical Co., St. Louis, MO. Purified human Stuart factor (Factor X), activated by Russell's viper venom (Factor Xa) (9) and purified humantrypsin-activatedplasma

thromboplastinantecedent(activatedPTA,FactorXIa) (10)werethe gifts of Dr. L. T. Goodnough, Case Western Reserve University. PF4 was thegiftof Dr. L. Culp, Case WesternReserveUniversity(1 1).

C!

(Cl esterase)was prepared asdescribed by Donaldson (12). Crude soybeanphosphatides(Centrolex "P," thegift of Central Soya Co., Fort Wayne, IN)weresuspendedat0.1% in 0.15Msodium chloride. Peripheral blood cellswereseparated by modifications of published methods, asdescribedbelow.Before testing,thesupernatant fluids of cellsuspensionswerefiltered through Millex GV 0.22 gm filters

(Mil-lipore Corp., Bedford, MA),asdescribed below.

Peripheralblood mononuclear cells (PBMC) and the supernatant fluids of PBMC suspensions wereprepared by anadaptation of B6yum's method (13). Typically, in replicate, 25 ml heparinized blood wasdiluted with 25 ml DPBS-CaMg. 10-ml aliquots were layered onto 3 ml Ficoll-Paque, and centrifuged at 450 g for 45 min. The PBMC layers were combined, washed twice with 40 ml DPBS-CaMg, once with 40 mlofanequal mixture of DPBS-CaMg and DPBSand once

with 40 mlof DPBS, pelleting the cells each time at 500gfor 10 min. Thewashedcells, suspended in DPBSat 5 X 106/ml, were

recentri-fuged in the same way. The mean viability of PBMC was93% (SD±6.8%).

The supernatant fluidwasfiltered throughaMillex GV

_0.22-gm

filter unit attached toapolypropylene syringe, replacing the filterafter

15 ml had been filtered. The PBMC, resuspendedat5X

106

cells/ml, and thefiltered supernatant fluidweresaved. Nolactic dehydrogenase wasdetected in the PBMC supernate in a sample containing 15

Mg

protein/ml, usingamethodsensitiveto1 U/liter ( 14).Insome experi-ments, the cellswererecentrifugedand resuspended repeatedly, saving successive supernatant fluids. PBMC and their supernatesweretested within an hour of separationafterstoragein an ice-water bath.

Concentratesof the supernatant fluid of PBMCwereprepared by negative pressuredialysis againstDPBS,orbypositive pressure filtra-tion throughanAmicon filter(ultrafiltrationcell, model 8400,

Ami-conCorp.) fitted witha PM 10 membrane. Concentratesweremade from the combined supernates of replicate preparations or from the combined supernates of successive washes ofasingle preparationof PBMC.

Thepossible presence of C1-INH, Clq,,B2-glycoprotein-I orPF4in

concentratedPBMC supernatant fluid(1 mgprotein/ml)was

exam-ined by immunodiffusion, using 1% agar(15).Thepresence of

glycos-aminoglycans containing uronic acidwas testedby measuringthe

concentration of uronic acid inthe same concentrate(16).

The suspensionsof PBMCwerecontaminated with platelets. In some experiments, the proportion of platelets was reduced by

- 85-95% bycentrifuging25 mlofheparinizedbloodat200g for15

min. The platelet-rich supernatewasremoved, and thecellswere

washed threetimes with DPBS-CaMg, centrifugingat200g.The

plate-let-depletedbloodwasusedtopreparePBMC.

To testtheeffectof trypsin on the inhibitoryproperties ofPBMC andPBMC supernate, 0.025 ml bovinetrypsin (0.05 mg/ml BS)orBS wasincubated with 0.1 ml PBMC, supernateorDPBSat370Cfor 2 h in 12 X 75 mm polystyrene tubes. Thereafter,0.025 mlLBTI(0.2 mg/ml BS) and 0.05 ml HF(0.1 U/ml in 0.5% BS-Alb)orBS-Alb alone, wereadded inrapidsuccession. After 10min, theamidolytic

activity of HFwasmeasuredasdescribed below.

Ammoniumsulfate-precipitablematerial in PBMC supernateswas

separated by addition of sufficient solid ammonium sulfatetoprovide

60% saturation. The mixturewasincubatedat4VC overnightand the

precipitatethatformed, separated by centrifugationat20C for 15 min

at 2,000 g, wasdissolved in sufficient DPBS to restoretheoriginal

volume and dialyzed for Ihagainst runningtapwaterandovernight against - 100 volof DPBS.

DFP-treated PBMC supernatant fluids were prepared by adding

1/100thvol0.1 MDFPinisopropanol(finalconcentration 1 mM). The mixturewasincubated for 90 min and thendialyzed against100 vol DPBS at4VC overnight.As acontrol, the supernatesweretreated in thesameway with 1/100thvolofisopropanol.

Cycloheximide-treated PBMC were prepared by suspending

PBMC inRPMI at aconcentrationof 5X106cells/ml. To oneoftwo

12-mlaliquots,0.12mlcycloheximide(5 mg/ml DPBS)wasaddedto

provideaconcentration of 50Mug/ml (18 gM).Thetwoaliquotswere

incubated with mildagitationfor 16 hat37°C and the cells tested for viability. The aliquotswerecentrifuged for 10 minat500 g and washed three times in DPBS. Thepelleted cellswerediluted in DPBSto5

X 106/ml,recentrifuged, the supernatant fluid saved, and the cells re-suspended in DPBSat5X 106/ml.

Sodium azide-treated PBMCwereprepared bysuspendingPBMC in RPMI at 107 cells/ml. 9.0-ml aliquotsweredilutedwith 9.0 ml

sodium azide(10mg/mlRPMI)orRPMI alone, incubated with mild

agitation for 20h at 37°C, washedasdescribed for

cycloheximide-treatedcells, and testedfor viability.

Crudecytosol andparticulatefractions of PBMCwereseparated

from 5mlwashed PBMC in DPBS (5X

106

cells/ml),snap-frozenin

polypropylenevials inadry ice-ethanol mixture and thawedat37°C,

repeating the process twicemore. The mixturewas centrifugedat

100,000 g for 1 hat20C,the supernatantcytosol fractionwas sepa-rated, and theparticulate fraction was resuspended in 5 ml DPBS and

recentrifuged.The supernatant solutionwaswithdrawn and the

partic-ulatefractionwassuspended in 5 ml DPBS.

Amonocyte-enriched fraction of PBMCwasprepared bya modifi-cation of the method of Fischer and Koran(17).PBMCwereseparated

from platelet-depleted normal blood, substituting 10 ml RPMI

con-taining2mML-glutamineand 10% human ABserumforDPBS in the final step. The cells weredilutedto5 X 106/mlin the samebuffer. Quadruplicate aliquotsof 11 mlwereplatedonto75cm2polystyrene

tissue cultureflasks(CorningGlassware,Corning,NY) and incubated for 70 minat37°C inahumidified atmosphere of 5% carbon dioxide and 95% air. Theflasks werethen washed three times with 5.0 ml

DPBSto removenonadherentcells. Adherent cellswereharvested by

adding5.0mlDPBS andscrapingtheflasks witharubberpoliceman, transferringthe cellsuspensionto a 50-ml polypropylene tube. The combined cellssuspensionsof thereplicatetubeswerecentrifugedat

600 g for 15 min. The sedimented cells, suspendedat5X

106/ml

in DPBS, were

recentrifuged,

the supernatant fluidwas removed and

saved, and the cells were resuspended at the same concentration. Crudecytosolandparticulatefractions of monocyteswerepreparedas

described forPBMC.

A human monocytoid cell line (U937), derived from ahuman

histiocytic lymphoma,wascultured bythe methodofSundstrom and

Nilsson(18).Thecellswerewashedandcentrifugedfor10 minat500 g in polystyrene tubes, the cellswerethensuspendedsuccessively,twice in DPBS-CaMg,once in anequalmixtureof DPBS-CaMgandDPBS,

andfinallyin DPBS,separatingthe cellseachtime bycentrifugation.

Thefinal cell suspension, >95% viable,was diluted in DPBSto 5

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monocyte-enriched cell fraction by centrifuging nonadherent PBMC at 500 g for 10min. The cells were washed three times in DPBS, diluted to 5 X 106/ml with DPBS and resedimented at 500 g for 10 min. The supernatantfluid and the sedimented cells, resuspended in DPBS to thesame concentration, were saved. Crude cytosol and particulate fractions of lymphocytes were separated as described for PBMC.

T andB lymphocytes were prepared by modifications of the methods of Greaves and Brown (19) and Weiner et al. (20). In brief, a cellfraction enriched in T cells was separated from a preparation of PBMC, substituting RPMI-HS for the final addition of DPBS. The cells werediluted to10'/mlin RPMI-HS. 6ml weretransferred to each of replicate polystyrene tissue culture flasks and incubated at370Cfor 1 hin ahumidifiedatmosphereof 5% carbon dioxide and 95% air. The

suspensionsof nonadherent cellswereremoved,eachflask was rinsed twicewith 2.5mlprewarmed RPMI-FBS, and the combined suspen-sions werecentrifuged at 500 gfor 10 min. The sedimented cells, suspended in 6.5 ml RPMI-FBS, werefiltered through 600 mg acid-washed nylon wool (scrubbed nylon, Fenwal Laboratories, Deerfield, IL) wetted with RPMI-FBS, refiltering the cells through the same

col-umnthreetimes. Nonadherent cellswerefilteredthroughthe column byapplying 10mlRPMI-FBS, prewarmedto370C. Theeffluentsof three such columnswerecombined, centrifugedat500 g for 10min,

the cell pelletwassuspended in 6.5mlDPBS,recentrifuged, andthe

pelletwashed threemoretimes with 20mlDPBS. The cellsuspension,

dilutedto5X Io6cells/ml,wasrecentrifuged,the supernatantfluidwas

saved and the cellswereresuspendedto5X106/ml.Theproportionof

Tcells wasidentified immunologicallybycytofluorography, using mouse monoclonalantibodiestothe OKT 11 marker (Ortho Diag-nostic Systems, Raritan, NJ) (21).

Afractionof PBMC enriched inBcellswasseparatedbywashing

thenylon wool column usedtoseparateTcellswith 50 ml RPMI-FBS and allowingthe column todrain. Adherent Bcellsweredislodged

mechanically, eluted with5 mlRPMI,andcentrifugedat500 g for 10 minat10C. The pelleted cellsweresuspended in 1mlofRPMI-FBS,

anddilutedto3X106 cells/ml.1 mlofthis crudeBcellsuspensionwas

mixed with 1 mlofa 1%suspensionofsheep erythrocytesin RPMI,

centrifugedat500 g for5minat40C,andincubatedat4VCovernight.

The pelleted cellswereresuspended and thecontentsof five such tubes werelayeredoverFicoll-Paqueat aratio of 8 ml cellsuspensionand 3 mlFicoll-Paque.The tubeswerecentrifugedat500 g for 30 min, the separated celllayer,enriched withBcells,waswashed threetimes in

DPBS,and the cellsweredilutedto5X

106/ml

andrecentrifuged.The supernatant fluidwassavedand the cellswereresuspendedin DPBS. Theproportion of B cellswasidentifiedby

cytofluorography,

using

goatpolyvalentimmunoglobulin againstsurfaceimmunoglobulin (PV antibodies,KallestadLaboratories,Minneapolis, MN) (21).

Granulocyteswereseparated from 25mlheparinizedvenousblood byamodification ofpublishedmethods(13, 22).The bloodwas di-lutedwithanequal volume ofDPBS-CaMg, layeredover 18 ml

Fi-coll-Paque,andcentrifugedat450 g for 40 min. The supernatant fluid

wasremoved and thethin

leukocyte

layer, 3ml,wastransferredto

eachoftwo50-mlsterilepolystyrene

centrifuge

tubes.Avolumeof 24 mlofdistilledwater wasaddedtoeachtubeand,after 20 s, 8 ml of0.67

M (3.6%) sodium chloridewasadded rapidlytoeach tube and the

contentsmixed. The tubeswerecentrifugedat250 g for 10min at2°C,

thepelleted granulocytes wereresuspended in 24 ml water andthe processrepeated. The cellswerethen suspended in 10 ml DPBS-CaMg

andrewashed, twice with DPBS-CaMg,oncewithanequal mixture of

DPBS and DPBS-CaMg, andfinally with DPBS. The pelleted cells were suspended in DPBSat5 X

106/ml.

The cell suspension and the supernatant fluid aftercentrifugationweresaved.

Erythrocyteswereseparated from 10mlcitrated venous blood by

centrifugation at250 g for 15 min. The supernatant, platelet-rich plasma was withdrawn and4 ml of thepelleted erythrocytes were

aspiratedfrom thebottomofthe tube, recentrifuged,andthebottom 3

mlof erythrocyteswerewithdrawn and washed three times with 3ml

DPBS, sedimentingthe cells each time at2,000gfor 10 min. The

erythrocyteswerethendilutedto2X I07 cells/mlwith DPBSandwas

recentrifuged at2,000g for 10 min. The supernatant fluid was

re-moved and saved, and the erythrocytes were rediluted to2 X 107

cells/ml.Noplateletsorleukocyteswereseenin smearedpreparations

stained withWright-Giemsastain.

Plateletswereseparated from 40 ml citrated blood by a modifica-tion of Kinlough-Rathbone's (23) method. The platelet-richplasma,

sedimentedat200 g for 10

min,

wasrecentrifugedatthesamespeedto

reduce contamination with erythrocytes. The platelet-rich plasmawas

mixed withanequal volume of DPBS-Alb, incubatedat370C for 15 min and centrifugedat1,800 g for 10 min. The pelleted plateletswere

washed twicemorein 10 ml of DPBS-Alb and suspended in DPBS (without albumin)at108cells/ml. This suspension and the supernatant fluid after recentrifugationweresaved.No erythrocytesorleukocytes

wereseeninsmearsof the platelet suspension stained with Wright-Giemsa stain.

The effect of cell suspensionsorthe supernatant fluid of cells

sus-pensions on activation of HF by ellagic acidwasmeasured by incubat-ing 0.1 ml HF (0.05 U/ml in BS-Alb) with 0.1 ml of the agent to be tested, diluted serially with DPBS, for 10 minat370C in 12 X 75mm

polystyrene tubes. Thereafter, 0.05 ml ellagic acid (2X10-5 M inBS)

wasadded to each tube. Incubation was continued for 60min, after which 1.0 ml HPPANwasadded. Incubationwascontinued for 60 min and the reaction was then stopped by addition of 0.3 ml glacial acetic acid. The amount of p-nitroaniline (p-NA) released from HPPAN was measured spectrophotometrically at 405 nmagainst blanks in which glacial acetic acid was added before substrate, in

com-parisontothe OD of p-NA solutions. Mixtures containing cellswere

centrifugedat800 g for 10min before measurement of OD. Controls included the substitution of appropriate buffers for the test solution, HF, or ellagic acid. The degree of inhibition by the test solutions was determined in comparison to the amidolytic activity ofarange of concentrations of HF. The coefficient of variation of theamidolytic

assay of HFwas3.2% (SD±3.1%, SE±1.0%) of any given value

ex-cluding observations in which the ODwas0.010orless. The coefficient of variation of the assay for inhibitory activitywas3.7% (SD±3.4%, SE±0.6%). Variations of this techniquearedescribed in thetext.All experimentswereperformed in replicate.

To test the effect of LBTI and SBTIonPBMC supernates, 0.1mlof supernate wasincubated with 0.05 ml LBTI orSBTI in BS, serially diluted with BS, for 10 minat37°C, 0.05 ml HF (0.1 U/ml BS-Alb)

wasadded andincubationwascontinued for 10min,after which 0.05 ml 2 X

lO-'

Mellagic acidwasadded. Afteranadditional 60min, 1.0

ml HPPANand 0.05 ml BSwereadded, and the assay continuedas

described above. As controls, BSwassubstituted for LBTIorSBTI in the initialmixture, and LBTIorSBTIwassubstituted for BS in the final mixture.

The effect of the supernatant fluid of cellsuspensionsonthe acti-vation of HF by glasswasmeasuredbymixing0.25ml HF(0.05 U/mI BS-Alb)orbovinealbumin alone and 0.25ml supernatantfluid, di-luted serially with DPBS, orDPBS alone. Aliquotsof 0.2 mlwere transferredto 10X75mmdisposable borosilicate glass tubes (Fisher ScientificCo.,Pittsburgh, PA).The tubeswereincubated for 60 minat

37°C,agitating the tubescontinuouslyinamechanical shaker. There-after0.1 ml DPBS and 1.0 ml HPPANwereadded and incubation and shakingwerecontinued for60min, after which 0.3mlglacial acetic acidwasadded. Theamountof p-NA releasedwasmeasuredas de-scribed above.

Thecapacity of PBMC supernates toinhibit the clot-promoting properties ofglasswastestedbyanearlier method (24).Induplicate, 10

X 75 mmdisposable glasstubes were coatedwith 0.2 mlsamples of supernateorBS. Volumes of0.1mleachofnormalpooled plasma and Centrolex Pwereincubated in these tubesat37°Cfor 1 min, and the

clottingtimewasmeasuredatthis temperature after addition of0.1 ml 0.025Mcalciumchloride,prewarmedat37°C.

To determinewhetherbindingtook place between HFandthe

inhibitoryagent(s) in PBMC supernates,a concentrateof PBMC

su-pernate wasboundcovalentlyto cyanogenbromide-activatedagarose

(CNBr-activated Sepharose 4B, Pharmacia FineChemicals)according

(5)

tothe manufacturer'sdirections,at aratio of 10 mgproteinto1 ggel.

A control gel was prepared in the sameway, omitting addition of PBMC supernate andblocking the active site with ethanolamine. A

volume of 2 ml of gel was packedinpolypropylene Econo-columns (i.d. 0.8 cm; Bio-RadLaboratories)andwashedwith DPBSbeforeuse. ImlHF(1.0 U/ml BS-Alb, 48U/mgprotein)wasfiltered through each column,eluting with DPBS. l-mlaliquotswerecollected in polysty-rene tubes and assayedfor coagulant HFactivityincomparisonwith thatof pooled normal plasma(25).

Theeffect of PBMC supernatesonthecoagulant action of throm-binwastested in 10 X 75 mm polystyrenetubesbymeasuringthe clotting timeat370Cofamixture of 0.1 mleachofpoolednormal human plasma, aconcentrate of supernate in DPBS, and bovine thrombin (5 U/ml). The presence offibrinogen in supernates was

examined in the same way, omitting plasma. Theeffect of PBMC supernates on the prothrombin time and kaolin-activated partial

thromboplastin timewassimilarlymeasured(25).

The effect of PBMC supernates ontheamidolytic properties of activatedPTA(26) andplasmin(25) and theesterolyticpropertiesof

Cl (27)wasdetermined bypublishedmethods. Theeffect of PBMC supernatesontheamidolyticpropertiesof activated Stuart factorwas

measuredbyanadaptation of methodIof0degardetal.(28),

incu-bating0.2 ml concentrated PBMC supernate(1.5 mgprotein/ml

DPBS)orDPBS aloneat

370C

for5minwith0.1mlactivated Stuart factor (2 U/ml 0.25% bovine albumin in BS, specific activity 1,200

U/mlprotein), in 12X 75 mmpolystyrene tubes.Thereafter,0.2ml I -3MS2222wasadded, incubationwascontinuedfor7min, and the reaction wasstopped by addition of 0.3 mlglacial acetic acid. The

amountof p-NA releasedwasmeasuredat405nmin comparisonto

blankstowhich acetic acidwasaddedbefore substrate.

CellcountingwascarriedoutwithaCoultercounter(model ZM, CoulterElectronics,Hialeah, FL). Theproportionof monocytesand

lymphocytes insuspensions of PBMCwasestimated by histochemical

stainingof monocytes fornonspecificesterase(17).Differentialcounts

ofgranulocyteswereperformed by Wright-Giemsa stainingof smeared cells.Tcells andBcellswereidentified withanautomated

flow-cytom-etry system(Coulter Epics V, CoulterElectronics)(21). Cellviability

was tested by trypan blueexclusion (29).

Protein assayswereperformed by the method of Lowry (30).

All centrifugations were at roomtemperature unless otherwise stated, at low speeds in a refrigerated centrifuge (model PR-2,

Damon/InternationalCentrifuge Co.,)at2°Cand atroom

tempera-tureinacentrifuge(model UV,Damon/InternationalCentrifugeCo.). Ultracentrifugation wasperformed in acentrifuge(model L5-65; Beckman Instruments, Inc., Fullerton, CA)at2°C.

Dialyses were performed in Spectro-Por 2 cellulose casings (6.4

mmdiam molecularweight cut-off 12-14,000; Spectrum Medical

In-dustries,

LosAngeles, CA).

Results

The inhibitory effect

ofPBMC suspensions and their

superna-tant

fluids

on

the

activation

of

HF

by ellagic

acid.

Suspensions

of PBMC inhibited

the

activation of

HFby

ellagic acid,

as measured by

amidolysis of

HPPAN. In atypical experiment,

PBMC,

at

initial concentrations of

6.25 X 105 cells/ml or more,

significantly inhibited

activation ofHF(Table I).

Simi-larly,

the supernatant

fluid of

PBMC cell suspensions inhibited

activation of

HF.

Inhibitory properties for

theactivation of HF

weredetected in

variable

but essentially similar amounts in the supernatant

fluids of nine further

washes

of

the PBMC

suspen-sion (data

not

shown).

The

inhibitory activity of

different

su-pernatant

fluid preparations

wasroughly

proportional

to

their

protein

content;a

dilution of

supernate that

inhibited

half the

amidolytic activity

of

the HF ina

mixture of

HFand supernate hada

protein

content

averaging

7.3

ug/ml (SD±2.72

Aig/ml).

The inhibitory properties ofplatelets, monocytes and

lym-phocytes on the activation of HF by ellagic acid. The

PBMC

used in the experiments

described

thusfarwere

contaminated

with platelets, containing, on the average, 13.3 (SD±7.0) platelets perPBMC (Table I). Platelet suspensions and their

supernatesinhibited activation of HF by ellagic acid, but this wassignificant only at relatively high concentrations of plate-lets(Table II).

Comparison of the data presented in Tables I and II

sug-geststhataportion of theinhibitory effect of PBMC suspen-sionswasduetotheircontentofplatelets. When PBMCwere

preparedfrom normal human blood that had been depleted of

- 95% of its platelet content,inhibition of the activation of

HFby PBMCwasreducedby - 50%, and by thesupernatesof

PBMC suspensions by - 75%. Thus, a significantportion of

theinhibitory properties of PBMC suspensions could be attri-buted to platelets. That platelets werenotsolely responsible for the inhibitory properties of PBMC suspensionswas demon-strated in studies of PBMC prepared from the blood of a pa-tient with severe thrombocytopenia. At concentrations of platelets that were not

inhibitory (Table

II), boththe cell sus-pension and the supernatant fluid inhibited activation ofHF

(Table I).

The PBMC were mixtures of -

80% lymphocytes

and 20%

monocytes. Amonocyte-enriched fraction of PBMC, prepared from platelet-depleted blood, and thesupernateof this

fraction

inhibited activation of HF (Table III). Similarly, the superna-tantfluidof a

suspension

of cultured

monocytoid cells

(U937)

inhibited activation

of HF

(Table

III).

A

lymphocyte-enriched

fraction of

PBMC,

prepared from blood that had been depleted of platelets, and the supernate of this fraction inhibited the activation of HF (Table III). Both T and B lymphocytes and their supernates were inhibitory

(Tables

IIIand

III).

The inhibitory properties ofgranulocytes and erythrocytes

and their

supernatant

fluids on the

activation

of

HF

by

ellagic

acid.

Granulocytes, separated

as

described,

and the superna-tantfluid of

granulocyte suspensions inhibited

activation of HF to

approximately the

same degree as PBMC suspensions (Table IV).

Neither

erythrocytes nor the supernatant fluid of erythro-cyte

suspensions had significant inhibitory properties

(Ta-bleIV).

Some

properties

of the inhibitory activity of

PBMC

super-nates. The

inhibitory properties

of

PBMC

suspensions

and their supernates were

directed against the activation of

HFand not at

the

amidolytic properties

of activated HF.

Addition of

cells

or supernatant

fluid after

HF

had

been

incubated with

ellagic acid for 60

min was

without

effect;

the

cells

and super-natant

fluid

themselves

did

not have

amidolytic

properties for

this substrate

(Table

I).

After ultracentrifugation, both

the cytosol and

particulate

fractions of PBMC inhibited the activation of HF by ellagic

acid,

butin

repeated experiments

the

cytosol appeared

tobe

relatively

more

inhibitory

(Table V).

Similarly,

both cytosol and

particulate fractions of lymphocytes

and monocytes were

inhibitory

(data

notshown).

The

inhibitory properties

of PBMC supernates were not reduced

by filtration of

supernatesthrough a Millex-GV

filter,

said to retain

particles

>0.22

Am

diam),

nor

by

dialysis

through

casings with

amolwt

cut-off

of

12-14,000.

(6)

re-TableI.

Effect

ofPeripheral Blood MononuclearCells on the Activation

of

HF by Ellagic Acid

Testmixture Amidolysis*

Cell suspension Supernatedilution

PBMC Platelets Cellsuspension Supernate Buffer

cells/ml cells/ml nmolp-NAreleased/ml/h

A. Normalblood

5X 106 9 X107 Undilute 2.8 0.5

2.5X 106 4.5X 107 1/2 3.4 7.5 -1.25X 106 2.25X107 1/4 6.9 18.2

6.25X10' 1.25X107 1/8 16.8 24.5 3.13X 10' 6.25 X106 1/16 24.1 25.4

0.0 0.0 00 - 27.6

5 X

106*

9 X

107*

Undilutet

1.9 0.8

5 X 106§ 9 X 107§

Undilutel

28.5 25.4

B.Bloodof a patient with aplastic anemia

5 X 106 2.7X106 Undilute 3.4 0.7 2.5X

106

1.35X 106 1/2 3.7 1.6 1.25X

106

6.75X 10' 1/4 12.3 12.0 6.25X 10' 3.38X 10' 1/8 15.9 18.7 3.13 X 10' 1.69X 10' 1/16 17.6 19.4

0.0 0.0 00 - 21.4

*_{Amidolytic activity generated}inamixture ofHF,testmixture(seriallydiluted withDPBS)andellagicacid(seeMethods). The test mixtures werePBMC (5X

106

cells/mi DPBS),the supernatant of the PBMCsuspensionandDPBS alone(buffer). (A)ThePBMC contained 9% mono-cytes and 91%lymphocytes,theplateletcountin the undiluted PBMC

suspension

was9X107 cells/mlor18plateletsperPBMC,and the pro-tein content of the supernatewas38ug/ml.(B)PBMCwerepreparedfrom the blood ofapatientwithaplasticanemia(peripheralblood

plate-letcount

5000/Ml).

The PBMCsuspension contained6% monocytes and 94%lymphocytesand theplateletcountin theundiluted PBMC

sus-pensionwas2.7X106cells/mlorapproximatelyone

platelet

for each 2 PBMC. The

protein

contentof the undiluted supernatewas16ug/ml.

In 12X75-mmpolystyrene tubes, 0.1 ml HF(0.05 U/ml,58U/mg protein)and 0.1-mltestmixturewereincubatedat370C for 10min,after which 0.05 ml4 X

10-'

Mellagicacidwasadded and incubationwascontinuedfor 60 min.

Thereafter,

0.1 ml DPBS and 1.0 ml HPPANwere

added,incubationwascontinued foranadditional 60 min, and the reactionwasstoppedbyaddition of 0.3mlglacialacetic acid. Theamount

of

p-nitroaniline

(p-NA)that hadbeen releasedwasmeasuredagainstsuitable blankstowhichglacialacetic acid had been added before HPPAN. *_Bovinealbumin in BS substituted for HF in the

original

mixture. I_DPBS_{substituted for PBMC}_or_{PBMC supernate in the}

origi-nalmixture and 0.1 ml PBMCorPBMC supernate addedtothemixture instead of DPBS after the60-minincubation of the mixture ofHF

and

ellagic

acid.

Table

II.

Effect

of

Platelets

on

theActivation

of

HF

by Ellagic Acid

Supernate

Platelets dilution Platelets Supernate Buffer

cells/ml nmolp-NAreleased/mil/h l08 Undilute 12.4 3.4

5x

107

1/2 19.0 9.8 2.5X107 1/4 20.9 18.0 1.25X 107 1/8 23.6 22.0 6.25 X

106

1/16 24.6 25.2

0.0 00 24.6

*_{Amidolytic activity generated in a mixture of HF, test mixture, and}

ellagicacid (see Methods). Thetestmixturesweresuspensions of platelets

(101

cells/ml),serially diluted with DPBS, the supernatant fluid separated from each suspension of plateletsandDPBS alone. The assays wereperformedasdescribed in the footnotetoTableI. Thespecific activityof theHF was58 U/mgprotein.

tained after incubation at room temperature or 370C

over-night

but

were

reduced

by incubation

at

higher

temperatures

for 30

min

by

approximately

one-fourth

at

560C and one-half

at

60'C,

and

essentially

completely by incubation

in a

boiling

water

bath

for this time.

Binding

of

HF to

the

inhibitory

agent(s) in PBMC

super-nates

could

not

be demonstrated. The

coagulant titer of

HF wasthe same, whether

filtered through

acolumnof agarose to

which PBMC

supernate had been

covalently

linked,

or

through

acontrol column

of

agarose.

Incubation of PBMC

or

PBMC

supernates

with trypsin

reduced

their

inhibitory properties, suggesting

that a

protein

component

participated

in

inhibition of the activation of

HF

(Table VI).

In agreement

with

this interpretation,

approxi-mately half of

the

inhibitory properties of

PBMC supernates were

precipitated

by ammonium sulfate

at

60% saturation.

No

evidence

was

obtained that the

inhibitory properties of

PBMC

supernatant

fluids

were

enzymatic.

The

inhibitory

properties of

PBMC supernate appeared to be

essentially

in-stantaneous

(Fig.

1).

When PBMC supernate was

incubated

(7)

Table III.

Effect

of

Monocytes

and

Lymphocytes

onthe Activation

of

HFby Ellagic Acid

Supernate

Cells dilution Cells Supernate Buffer

cells/ml

A.Adherent cells 5x 106 2.5X 106 1.25X106

6.25 X

10'

0.0

B.Monocytoid cells

C.Nonadherent cells 5X 106 2.5 x 106 1.25 X 106 6.25 X

10'

0.0 D. B cells

SX106 2.5X 106 1.25X 106 6.25X 105

0.0 E.Tcells

S X 106 2.5>X 106 1.25 x 106 6.25X 105

0.0

nmolp-NA released/ml/h

Undilute 1/2

1/4 1/8

00

Undilute 1/2 1/4

1/8

00

Undilute 1/2 1/4 1/8

00

0.6 0.1 0.6 13.5

0.3 0.0 6.4 21.8

1.5

3.8

11.5

20.9

0.0 1.0 10.3 19.3

0.9 0.8 10.6 7.4

0.0 0.7 1.6 8.0

0.0 2.6 19.6 25.6

0.2 3.9 13.5 19.7

1.3 11.8 18.2 21.3

32.1

22.9

28.9

19.8

22.0

*

_Amidolytic

_{activity generated in a mixture of HF,}

testmixture

(se-riallydiluted with DPBS) andellagicacid (see Methods). The test

mixturesinAwerepolystyrene-adherentcells, at an initial concen-tration of 5X 106cells/ml,prepared from PBMC that had been sepa-ratedfrom platelet-depleted blood, and thesupernateof the initial adherent cellsuspension;the adherent cell suspension contained 94% monocytes, 6%lymphocytesand2.3X 10'platelets/ml or 4.6 plate-lets per adherent cell. The testmixturein B was the supernate of a

suspensionof human monocytoid cells (U-937) containing 5X 106 cells/ml.ThetextmixturesinC were nonadherentcells,at aninitial concentration of 5X

106

cells/ml, prepared from PBMC that had

beenseparatedfrom platelet-depleted blood, and the supernate of the initial nonadherentcellsuspension; the nonadherentcellsuspension contained 1.5percent monocytes, 98.5 percentlymphocytes,and 2.4

X _{106 platelets/ml, or one platelet per 2 nonadherent}_cells._{The test}

mixtures inDandEwereBand T cells and their supernates,

respec-tively.TheBcell preparation contained 85 per cent B cells, and the

Tcell preparation, 81 percentTcells. The assay was performed as described in the footnote to TableI.The specific activity of the HF

usedin A, C, D,and E was58 U/mg protein, and in B, 98 U/mg protein. A,B,C,D, and E represent separate experiments.

Table IV.

Effect

ofGranulocytes

and

Erythrocytes

onthe Activation

of

HF

by Ellagic

Acid

Test mixture Amidolysis*

Supernate

Cells dilution Cells Supernate Buffer

cells/ml nmolp-NAreleased/ml/h

A.

Granulocytes

5X 106 Undilute 0 3.3 2.5X 106

1/2

0 8.9 1.25X 106

1/4

3.5 15.9 6.25X 105 1/8 4.2 18.2 3.12X 105

1/16

11.8 20.1 1.56X 105

_1/32

15.8 20.3

0.0 00 20.7

B.

Erythrocytes

2X 107 Undilute 19.9 20.7

0.0 00 21.5

*

Amidolytic

activity generatedin 12X75mm

_polystyrene

tubes in

amixture of0.1ml HF(0.05

U/ml,

58U/mg protein),0.1 ml test

mixture, seriallydiluted withDPBS,and0.05 ml 2 X10og Mellagic

acid

(see

Methods).Thetestmixtures includedneutrophils(5 X 106

cells/ml

DPBS), erythrocytes (2X

107 cells/ml

DPBS),and the

super-natesof these cell suspensions. Thegranulocytepreparation

con-tained 94%segmented neutrophils,4% eosinophils and 2%

lympho-cytes. Noplateletswerediscerned in stainedsmearsof the

granulo-cyteorerythrocytesuspensions. The protein content of the undiluted

(100%)supernatant of thegranulocyte suspensionwas

17Atg/ml,

and oftheerythrocyte suspension,<8

ug/ml.

with

HF

for

varying

intervals before addition

of

ellagic acid, its

inhibitory activity gradually

decreased. In

contrast,

when

su-pernatewas

incubated with

ellagic

acid

for

a

variable

period

Table V.

Effect

of CytosolandParticulate Fractions of

PBMC on theActivation ofHFby Ellagic Acid

Amidolysis*

Test mixture Particulate

dilution Cells Cytosol fraction Buffer

Undilute 3.7 1.8 4.8 1/2 2.2 0.6 4.6

1/4 1.9 1.9 21.0

1/8 5.9 1.3 21.9

1/16 15.6 13.5 20.2

1/32 18.0 17.7 19.8

00 20.4

*_{Amidolytic activity generated in 12}_X_{75 mm polystyrene tubes in}

amixture of HF,testmixture (serially diluted with DPBS). Thetest

mixtureswerePBMC(5 x 106 cells/ml DPBS), the cytosol fraction ofPBMC separated by ultracentrifugation of freeze-thawed PBMC, the particulate fraction after ultracentrifugation, diluted with PBMC

tothe original volume, and DPBS alone (buffer). The PBMC

con-tained 20% monocytes and 80% lymphocytes. Amidolytic activity

wasmeasuredasnoted in the footnote to Table I. The specific

activ-ityofthe HFwas100 U/mg protein.

(8)

Table VI.

Effect

of

Trypsin

on the

Inhibitory

Properties

of

PBMC and PBMC

Supernate

Testmixture Trypsin HF Amidolysis*

nmol p-NA released/ml/h

DPBS 0 + 15.8 DPBS + + 17.8

DPBS + 0 0

PBMC 0 + 4.6 PBMC + + 11.0

Supernate 0 + 5.1

Supernate + + 14.1

* In 12X75mmpolystyrenetubes,

0.1

ml DPBSorPBMC or PBMC supernate wasincubated with0.025 ml bovine trypsin (0.05

mg/ml)orBSat370C for2 h.Thereafter,0.025 mlLBTI, 0.2

mg/ml

and 0.05 ml HF(0.1 U/ml)orBS-Albalonewereadded inrapid

suc-cession and, after 10 min, 0.05 mlellagicacid, followed,after 1 h, by 1 ml HPPAN.Afteranadditional60 min, the reaction was stopped

by additionof 0.3 mlglacial acetic acidand theamount

ofp-nitroan-iline (p-NA) releasedwasmeasuredagainstblankstowhichglacial acetic acidwasaddedbefore additionof substrate. The PBMCwere

suspendedat aconcentration of5X

106/ml

DPBS;the supernate of PBMCwasin thesamebuffer. Trypsin, LBTI,andellagicacidwere

dissolved

in BS.The specific

activity

of

the HF

was100 U/mg protein.

before addition of

HF,

it

retained

its

inhibitory properties

for

atleast 60

min.

At the

concentrations

tested,

the inhibitory

properties

of

PBMC supernatant fluids

were not

impaired

by incubation with 1 mM

DFP. Nor

was

inhibition of the

activa-tion ofHFprevented

by

addition of0.5 mM EDTAor

0.5

mM

benzamidine

tothe initial

mixture of

HF

and

supernate.

Ap-propriate concentrations ofSBTI

or

LBTI

are

known

to inhibit the

amidolytic properties

of

ellagic

acid-activated HF

(31).

At

concentrations

of 30

ig/ml

orless in the

mixture

ofPBMC

supernate,

HF and

ellagic

acid, concentrations

ofSBTI or

LBTI that did

not

affect

amidolysis by activated HF,

these agentsdid not

block the

inhibitory properties

of PBMC

super-nates.

The

inhibitory properties

of PBMC supernates could

not be identified with other

agents

that

might

influence the

activa-Figure 1.

Time

course of

inhibition

byPBMC

super-P Efi x~x~x x x x natesof the activation of

as v 20[ ~ _HF

by

ellagic

acid. In 12 101 H0 . , . X 75 mm polystyrene

z tubes, O. mlPBMC super-< nate

(diluted

50% with

2.5

5 10 20 40

_DPBS)

wasincubatedfor

INITIA INCUBATION PERIOD~min) 10sto40 minwith 0.05 iNITIAL INCUBATION PERIOD (minI mlellagic acid (2 X

_10-1

M), after which0.1ml HF (0.05 U/ml, 58 U/mg protein) was added. Themixturewasincubatedforanadditional 60 min, 1.0ml HPPAN wasadded, andincubationwascontinued for 60 min (!). Con-versely,0.1 ml supernate wasincubatedwith0.1ml HFfor

lO

sto 40 min,

after

which 0.5mlellagicacidwasadded and the procedure

continuedasbefore(o). The reactionwasstoppedbyaddition of 0.3 mlglacialacetic acid and theamountofp-NA releasedwas

mea-sured(see Methods).The

amidolytic

activitygeneratedin mixtures

of0.1 ml HF and 0.1 mlDPBS,incubated for 10sto40 min before addition of

_ellagic

_acid,

wasalso measured

(x).

tionof

HF.

Thus,

the

thrombin time

was

unaltered

ina

mix-ture of

0.2

ml each of normal

_plasma,

concentrated PBMC

supernate

(56 ,ug

protein/ml)

and bovine thrombin

(5

U/ml),

suggesting

that the

_inhibitory

effects of PBMC

supernateswere

not

attributable

to

heparinlike

agents.

Supporting

this

_{interpretation,}

no

uronic acid

was

detected

inaconcentrate

of PBMC

supernate

containing

1mg

protein/

ml.

Moreover,

the addition of

PF4,

at

concentrations

as

high

as75

_,ug/ml

ina

mixture

of

PBMC supernate and

HF

did

not

neutralize the

_{inhibitory properties}

ofthe

supernate.

Rather,

as

demonstrated

in

experiments

in

which its

concentration was

reduced

to

0.24 isg/ml,

PF4 itself inhibited the

activation

of

HF

_{by ellagic}

_acid,

and its

_{inhibitory activity}

was

additive

to

that of PBMC

supernates.

The

inhibitory

properties

of

PBMC

supernates

could

not

be

attributed

to

_PF4,

which

could

not

be

demonstrated

upon

immunodiffusion

against

antiserum

against

PF4;

a

crude

IgG

fraction of this antiserum did

not

neutralize

the

inhibitory

properties

of

PBMC

supernates

(Table

VII).

PBMC supernates

didnotcontain

_{material coagulable with}

thrombin.

They

didnot alter the

prothrombin

time

or

acti-vated

_{partial thromboplastin}

time

of

normal

plasma (data

not

shown).

Nor did

they

inhibit

the

amidolytic properties

of CI

(data

not

shown);

C!

inhibitor

wasnot

demonstrable by

im-munodiffusion,

anda

crude

immunoglobulin fraction

of

spe-cific

antiserum

did not

reduce the

inhibitory

properties of

PBMC

supernates

(Table

VII).

TableVII.

_Effect

_of

Antiserumsonthe

_Inhibitory

Properties

of

PBMC

Supernate

Amidolysis*

A. Buffer

PBMC supernate+buffer

PBMC supernate+normal goat

IgG

PBMC supernate+goatanti-Clq

IgG

PBMC supernate+normal rabbit

_IgG

PBMC supernate+rabbit

anti-02-glycoprotein-I

IgG

B. Buffer

PBMC supernate+buffer

PBMCsupernate+ normalgoat

IgG

PBMC supernate+goatanti-PF4

IgG

PBMCsupernate+normal rabbit

_IgG

PBMC supernate + rabbit

anti-CI-INH

22.6

11.9

9.9

9.2 9.2

10.6

21.5

11.9

13.5 14.7 12.9 14.1

* In 12X75mmpolystyrene tubes,0.05 ml PBMC supernateor

DPBS(buffer)wasincubated for 30minat370C with 0.05 mlIgG

orbuffer,after which0.1mlHF(0.05 U/ml)wasadded, followed, after 10

min,

by0.05 mlellagicacid. The evolution of amidolytic

ac-tivity

wasthen determinedasdescribed in Methods. (A) Thespecific

activity

ofHFwas58U/mg protein,theproteincontent of the PBMC supernatewas22$g/mland the initial concentration of

ellag-ic acidwas2X

10-'

M.(B)The specific activity of HF was 43U/mg protein,the proteincontentof the PBMC supernate was 15ug/ml,

and the initial concentration of ellagic acid (of another lot) was 4

X

10-4

M. The initialproteinconcentration ofIgGin Awas2.2

(9)

Clq and j32-glycoprotein-I inhibit theactivation of HF(32, 33).Neither of these proteins couldbedemonstratedin a

con-centrate of PBMC upon immunodiffusion against antisera against this protein. Further,a crude

immunoglobulin

fraction of these antiserums didnotneutralize the inhibitory effect of PBMC supernates against the activation ofHF(Table VII). At veryhigh

concentrations,

PBMC supernates had some inhibi-toryactivity against human urokinase-activated plasmin; a

preparation of PBMC supernate (508 ,ug

protein/ml)

reduced the amidolytic activity of this enzymefrom 37.0 to 29.4nmol

p-NA

released/ml per h by the method described.

Incubation of PBMC for 16 h with cycloheximide (50

,gg/ml) sharply

reduced the

inhibitory properties

ofPBMC

su-pernates,which contained strikinglylessprotein thanthe

su-pernates of untreated

cells,

as

though

protein synthesis

had

been

depressed

(Table

VIII).

Similarly,

PBMC treated with

cycloheximide were less

inhibitory

than untreated

cells,

but the difference was less pronounced.

Incubation

of PBMC

overnight

with sodium azide at a

Table VIII. Effect of

Cycloheximide

and Sodium Azide on the Inhibitory

Properties

ofPBMC and Their Supernates

Amidolysis*

Treated Treated

Test mixture dilution PBMC PBMC Supernate supernate Buffer

nmol p-NAreleased/mil/h

A.Cycloheximide

Undilute 2.9 1.6 0.3 14.3 1/2 1.9 3.7 0.6 22.8 1/4 1.4 15.8 2.1 25.6 1/8 7.0 22.2 12.4 26.4 1/16 18.4 24.4 22.5 24.4

o 27.4

B.Sodium azide

Undilute 2.1 1.1 0.3 9.9 1/2 0.9 4.8 0.2 19.3 1/4 0.9 18.3 0.8 22.3 1/8 7.8 21.7 10.8 22.6 1/16 16.9 22.6 20.6 22.9

_-

25.3

InB, PBMC and sodiumazide-treatedPBMCandtheirsupernates were prepared asdescribed inMethods. Tested by trypan blue

exclu-sion,the PBMC in theoriginal suspensionwere96%viable. After in-cubationfor 20 h, PBMC treated with sodium azidewere3%viable, and untreated PBMCwere92%viable. The PBMC and their super-nates weretestedfor their capacitytoinhibit the activation ofHFby

ellagicacidasin TableI(seeMethods).Theproteincontentof the

supernateof untreated cellswas78

Mg/ml;

noproteinwasdetectedin thesupernateof treated cells. TheHFpreparation used hadaspecific

activityof 56 U/mg protein.

*_{In A, PBMC and}_{cycloheximide-treated}_{PBMC and their}

super-nates were prepared asdescribed inMethods. Tested by trypan blue

exclusion,thePBMC intheoriginal suspensionwere91%viable. After incubation for 16h,PBMC treatedwithcycloheximide were

33%viable,and untreated PBMC were 50% viable. The PBMC and

theirsupernates weretestedfor their capacitytoinhibitthe

activa-tionofHFby ellagic acidas inTableI(see Methods). Theprotein

contentof thesupernateof untreatedcellswas 77

Ag/ml,

and of

cy-cloheximide-treated cells, 10 ,ug/ml. TheHFpreparationused had a

specific activity of 58 U/mg protein.

final

concentration of 0.5

mg/ml,

sufficienttokill 97% of

cells,

greatly

reduced the

inhibitory

properties

of

both

the cells

and

the supernatant

fluid,

whichnowcontainedno

detectable

pro-tein

(Table

VIII).

The

inhibitory

properties

of

PBMCorPBMC supernates

for

the activation

of

HF

by glass

and

by sulfatides.

Conceiv-ably,

in the

experiments

recorded thus

far, inhibition

ofthe activation of HFmayhave been due toa

specific interaction

between

PBMCorPBMC

supernatant

fluid and

ellagic

acid. Thesame

inhibitory characteristics

were,

however,

demonstra-ble when activation of HFwascarried out

by

exposureto

glass

(Table IX). Consistently,

at

higher dilutions,

PBMC super-nates enhanced

amidolysis,

a property not observed in the

absence

of

HF. This enhancement

of

amidolysis

was not blocked

by

treatment ofthe supernates with 1.0 mM DFP

(data

not

illustrated).

The plasmas

of

individuals with Hageman

traitor

plasma

prekallikrein deficiency (Fletcher trait),

and several

ill-defined

fractionsornormal

plasma,

containagentsthat adhereto

glass

andinhibit its

capacity

toinitiate clotting (24, 34, 35). PBMC

supernates,

however,

did not appear to share this property. Coating glass tubes with the plasma ofapatient with Hageman trait lengthened the partial thromboplastin time of normal

plasma

from79.5s,testedin tubes coated with BS,to 107.8s.

Incontrast,the

clotting

time of tubes coated with PBMC su-pernatantfluid (15Mgprotein/ml)was74.8s.

Table IX.

Effect

ofPeripheral Blood MononuclearSupernates onthe Activation

of

HF

by

Glass and

Sulfatides

Amidolysis PBMC supernate

dilution Glass* Sulfatidest

nmolp-NAreleased/ml/h

Undilute 2.0 1.3

1/2 8.1 1.3

1/4 13.8 12.6 1/8 12.8 27.8

00 10.9 31.0

0o§ 0.5

Undilutell

11.3

* Mixtures of 0.25 ml each ofa concentrateofPBMC supernate,

di-lutedserially in DPBS, andHF(58U/mgprotein, 0.05U/ml) were

prepared in 12X75mmpolystyrenetubes.Aliquots of0.2ml were

transferredto10X75mmglass tubes andincubatedat370Cfor60 minwith continual shaking. Then, 0.1 mlDPBS and 1.0 ml HPPAN wereaddedtoeachtube andincubationwascontinued foran

addi-tional 60min.Thereactionwasstoppedby addition of 0.3mlglacial acetic acid and theamountofp-NAthathad been releasedwas

mea-suredagainstsuitable blankstowhichglacial acetic acidhadbeen added before HPPAN.