American Journal of Hematology 41 :92-96 (1992)

Procoagulant Activity of Endotoxin or Tumor Necrosis Factor Activated Monocytes Is Enhanced by IgG From Patients With Lupus Anticoagulant Jean-Franqois Schved, Jean-Christophe Gris, Valerie Ollivier, Jean-Luc Wautier, Gerard Tobelem, and Jacques Caen Laboratoire d’Hematologie, HBpital de Nimes, Nimes (J.F.S., J.C.G., V.O.), and lnstitut des Vaisseaux et du Sang, HBpital Lariboisiere, Paris (J.L.W., G.T., J.C.), France

The effect of lupus anticoagulant(LA) positive plasma on the expression of human monocyte procoagulant activity (PCA) was studied. LA positive plasma were able to enhance the endotoxin or TNF alpha induced monocyte associated PCA. The monocyte PCA had the characteristicof tissue factor activity (factor VII, factor X dependence). The enhancement of monocytePCA could be confirmed using purified LA positiveIgG. The stimulating effect was supported by the F(ab’)2 fragments. o 1992 Wiley-Liss, Inc. Key words: lupus anticoagulant, monocyte, tissue factor, endotoxin, tumor necrosis factor

INTRODUCTION

PATIENTS AND SUBJECTS

Blood samples obtained from 12 patients, 4 males and Lupus anticoagulant (LA) was defined as autoantibody that interferes with phospholipid dependent coagulation 8 females, mean age 36 years (range: 22-61), were studin in vitro tests [ 11. The presence of Lupus anticoagulant ied. Five patients had a history of thromboembolism. At is known to be associated with the occurrence of venous the time of blood withdrawal, none of the patients was and arterial thrombosis [2,3]. Several mechanisms have suffering from infectious disease, none was treated with been proposed to explain the frequency of LA-associated heparin, oral anticoagulant, or corticosteroid, and the thrombosis: inhibition of the release of prostacyclin obtained blood samples were negative for cryoglobuline[4,5], deficiency of protein C activation or impairment of mia. Fifteen healthy control subjects (nine males and six catalytic function of activated protein C [6-81, and impairment of fibrinolysis [9,1O]. Recently, it has been females), mean age 32 years (range: 24-45), were from suggested that sera from patients with systemic lupus laboratory staff or blood donors. None was under drug erythematosus can increase the endothelial procoagulant therapy. activity (PCA) expression induced by tumor necrosis factor alpha [ 1 11. The cellular PCA is assumed to be a tissue MATERIAL AND METHODS factor activity since it requires factor VII for inducing coagulation and is dependent on factor X and V, but not Blood samples for determination of LA were collected by venipuncture in plastic tubes containing 3.8% trisoon factor IX. Monocytes can develop procoagulant activity (PCA) in dium citrate. Platelet poor plasma was obtained by two vitro in response to endotoxin [ 121, immune complexes centrifugation steps performed at 3,OOOg for 20 min. [ 131, tumor necrosis factor (TNF) [ 141, and tuftsin [ 151. The induction of this tissue factor activity may play a role Received for publication August 6 , 1991; accepted February 27, 1992. in thrombosis and inflammation. In this study, we have investigated the influence of LA Address reprint requests to Dr. J.F. Schved, Laboratoire positive plasma from 12 patients on PCA induced by d’Hematologie et Irnmunologie, HBpital de Nimes, BP 26, 30006 Nimes Cedex, France. endotoxin or TNF alpha-stimulated human monocytes. 0 1992 Wiley-Liss, Inc.

Lupus Anticoagulant and Monocytes

Serum was obtained from blood collected into glass tubes without anticoagulant. Platelet poor plasma and serum were stored at -80°C until use. Lupus Anticoagulant Activity

For each patient, kaolin clotting time [ 161, tissue thromboplastin inhibition test (thromboplastine non calcique, La Technique Biologique, Paris, France; final dilution MOO”), and the platelet neutralization procedure [I71 were performed. All patients included in the study had a positive tissue thromboplastin inhibition test and a positive kaolin clotting time (patient/control ratio and patient + control 1:1 mixture/control ratio were superior to 1.2 for both assays) and the platelet neutralization procedure was positive (correction of the prolonged activated partial thromboplastin time of 5 sec or greater by the platelet suspension compared with the saline control). Antiphospholipid antibodies (Asserachrom@ APA, Stago, Asnieres, France) were present in all the samples from the patients with LA. IgG Purification

IgG were purified from patients or control plasma with protein A Sepharose 4B chromatography (Pharmacia, Uppsala, Sweden) according to the instructions of the manufacturer. IgG depleted plasma were kept. F(ab’)2 fragments were prepared after pepsin digestion using unsolubilized pepsin (Pepsin Sepharose 4B, Sigma Chemical Co., St Louis, MO). IgG were aggregated by heating purified control IgG at 63°C for 20 min. IgG concentrations were measured by laser nephelometry (BNA, Behringwerke, Marburg, FRG). LA activity of purified IgG and F(ab’)2 was tested by mixing normal plasma with IgG (final concentration: 5 mg/ml) or F(ab’)2. Purified IgG and F(ab’)2 were treated with an endotoxin removing gel (Detoxi-Gel@,Pierce, Rockford, IL). All samples used were free of detectable amounts of endotoxin, as determined by an endotoxin assay (Coatesto endotoxin, Kabi, MoIndal, Sweden). Monocyte Isolation

Monocyte separation was performed as previously described [ 151. Monocytes were identified by nonspecific esterase and chloroacetate esterase staining. The percent of monocytes was between 15 to 25% before adhesion and 80 to 90% after removing the nonadherent cells. Monocyte preparations were incubated in RPMI 1640glutamin (RPMI) containing 20% of normal plasma, LA plasma, or IgG for various period of time to define the most appropriate conditions. We chose a 4 hr incubation period for plasma and a 1 hr period for IgG or F(ab’)2. After incubation with plasma or IgG (final concentrations: 0.4 mg/ml), the monocytes were washed twice in

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Hank’s balanced salt solution (HBSS) and then incubated for 4 hr in RPMI, in RPMI plus endotoxin (10 pg/ml) (LPS butanol extracted E Coli, Sigma Chemical Co., St Louis, MO), or in RPMI plus TNF alpha (10 ng/ml) (alpha TNF, Boehringer, Mannheim, FRG). In some experiments T-depleted mononuclear cells were used: the T lymphocytes were removed by incubation with sheep red blood cells to allow the formation of rosettes. The T-depleted cells contained less than 5% T lymphocytes as assessed by flow cytometry using antiCD3 monoclonal antibodies. Procoagulant Activity Assays

After incubation, the monocytes were washed twice in HBSS. The monocytes were detached using a rubber policeman and disrupted by sequential freezing, thawing, and sonication. The procoagulant activity of the lysates was determined using a one-stage clotting assay. The lysates (50 1.1) resuspended in buffer were added to normal plasma (50 pl) and incubated for 2 min at 37°C. Fifty microliter of 25 mM CaC12 solution were added and the time to clot formation was measured in duplicate on an automated coagulometer (ST888, Stago, Asnieres, France). Assay specificity was established by incubating samples with different plasma deficient in factor VII, factor IX, factor V, or factor X . Rabbit brain thromboplastin (Technique Biologique, Paris, France) was used to establish a standard curve: a 100 units/ml activity corresponded to a clotting time of 28 sec. Amidolytic Assays

Tissue factor activity of cell lysates was assayed using an adaptation of a commercially available kit (Stachromo VII, Stago, Asnieres, France). Purified bovine factor X (50 pl of a 0.5 plasma equivalent univml solution) were added to 50 pl prewarmed dilution normal plasma (130). After incubation of the mixture, 50 pl of monocyte lysate were added. A CBS 48.03 chromogenic substrate 5 mM solution in 30 mM CaC12 was then added and changes in absorbance (405 nm) were recorded every 28 sec for various ranges of time depending on the tested thromboplastin dilution. Rabbit brain thromboplastin (Technique Biologique, Paris, France) was used to establish a standard curve. This allowed to attribute a theoretical thromboplastin dilution to an experimental change in absorbance. As a control, factor Xa activity was determined in plasma deficient in factor VII, factor X, or factor V. Statistics

Results were expressed as median and range. For statistical analysis, the Mann-Whitney U test and the Wilcoxon’s paired rank sum test were used. A P value c0.05 was considered as significant.

Schved et al.

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Effect of IgG Containing LA Activity on Monocyte PCA p.0.007 Results of kaolin clotting time obtained by mixing 30 p-0.006 normal plasma with purified IgG (5 mg/ml) evidenced that IgG purified from LA positive plasma contained LA NS activity (control IgG: 89 sec; IgG from LA positive patients: respectively, 157, 175, and 164 sec). Control IgG or LA positive IgG alone (final concentrations 0.4 mg/ml) did not induce any significant procoagulant activity when compared to RPMI alone (1.8, 1-2.5 0’ , , I PCA units). PCA induced by endotoxin was significantly NP LAP NP*LPS LAP*LPS NP+TNF LAP+TNF higher when monocytes were prior incubated with LA positive IgG than when incubated with control IgG (Fig. extreme values n median 2A). TNF alpha induced PCA was also enhanced after Fig. 1. Effect of lupus anticoagulant positive plasma on preincubation with LA positive IgG and the obtained spontaneous, endotoxin or TNF alpha-induced monocyte activities were higher than after control IgG (Fig. 2B). procoagulant activity NP: normal plasma, n = 15. LAP: lupus Control or LA positive aggregated IgG were without efanticoagulant positive plasma, n = 12. Endotoxinconcentrafect on endotoxin or TNF alpha induced PCA. tion: 10 pg/ml (LPS). TNF alpha concentration: 10 ng/ml. F(ab’)2 from patients or from control did not induce any significant PCA on unstimulated monocytes. Endotoxin or TNF alpha-stimulated monocytes significantly expressed more PCA in the presence of F(ab’)2 from the RESULTS patients than from the controls (Fig. 2C). Effect of LA Positive Plasma on Monocyte PCA No significant PCA could be detected when procoaguInduced by Endotoxin or Alpha TNF (Fig. 1) lant assays were performed using plasma deficient in A significant PCA was found after a 4 hr incubation factor VII, factor X, or factor V. On the other hand, time of monocytes with endotoxin or TNF alpha. In the factor IX deficient plasma gave results identical to norabsence of incubation, PCA was not detectable. Mono- mal plasma. cytes generated a low PCA after incubation in plasma alone: 6.7 PCA units (range 4.1-9.3) with control Effect of T Lymphocyte Depletion on plasma, 9.5 (5-14.9) with LA positive plasma. PCA Activity The endotoxin induced PCA were higher when monoAfter T lymphocyte depletion using rosetting, no PCA cytes were preincubated with plasma from LA positive expression could be detected in monocyte lysates after patients (20.10 PCA units, 8.e32.9) than with normal endotoxin stimulation, whatever the monocytes were preplasma (13.2, 7.5-15.4) ( P = 0.007). Similarly, PCA incubated in plasma, IgG or F(ab’)2. measured after monocyte stimulation induced by TNF alpha were higher when monocytes were incubated with Amidolytic assays LA positive plasma (16.35, 9.7-24.6) than when monoProcoagulant activities generated by monocytes after cytes were incubated with normal plasma (10.9, 9.6-12) incubation in normal or LA positive F(ab’)2 fragments ( P = 0.006). alone were within the same ranges. The activity induced The enhancement of endotoxin or TNF alpha induced by addition of endotoxin was much more pronounced monocyte PCA was found when factor IX deficient when monocytes were primed with LA positive F(ab’)2 plasma was used for procoagulant assays. No significant than with control F(ab’)2 (Fig. 3). This PCA was a tissue PCA was found when procoagulant assays were perfactor activity, only depending on factor VII and factor X. formed using plasma deficient in factor VII, factor X, or factor V. Tissue factor activity of endotoxin or TNF alpha-inDISCUSSION duced PCA was also assessed by amidolytic assays using Monocytes are known to produce tissue factor which deficient or normal plasma. Normal plasma, plasma deficient in factor IX or factor V, gave identical results. No can trigger the coagulation cascade throught different significant PCA was obtained when plasma deficient in pathways depending on the inducer [18]. The procoagufactor VII or factor X were used. The effect of LA posi- lant activity generated by monocytes in our study had the tive plasma on endotoxin or TNF alpha induced PCA was properties of tissue factor since it was dependent on the presence of factor VII and X [ 121. suppressed by prior IgG depletion. Procoagulant activity (A.U.)

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Lupus Anticoagulant and Monocytes Tissue Factor Units

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Fig. 3. Amidolytic assay: effect of lupus anticoagulant positive IgG F(ab’)2 fragments on endotoxin-inducedtissue factor activity of monocytes. Pi: IgG F(ab’)2 fragments from patient 1. Endotoxin concentration: 10 pglml (LPS).

anticoagulant. PCA produced by unstimulated monocytes was similar whether the cells were incubated with N P1 P2 P3 N+TNF Pl+TNFP2+TNFP3*TNF control or patient plasma. On the opposite, a significant Procoagulant activity (A.U.) increase of TNF alpha or endotoxin-induced PCA was 8 D.0.008 obtained after incubation of monocytes with plasma or II c * 0.00 6 I purified IgG from patients with LA as compared to nor6 mal plasma. IgG depleted plasma from patients were also NS depleted of the priming activity. F(ab’)2 fragments preNS pared from patient IgG exhibited the priming effect on 4monocyte PCA. Taken together these data indicate that -Ithe enhancement of PCA induced by endotoxin or TNF alpha was supported by antibody specificity associated to IgG fractions from patients with LA. No PCA enhance0‘ I ment was found using LA positive IgG in the absence of R F F1 R+LPS F+LPS Fl+LPS endotoxin or TNF alpha-induced monocyte stimulation. LA positive antibodies are known to react with negatively extreme values 0 median charged phospholipids. The priming activity of patients Fig. 2. Effect of lupus anticoagulant positive IgG (0.4 mgl plasma and IgG observed in our study can therefore be ml) or lgG F(ab‘)2 fragments on spontaneous, endotoxin, or mediated by antiphospholipids antibodies. Thus, antiTNF alpha-induced monocyte procoagulant activity. A: IgG, endotoxin. B: IgG, TNF alpha. C: F(ab’)2 fragments, endo- gens recognized by the IgG onto the monocyte memtoxin. N: control IgG, lupus anticoagulant negative. Six as- branes could be phospholipids. The role of beta 2 glycosays for each experiment. P1, P2, P3: lupus anticoagulant protein I, which has recently been described to be positive IgG from patients 1, 2, and 3. Six assays for each required for antibody-phospholipid interaction [ 191, deexperiment. R: RPMl medium alone. Six assays were per- serves here to be studied. The interaction of patient IgG formed for each experiment. F: control IgG F(ab’)2 fragments. Six assays were performed for each experiment. F1: with phospholipids of monocyte surface could induce lupus anticoagulant positive IgG F(ab‘)2fragments from pa- membrane changes leading to an increased PCA exprestient l.Six assays were performed for each experiment. En- sion after endotoxin or cytokine stimulation. A recent dotoxin concentration: 10 pg/ml (LPS). TNF alpha concen- study [20] reported that monocytes from LA positive or tration: 10 nglml. negative patients with systemic lupus erythematosus generated more PCA than control monocytes suggesting that In our study, the ability of control blood monocytes to monocytes from SLE patients were primed in vivo, while generate endotoxin or TNF alpha-induced PCA was mod- purified IgG from patients had the same effect than conified after priming by plasma from patients with Lupus trol IgG. In our study, IgG alone did not induce signifi0’

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cant PCA. None of our patients had systemic lupus erythematosus. The higher incidence of thrombosis in patients with LA [2,3] is still unexplained. The effects of LA on various cell functions have been studied, particularly on endothelial cells [4-7,111, on monocytes [203 containing models. In vivo, it has been suggested that monocyte PCA generation could be associated with thrombotic events: monocytes obtained from patients with meningococcal infection [21] or with thromboembolic complication [22] have increased PCA. This suggests that an increased monocyte PCA could play a role in activating the blood coagulation system in vivo in response to inflammatory stimuli [23]. In patients with Lupus anticoagulant may occur a twostep enhancement of monocyte procoagulant activity consisting in a priming by LA immunoglobulins followed in some circumstances by endotoxin or cytokine-induced cellular activation. REFERENCES 1, McNeil HP, Chesterman CN, Krilis SA: Anticardiolipin antibodies and lupus anticoagulants comprise separate antibody subgroups with different phospholipid binding characteristics. Br J Haematol 73506, 1989. 2 . Boey M, Colaco CB, Gharavi A E Thrombosis in systemic lupus erythematosus: Striking association with the presence of circulating anticoagulant. Br Med J 287:1021, 1983. 3. Glueck HI, Kant KS, Weiss MA: Thrombosis in systemic lupus erythematosus. Arch Intern Med 145:1389, 1985. 4. Cameras LO, Machin SJ, Vermylen J: Arterial thrombosis, intrauterine death and ‘‘lupus’’ anticoagulant: Detection of immunoglobulin interfering with prostacyclin formation. Lancet i:244, 1981. 5 . Schorer AE, Wickham NWR, Watson KV: Lupus anticoagulant induces a selective defect in thrombin mediated endothelial prostacyclin release and platelet aggregation. Br J Haematol71:399, 1989. 6. Comp PC, De Bault LE, Esmon NL: Human thrombomodulin is inhibited by IgG from 2 patients with non specific anticoagulants. Blood 62:1099, 1983. 7. Cariou R, Tobelem G, Soria C: Inhibition of protein C activation by endothelial cells in the presence of lupus anticoagulant. N Engl J Med 314:1193, 1986. 8. Marciniak E, Romond H: Impaired catalytic function of activated

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