Reduced Free Protein S Levels in Patients with Inflammatory Bowel Disease

Reduced Free Protein S Levels in Patientswith Inflammatory Bowel DiseasePrevalence, Clinical Relevance, and Role of

Anti-Protein S Antibodies

SIMONE SAIBENI, MD, MAURIZIO VECCHI, MD, CARLA VALSECCHI, PhD,ELENA M. FAIONI, MD, CRISTINA RAZZARI, MD, and ROBERTO DE FRANCHIS, MD

We evaluated free plasma levels of protein S, a natural anticoagulant factor, the prevalenceof anti-protein S antibodies, a possible cause of protein S deficiency, and their correlationwith anti-phospholipid antibodies in 53 patients with inflammatory bowel disease (IBD) and53 age- and sex-matched controls. Mean free plasma protein S levels (6 SD) were significantlylower in IBD patients (0.98 6 0.32 IU/ml) than in controls (1.06 6 0.28 IU/ml) (P , 0.05);only one patient showed protein S deficiency. Specific antibodies to protein S were found infour IBD patients (7.5%) and in one control (1.9%) (P 5 NS). Five IBD patients (9.4%) andnone of the controls showed anti-phospholipid antibodies (P , 0.06). No correlation wasfound between free protein S levels and anti-protein S antibodies or between anti-protein Sand anti-phospholipid antibodies. In conclusion, free plasma protein S levels are slightly butsignificantly decreased in IBD patients. The prevalence of anti-protein S and anti-phospholipid antibodies is increased in IBD patients. Anti-protein S antibodies do not appearto determine low protein S levels or to overlap with or belong to anti-phospholipid antibodies.

KEY WORDS: inflammatory bowel disease; thrombosis; protein S; antibodies.

Among the extraintestinal manifestations of inflam-matory bowel disease (IBD), thromboembolic eventsplay a relevant role both for the associated highmorbidity and for the young age of affected patients(1, 2). Although the increased risk for thrombosis inIBD has been recognized for a long time (3), theprevalence of these events has not been fully estab-lished. Clinical studies report a prevalence of throm-boembolic accidents varying between 1.2% and 7%

(3, 4), whereas autopsy studies report a prevalence of39% (1).

The pathogenetic mechanisms of such complica-tions are also not fully understood: several authorsindicate that qualitative and/or quantitative changesin platelets (5), coagulation profiles (6–9), fibrinolysis(10), and prothrombotic risk factors (11) occur inIBD. Although data from the literature are contro-versial, it is uniformly accepted that IBD is charac-terized by a hypercoagulability state and/or by a pro-thrombotic state.

In recent years, several case reports have suggesteda correlation between cases of thrombosis in IBD andlow levels of circulating protein S (12–16), an impor-tant natural anticoagulant. Reduced protein S levelshave also been reported in series of Crohn’s disease

Manuscript received May 30, 2000; accepted October 2, 2000.From the Gastroenterology and Gastrointestinal Endoscopy Ser-

vice and Angelo Bianchi Bonomi Haemophilia and ThrombosisCenter, IRCCS Ospedale Maggiore and Department of InternalMedicine, University of Milan, Italy.

Address for reprint requests: Dr. Maurizio Vecchi, Gastroenter-ology Service, IRCCS Ospedale Policlinico, Via Pace 9-20122Milan, Italy.

Digestive Diseases and Sciences, Vol. 46, No. 3 (March 2001), pp. 637–643

637Digestive Diseases and Sciences, Vol. 46, No. 3 (March 2001)0163-2116/01/0300-0637$19.50/0 © 2001 Plenum Publishing Corporation

patients without thrombosis (9, 17). In these studies,the observed low protein S levels were attributed tovitamin K deficiency (13) or to the ongoing inflam-matory process causing increased levels of C4bBP(12), an acute-phase reactant that binds to circulatingprotein S.

A further cause of protein S deficiency has beenrecently described in a pediatric patient (18) andsubsequently confirmed in other reports (19, 20).According to these studies, low levels of protein Smay also originate from the production of specificanti-protein S autoantibodies, resulting in a reductionof its activity and antigenic levels.

It has been suggested that such anti-protein S an-tibodies may arise by cross-reactivity with viral orbacterial epitopes (18, 21, 22) or that they might bepart of a nonspecific immune response. It has alsobeen suggested that antibodies to protein S mightbelong to the group of anti-phospholipid antibodies(23, 24) that are frequently observed in IBD (25, 26).

The aim of the present study was to evaluate freeplasma protein S levels, the prevalence and signifi-cance of anti-protein S antibodies and their correla-tion with antiphospholipid antibodies in patients withIBD.

MATERIALS AND METHODS

Patients. Twenty-four patients with ulcerative colitis(UC) (14 men, 10 women; mean age 6 SD, 41.3 6 15.2years) and 29 with Crohn’s disease (CD) (10 men, 19women; 39.1 6 13.8 years), consecutively referred to ouroutpatient gastrointestinal clinic between January and No-vember 1999, were prospectively enrolled in the study.Twelve ulcerative colitis patients had quiescent disease, sixhad mild active disease, and six had moderately activedisease according to Truelove and Witts criteria (27).Twenty-three Crohn’s disease patients had quiescent dis-ease and six had active disease, defined as a Crohn’s diseaseactivity index (CDAI) .150 (28). Two ulcerative colitispatients and one Crohn’s disease patient had a history ofprevious thrombosis. The clinical characteristics of the IBDpatients are shown in Table 1.

Fifty-three sex and age matched healthy controls werealso included in the study. They were randomly picked froma larger cohort of healthy individuals (approximately 900)with a negative personal and family history of thrombosiswho had been previously enrolled as normal controls forseveral hemostatic parameters.

Free Protein S. Protein S was measured as free protein Sconcentration according to a modification of the methodoriginally described by Comp et al (29). Plasma (200 ml),stored at 280°C and never thawed previously, was mixedwith 100 ml of a solution of PEG 6000 (10.5% in 50mmol/liter Tris HCl, pH 7.4, 100 mmol/liter NaCl), andshaken vigorously on an orbital shaker for 60 min at 4°C.The mixture was then centrifuged at 12,000 g for 5 min and

the supernatant separated, frozen, and stored at 280°C.Free protein S concentration was measured by ELISA bystandard procedures. Incubation times were 18 hr with theantigen and 2 hr with the secondary antibody (both primaryand secondary rabbit polyclonal anti-protein S antibodies(the latter peroxidase labeled) were purchased from DakoA/S, Glostrup, Denmark). Levels of free protein S in thesamples were calculated by extrapolation from a standardcurve constructed with serial dilutions of a reference plasmaprocessed as the patient and control samples (the referenceplasma was a pool of plasmas from 40 healthy donors, 20men and 20 women). A control plasma of known concen-tration was included in each assay. The reference plasmawas calibrated against the international standard for proteinS (1st International Standard for protein S, NIBSC 93/590).Results of measurements are therefore reported as inter-national units per milliliter.

Anti-Protein S Antibodies. Anti-protein S antibodieswere evaluated by means of an ELISA method using com-mercially available pure protein S (Enzyme Research Lab-oratory, Southbend, Indiana, USA) as the coated antigen.Protein S at a concentration of 2.5 mg/ml, in Tris NaCl 0.12mol/liter, pH 7.4, was coated on ELISA plates overnight at4°C. Diluted sera (1:250 in Tris NaCl 0.12 mol/liter, 2%BSA) were then incubated for 1 hr at 22°C. The immunereaction was revealed by subsequent incubations with rabbitanti-human IgG for 1 hr at 22°C and HRP-labeled goatanti-rabbit IgG for 1 hr at 22°C followed by the appropriatechromogenic substrate for 3 min. The optical density (OD)reading was then obtained at l 5 492 nm. Results areexpressed as the percentage of OD readings of a knownhigh titer positive serum used as reference curve. A cutoffvalue for the presence of anti-protein S antibodies was setat OD readings higher than 12% (corresponding to the 95thpercentile of the distribution of OD readings in normals).

Immunoblotting. To define the specificity of the immunereaction, immunoblotting was performed as follows: humanpurified protein S (1 mg/lane) and human prothrombin (1mg/lane) were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (10% polyacrylamide)

TABLE 1. CHARACTERISTICS OF IBD POPULATION

Crohn’sdisease

(N 5 29)

Ulcerativecolitis

(N 5 24)

Men/women 10/19 14/10Age (mean 6 SD, yr) 37.2 6 12.6 41.4 6 15.2Duration of disease

(mean 6 SD, months)74.2 6 56.9 75.2 6 54.2

Disease activityQuiescent 23 12Mild 6Moderate 6CDAI .150 6

Location/extensionIleum 9Ileum 1 colon 11Colon 9Pancolitis 5Left colitis 12Proctosigmoiditis 7

SAIBENI ET AL

638 Digestive Diseases and Sciences, Vol. 46, No. 3 (March 2001)

and then transferred to nitrocellulose membranes (0.45-mmpore diameter) for electroblotting at fixed voltage (85 V)for 1 hr at 4°C. After blocking, the membrane was cut instrips and exposed for 2 hr to the purified IgG fraction ofIBD patient plasma. After thorough washing the strips wereincubated for 2 hr with phosphatase-labeled goat anti-human IgG followed by the addition of the chromogenicsubstrate. Prothrombin was run in parallel with protein S asa control for specificity of the antibody reaction.

Anti-Phospholipid Antibodies. These were measured byELISA. Coating of microtiter 96-well plates (Falcon) wasperformed with cardiolipin (purchased from Sigma) dilutedin ethanol (50 mg/ml for 18 hr at 4°C). Incubation with 50 mldiluted plasma (1:50 in 0.15 mol/liter phosphate-bufferedsaline, pH 7.3, 10% BSA) or standards (serum calibratedagainst the standard of Harris) was for 2 hr at 22°C. Afterwashing, 50 ml goat anti-human IgG (g-chain specific) al-kaline phosphatase conjugated antibodies were added toeach well and incubated for 90 min at 4°C. After washing,the substrate was added and the color reaction proceededfor 60 min, then was stopped by addition of 3 M NaOH.Optical density readings were carried out at l 5 405 nm.Results were extrapolated from the curve generated by thestandards. The lower limit for considering the samplespositive was 10 units/ml for IgG anticardiolipin antibodiesand 10 units/ml for IgM anticardiolipin antibodies.

Lupus Anticoagulant. This was determined according torecently published guidelines (30).

Other Measurements. Full blood count, acute-phase re-actants [erythrocyte sedimentation rate (ESR), C-reactiveprotein (CRP), orosomucoid], renal and hepatic function,electrophoresis of serum proteins were measured in allpatients.

Statistical Analysis. Statistical analysis was performed bymeans of Mann-Whitney test and Fisher’s exact test.

RESULTS

The distribution of free plasma protein S levels isshown in Figure 1. Mean free plasma protein S levelswere slightly but significantly lower in patients withIBD (0.98 6 0.32 IU/ml) than in healthy controls(1.06 6 0.28 IU/ml) (P , 0.05). However, of allpatients studied, only one affected by ulcerative colitishad protein S deficiency. At the time of enrollment hewas in a quiescent phase of disease (although with aslight increase of acute-phase reactants) and he wasknown to have had previous deep vein thrombosis.

Stratification of IBD patients according to diagno-sis, disease activity, extension/location or duration ofdisease showed no correlation with free protein Slevels.

The mean OD readings obtained in ELISA deter-minations of anti-protein S antibodies did not signif-icantly differ between IBD patients (7.1 6 2.7%) andhealthy controls (6.5 6 1.6%, P 5 NS). No differencewas found when IBD patients were divided accordingto diagnosis, disease activity, location, or duration of

the disease. The presence of anti-protein S antibod-ies, defined as an OD reading higher than 12% of aknown high titer positive control, was observed in 4 of53 IBD patients (2 with ulcerative colitis, 2 withCrohn’s disease) (7.5%) and in 1 of 53 controls(1.9%). This difference did not reach a statisticalsignificance. Immunoblotting experiments performedto assess the specificity of the immune reactionshowed that purified IgG fractions from ELISA-positive patients reacted with blotted protein S andnot with prothrombin, another purified homologousvitamin K-dependent protein (Figure 2).

Antibodies to protein S were not found in the onlypatient with protein S deficiency; no difference wasfound in free plasma protein S levels between IBDpatients with or without circulating anti-protein Santibodies.

The presence of anti-phospholipid antibodies wasobserved in 5 of 53 IBD patients (9.4%), but in noneof control subjects (P , 0.06). Three patients withanti-phospholipid antibodies were affected by ulcer-ative colitis and two by Crohn’s disease. All threeulcerative colitis patients showed the presence ofanti-cardiolipin antibodies only; one patient had ahistory of previous deep vein thrombosis, and he wasin a quiescent phase of disease at the time of enroll-ment. One Crohn’s disease patient with anti-phospholipid antibodies showed the presence of bothanti-cardiolipin antibodies and lupus anticoagulant,while the remaining Crohn’s disease patient, withonly anti-cardiolipin antibodies, had urticarioid vas-culitis. No difference in free plasma protein S levelswas found between IBD patients with anti-cardiolipinantibodies and IBD patients without anti-cardiolipinantibodies.

None of the IBD patients with anti-protein S anti-bodies showed anti-phospholipid antibodies. Theseresults are summarized in Table 2.

DISCUSSION

Several recent reports have suggested that proteinS deficiency might play a relevant role in the hyper-coagulability state and the increased risk for arterialand venous thrombosis observed in IBD.

Protein S is a vitamin K-dependent plasma proteinwith indirect anticoagulant activity: indeed, it func-tions as a cofactor to activated protein C, whichinactivates coagulation factors Va and VIIIa and in-creases plasminogen activator levels (31). Two formsof protein S exist in plasma: one is bound to C4b-binding protein (C4bBP), a plasma protein involved

ANTI-PROTEIN S ANTIBODIES IN IBD

639Digestive Diseases and Sciences, Vol. 46, No. 3 (March 2001)

in the regulation of complement activity and acting asan acute phase reactant, and one is free. The latter isthe active form and normally represents approxi-mately 40% of total circulating protein S antigen.

A decrease in free protein S levels may lead to adecrease in the ability to inhibit blood clot formation,resulting in a prothrombotic state. Indeed, inheriteddeficiency of protein S is associated with an increasedrisk of developing thromboembolic disease, estimatedaround 5- to 10-fold that of noncarriers (32, 33). Thelink between acquired protein S deficiency andthromboembolic disease is more uncertain, and this

uncertainty is probably due to the fact that in theconditions more frequently associated with acquiredprotein S deficiency, such as disseminated intravascu-lar coagulation, malignancy, oral anticoagulant ther-apy, liver failure, and vitamin K deficiency (34), othercoagulation defects usually coexist. Furthermore, thepossible role of increased C4bBP levels in inducingfree protein S deficiency (35) has also been debated(32, 36). On the other hand, thrombosis due to ac-quired protein S deficiency induced by anti-protein Sautoantibodies has been recently reported.

In the present series, IBD patients show that free

Fig 1. Free plasma protein S levels (IU/ml) in IBD population and in the control group.

SAIBENI ET AL


plasma protein S levels are decreased. This findingconfirms the observation by Aadland et al (17) thatfree plasma protein S levels are significantly lower inpatients with Crohn’s disease than in controls. Fur-thermore, our data suggest that this may be the casealso for ulcerative colitis patients, since in the presentstudy free plasma protein S levels did not show anysignificant correlation with diagnosis, disease activity,or location or duration of disease. This is not inagreement with the observation by Hudson et al (9)that free plasma protein S levels are higher in CDpatients in an activity phase of the disease than in CDpatients with quiescent disease.

The clinical significance of this slight but significantreduction is uncertain: overt free protein S deficiency

was observed only in one of 53 IBD patients (1.9%),a young man with ulcerative colitis and a previousthrombotic event, in which a relevant role of thisdeficiency can be supposed, as also suggested byseveral other reports (12–16).

A role in inducing protein S deficiency and relatedthrombosis recently has been attributed to the pro-duction of anti-protein S antibodies, which may resultfrom cross-reactivity with viral or bacterial epitopes(22), be part of a nonspecific immune response orbelong to the anti-phospholipid antibodies family (23,24).

Since IBD patients are characterized by the pro-duction of several autoantibodies and anti-phospho-lipid antibodies have also been observed with in-

Fig 2. Immunoblotting of protein S and prothrombin. Protein S (lanes 2 and 5) and prothrombin (lanes 3 and 6) were run on SDS-PAGEat 1 mg/lane as described in Materials and Methods. After transfer to a nitrocellulose membrane, the IgG fraction of a patient with noanti-protein S antibodies (lanes 2 and 3) or with anti-protein S antibodies (lanes 5 and 6) by ELISA determination was incubated with themembrane. Lanes 1 and 4 are the molecular weight standards, as indicated.

TABLE 2. FREE PLASMA PROTEIN S LEVELS, ANTI-PROTEIN S ANTIBODIES AND ANTI-PHOSPHOLIPIDANTIBODIES IN IBD PATIENTS AND IN HEALTHY CONTROLS

IBD patients Controls P

Free plasma protein S levels (mean 6 SD, IU/ml) 0.98 6 0.32 1.06 6 0.28 ,0.05Protein S deficiency 1/53 (1.9%) 0/53 (0%) NSAnti-protein S antibodies 4/53 (7.5%) 1/53 (1.9%) NSOptical density readings (mean 6 SD, %) 7.1 6 2.7 6.5 6 1.6 NSAnti-phospholipid antibodies 5/53 (9.4%) 0/53 (0%) ,0.06



creased frequency in such patients (25, 26), weevaluated for the first time the prevalence of anti-protein S antibodies and their possible role in induc-ing protein S reduction.

Anti-protein S antibodies were found by ELISA in7.5% of the patients. The prevalence of these anti-bodies in IBD patients did not significantly differ fromcontrols, and no difference in their prevalence wasobserved when IBD patients were stratified accordingto diagnosis, disease activity, or extension/location orduration of disease.

The fact that in these patients the anti-protein Sactivity detected by ELISA was indeed due to thepresence of specific autoantibodies is proven by theimmunotransblot findings. However, the role playedby anti-protein S antibodies in our IBD populationremains to be clarified. Indeed, the presence of theseautoantibodies does not appear to lead to a significantdecrease in free plasma protein S levels: the only IBDpatient with protein S deficiency did not show anti-protein S antibodies, whereas the mean free plasmaprotein S level observed in IBD patients who carriedanti-protein S antibodies was similar to that observedin IBD patients without anti-protein S antibodies.

Our data confirm, in agreement to other reports(25, 26), an increased prevalence of anti-phospholipidantibodies among IBD patients: this class of antibod-ies was found in 5 of 53 IBD patients (9.5%) and innone of the controls; this difference was very close tostatistical significance (P , 0.06). Of five IBD pa-tients who showed anti-phospholipid antibodies,three had ulcerative colitis and two Crohn’s disease.The presence of anti-phospholipid antibodies was notrelated to anti-protein S nor did it appear to inducechanges in protein S levels. These data appear toindicate that, in IBD patients, anti-protein S antibod-ies do not belong to the anti-phospholipid antibodyfamily.

In conclusion, we describe for the first time thepresence of anti-protein S antibodies in some patientswith inflammatory bowel disease. However, these donot appear to be the cause of the decreased protein Slevels and the thrombotic tendency observed in IBD.

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Reduced Free Protein S Levels in Patients with Inflammatory Bowel Disease