Blood, Vol 70, No 1 (July), 1987: pp 69-76 69

Immunological Specificity and Mechanism of Action of

IgG Lupus Anticoagulants

By Vittorio Pengo, Perumal Thiagarajan. Sandor S. Shapiro, and Marilyn J. Heine

Although observations have implied that lupus anticoagu-

lants have immunologic specificity toward anionic phos-

pholipids. this assumption has been directly demonstrated

in only one patient with a monoclonal 1gM paraprotein. We

tested the generality of this hypothesis directly by isolating

five lgG lupus anticoagulants from patients with lupuslike

syndromes and/or thrombosis. lgG lupus anticoagulant

fractions were isolated free of other plasma proteins and

free of contaminating phospholipid by adsorption to and

elution from cardiolipin-cholesterol-dicetyl phosphate

liposomes. followed by chromatography on protein A-

Sepharose. Cardiolipin liposomes. but not phosphatidyl-

choline liposomes, were capable of removing all, or nearly

all. lupus anticoagulant activity from patient plasma. The

affinity-purified lgG preparations reacted with cardiolipin,

L UPUS ANTICOAGULANTS are acquired inhibitors

of coagulation tests that occur in systemic lupus ery-

thematosus (SLE), in various other clinical settings, and in

apparently normal persons.’

Because the lupus anticoagulant effect is noted in all

phospholipid-dependent coagulation tests2�6 and because of

the correlation between the presence of lupus anticoagulants

and reactivity against cardiolipin in solid-phase immunoas-

says,7�” lupus anticoagulants have been widely assumed to be

antibodies reactive against anionic phospholipids. Actually,

the immunologic nature and mechanism of action of the

Iupus anticoagulant effect have only been demonstrated in

one patient.’2 In 1980, our laboratory’2 clarified the mecha-

nism ofaction ofan 1gM A paraprotein with lupus anticoagu-

lant activity. The purified paraprotein showed immunologic

reactivity against the anionic phospholipids phosphatidylser-

me, phosphatidylinositol, and phosphatidic acid, but did not

react with the neutral phospholipids phosphatidylcholine or

phosphatidylethanolamine. This protein inhibited the bind-

ing of prothrombin and factor X to phospholipid micelles,

thereby accounting for the prolongation of phospholipid-

dependent coagulation tests.

Since this initial work, it has been widely assumed,

although not rigorously demonstrated, that all lupus antico-

agulant activity is due to the same mechanism. However,

other authors have stressed the heterogeneity of the lupus

anticoagulant patient population, as well as the apparent

variability in the response of lupus anticoagulant tests to the

presence of platelets,’3 to argue against a common mecha-

nism ofaction. Interestingly, neither the original patient, nor

four other 1gM lupus anticoagulants we subsequently stud-

ied, nor 1gM lupus anticoagulants reported by others,’4”5

have been associated with thrombosis. Most lupus anticoagu-

lants, on the other hand, occur in a setting of polyclonal

increases in immunoglobulins and are associated with a high

incidence of thrombosis.”62’ To assess whether there is

indeed a general mechanism for the lupus anticoagulant

effect, a technique for isolating lupus anticoagulants from

plasma of patients without monoclonal paraproteins is neces-

sary. The preparation must be a pure immunoglobulin free of

contaminating phospholipid. We developed a technique that

phosphatidylserine. phosphatidylinositol. and phosphatidic

acid, but not with phosphatidylcholine or phosphatidyl-

ethanolamine. and inhibited calcium-dependent binding of

prothrombin and of factor X to phosphatidylserine-coated

and to cardiolipin-coated surfaces. F(ab’)2 fragments

retained Iupus anticoagulant activity and bound to cardioli-

pin in an enzyme-linked immunosorbent assay (ELISA).

Anticardiolipin and lupus anticoagulant activity were bothpresent in acidic fractions on isoelectric focusing. These

data strongly suggest that most, if not all, lupus anticoagu-

lants are antibodies that have immunologic specificity

towards anionic phospholipids. thereby blocking the calci-

um-mediated binding of vitamin K-dependent coagulation

factors to coagulation-active phospholipid surfaces.

S 1987 by Grune & Stratton, Inc.

uses affinity purification on cardiolipin liposomes followed

by protein A-Sepharose chromatography to prepare phos-

pholipid-free purified lupus anticoagulants. We isolated five

IgG lupus anticoagulants, four from patients with a history

of thrombosis, and examined the immunologic nature of their

interaction with phospholipids and the mechanism of their

inhibition of phospholipid-dependent coagulation tests.

MATERIALS AND METHODS

Human thrombin was a gift from Dr John Fenton, New York

State Department of Health Laboratories, Albany. Pepsin waspurchased from Worthington Biochemical, Freehold, Ni. Protein

A-Sepharose, Sephacryl G-75 (Superfine) and Sephacryl 5-300

were obtained from Pharmacia Fine Chemicals, Piscataway, NJ.

Ampholines were purchased from LKB, Bromma, Sweden. Phos-phatidylserine, phosphatidylinositol, phosphatidylethanolamine,

phosphatidic acid, and phosphatidylcholine were obtained from

Supelco, Bellafonte, PA. Cardiolipin, dicetyl phosphate, cholesterol,alkaline phosphatase-labeled goat antibodies to human lgG. human1gM, and rabbit IgG, as well as p-nitrophenyl phosphate and bovineserum albumin (BSA) (fraction V) were purchased from SigmaChemical, St Louis. The purity of phospholipids was checked by

thin-layer chromatography.2’ Russell viper venom was obtained

from Burroughs Wellcome, Raleigh, NC, and Thrombofax waspurchased from Ortho Diagnostic Systems, Raritan, NJ. Antisera tohuman IgG, IgA, and 1gM were obtained from Meloy Laboratories,

From the Cardeza Foundation for Hematologic Research.

Department of Medicine, Jefferson Medical College of Thomas

Jefferson University, Philadelphia.Submitted December 5. 1986; accepted February 1 1. 1987.Supported in part by Grants No. HL-09163. AG-04861 (5.5.5.).

HL-27278 (P. T.), and T32-AM-07084 (M.J.H.)from the National

Institutes ofHealth; an Established Investigator Award (84-190); a

grant-in-aid from the American Heart Association (P. T.); and the

Sheryl N. Hirsch Award ofthe Lupus Foundation of Philadelphia

(5.5.5.). V.P. is a Visiting Research Fellow from the Cardiology

Department. University ofPadua. Italy.Address reprint requests to Dr Sandor S. Shapiro. Cardeza

Foundation, Jefferson Medical College. 1015 Walnut St. Philadel-

phia, PA 19107.© 1987 by Grune & Stratton. Inc.

0006-4971/87/7001-0008$3.00/0

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70 PENGO ET AL

Springfield. VA. Standard laboratory chemicals were supplied byFisher Scientific, Fair Lawn, NJ. Carrier-free 1251 was obtained fromCambridge Nuclear, Billenica, MA.

JgG Purification

Lupus anticoagulant plasma was clotted by incubation with 2

U/mL of human thrombin for 2 hours at 37#{176}.Fibnin was removed bycentnifugation at 4#{176}Cfor 10 minutes at 14,000 g. y-Globulins wereprecipitated from the supernatant serum by addition at 4#{176}Cof an

equal volume of saturated ammonium sulphate. After 25 minutes of

stirring, the precipitate was recovered by centnifugation in the coldfor 10 minutes at 14,000 g. The precipitate was dissolved in

approximately one-seventh the original plasma volume, using Tnis-buffered saline (TBS: 0.02 mol/L of Tnis, 0. 1 5 mol/L of NaCI, pH

7.4), and applied to a 2.5 x 90-cm column of Sephacryl 5-300equilibrated with TBS. Fractions of 2.5 mL were collected and testedqualitatively for IgG, 1gM. and IgA by Ouchterlony double diffusion

and for lupus anticoagulant activity by their ability to prolong thedilute Russell viper venom time (RVVT) of normal plasma.23’�4

Fractions containing lupus anticoagulant activity were pooled and

applied to a I x 5-cm column of protein A-Sepharose equilibratedwith TBS. The lupus anticoagulant activity, in every case, wasretained on the column and was eluted using a buffer consisting of

0.1 mol/L of glycine, 0.5 mol/L of NaCl, pH 3, and immediatelyneutralized with I mol/L of Tnis buffer, pH 8.4. Eluates were

dialyzed against TBS before being tested for lupus anticoagulantactivity. Control lgG was prepared from normal plasma in anidentical manner. IgG prepared in this manner is free of other

immunoglobulins and shows no bands other than IgG on sodiumdodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)

after staining with Coomassie blue.

F(ab’)2 Preparation

After dialysis against acetate buffer (0.2 mol/L of Na acetate, 0.2mol/L of NaCI, pH 4), purified IgG (2 to 3 mg/mL) was incubated

at 37#{176}Cfor 20 hours with freshly prepared pepsin at a weight ratio of50: 1 25 At the end of the 20-hour incubation, no intact IgG wasdetectable by SDS-PAGE. The reaction was stopped by adding solid

Tnis (5 to 10 mg). The digested material was dialyzed against TBSand applied to a 1 x 5-cm protein A-Sepharose column. Theflowthrough, containing the F(ab’)2 fragments, was concentrated by

Amicon ultrafiltration using PM-l0 membranes (Amicon, Lexing-ton, MA) and tested for lupus anticoagulant and anticardiolipinactivities. The protein A-Sephanose flowthrough was free of whole

IgG and Fe-fragments, as judged by SDS-PAGE and by doublediffusion against antiserum to human IgG (Fe-specific).

ELISA for Anticardiolipin Antibodies

One microgram of cardiolipin in 25 j�L of ethanol was added toeach well of a 96-well microtiter plate (Falcon #391 1, BectonDickinson, Oxnard, CA) and evaporated under a stream of nitrogenfor 20 minutes, after which the wells were washed twice with I 00 �zL

of phosphate-buffered saline (PBS: 8 g/L of NaCI, 0.2 g/L ofKH,P04. 2.9 g/L of Na2HPO4 . l2H20, 0.2 g/L of KCI, pH 7.4).All subsequent steps were carried out at room temperature. One

hundred microliters of 10% fetal calfserum (FCS) in PBS was thenadded to each well and incubated for 2 hours to block nonspecific

binding. After the plates were washed six times with PBS, 100 j.tL of

plasma diluted in PBS containing 10% FCS was added to each well

and incubated for I hour. The wells were washed six times with PBS,after which 100 �.iL of a 1:1000 dilution of alkaline phosphatase-

labeled goat anti-human IgG or goat anti-human 1gM was added

and incubated for I hour. The plates were then washed six times

more in PBS, and 100 �iL of freshly prepared p-nitrophenyl phos-

phate solution (1 mg/mL in 10% diethanolamine buffer, pH 9.8)was added to each well. After I -hour incubation, the absorbance was

measured at 405 nm, using a Titertek Multiscan plate reader (FlowLaboratories, McLean, VA). In experiments using F(ab’)2, 100 �zL

of a 1:400 dilution of rabbit anti-human F(ab’)2 (Jackson Laborato-

nies, Avondale, PA) was used as a second antibody, and alkalinephosphatase-labeled goat anti-rabbit IgG was used as the developingantibody.

The ELISA assay for antibody activity against other phospholi-pids was performed in a similar manner, except that: (a) Two

micrograms of phosphatidylsenine, phosphatidylinositol, phospha-tidic acid, phosphatidylethanolamine, or phosphatidylcholine in 10

�L ofchloroform was added to each well ofa 96-well microtiter platecontaining 30 zL of ethanol; (b) when phospholipid reactivity of

eluates from cardiolipin liposomes was studied, 100 �iL-aliquots ofeluate were used; and (c) the reaction was stopped after I 5 minutes.

Isoelectric Focusing

Forty grams of Sephadex G-75 was washed and prepared asdescribed by the manufacturer, and finally suspended in 90 mL of

d#{231}ionized water. To the Sephadex was added: 2.5 mL of Ampholine,

pH 5 to 7; 2.5 mL of Ampholine. pH 7 to 9; and S mL of purified

patient IgG (10 mg/mL in 1% glycine). After gentle stirring, the

mixture was poured into a 10 x 20-cm glass tray, and excess waterwas evaporated under a stream of air. Focusing was performed at10#{176}for 1 5 hours at 8 W constant power. The gel was then cut into six

fractions. A small amount of each fraction was resuspended in

deionized water for pH measurement; the remainder ofeach fractionwas resuspended in 5 mL ofTBS in a small column, and the proteinswere eluted by washing the column with 5 mL of TBS. Column

eluates were dialyzed against TBS and tested for lupus anticoagu-lant and anticardiolipin activities.

Analytical isoelectnic focusing was performed in an LKB Multi-phor apparatus on 5% polyacrylamide gels containing 2.2% (wt/vol)Ampholine, pH 5.5 to 8.5. Purified IgG or liposome eluates weredialyzed against deionized water, applied to the gel, and run at 50mA with a constant power of 25 W. The gels were fixed for I hour in

a solution of 5% tnichloroacetic acid and 5% sulfosalicylic acid and

stained using the Bio-Rad silver-staining kit (Bio-Rad Laboratories,

Richmond, CA).

Immunoaffinity Purification ofAnticardiolipin Antibodies

Liposomes were prepared essentially according to the method of

Kinsky.” A mixture of cardiolipin, cholesterol and dicetyl phosphate(molar ratio I 0: 1 5:2) in chloroform was dried in a stream of nitrogen

in a 25-mL glass tube. The lipids were resuspended in 1 mL of 0.15

mol/L of NaCl by vigorous agitation, using a vortex mixer. Afterresuspension, the concentration of phospholipids was 5.3 to 5.9mg/mL. Ten milliliters each of saline and plasma were added to I

mL of liposome suspension. and the mixture was incubated at 4#{176}Cfor 30 minutes. After centnifugation at 27,000 g for 10 minutes at4#{176}C,the precipitate was washed three times with 0. 1 5 mol/L ofNaCl. The precipitate was resuspended by vigorous agitation in 2

mL of 3 mol/L of NaCI, following which the suspension was addedto a separatory funnel containing 10 mL of ethyl ether.27 Aftersettling, the clear aqueous layer was removed from the bottom of the

funnel, centrifuged, and dialyzed in the cold against three changes ofTBS. The dialyzed eluate was tested for lupus anticoagulant activity

by the dilute RVVT, for immunoglobulin class by Ouchterlonydouble diffusion, and for reactivity against phospholipids byenzyme-linked immunosorbent assay (ELISA). The eluate wasfurther purified by protein A-Sepharose column chromatography, as

previously described, and retested for purity and for biological

activities.

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MECHANISM OF LUPUS ANTICOAGULANTS 71

Labeling ofCardiolipin with !25J

Cardiolipin was radioiodinated directly by the chloramine-T

method, as described by Antonov and colleagues.28 Thin-layer

chromatography demonstrated that -60% of the added radioactivity

had been incorporated into cardiolipin. Most of the remainder

traveled with the solvent front. Labeled cardiolipin had the samemobility as standard cardiolipin. The spot containing the radioactive

cardiolipin was extracted from the silica gel plate as described

previously.22 When re-run on thin-layer chromatography, this

extracted labeled material showed a single spot with the mobility of

standard cardiolipin; no radioactivity was present in the solvent

front. On the basis of phosphorus content,29 the specific radioactivityof ‘25I-cardiolipin was calculated as 26 mCi/mmol.

Coagulation Studies and Assessment of Lupus

Anticoagulant Activity

Venous blood was collected in one-tenth volume of 3.8% tnisodium

citrate, and plasma was obtained by centnifugation in the cold at

2,300 g for 15 minutes. Routine coagulation studies were performed

as previously described.’2 Lupus anticoagulant activity of patient

plasma was assessed by the dilute RVVT, as previously

described.23’24 In our laboratory, the dilute RVVT of normal plasmais 25.6 ± 2.6 seconds ( ± 2 SD). Patients were not considered to have

lupus anticoagulants unless their dilute RVVT was >30 seconds andwas not corrected by addition of an equal volume of normal plasma.

The anticoagulant activity of IgG or of F(ab’)2 was measured in the

dilute RVVT by adding 200 �L of the dialyzed test material, instead

of the usual 100 ML, to this system. Addition of 200 �tL of TBS aloneto normal plasma gives a dilute RVVT of 27 to 30 seconds.

Prothrombin and Factor X Binding to Phospholipid

Prothrombin was purified from normal plasma by a slight modifi-cationt#{176}of the method of Morrison and Esnouf,3’ and labeled with1251 by the lodogen method32 to a specific activity of 231 Ci/mmol.

Factor X was purified according to the method of Miletich andco-workers,3’ with some modifications described previously,’2 and

labeled with 1251 by the lodogen method to a specific activity of 60Ci/mmol. Two micrograms of phospholipid was added to each well

of a microtiter plate and dried and washed as described above. Two

hundred microliters of TBS containing I mg/mL of BSA was thenadded, to block nonspecific binding, and the plates were incubatedfor I hour. After plates were washed once with TBS, 150 �zL of TBS

containing I mg/mL of BSA and 2 mmol/L ofcalcium was added,

followed immediately by 50 �L of immunoaffinity-punified IgG (134

zg/mL) or control IgG ( I 50 �.�g/mL) in TBS. The plates wereincubated for 1 hour at room temperature, after which 10 ML of

labeled prothrombin (I .4 x 106 cpm) or factor X (I .7 x 106 cpm)was added. After 10-minute incubation at room temperature, the

wells were aspirated dry, and the bottoms of the wells were punched

out and counted in an Isodyne ‘y-counter (Model 1 185, GD. Searle,

Chicago). Maximum binding of prothrombin was determined by

addition of TBS-albumin-calcium buffer alone, in the absence of any

IgG. As a control, prothrombin and factor X binding were measuredin the presence of 2 mmol/L of EDTA and were 3% and I 1% of

maximum binding, respectively. Residual binding, measured in the

presence of a 50-fold excess of cold prothrombin or factor X, wassubtracted from total binding, to give “specific” binding.

Patients

Patient no. I. The patient was a 35-year-old man with a 10-yearhistory of a manic-depressive illness for which he had been treated

with lithium, chlorpromazine, and other tnicyclic antidepressants.

He had not received any medication except lithium in the 6 months

prior to study. He had had several episodes of deep vein thrombosis

as well as at least one probable pulmonary embolism. He had a

normal platelet count, and his serological test for syphilis (RPR) was

negative. At the time of the study, he was receiving warfanin.

Patient no. 2. The patient was a 39-year-old woman with SLE of

8-year duration who had end-stage renal disease and was onhemodialysis. She had had a cerebrovascular accident at the age of

35, and also had a history of possible pulmonary emboli. She had

occlusion of arteniovenous shunts in the forearm on at least two

occasions. Her platelet count was in the low-normal range. She had a

false-positive serological test for syphilis (RPR) at the time of

diagnosis, but not at the time of study, when she was receiving 20 mg

of prednisone every other day.Patient no. 3. The patient was a 32-year-old woman with a

history of three spontaneous abortions, after which she delivered a

full-term live baby while on steroid therapy. She had a normal

platelet count. She had had a false-positive serology (RPR) since her

teens, but had no other signs of SLE.Patient no. 4. The patient was a 49-year-old man with a 10-year

history of SLE. He had an acute episode of right ileofemoral arterial

occlusion I year earlier, at which time a lupus anticoagulant was

discovered. He had a normal platelet count and a negative serologi-

cal test for syphilis (RPR).Patient no. 5. The patient was a 58-year-old woman with renal

disease and possible SLE. She had no history of thromboembolic

phenomena. She had a normal platelet count and a false-positive

serology (RPR). At the time ofthe study, she was receiving 20 mg of

prednisone daily.

As shown in Table I , all five patients had a prolonged dilute

RVVT, which did not normalize on addition of an equal volume of

normal plasma. Four of the five patients had a prolonged activated

partial thromboplastin time (APTT), whereas only one (patient no.

1, receiving warfarin) had a prolonged prothrombin time (PT).

RESULTS

Immunoglobulin Nature ofthe Lupus Anticoagulant and

Anticardiolipin Activities

Lupus anticoagulant activity in all five patients gel-

filtered on Sephacryl 5-300 in the IgG peak. Three patients

(patients no. I, 3, and 5) had, in addition, a very small

amount of inhibitory activity associated with the 1gM peak.

The maximum prolongation of the dilute RVVT in 1gM

fractions was 2 seconds, whereas the maximum prolongation

in the IgG peak was I 3 to 14 seconds. No prolongation of the

RVVT was observed in any of the Sephacryl 5-300 fractions

of control plasma. The IgG peaks were further purified by

chromatography on protein A-Sepharose. Glycine eluates of

the protein A-Sepharose columns contained the inhibitory

activity, whereas the flowthrough fractions had no lupus

anticoagulant activity.

IgG anticardiolipin activity was detected in all five patient

Table 1 . Screening Coagulation Tests

Patient

1 2 3 4 5 Normal

APTT (s) 90.0 27.0 42.6 38.4 42.0 23-36

PT(s) 23.2 11.7 13.2 12.2 12.0 11.5-13.5

Dilute RVVT (5)

Patient 38.2 33.4 34.0 36.5 46.3 23-28.2

Patent+normal(1:1) 36.6 30.4 29.9 32.0 42.7 23-28.2

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72 PENGO ET AL

After protein A-Sepharose chromatography.

plasmas; in addition, patients I , 3, and 5 had weak 1gM

anticardiolipin activity.

F(ab’), fragments prepared from patient IgG prolonged

the dilute RVVT of normal plasma in a dose-dependent

manner; normal F(ab’)2 had only a minimal effect on this test

(Fig IA). Patient F(ab’)2 fragments also bound in a dose-

dependent manner to cardiolipin in the ELISA, whereas

control F(ab’)2 showed only minimal binding (Fig 1 B).

Preparative isoelectnic focusing of purified IgG from

patient 1 (Fig 2) demonstrated a peak of lupus anticoagulant

and anticardiolipin activity in the first fraction, pH 5.8; in

contrast, IgG was distributed throughout all six fractions

relatively equally.

Immunoaffinity Purification and Properties of Lupus

Anticoagulant and Anticardiolipin Antibodies

As shown in Table 2, when IgG-containing fractions from

a Sephacryl 5-300 column (patient no. 5) were incubated

with cardiolipin liposomes, nearly all lupus anticoagulant

icti� ity was adsorbed. When phosphatidylcholine liposomes

were used instead, no lupus anticoagulant activity was

004 012 020 028

F(ob’)2 CONCENTRATION (42101

Fig 1 . (A) Effect of F(ab’), fragments and whole lgG on the

dilute RVVT of normal plasma. Control F(ab’),. --; patientF(ab’),. #{149}-#{149};patient lgG. A- . -A. Patients are identified bynumber. (B) Anticardiolipin activity of F(ab’), fragments.

DILUTE �I�_I___I__�I___I___U.___M�______BUFFER

I .0

ANTI’ 0.8CARDIOLIPIN 0.6

ACTIVITY 04(A405) 0.2

3PROTEIN 21

CONCENTRATION1mg/mi) �Fn ri 11 ri ii �

pH

‘2 3 45 6

FRACTIONS

Fig 2. Preparative isoelectric focusing on Sephadex G-75 oflgG from patient no. 1 . purified on protein A-Sepharose.

adsorbed. The cardiolipin liposome eluate contained lupus

anticoagulant activity, whereas the phosphatidylcholine lipo-

some eluate contained none.

Cardiolipin liposome eluates were prepared from patient

and normal plasmas (Table 3). Eluates from patient plasma,

in contrast to eluates from normal plasma, possessed lupus

anticoagulant activity. When tested by Ouchterlony double

diffusion, patient eluates contained IgG and albumin,

whereas control eluates contained only albumin. Patient and

control eluates were negative for 1gM, IgA, and lipoproteins

by the same method. Final purification of patient liposome

eluates was achieved by chromatography on protein

A-Sepharose. Eluates from this column were pure IgG

preparations, as judged by double diffusion and by silver

staining of SDS-PAGE gels. To estimate the cardiolipin

contamination of the affinity-purified IgG, we purified one

patient plasma with liposomes containing ‘25I-cardiolipin.

The starting liposome preparation contained 216,250 cpm.

The centrifuged, dialyzed liposome eluate contained 219

cpm. After protein A-Sepharose chromatography, the eluted

IgG contained only 42 cpm, representing < I mol of cardioli-

pin/10’#{176}mol of IgG.

The affinity-purified antibodies prolonged the dilute

RVVT of normal plasma in a dose-dependent manner, as

shown in Fig 3. A final concentration of as little as 3.3

sg/mL of this purified antibody produced a measurable

increase in the clotting time. Control IgG had no effect.

Based on the anticoagulant activity of the affinity-purified

IgG, we estimate that lupus anticoagulants in these patients

account for no more than 0.2% of the total plasma IgG.

Table 2. SpecifIcity of Liposo me Purification

Test MaterialDilute RVVT

(s)

Sephacryl S-300 lgG pool

Liposome eluate

Cardiolipin

Phosphatidylcholine

Liposome supernatant

Cardiolipin

Phosphatidylcholine

Buffer

38.5

42.3

29.9

31.7

41.1

28.8

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pH 8.5

MECHANISM OF LUPUS ANTICOAGULANTS 73

Fig 3. Effect of affinity-purified patient lgG and control lgG onthe dilute Russell viper venom time (RVVT) of normal plasma.

Table 3. Lupus Anticoagulant Activity of Liposome Eluates From

Patient and Normal Plasma

Patient

A2�Eluate

Dilute RVVT(Eluate/Buffer. s)

1 0.237 34.4/27.9

2 0.991 38.8/27.9

3 0.291 36.9/28.4

A 0.044 26.9/27.8

B 0.114 29.4/28.4

With the ELISA test system, the cardiolipin liposome

eluates reacted with cardiolipin and, as shown for patient I in

Fig 4, also reacted with phosphatidylserine, phosphatidylino-

sitol, and phosphatidic acid, but not with phosphatidyletha-

nolamine, phosphatidylcholine, or dicetyl phosphate. Identi-

cal results were obtained in all four patients tested (patients

no. 1, 2, 3, and 5). Normal IgG, at approximately the same

concentration as in patient eluates, showed no reactivity

against any phospholipids. In addition, liposome eluates from

normal plasma contained only trace amounts of protein and

showed little or no reactivity.

Analytical isoelectric focusing of affinity-purified, phos-

pholipid-free IgG from patients no. 2 and 3 (Fig 5) showed

several bands ofacidic p1; in contrast, the bulk ofnormal IgG

focused in the alkaline range.

Inhibition ofProthrombin and Factor X Binding to

Phosphatidylserine-coated Surfaces

The calcium ion-dependent binding of ‘25I-prothrombin to

phospholipid-coated surfaces was studied in microtiter

plates. As expected, prothrombin bound to phosphatidyl-

serine-coated wells ( 136 to 205 fmol/well, in eight expeni-

ments), and, to a much lesser extent, to cardiolipin-coated

wells (27 and 44 fmol/well, in two experiments). In contrast,

prothrombin binding to phosphatidylcholine-coated wells

was only 6.2 and 7.0 fmol/well in two experiments, a figure

similar to the value obtained for nonspecific binding to

phosphatidylsenine-coated wells in the presence of EDTA (6

to 9 fmol/well). As shown in Fig 6A, immunoaffinity-

purified patient IgG inhibited the binding of prothrombin to

phosphatidylsenine-coated wells by 73% and 66%, respec-

PATIENT ELUATE CONTROL ELUATE CONTROL (gO

fT�:�T..: �

�----�-�.I 8 I 4 12 8 I 4 I � I B I 4 I 2

DILUTIONS OF ELUATE

Fig 4. Antiphospholipid activity (ELISA) of cardiolipin lipo-

some eluates. Because control eluates contained essentially nolgG. reactivity of purified normal lgG is also shown.

tively. Purified IgG from patient no. I (not shown in Fig 6),

tested only at a much lower protein concentration (23

�zg/mL), inhibited prothrombin binding by 25.5%. Binding

of factor X was also inhibited by patient IgG, although to a

slightly lesser extent than prothrombin (Fig 6B). Three

control IgG preparations showed no inhibition of prothrom-

bin or factor X binding. Binding to cardiolipin-coated wells

was also inhibited by patient eluates.

DISCUSSION

Although earlier observations implied that lupus anticoag-

ulants have as their locus of action the negatively charged

phospholipids,”3’56’34 this assumption has been directly dem-

onstrated in only one patient.’2 On the basis of this case, the

correlation that has been observed between the presence of

anticardiolipin activity and lupus anticoagulant activity,7�”

as well as some more recently described absorption studies,35

all lupus anticoagulants have been widely assumed to operate

by the same mechanism. We tested the generality of this

hypothesis directly in five patients with lupuslike syndromes

and/or thrombosis, whose lupus anticoagulants were largely

IgG.

pH 5.5

I 2 3

Fig 5. Analytical isoelectric focusing of normal IgG (lane 1 ) andimmunoaffinity-purifled lgG from patients no. 2 and 5 (lanes 2 and3).

For personal use only.on April 9, 2019. by guest www.bloodjournal.orgFrom

80

60

20

23

BUFFER PATIENTS

80

60

40

20

I z

BUFFER PATIENTS

120A cardiolipin liposome eluates invariably contain significant

- amounts of albumin, a further purification step by protein‘00 - - - A-Sepharose chromatography was added.

0

Using these preparations, we were able to obtain a gooddose-response relationship between lupus anticoagulant

activity and IgG concentration and to detect lupus anticoag-

ulant activity at IgG concentrations as low as 3 �ig/mL.

Thus, the dilute RVVT, as normally performed on plasma,

can detect lupus anticoagulants at plasma concentrations of

-

�I�1I:!:::::!1 r-i

- .� � �CONTROLS EDTA

�l0�ig/mL.

The mechanism of prolongation of phospholipid-depen-

dent coagulation tests in the patients we studied is similar to

that of the patient previously reported.’2 Our data clearly

show inhibition by patient affinity-purified IgG of prothrom-

bin binding to phosphatidylserine surfaces and, to a lesser

120 B extent, a similar inhibition of factor X binding. The reason

for this difference in inhibition is not presently known. Our

100 data further indicate that prothrombin can bind to cardioli-

pin surfaces, although to a much lesser extent than to

phosphatidylserine surfaces, and that this binding is also

inhibited by the affinity-purified IgG.

Although all six lupus anticoagulants we studied in this

and the previous’2 report are clearly antibodies with anionic

phospholipid specificity, these anticoagulants probably rep-

resent only a subpopulation of antibodies to anionic phos-

pholipids. Several studies suggest that there may be more

than one type of anticardiolipin antibody,’#{176}’38�#{176}and we have

0

CONTROLS EDTA

studied patients with true-positive and false-positive serolog-

ies whose anticardiolipin antibodies did not possess lupus

anticoagulant activity. Furthermore, the presence of severalFig 6. Effect of affinity-purified antibodies on binding of

1’l-prothrombin (A) and 1’l-factor X (B) to phosphatidylserine-

coated wells. Patients are identified by number. Controls arepurified normal lgG at the same concentration as affinity-purifiedpatient lgG.

We demonstrated that both lupus anticoagulant and anti-

cardiolipin activities are mediated by the F(ab’)2 portion of

IgG and therefore are immunologic in nature. We demon-

strated that all, or nearly all, lupus anticoagulant activity can

be adsorbed on cardiolipin-containing liposomes, but not on

phosphatidylcholine-containing liposomes. We further dem-

distinct bands on isoelectric focusing of the affinity-purified

IgG preparations from our patients raises the possibility that. . . . . . .

not all affinity-purified IgG anticardiohpin antibodies pos-

sess lupus anticoagulant activity.

In addition to anticardiolipin antibodies, other phenomena

have been correlated with the presence of a lupus anticoagu-

lant, including biologic false-positive serology,”3’�#{176}’4’ throm-

bocytopenia,7’2#{176} a history of recurrent abortions,4253 and a

high incidence of thromboembolic phenomena.’7’42’43 Three

of our patients had false-positive serologies, and only 1 may

have had thrombocytopenia, whereas 4 of the 5 had either a

history of thrombosis or of recurrent abortion. Because this is

onstrated that with a combination of cardiolipin-liposome

adsorption, high molanity NaC1 and ether elution, and

protein A-Sepharose chromatography, it is possible to obtain

pure, phospholipid-free, IgG fractions from patient plasma

that interact with anionic but not neutral phospholipids and

also act as lupus anticoagulants. Recently, Harris and asso-

ciates” reported a cardiolipin liposome purification proce-

a highly selected group, no conclusions can be drawn con-

cerning incidence of these clinical findings. Because all

patients had anticardiolipin antibodies, it is surprising that

two had negative serologies. Even a high concentration (56

.�g/mL) of affinity-purified IgG from one patient did not

produce a positive RPR serology, suggesting that antibody

concentration alone may not explain this observation. In this

dune using I mol/L of sodium iodide as the elutant. We connection, it is of interest that the two patients with positive

previously used sodium iodide as an elutant from phospha-

tidic acid36 and phosphatidylsenine37 liposomes, but found

serologies at the time of our study had some 1gM lupus

anticoagulant and 1gM anticardiolipin activity. Because

that these preparations were heavily contaminated with many flocculation tests are more sensitive to 1gM than to

phospholipid, making some subsequent testing procedures

difficult to evaluate. For example, the presence of free and

IgG antibodies,54 a biologic false-positive serology may

correlate better with levels of 1gM than of IgG anticardioli-

IgG-bound phospholipid in the liposome eluates interfered

with coagulation tests and was capable of producing spurious

results on isoelectric focusing. For this reason, we developed

pin antibodies.

The high correlation between lupus anticoagulants and

thrombocytopenia raises the question of the interaction of

the present liposome elution procedure. In addition, since lupus anticoagulants with platelets. The strong correlation

74 PENGO ET AL

‘3z02

0

Za:

a:#{176}‘U.

a:� 40

0320-I

20

a:000

H

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MECHANISM OF LUPUS ANTICOAGULANTS 75

with thromboembolic phenomena and the possible effect of

lupus anticoagulants on endothelial cell prostacyclin produc-

tion47 raise similar questions concerning the interaction of

these antibodies with endothelial cells. It will be of interest to

determine whether purified lupus anticoagulants interact

with platelets or endothelial cells, or whether the clinical

phenomena observed depend on other factors.

ACKNOWLEDGMENT

We wish to thank Drs S. Cowchock, R. Zalusky, and N. Jermano-vich for providing patient plasma samples; Dr I. Alam for the

phosphorus determinations; S. McCord for expert technical assis-

tance; and Eliza Batlle and Rosemanie Silvano for typing the

manuscript. We also wish to thank Dr S. Cowchock for herassistance in setting up the anticardiolipin ELISA assay.

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1987 70: 69-76  

V Pengo, P Thiagarajan, SS Shapiro and MJ Heine anticoagulantsImmunological specificity and mechanism of action of IgG lupus 

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