Journal of Thrombosis and Haemostasis, 3: 12431249

ORIGINAL ARTICLE

Anti-protein S antibodies following a varicella infection: detection, characterization and inuence on thrombin generation
V. REGNAULT,* F. BOEHLEN, H. OZSAHIN, D. WAHL,* P. G. DE GROOT, T. LECOMPTE* and P . D E M O E R L O O S E
*Inserm 734 and Hospital of Nancy, France, Hemostasis Unit, Department of Medicine and Hematology, and Department of Pediatrics, University Hospital, Geneva and Faculty of Medicine, Switzerland, and Hematology Department, University Medical Center, Utrecht, The Netherlands

To cite this article: Regnault V, Boehlen F, Ozsahin H, Wahl D, de Groot PG, Lecompte T, de Moerloose P. Anti-protein S antibodies following a varicella infection: detection, characterization and inuence on thrombin generation. J Thromb Haemost 2005; 3: 12439.

Summary. Postinfectious purpura fulminans is a rare disease. Varicella is one of the precipitating conditions and we recently observed such a case. The 4-year-old child was found to have a severe transient protein S deciency. By enzyme-linked immunosorbent assay and surface plasmon resonance we rst demonstrated that anti-protein S antibodies were present and also transient. Next we characterized the epitopes against which these antibodies were directed and found that they predominantly recognized the N-terminal part of protein S. Finally we showed by thrombography a transient dramatic hypercoagulable state as a result of thrombin being unregulated by the dynamic protein C inhibitory system: in vitro thrombin generation, in response to a low concentration of tissue factor, was almost insensitive to activated protein C up to 25 nmol L)1 on day 4 while it was normally sensitive on day 42. For the rst time, we demonstrated a temporal relationship between protein S deciency, antibodies to protein S and hypercoagulability, thus supporting the pathogenic role of these antibodies. Keywords: antiphospholipid antibodies, protein S, purpura fulminans, surface plasmon resonance, thrombography, varicella. Introduction Severe thromboembolic disease and purpura fulminans, although rare, are well-described complications of varicella
Correspondence: Philippe de Moerloose, Hemostasis Unit, University Hospital of Geneva, 1211 Geneva 14/Switzerland. Tel.: +41 22 27 29 751; fax: +41 22 37 29 777; e-mail: Philippe.de Moerloose@hcuge.ch Received 3 December 2004, accepted 11 January 2005 2005 International Society on Thrombosis and Haemostasis

[1,2]. These complications are usually associated with a decrease in protein C (PC) and most often protein S (PS). The importance of these two inhibitors in hemostasis is well known because severe inherited deciency is associated with neonatal purpura fulminans, and heterozygous PC or PS deciency is associated with venous thromboembolism. During recovery from varicella, DAngelo et al. rst reported the occurrence of thrombosis with a transient deciency of PS ascribed to the presence of a circulating autoantibody to PS [3]. Since then, a few reports have conrmed the presence of anti-PS antibodies after varicella associated with severe thrombotic complications [47]. Indeed Levin et al. identied, in ve children with purpura fulminans after varicella, anti-PS antibodies by dot blotting and Western blotting and quantied them serially by enzyme-linked immunosorbent assay (ELISA) [4]. Post-varicella antibodies to PS associated with purpura fulminans were demonstrated by Manco-Johnson and colleagues with plasma, isolated immunoglobulin G (IgG) and PS afnity-puried antibodies [5]. Peyvandi et al. also observed PS deciency, presumably autoimmune, in a patient with deep vein thrombosis after varicella [6]. Josephson et al. determined the prevalence of anti-PS antibodies, not only in children with varicella complicated by purpura fulminans and/or thromboembolism but also in children with varicella and without such complications [1]. Finally, characterization of epitopes on PS was reported by one team [7]. Anti-PS antibodies have also been found in children with purpura fulminans after infections other than varicella [8,9]. An increase in prothrombin fragment 1 + 2 plasma concentrations has been shown [1,5,10]. These studies showed an association of PS deciency and systemic activation of coagulation but the link with anti-PS antibodies remains elusive. Moreover, as transient anti-PS autoantibodies after varicella are common, but not predictive of thrombotic complications [1,10], it is important to study whether they could have a direct impact on coagulation. In this report we

1244 V. Regnault et al

demonstrate for the rst time an in vitro hypercoagulable state temporarily associated with autoantibodies to PS and PS deciency, very likely leading to the observed purpura fulminans. Materials and methods
Case report

Coagulation tests

We recently reported this case [11]. Briey, a previously healthy 4-year-old boy was admitted (day 0; D0) with fever, varicella and leg pain. On D1 he developed extensive ecchymoses with necrotic centres over his legs and the diagnosis of postinfectious purpura was made. In the next days, skin necrosis was obvious. At D0, the International Normalized Ratio (INR) was 2.38, activated partial thromboplastin time (APTT) was 43.4 s (normal range 25.031.0 s) and brinogen was 0.3 g L)1. Fresh frozen plasma, steroids and immunoglobulins were rst given and heparin therapy was started on D4 (after blood drawing for coagulation studies). During the following days, there was an improvement with no further extension of the cutaneous lesions. Heparin therapy was discontinued after 19 days. The patient made a complete recovery.
Proteins and reagents

Platelet-poor plasma was obtained by centrifugation of citrated blood at 1750 g for 10 min. Plasma was stored at ) 70 C in small aliquots until tested. Plasma coagulation tests were determined by standard methods with a Behring Coagulation System (BCS) analyzer (Dade Behring, Marburg, Germany). Levels of total and free PS antigens were measured by ELISA (Asserachrom Total and Free PS, Diagnostica Stago). Factor V Leiden, Factor II and 20201 A mutations were analyzed as previously described [16]. D-dimer was measured using Vidas New D-dimer assay (bioMerieux, Marcy-lEtoile, France) [17]. The presence of lupus anticoagulant was tested with PTT LA (Diagnostica Stago), dilute Russell viper venom time (Dade Behring) and Staclot-LA test (Diagnostica Stago). Anticardiolipin (aCL) and anti-b2-glyco-protein I (anti-b2GPI) antibodies were measured by ELISA [18,19].
Surface plasmon resonance (SPR) analysis

Bovine serum albumin (BSA) and all chemicals were from Sigma (St Louis, MO, USA). IgG was puried by afnity chromatography on protein GSepharose (Pharmacia Biotech, Uppsala, Sweden). IgG concentration was determined by an in-house sandwich ELISA using the GG-7 monoclonal anti-human IgG (Fc-specic) antibody (Sigma) as capture antibody and a peroxidase-conjugated goat anti-human IgG (Fc-specic) antibody (Sigma) as conjugated antibody. Puried PS and PS-decient plasma were purchased from ` res, France). Recombinant PS, the Diagnostica Stago (Asnie rst 242 amino acids of PS (mini-PS) and the sex-hormone binding globulin (SHBG)-loop were prepared in-house as previously described [7,12]. Factor X was puried from plasma according to Koppelman et al. [13]. Peroxidaseconjugated goat anti-human IgM and IgG were from Biosource (Camarillo, CA, USA). Human activated PC (APC) was prepared in-house as previously described [14]. Platelet microvesicles were prepared from human washed platelets as previously reported [15] using 10 lmol L)1 ionomycin as platelet activator. Platelet microvesicles were washed three times at 13 000 g for 45 min at 4 C using HBS buffer (20 mM HEPES, 140 mmol L)1 NaCl, pH 7.35 containing 5 g L)1 BSA) Recombinant human tissue factor (Innovin) was obtained from Dade Behring (Marburg, Germany). The uorogenic substrate of thrombin, Z-GlyGly-Arg-AMC, was from Bachem (Bubendorf, Switzerland) and the calibrator with a known thrombin-like activity was obtained from Synapse BV (Maastricht, the Netherlands). STA-Staclot PS was from Diagnostica Stago.

All experiments were performed using the Biacore X instrument (Biacore, Uppsala, Sweden) at 25 C with a ow-rate of 5 lL min)1, as previously reported [20]. Puried PS, mini-PS or SHBG-loop (20 lg mL)1 in 10 mmol L)1 sodium acetate, pH 3.0) was covalently coupled to a CM5 sensor chip using standard amine coupling chemistry [21]. HBS (10 mM HEPES, 150 mmol L)1 NaCl, 0.005% (v/v) Tween-20, pH 7.4) was used as running and dilution buffer and a pulse of 10 mmol L)1 NaOH for regenerations. Either plasma (1 : 5 dilution) or puried IgG (adjusted to a concentration of about 1.25 mg mL)1) were injected over the biosensor surfaces. Results were expressed in resonance units (RU) [22]. Nonspecic binding to the matrix was eliminated by subtracting the signal obtained on a blank surface (activated carboxymethyldextran saturated with ethanolamine). Dissociation rate constants were calculated by non-linear tting of the primary sensorgram data using BIAEVALUATION 3 software (Biacore) assuming a single averaged dissociation constant.
ELISA against PS, mini-PS and SHBG

The binding of patient plasma IgG and IgM to PS and other proteins was also determined by ELISA as previously described [7]. Puried plasma total PS, recombinant wild-type PS, miniPS and SHBG-loop were coated. Factor X or albumin were also coated and served as controls. The 96-well plates were incubated overnight and next washed with TBS, 0.1% (v/v) Tween-20, 3 mmol L)1 CaCl2, and blocked with the same buffer containing 3% BSA. Patient serum (1 : 50 dilution) was added and incubated at 37 C for 2 h. After washing, peroxidase-conjugated goat anti-human IgM and IgG were added and incubated at 37 C for 1 h. Color development was monitored at 405 nm. Pooled normal human plasma served as a control. Results are expressed as absorbance found with the patient plasmas minus the absorbance found with control plasma.
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Anti-protein S antibodies after varicella infection 1245 Fluorogenic measurement of thrombin activity

800 Binding to protein S (RU) 700 600 500 400 300 200 100 0 0 5 10 15 20 25 30 35 40 45 12 control plasma on day plasmas IgG
4-7 30-42 control

Calibrated automated thrombography was performed at 37 C according to Hemker et al. in a microtiter plate uorometer (Fluoroskan Ascent, ThermoLabsystems, Helsinki, Finland) using the dedicated software program (THROMBINOSCOPE 2.106, Synapse BV), as previously reported [23]. Briey, thrombograms were recorded in citrated platelet-poor plasma centrifuged at 13 000 g to discard endogenous microvesicles. Platelet microvesicles at 4 lmol L)1 phosphatidylserine equivalents were added as a source of cellular surfaces. Coagulation was triggered by recalcication (nal concentration in CaCl2: 16.7 mmol L)1) in the presence of 0.5 pmol L)1 recombinant human tissue factor. Experiments were carried out in the absence or in the presence of APC at various nal concentrations (0.85, 1.7, 6.7, 13.9, 25 nmol L)1). The molar amount of active thrombin present in clotting plasma was calculated from the signal of wells in which thrombin was generated and the calibration signal [24]. The total amount of thrombin activity (i.e. the endogenous thrombin potential ETP) was assessed as the area under the curve. Results

Fig. 1. Binding of proteins to immobilized protein S on biosensor surface. Diluted plasma from the patient (day 4 to day 42) and 12 controls, or total puried IgG (IgG4)7 or IgG30)42) from the patient or from a healthy subject (control) was injected over commercially available puried PS covalently coupled to the dextran matrix using amine coupling chemistry; results are expressed as resonance units (RU) and are means of at least three determinations, the bars indicate SD.

Detection of antibodies to PS Coagulation results

There was no deciency in PC and antithrombin as well as no factor V Leiden or factor II 20210 A mutations. However, at the time of admission, PS was virtually undetectable and failed to respond to infusions of freshfrozen plasma (PS at D4 < 5%), despite correction of other coagulation factors (at D4, INR 1.0, APTT 23 s, brinogen 2.5 g L)1, factor V 88%, factor X 100%). The levels of PS progressively normalized with also a correction of D-dimer levels (Table 1). IgG [8.8 G isotype phospholipid units (GPL), cut-off: 5 GPL] and IgM [10.2 M isotype phospholipid units (MPL), cut-off: 3 MPL] aCL as well as antib2GPI IgM antibodies at low levels were found but no lupus anticoagulant was detectable. Anti-cardiolipin and anti-b2GPI antibodies disappeared after 1 month. Both the patients parents were tested for free and total antigen PS; their levels were within the normal ranges.

Binding of plasma components of the patient to immobilized PS was investigated using SPR eight times over a 42-day follow-up (Fig. 1). High binding (700 RU) was observed on D4 whereas signals ranging from 0.35 to 10 RU were obtained with plasmas from healthy donors. The binding decreased over time, in correlation with the increase in PS concentration. These results were suggestive of antibodies to PS. Plasmas from day 4 to day 7 on the one hand and from days 30 and 42 on the other hand were pooled and total IgG was puried. Binding signals for these two IgG preparations (IgG4)7 and IgG30)42), when adjusted at the same concentration as diluted plasma, were 460 and 105 RU, respectively, conrming the presence of IgG directed to PS (binding signal for control IgG: 45 RU). The dissociation phase showed good curve tting with the monoexponential decay model and a ko value of 4.7 10)3 s)1 was calculated. Addition of puried PS to IgG4)7 prior to injection over immobilized PS resulted in a concentration-dependent inhibition of IgG4)7 binding.
ELISA results

Table 1 Evolution of the total and free protein S antigen levels (%) and of D-dimer (ng mL)1) Day 1 4 6 8 10 14 21 25 Free protein S <5 <5 <5 9 26 46 56 59 Total Protein S <5 <5 8 23 37 61 80 81 D-dimer > 10 000
ND

5068
ND ND

1366 618 426

D0 is the day of admission to the hospital. 2005 International Society on Thrombosis and Haemostasis

Figure 2 shows the changes in the antibody titer and in the level of total PS in the days after the admission. The increase in plasma PS level mirrored the decrease in the antibody titer. As shown in Fig. 3, on day 0, the patient displayed IgG and IgM autoantibodies against PS. The IgG antibodies were only directed against mini-PS. This result was conrmed using SPR: for IgG4)7 similar levels of binding and ko values (4.7 10)3 and 3.3 10)3 s)1) were obtained with immobilized PS and mini-PS, respectively, while no binding occurred on SHBGloop; for IgG30)42, binding level to immobilized mini-PS was

1246 V. Regnault et al

0.7 IgG binding to protein S (absorbance at 405 nm) 0.6 0.5 0.4 0.3 0.2 0.1 0.0 0 5 10 15 20 25 30 35 40 45 Day

100 Total protein S (%) 80 60 40 20 <5

concentration of 9 mg mL)1 similar to the basal IgG concentration in the patient plasma (i.e. 10.8 mg mL)1 risen to 22.5 mg mL)1 after administration of immunoglobulins), the addition of IgG30)42 did not affect the biological activity of a control plasma whereas the addition of IgG4)7 resulted in a  15% decrease in PS activity.
Phenotyping the hemostatic system by thrombography

Fig. 2. Binding of IgG to immobilized protein S in ELISA and protein S concentration. Closed symbols represent binding of IgG to microtiter plates coated with PS. Total PS level (e) was determined by ELISA using a commercial assay (Diagnostica Stago).

 four-fold less important than those of IgG4)7, ndings were similar for immobilized PS. IgM antibodies recognized predominantly mini-PS but also weakly recognized the SHBGloop of PS (Fig. 3B). No antibodies were found against factor X or albumin. No differences were found when plasma or recombinant PS was used as antigen (data not shown). Titers of the antibodies rapidly decreased (Fig. 3C,D) and completely disappeared after 3 weeks.
Interference of anti-PS autoantibodies with the PC system

The APC cofactor activity of PS in a plasma system was studied using a commercial clotting assay. At a nal

In plasma preparations from healthy subjects, we have reported that the addition of APC led to a concentrationdependent decrease in ETP [23]. Almost no inhibitory effect of added APC on ETP was observed with plasma from D4 (Fig. 4, left panel) whereas a phenotype similar to those of healthy subjects was observed with plasma from D42 (Fig. 4, right panel). These effects are best rendered by two parameters: the ETP of the uninhibited sample and the concentration of APC at which 50% inhibition would occur (IC50-APC), as previously suggested [25]. In the absence of APC similar ETP values were obtained for these two plasmas (1607 and 1492 nmol L)1, respectively). By contrast, the IC50-APC was not reached (> 25 nmol L)1) for plasma collected on D4 whereas the IC50-APC was of 4.9 nmol L)1 for plasma collected on D42. For comparative purposes, the APC responses of a commercial PS-decient plasma with and without 10 lg mL)1 (corresponding to 40%) puried free PS added were studied. As shown in Fig. 5, there was only a slight inhibitory effect in the absence of free PS (IC50-APC > 25 nmol L)1) while the addition of puried PS to reach the level of free PS in plasma restored a normal response (IC50-APC of 2.7 nmol L)1).

Binding of IgG autoantibodies (absorbance at 405 nm)

1.2 1.0 0.8 0.6 0.4 0.2 0.0 protein S mini protein S SHBGloop

Binding of IgM autoantibodies (absorbance at 405 nm)

1.2 1.0 0.8 0.6 0.4 0.2 0.0 protein S mini protein S SHBGloop

factor X

factor X

Binding of IgG autoantibodies (absorbance at 405 nm)

Binding of IgM autoantibodies (absorbance at 405 nm)

1.2 1.0 0.8 0.6 0.4 0.2 0.0 0 5

1.2 1.0 0.8 0.6 0.4 0.2 0.0

10 15 20 25 30 35 40 45

10 15 20 25 30 35 40 45

Day

Day

Fig. 3. Characterization of anti-protein S antibodies. ELISA trays were coated with plasma PS (d), mini-PS (s), SHBG-loop (e) or factor X (m) and subsequently incubated with patient plasma (1 : 50 dilution). The binding of antibodies was revealed with peroxidase-labeled goat anti-human antibodies and results are expressed as absorbance with the patient plasma minus absorbance with control plasma; Values with control plasma using antihuman IgG and IgM, respectively: 0.153 and 0.091 for plasma PS; 0.144 and 0.109 for mini-PS; 0.312 and 0.078 for SHBG-loop; 0.109 and 0.070 for factor X. (A) IgG on day 0; (B) IgM on day 0; (C) Time-course of the level of IgG; (D) Time-course of the level of IgM. 2005 International Society on Thrombosis and Haemostasis

Anti-protein S antibodies after varicella infection 1247

300 250 Thrombin (nM) Thrombin (nM) 200 150 100 50 0 0 10 20 30 Time (min) 40

300 250 200 150 100 50 0 0 10 20 30 Time (min) 40

Fig. 4. Thrombograms with platelet-poor plasma (PPP) of the patient. Left: plasma on day 4; right: plasma on day 42. Supernatant of PPP centrifuged at 13 000 g was triggered with 0.5 pmol L)1 tissue factor, CaCl2 and platelet microvesicles were used as source of cellular surface. Added APC concentrations: 0, 0.85, 1.7, 6.7, 13.9 and 25 nmol L)1.

2000 1800 1600 1400 ETP (nM.min) 1200 1000 800 600 400 200 0 0 5 10 15 20 APC concentration (nM) 25

Fig. 5. Eect of protein S deciency on the inhibition of ETP by APC; the same experimental conditions as in Fig. 4. ETP values at the studied APC concentrations (0, 0.85, 1.7, 6.7, 13.9 and 25 nmol L)1) for the patients plasmas (circles) on day 4 (gray) and on day 42 (light gray) were compared with those obtained for a commercial PS-decient plasma (triangles down) with (dark gray) or without (black) 10 lg mL)1 added puried PS (corresponding to 40%). ETP values (nmol L)1 min)1) in the absence of APC: PS-decient plasma: 1828; PS-decient plasma with puried PS: 1600; the patient on day 4: 1607; the patient on day 42: 1492. IC50-APC values (nmol L)1): PS-decient plasma: > 25; PS-decient plasma with puried PS: 2.7; the patient on day 4: > 25; the patient on day 42: 4.9.

Discussion About 50 cases with purpura fulminans post-varicella have been reported. Most cases were associated with a transient
2005 International Society on Thrombosis and Haemostasis

and severe PS deciency as a result of anti-PS antibodies [17]. These autoantibodies were found to be IgG and/or IgM [3,5]. However, data about ne characterization of the epitopes recognized and functional properties of these autoantibodies are scarce. Moreover in vitro demonstration of the mechanisms leading to in vivo thrombotic complications is still lacking. Our data conrm that the acquired PS deciency is most likely the result of the presence of anti-PS antibodies because the levels of PS in the plasma of the patient mirrored the levels of antibodies. Using ELISA we detected in our patient both IgG and IgM to PS. However, since similar levels of binding were obtained using SPR with plasma and total puried IgG, we may infer that the antibodies were mainly IgG. It is possible that antiPS IgM were low-avidity antibodies that were not detected under ow conditions at low PS density (SPR) whereas a positive signal was obtained under the conditions of ELISA (in which high antigen density is less physiologically relevant and conditions are static). The SPR signal is proportional to the concentration of IgG. Thus, assuming a 21-day half-life for IgG and the rapid offset of production of IgG because of treatment with steroids and immunoglobulins, the rapid disappearance of IgG raises the hypothesis of formation and subsequent elimination of immune complexes in the uid phase, in agreement with DAngelo et al. and Manco-Johnson et al. [4,5]. Both the severe PS deciency and the inhibition of IgG binding to immobilized PS by PS in uid phase supported this hypothesis. The autoantibodies are mainly directed against the N-terminal part, conrming the previous results of van Ommen et al. [7] who showed that at least two epitopes are present on PS for the autoantibodies; however, in this patient, the predominant epitope was present in the rst 242 amino acids. One of the possible explanations regarding the higher IgG signal with miniPS than with PS might be the higher coating density of mini-PS on ELISA plates. Another explanation might be that the epitope for the antibodies is more readily accessible on mini-PS because of an altered conformation or the absence of SHBG-loop. It has been reported that APC could wrap around PS, making contact with solvent-exposed charged and hydrophobic/ aromatic residues located on the same face of mini-PS [26].

1248 V. Regnault et al

The modest effect of puried IgG added to normal plasma on APC cofactor activity of PS in a clotting assay suggested that antibodies, which were mainly directed against the rst 242 residues, were not directed to epitopes on the same face as that involved in APCPS interaction. An increase in F1 + 2 prothrombin fragment in patients with varicella and thrombotic complications has already been reported [1,5,10], suggesting that these patients generate more thrombin in vivo. The calibrated automated thrombography methodology enabled us to display a hypercoagulable state in vitro, which disappeared along with IgG to PS, hence providing a sufciently strong incentive to invoke a link between this state and the presence of such antibodies. Using this integrative assay probing the function of the PC system, in vitro thrombin generation was almost insensitive to APC. Since no PS was detectable in this plasma, steric hindrance at the phospholipid surface as a result of the presence of IgGPS complexes is very unlikely. The physiological plasma concentration of APC is far below the Kd of this protein for negatively charged phospholipid membranes; binding afnities of PC were reported to be 1001000-fold lower than those of the other vitamin K-dependent proteins [27]. By contrast, PS, the concentration of which (free form) in plasma is around 130 nmol L)1, is one of the vitamin K-dependent proteins with the highest afnity for phospholipid membranes, its dissociation constant being in the nanomolar range [28]. Using puried coagulation factors it has been shown that the formation of a 1 : 1 stoichiometric APCPS complex increases the afnity of APC for membranes and APC-dependent cleavage of factor Va [29]. Consistent with this, resistance to APC was indicated with a commercial PS-decient plasma. Altogether these data add further evidence for a crucial contribution of PS to the APC anticoagulant function. There are still many unresolved issues concerning the dramatic thromboembolic complications after varicella. The rst one deals with the origin of such autoantibodies. It is possible that there is an epitope mimicry between the infecting virus and PS; Manco-Johnson and colleagues [5] showed binding of anti-PS antibodies of a patient to a triplet of varicella peptides but another study did not nd a crossreactivity [3]. Another very intriguing question is the reason why some individuals develop such complications. Most authors have found that the severity of the clinical manifestations correlated with the concentrations of anti-PS antibodies and the severity of PS deciency [1,5,7,10]. In our patient, PS was undetectable. It is also possible that a coinfection with another infectious agent plays a role, i.e. co-infection with Streptococcus is frequently reported. Another explanation could be, as in our patient and in many reports, the simultaneous presence of antiphospholipid antibodies (aPL) [15,7,10,30]. This raises the hypothesis of a very robust but transient and non-specic immunologic response following varicella. Most aPL occurring in children are transient and believed to be associated with viral or bacterial infections and they are usually not thrombogenic [10,31]. Anticardiolipin antibodies have been associated with functional impairment of

the PC anticoagulant pathway [32]. So aPL may contribute to the thrombotic risk, as well as to a concomitant presence of a thrombophilic disorder not found in our patient [9]. We have previously demonstrated APC insensitivity in lupus anticoagulant patients with or without aCL [33], while we did not observe such a dramatic prole in patients with aCL alone (data to be published). Thus in the patient reported here, although aCL have been detected, they are not likely to explain the almost complete insensitivity of ETP to APC. Recognition that post-varicella thromboembolic complications are mainly caused by transient autoimmune-mediated PS deciency may enable treatment to be administered on a rational basis. It suggests indeed that heparin should be promptly administered. Furthermore, since it is an autoimmune process, immunoglobulins, immunosuppressive agents and plasmapheresis may be discussed [34]. Fresh frozen plasma or vitamin K concentrates containing PS are usually ineffective and potentially dangerous (increase of inhibitors titers as well as serum sickness) [4,10]. In our patient the simultaneous administration of steroids and immunoglobulins may have played a role in the rapid reduction of anti-PS antibodies. We conclude that acute varicella infection may lead to antiPS antibodies and mediated PS deciency, accompanied by a very strong in vitro thrombin generation because of its nonregulation by the PC system. Our ndings strongly suggest the interest of a global assay probing the effect of pro- and anticoagulant factors on thrombin generation to discriminate among patients with anti-PS antibodies those who are at risk of thrombotic complications. Acknowledgements gion This study was supported in part by grants from the Re Lorraine, the District of Nancy and the Henri Poincare Nancy 1 University and the Swiss National Research Funds 32067746.02. We are grateful to Jean-Pierre Max (Inserm U ` le Pommereuil 734) for skillful technical assistance, to Miche (Rennes, France) and Guido Reber (Geneva, Switzerland) for helpful comments and suggestions. References
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2005 International Society on Thrombosis and Haemostasis