antiphospholipid antibodies predict imminent vascular events independently from other risk factors...

Antiphospholipid antibodies predict imminent vascular eventsindependently from other risk factors in a prospective cohort

Carolyn Neville1,*, Joyce Rauch2,*, Jeannine Kassis3, Susan Solymoss4, LawrenceJoseph1,5, Patrick Belisle1, Jerrold S. Levine6, and Paul R. Fortin7,8

1Division of Clinical Epidemiology, Department of Medicine, McGill University Health Centre,Montreal, Quebec, Canada2Division of Rheumatology, Department of Medicine, McGill University Health Centre, Montreal,Quebec, Canada3Department of Hematology, Maisonneuve-Rosemont Hospital, University of Montreal, Montreal,Quebec, Canada4Division of Hematology, Department of Medicine, McGill University Health Centre, Montreal,Quebec, Canada5Department of Epidemiology and Biostatistics, McGill University, Montreal, Quebec, Canada6Section of Nephrology, Department of Medicine, University of Illinois at Chicago, and Section ofNephrology, Department of Medicine, Jesse Brown Veterans Administration Medical Center,Chicago, Illinois, USA7Division of Health Care and Outcome Research, Toronto Western Research Institute, Universityof Toronto, Toronto, Ontario, Canada8Division of Rheumatology, Department of Medicine, University Health Network, University ofToronto, Toronto, Ontario, Canada

SummaryAntiphospholipid antibodies (aPL) are associated with vascular events, but the magnitude of thisrisk, alone, or in combination with other atherogenic and thrombophilic risk factors, remainsunclear. A prospective cohort of 415 persons was studied for arterial and venous events (AE andVE) over a median time of 7.4 years. aPL and coagulation abnormalities were measured uponbeginning of the study and annually for the first four years. Within the cohort, a nested case-control study was conducted to investigate the role of endothelial and inflammatory markers inpredicting new vascular events. Forty-five individuals had new vascular events: 18 occurredduring the first year of follow-up. The proportion of event-free survivors at eight years was 90%(95%CI = 87%, 94%) for aPL-negative and 72% (60%, 85%) for aPL-positive individuals,respectively. Predictors for new AE were previous AE (HR=5.7 [2.7, 12.0]), diabetes (5.6 [2.4,13.2]), aPL positivity (2.6 ([1.2, 5.9]), and age (1.04 [1.01, 1.07]). New VE were predicted byprevious VE (6.1 [1.9, 19.9]), anti-β2-glyco-protein I (aβ2GPI) positivity (5.8 [1.4, 24.1]),activated protein C resistance (APCR) (4.1 [1.1, 15.1]), and gender (3.7 [1.1, 12.9]). In the nestedcase-control study, similar predictors were observed for AE, while abnormal APCR (OR=5.5 [1.1,26.6]) and elevated von Willebrand factor (vWF) (OR=5.0 [1.2, 19.8]) best predicted VE. We

© 2009 Schattauer GmbH, Stuttgart

Correspondence to: Paul R. Fortin MD, MPH, FRCP(C), Director of Clinical Research, Arthritis Centre of Excellence, TorontoWestern Hospital, Room MP-10–304, 399 Bathurst Street, Toronto, Ontario, Canada, M5T 2S8, Tel.: +1 416 603 6267, Fax: +1 416603 6288, [email protected].*These authors contributed equally to this research.

PubMed Central CANADAAuthor Manuscript / Manuscrit d'auteurThromb Haemost. Author manuscript; available in PMC 2012 September 06.

Published in final edited form as:Thromb Haemost. 2009 January ; 101(1): 100–107.

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demonstrate that aPL independently predict new vascular events and discriminate betweenindividuals with and without events in the first two years of follow-up, indicating that aPL areassociated with a short-term risk of developing new and recurrent vascular events.

KeywordsAntiphospholipid antibodies; thrombosis; anti-beta2-glycoprotein I; activated protein C resistance;von Willebrand factor

IntroductionAntiphospholipid antibodies (aPL) are associated with arterial and venous events (AE andVE), but it remains unclear whether the risk they confer is independent of otheratherosclerotic or thrombophilic risk factors. We have established a prospectively followedcohort that is well-characterized with respect to demographic, clinical, and laboratoryinformation, measurement of baseline aPL profiles, atherosclerotic and thrombophilic riskfactors, and ascertainment and validation of new and previous AE or VE. aPL measurementswere performed prior to the occurrence of new vascular events, and the analysis accountedfor a history of prior vascular events. Our objective was to test whether aPL predictimminent vascular events independently of other atherogenic or thrombophilic risk factors ina prospective study, and to study the additional risk conferred by the presence ofinflammatory and endothelial markers in a nested case-control study.

MethodsPopulation

MAPS cohort—The Montreal Antiphospholipid Antibody Study (MAPS) is a cohort of415 persons followed prospectively for the development of new vascular events. The lowfrequency (1–5%) of aPL positivity in the general population makes a population-basedprospective study impractical. We chose instead to identify a readily available sample ofpersons at higher risk for aPL positivity matched to a control population and to observe themprospectively for the development of AE or VE. Participants were recruited fromconsecutive persons attending the outpatient blood test centres at two Montreal universityhospitals (The McGill University Health Centre and Hôpital Maisonneuve-Rosemont). Weselected a group of individuals requiring aPL testing (anticardiolipin antibody [aCL] and/orlupus anticoagulant [LA]) (n=207) and thus at high suspicion for the presence of an aPL,and matched these individuals by age, gender, and centre to a second group requiring acomplete blood count (CBC) (n=208) who were not suspected to have aPL. The recruitmentprocess has been described in detail elsewhere (1). For the current study, one individual wasexcluded because of a missing blood sample (N=414). The study design and consentprocedures were reviewed and approved by the Research Ethics Boards of both institutionsaccording to the principles of the Declaration of Helsinki.

Nested case-control study population—To study the role of inflammatory (highsensitivity C-reactive protein [hsCRP]) and endothelial (D-dimers [DD], fibrinogen [FIB],factor VIII, [FVIII] and von Willebrand factor [vWF]) prothrombotic risk factors, we chosea nested case-control study design. Each of the 45 cases with new vascular events (de novoor recurrent vascular event since entry into the cohort) was age-, gender-, and visit date-matched with four controls without new vascular events (exceptions: six cases had threecontrols and two cases had two controls; total controls=170). Assays were performed onblood samples drawn closest to and prior to the date of the new vascular event.

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Clinical dataClinical data at baseline included: demographic parameters (age, gender, race, education);medications; comorbidities (thyroid gland disease, diabetes mellitus (DM), hypertension[HBP], and systemic lupus erythematosus [SLE]); history of pregnancy morbidity andvascular events; family history of cardiovascular disease (CVD) defined as cerebrovascularaccident (CVA), transient ischemic attack (TIA), myocardial infarction (MI), or angina infirst-degree relatives; and smoking. Follow-up data collected over each six month periodincluded: new AE or VE, new comorbidities, and medications. The primary outcome wasdefined as any new AE or VE. AE were classified as CVA, TIA, MI, angina, or other sitesof arterial thrombosis. VE were classified as deep vein thrombosis (DVT), pulmonaryembolism (PE), or other sites of venous thrombosis. All previous events were confirmed bymedical record review by a physician blinded to aPL status. All new reported events wereconfirmed by a panel of physicians. Criteria for confirmation of a vascular event included apositive diagnostic test and/or a documented clinical diagnosis by the treating physician.Only confirmed events were used in the analyses.

Laboratory testsaPL assays—Participants were tested for IgG and IgM aCL, LA, and IgG and IgM anti-β2-glycoprotein I antibodies (aβ2GPI) using serum (aCL, aβ2GPI) or plasma (LA) that hadbeen aliquotted and stored frozen at −70°C. aCL was measured using the Louisville assay(Louisville APL Diagnostics, Inc., Louisville, KY, USA). LA was detected using a diluteactivated partial thromboplastin time (APTT) assay (Automated APTT, bioMérieux Canada,Inc., Montreal, QC, Canada), in which the APTT reagent was diluted 1/10 in 20 mM HEPESbuffer, pH 7.4, containing 15 mM NaCl, as previously described (2). Plasma samples weremixed 1:1 with Verify 1 coagulation control plasma (bioMérieux Canada, Inc., Canada) tocorrect for coagulation factor deficiencies. Confirmation of LA activity was performed byneutralization with hexagonal phase phosphatidylethanolamine, as previously described (2).aβ2GPI was measured by ELISA as previously described (3), except plates were coated witheither 15 μg/ml human β2GPI (Crystal Chem, Downers Grove, IL, USA) or gelatin (forcontrol wells) at 4°C, and alkaline phosphatase-conjugated goat anti-human IgG or IgM(Sigma-Aldrich, St Louis, MO, USA) was used. Sera were considered positive if theyexceeded the normal cut-off value (<0.7 OD405 units) for the assay, which was based on themean + 10 SD (for IgG), or mean + 5 SD (for IgM), of 25 healthy control sera. Positiveresults were confirmed by repeat testing on human β2GPI, and binding specificity wasconfirmed by lack of significant binding (≤0.2 OD405 units) to control wells coated with anirrelevant antigen (gelatin) and blocked similarly to β2GPI-coated wells. Tests wereconsidered to be aCL-positive if >40 U/ml for aCL (IgG or IgM); aβ2GPI-positive if ≥0.7OD405 units for aβ2GPI (IgG or IgM); and LA-positive if ≥6.0 seconds above the controlplasma for LA, and >8.0 seconds above the control for confirmation of LA.

Coagulation factor assays—Activated protein C resistance (APCR) phenotype wasdetermined as previously described (1) using APTT reagent and APC/CaCl2 (Chromogenix,DiaPharma, West Chester, OH, USA). APCR ratio was defined as the ratio of the clottingtime in the presence and absence of APC/CaCl2, respectively. This assay, in contrast tocommercial APC™ Resistance V assays, detects an acquired APCR phenotype in additionto factor V Leiden mutation. An APCR ratio >2.0 was classified as a normal APC response.Homocysteine (tHcy) was measured in plasma using a quantitative assay on the IMxInstrument Axis patented enzymatic conversion method (1). Functional ATIII was measuredby amidolytic assay based on Xa inhibition (Chromogenix, DiaPharma). Functional proteinC was measured by amidolytic assay (Berichrom Protein C assay, Behring Diagnostic,Montreal, QC, Canada). Functional protein S was measured by immunoassay assay (ProteinS Liatest, Diagnostic Stago, Abbott Laboratories, Mississauga, ON, Canada).

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Endothelial and inflammatory markers—DD and vWF antigen (vWF) were measuredby an immuno-tur-bidometric method (Liatest, Diagnostica Stago, Abbott Laboratories,Mississauga, ON, Canada). Functional FIB was assayed using the Clauss method(Diagnostica Stago, Inc., Abbott Laboratories, Mississauga, ON, Canada). FVIII activitywas quantitated in a standard one-stage clotting assay (Diagnostica Stago, Inc., AbbottLaboratories, Mississauga, ON, Canada).

Genetic studiesThe following genetic studies were done on banked DNA using standard procedures: factorV Leiden mutation (amplification of genomic DNA with the polymerase chain reaction anddigestion of fragments with MnlI) (4); and prothrombin gene G20210A mutation (genomicDNA amplification with PCR and digestion of fragment with HindIII) (5).

Other assayshsCRP was performed using IMMAGE Immunochemistry System (Beckman Coulter, Inc.,Fullerton, CA, USA) and low-density lipoprotein (LDL was done on the Hitachi 917instrument with kits from Roche Diagnostics (Laval, QC, Canada).

Data analysisAll data were double-entered into Medlog (Medlog Systems, Incline Village, NV, USA),and discordant entries were verified and corrected. Means, standard deviations, and mediansfor continuous variables, and proportions for dichotomous variables, were calculated. Weanalyzed all of our models using two different cut-off values for IgG/IgM aCL: a lower cut-off using the threshold of 15 PL units and a higher cut-off value of 40 PL units (definition ofmoderate to high aCL according to the classification criteria for antiphospholipid syndrome(APS) [6]). As analyses using the two different cut-off value for aCL did not differqualitatively, only the results for the more rigorous aCL cut-off value (>40 PL units) arepresented. Kaplan-Meier and proportional hazard survival analyses were used to comparethe time to new AE or VE in aPL-positive versus aPL-negative individuals. Both univariateand multivariate models were run, and parameter estimates compared to assess possibleconfounding variables. Variables included in the regression analyses were time to first AEor VE as outcome variables, and aPL positivity (aCL IgG/IgM >40, confirmed LA, and/oraβ2GPI IgG/IgM) as the predictor variable. Covariates for new AE were: age, gender, SLE,history of previous AE, anticoagulation therapy, APCR, LDL cholesterol, family history ofCVD, smoking, hypertension, diabetes, hsCRP, and hyperhomocysteinemia. Covariates fornew VE were: age, gender, SLE, history of previous VE, anticoagulation therapy, APCR,ATIII, factor V Leiden, and PT mutation.

Analyses for nested case-control studyUnivariate regressions for case-control studies were performed using new AE or VE as theoutcome variable and aPL positivity, DD, FVIII, vWF, FIB, hsCRP, APCR, tHcy, familyhistory of CVD, smoking, SLE, and DM as predictor variables. Variables that were shownto be possible predictors in the univariate analysis were then evaluated as predictor variablesin two separate logistic regression models using either new AE or new VE as outcomevariables. Analyses were performed using SAS (The SAS System for Windows, 2002; TheSAS Institute Inc., Cary, NC, USA).

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ResultsBaseline characteristics

Baseline characteristics of the prospective cohort and nested case-control population (Table1) show a predominantly middle-aged female Caucasian population. No clinically importantdifferences in age, gender, education, or ethnicity were observed based on aPL positivitystatus, but SLE, hypertension, family history of CVD, and smoking were more frequent inthe aPL-positive group. Similarly, prednisone, hydroxychloroquine, anti-platelet, andanticoagulant drug usage was higher in this group. aPL positivity was observed in 59 (14%)individuals. Of these, 41 had only one aPL (10 with aCL, 25 with LA, and six with aβ2GPI);10 had two aPL (three with aCL and aβ2GPI, and seven with aCL and LA); and eight werepositive for all three antibodies. None of the subjects was positive for both LA and aβ2GPI.Previous history of vascular events was confirmed in 68 participants: 40 had AE and 39 hadVE (11 had both). There was an important difference in the percentage of aPL-positiveversus aPL-negative participants with confirmed previous events: AE (24% vs. 7%) and VE(27% vs. 7%). When compared to the overall cohort, the subjects selected for the nestedcase-control study (i.e. those with new events since entry into the study) were older andreported a family history of CVD more frequently. In addition, the nested case-control studycases had a greater proportion of smokers, diabetics, and individuals with aPL positivity,acquired APCR, and elevated levels of hsCRP.

Prospective cohortNew vascular events—Forty-five individuals had 66 new vascular events during themedian 7.4 years of follow-up: 21 individuals developed a new event, while 24 had arecurrence. Table 2 describes the distribution of new events. Overall, there was a greateroccurrence of AE than VE. Individuals with previous events were more likely to incur afuture event. Of 68 individuals with previous events, 24 (35%) went on to develop a newevent compared to 21 (6%) of 346 individuals without previous events (OR=8.4 [4.3, 16.4]).Forty-nine of the individuals with previous events were taking anticoagulant and/orantiplatelet therapy: 27 were on coumadin or heparin, 27 were on clopidogrel oracetylsalicylic acid (ASA), and five were on both anticoagulant and antiplatelet therapy.More importantly, of the 45 individuals with new events, 24 were on anticoagulant/antiplatelet therapy: 11 were on Coumadin or heparin, 15 were on clopidogrel or ASA, andtwo were on both. Although the numbers are small, persons with previous events were morelikely to incur the same type of event. Of the 13 persons with previous AE only, nine (69%)incurred a new AE. Similarly, of the eight persons with previous VE, only five (63%)incurred a new VE. Of the small number of individuals (n=3) with both previous AE andVE, two (67%) incurred a new AE. Three individuals developed both AE and VE as newevents; two (67%) had incurred a previous AE.

Table 3 provides a description of the yearly cumulative events observed. The majority ofindividuals (n=38) had a single new event during the eight-year follow-up, but 11individuals had two new events and two individuals had three new events. Most (39.4%) ofthe new events occurred within the first year of follow-up.

Figure 1 shows the Kaplan-Meier survival curve (with 95% confidence interval) for newvascular events in individuals with and without aPL. The proportion of event-free survivorsat eight years was 90% (95% CI= 87%, 94%) for aPL-negative and 72% (60%, 85%) foraPL-positive individuals. Similarly, the proportion of AE-free and VE-free survivors at eightyears was 93% (90%, 96%) versus 79% (60%, 85%), and 97% (95%, 99%) versus 86%(77%, 97%) for aPL-negative versus aPL-positive individuals, respectively (data notshown).

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Predictors of new vascular events—Predictors (hazard ratio [HR] [95% CI]) for newAE were previous AE (5.7 [2.7, 12.0]), DM (5.6 [2.4, 13.2]), aPL positivity (2.6 [1.2, 5.9]),and age (1.04 [1.01, 1.07]). New VE were predicted with previous VE (6.1 [1.9, 19.9]),aβ2GPI IgG/IgM (5.8 [1.4, 24.1]), APCR (4.1 [1.1, 15.1]), and male gender (3.7 [1.1, 12.9]).

Nested case-control studyResults of univariate and multivariate regression analyses are reported in Table 4. In theunivariate models for AE, DM, previous AE, smoking, aPL positivity, and hsCRP werepredictors of new AE. Previous AE (OR=10.4 [2.8, 39.0]), DM (9.9 [1.5, 64.6]), smoking(4.4 [1.3, 14.4]), and aPL positivity (3.8 [1.0, 14.7]) remained independent predictors in themultivariate analysis. For VE, the univariate analyses identified the following predictors:previous VE, elevated vWF, abnormal APCR, aPL positivity, and elevated FVIII. As FVIIIwas highly correlated with vWF (Pearson correlation coefficient = 0.68), these variableswere run in separate multivariate models. In the multivariate analysis with vWF, abnormalAPCR (5.5 [1.1, 26.6]) and elevated vWF (5.0 [1.2, 19.8]) were independent predictors forVE. When vWF was replaced by FVIII in the same multivariate models, APCR was retainedas a predictor for VE (10.9 [1.6, 76.3]) and FVIII showed an OR of 2.0 (CI=0.9, 4.6) (resultsnot shown).

DiscussionWe found in our prospective cohort study that aPL discriminates between those with andwithout new vascular events as early as the second year of follow-up, and is therefore a goodpredictor of imminent vascular events. We also observed that recurrent AE is more likely todevelop in those with previous AE, and recurrent VE in those with previous VE. When riskfactors were considered in multivariate survival models, aPL positivity independentlypredicted AE. Age, DM, and previous AE were also independent predictors of AE in boththe prospective and nested case-control analyses. Multivariate models for VE in the entireprospective cohort revealed that aβ2GPI, APCR, previous VE, and male gender wereindependent predictors. The results for VE in the nested case-control study differedsomewhat and showed acquired APCR and vWF to be the only independent predictors.

The prospective studies on aPL and vascular events published to date are heterogeneous intheir study populations, designs, statistical methods, as well as in their definitions andchoices of predictor, outcome, and other variables. We reviewed 19 prospective studies (7–25) with at least one aPL defined as predictor variable, a vascular event as outcome variable,and the use of multivariate analytical methods. Eleven studies (7–17) demonstrated that aPLpositivity was an independent predictor of a vascular event, while eight studies (18–25)reported that it was not. Thus, published prospective studies have yielded conflictingfindings, and the different study populations, designs, definitions (predictor, outcome, andother variables), and statistical methods render cross-study comparison difficult.

In the present study, we have attempted to address the problems of some of the previousprospective cohort studies, as well as their comparability. We have chosen a population seenat two tertiary care hospitals for aPL testing and matched them by age, gender, and site withpersons seen for a CBC. This study design maximized recruitment of persons at a highsuspicion for aPL positivity. We have followed the cohort prospectively for an average of7.4 years and have used a single laboratory for measurement of a given risk factor. We havealso carefully documented our outcome (vascular events), and confirmed events individuallythrough chart review and confirmation by a panel of experts.

We faced some limitations in this study. The use of a convenience sample limitsgeneralizability to similar patients seen in academic outpatient services, but allows

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nevertheless the demonstration that aPL is independently associated with a higher risk ofdeveloping a new vascular event. Treatment with anticoagulation or antiplatelet agents couldnot be standardized in this longitudinal observational cohort. We observed a high rate ofrecurring vascular events in treated patients with previous events, as previously documentedby Wittkowsky et al. (26). Since 49/68 (72%) of individuals with previous vascular eventswere anticoagulated or were on antiplatelet agents, it is likely that we are underestimatingthe risk associated to the presence of aPL. Similarly, undocumented self-medication (e.g.ASA) of aPL-positive individuals with no previous vascular events would also lead to anunderestimation of the true risk associated with aPL. The limited number of new vascularevents, particularly VE, resulted in inconclusive findings for some of the thrombophilic riskfactors because of wide confidence intervals (e.g. FVIII [OR=2.0 (0.9, 4.6)] in the survivalanalysis for VE). Furthermore, although aPL best predicted AE, we could not determinewhich individual aPL was most predictive. LA, in particular, would have been interesting toevaluate as an independent variable, and we acknowledge that the use of the recommendedminimum of two or more LA-sensitive assays (27) may have expanded the number of LA-positive individuals in our study and permitted this analysis. Our number of new vascularevents was also insufficient to determine the effect of aPL persistence, number of aPLpresent, or the combined effect of aPL and other thrombophilic risk factors. We havepreviously reported that increased aCL titre and the concomitant presence of multiple aPLare associated with an increased risk of having had a previous vascular event (28), and thatthe odds ratio associated with a previous event increased 1.46-fold with each additionalprothrombotic risk factor (29). We have also shown that the persistence of aCL positivitywas associated with an increased risk of having LA or aβ2GPI in this cohort (30). In light ofour present findings, persistence of aPL positivity, number of aPL, and a combination ofaPL and other risk factors, such as antiphosphatidyletanolamine (31), would be interesting toevaluate in a larger cohort.

It is important to note that aβ2GPI was useful and superseded aPL positivity in predictingVE, validating the recent inclusion of aβ2GPI as one of the laboratory criteria forclassification of APS. Use of the nested case-control design allowed us to evaluateadditional atherosclerotic and thrombophilic risk factors, including APCR, hsCRP, DD, FIB,FVIII, and vWF. Here, we demonstrate that APCR and vWF are risk factors in thedevelopment of VE and are independent of aβ2GPI or aPL. APCR performed consistently inboth the cohort (HR=4.1 [1.1, 15.1]) and nested case-control (OR=5.5 [1.1, 26.6]) analyses.This confirms our previous findings in a retrospective analysis of this cohort, which showedthat aPL-positive individuals with an abnormal APCR phenotype had a three-fold greaterrisk of having had a previous VE than individuals with neither aPL nor APCR (1). Previousstudies have convincingly linked acquired APCR to the presence of inflammatory andautoimmune diseases, low protein S, aPL, and anti-protein S antibodies (32–34). We believethat acquired APCR captures a functional abnormality, possibly mediated by aPL.

In summary, we have found that aPL positivity affects vascular event-free survival, and thatthe difference between the aPL-positive and aPL-negative groups appears early and issignificant within two years of follow-up. These data demonstrate that the risk of newvascular events in aPL-positive individuals is significant even in individuals who have notpreviously had an event. In individuals who had incurred a previous event, there was atendency for the same type of event (AE or VE) to recur, suggesting that predisposingfeatures for AE and VE may differ. This is further supported by our findings that AE werepredicted by traditional cardiovascular risk factors, while VE were predicted bythrombophilic and/or endothelial risk factors. The existence of distinct risk factors for AEversus VE suggests that patients developing these events may represent distinct populations,and that effective prophylaxis of AE and VE, apart from anticoagulation, may not beequivalent. Such differences may also help to explain the conflicting results found in earlier

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prospective studies on aPL and vascular events. Definitive evidence-based studies areneeded to guide clinicians in the preventive treatment of persons with aPL, whether in theabsence or presence of a preceding AE or VE.

AcknowledgmentsFinancial support: This study has been funded in part by operating grants from The Arthritis Society (97/0007) andthe Canadian Institutes of Health Research (MOP-49509 and MOP-64336 [PRF, JK, JR] and MOP-42391 [JR]).PRF is a Distinguished Senior Investigator of The Arthritis Society with additional support from the ArthritisCentre of Excellence, University of Toronto.

We are grateful to Martine Le Comte for the follow-up telephone interviews and entry of data; Rebecca Subang,Marie-Louise Alonso, and Karine Na-deau for their technical assistance; and Drs. Martin Veilleux, Sylvy LaCh-ance, and Suzanne Morin for their work in reviewing all reported vascular events.

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22. Ahmed E, Stegmayr B, Trifunovic J, et al. Anticardiolipin antibodies are not an independent riskfactor for stroke: an incident case-referent study nested within the MONICA and Vasterbottencohort project. Stroke. 2000; 31:1289–1293. [PubMed: 10835446]

23. Levine SR, Salowich-Palm L, Sawaya KL, et al. IgG anticardiolipin antibody titer > 40 GPL andthe risk of subsequent thrombo-occlusive events and death. A prospective cohort study. Stroke.1997; 28:1660–1665. [PubMed: 9303006]

24. Calvo-Alen J, Toloza SMA, Fernandez M, et al. Systemic lupus erythematosus in a multiethnic UScohort (LUMINA) – XXV. Smoking, older age, disease activity, lupus anticoagulant, andglucocorticoid dose as risk factors for the occurrence of venous thrombosis in lupus patients.Arthritis Rheum. 2005; 52:2060–2068. [PubMed: 15986376]

25. van Goor MP, Alblas CL, Leebeek FW, et al. Do antiphospholipid antibodies increase the long-term risk of thrombotic complications in young patients with a recent TIA or ischemic stroke?Acta Neurol Scand. 2004; 109:410–415. [PubMed: 15147465]

26. Wittkowsky AK, Downing J, Blackburn J, et al. Warfarin-related outcomes in patients withantiphospholipid antibody syndrome managed in an anticoagulation clinic. Thromb Haemost.2006; 96:137–141. [PubMed: 16894455]

27. Brandt JT, Triplett DA, Alving B, et al. Criteria for the diagnosis of lupus anticoagulants: anupdate. On behalf of the Subcommittee on Lupus Anticoagulant/Antiphospholipid Antibody of theScientific and Standardisation Committee of the ISTH. Thromb Haemost. 1995; 74:1185–1190.[PubMed: 8560433]

28. Neville C, Rauch J, Kassis J, et al. Thromboembolic risk in patients with high titre anticardiolipinand multiple antiphospholipid antibodies. Thromb Haemost. 2003; 90:108–115. [PubMed:12876633]

29. Hudson M, Herr AL, Rauch J, et al. The presence of multiple prothrombotic risk factors isassociated with a higher risk of thrombosis in individuals with anticardiolipin antibodies. JRheumatol. 2003; 30:2385–2391. [PubMed: 14677182]

30. Neville C, Rauch J, Kassis J, et al. The persistence of anticardiolipin antibodies is associated withan increased risk of the presence of lupus anticoagulant and anti-beta2-glycoprotein I antibodies.Rheumatology (Oxford). 2006; 45:1116–1120. [PubMed: 16510527]

31. Sanmarco M, Gayet S, Alessi MC, et al. Antiphosphatidylethanolamine antibodies are associatedwith an increased odds ratio for thrombosis. A multicenter study with the participation of the

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European Forum on antiphospholipid antibodies. Thromb Haemost. 2007; 97:949–954. [PubMed:17549297]

32. Tomas JF, Alberca I, Tabernero MD, et al. Natural anticoagulant proteins and antiphospholipidantibodies in systemic lupus erythematosus. J Rheumatol. 1998; 25:57–62. [PubMed: 9458203]

33. Nojima J, Kuratsune H, Suehisa E, et al. Acquired activated protein C resistance is associated withthe coexistence of anti-prothrombin antibodies and lupus anticoagulant activity in patients withsystemic lupus erythematosus. Br J Haematol. 2002; 118:577–583. [PubMed: 12139749]

34. Meesters EW, Hansen H, Spronk HM, et al. The inflammation and coagulation cross-talk inpatients with systemic lupus erythematosus. Blood Coagul Fibrinolysis. 2007; 18:21–28.[PubMed: 17179822]

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What is known about this topic?

• Antiphospholipid antibodies are associated with arterial and venous thrombosisbut it remains unclear whether the risk they confer is independent of otheratherosclerotic or thrombophilic risk factors.

• To date, published prospective studies have yielded conflicting findings on theimportance of aPL as a predictive factor of thrombovascular events, and thedifferent study populations, designs, definitions (predictor, outcome, and othervariables), and statistical methods render cross-study comparison difficult.

• Previous studies have convincingly linked acquired activated protein Cresistance to the presence of inflammatory and autoimmune diseases, lowprotein S, aPL, and anti-protein S antibodies.

What does this paper add?

• By measuring the aPL prior to the occurrence of new thrombovascular events,we demonstrate that the presence of antiphospholipid antibodies (aPL) increasesthe risk of later developing a subsequent thrombovascular event and that thiseffect is seen as early as two years of follow-up. At eight years of follow-up, thethrombovascular-free survival was 90% (95% CI=87%, 94%) for those withoutaPL versus 72% (95% CI=60%, 85%) for those with aPL.

• For arterial events, aPL was an independent predictor with other importantpredictors including previous arterial events, diabetes, and age.

• For venous events, anti-β2-glycoprotein I was the best independent aPLpredictor; other important predictors included activated protein C resistance andgender.

• A nested-case control study confirmed the importance of the same predictors forarterial events and identified new potentially important risk factors ofthrombosis for venous events, including activated protein C resistance and vonWillebrand factor (vWF).

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Figure 1. Vascular event-free survival curve (with 95% confidence interval) for individuals with(solid line) and without (dashed line) antiphospholipid antibodies (aPL) over an averageprospective follow-up of 7.4 yearsThe two curves for the number of vascular event-free individuals separate early (within thefirst year of follow-up) and the 95% confidence limits are distinct at two years of follow-up.

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Tabl

e 1

Bas

elin

e ch

arac

teri

stic

s an

d va

scul

ar e

vent

s in

the

MA

PS c

ohor

t (N

=41

4) a

nd in

the

nest

ed c

ase-

cont

rol s

tudy

pop

ulat

ion

(N=

215)

.

MA

PS

coho

rtN

este

d ca

se-c

ontr

ol p

opul

atio

n

[N (

%)

unle

ss s

tate

d ot

herw

ise]

aPL

-neg

ativ

eN

= 3

55aP

L-p

osit

ive

N =

59

Tot

alN

= 4

14^

Cas

esN

=45

Con

trol

sN

=170

Tot

alN

=215

Age

in y

ears

, mea

n (S

D)

46.5

(14

.4)

45.9

(12

.5)

46.4

(14

.1)

52.2

(12

.5)

50.7

(12

.7)

51.0

(12

.6)

Edu

catio

n in

yea

rs, m

ean

(SD

)12

.9 (

2.7)

12.5

(2.

6)12

.9 (

2.7)

12.0

(2.

7)13

.3 (

2.7)

13.0

(2.

8)

Gen

der

– fe

mal

e29

5 (8

3.1)

49 (

83.1

)34

4 (8

3.1)

34 (

75.6

)13

3 (7

8.2)

167

(77.

7)

Rac

e –

Cau

casi

an29

4 (8

2.8)

55 (

93.2

)34

9 (8

4.3)

41 (

91.1

)14

5 (8

5.3)

186

(86.

5)

Smok

ing

95 (

26.8

)20

(33

.9)

115

(27.

8)19

(42

.2)

37 (

21.8

)56

(26

.1)

Dia

bete

s19

(5.

4)4

(6.8

)23

(5.

6)8

(17.

8)3

(1.8

)11

(5.

1)

Hyp

erte

nsio

n50

(14

.1)

16 (

27.1

)66

(15

.9)

13 (

28.9

)30

(17

.7)

43 (

20.0

)

SLE

63 (

17.8

)22

(37

.3)

85 (

20.5

)11

(24

.4)

43 (

25.3

)54

(25

.1)

Fam

ily h

isto

ry o

f C

VD

*16

3 (5

0.9)

37 (

67.3

)20

0 (5

3.3)

27 (

62.8

)94

(58

.8)

121

(59.

6)

LD

L-c

hole

ster

ol m

ean

(SD

)3.

0 (0

.9)

3.3

(0.9

)3.

1 (0

.9)

3.0

(0.8

)3.

1 (1

.0)

3.1

(0.9

)

Thy

roid

gla

nd d

isea

se53

(15

.0)

6 (1

0.2)

59 (

14.3

)3

(6.7

)28

(16

.5)

31 (

14.4

)

Pred

niso

ne27

(7.

6)9

(15.

3)36

(8.

7)5

(11.

1)15

(8.

8)20

(9.

3)

Hyd

roxy

chlo

roqu

ine

44 (

12.4

)10

(16

.9)

54 (

13.0

)5

(11.

1)27

(15

.9)

32 (

14.9

)

Ant

i-pl

atel

et a

gent

s**

42 (

11.8

)19

(32

.2)

61 (

14.7

)13

(28

.9)

26 (

15.3

)39

(18

.1)

Ant

icoa

gula

nts*

*16

(4.

5)18

(30

.5)

34 (

8.2)

11 (

24.4

)12

(7.

1)24

(11

.2)

Est

roge

n th

erap

y***

40 (

13.6

)5

(10.

2)45

(13

.1)

5 (1

4.7)

20 (

15.0

)25

(15

.0)

Prev

ious

vas

cula

r ev

ents

43 (

12.1

)25

(42

.4)

68 (

16.4

)24

(53

.3)

21 (

12.4

)45

(20

.9)

A

rter

ial e

vent

s (A

E)

26 (

7.3)

14 (

23.7

)40

(9.

7)17

(37

.8)

13 (

7.6)

30 (

14.0

)

V

enou

s ev

ents

(V

E)

23 (

6.5)

16 (

27.1

)39

(9.

4)11

(24

.4)

11 (

6.5)

22 (

10.2

)

Preg

nanc

y m

orbi

dity

****

17 (

5.7)

42 (

85.7

)59

(17

.1)

9 (2

6.5)

24 (

18.0

)33

(19

.8)

APC

R+

(≤2)

79 (

22.7

)27

(47

.4)

106

(26.

2)17

(37

.8)

36 (

21.6

)+ +

53 (

25.0

)

A

cqui

red

60 (

17.2

)23

(40

.4)

83 (

20.5

)15

(33

.3)

27 (

16.2

)42

(19

.8)

In

heri

ted

16 (

4.6)

2 (3

.5)

18 (

4.4)

2 (4

.4)

6 (3

.6)

8 (3

.8)

aPL

pos

itive

0 (0

)59

(10

0)59

(14

.2)

15 (

33.3

)21

(12

.4)

36 (

16.7

)


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MA

PS

coho

rtN

este

d ca

se-c

ontr

ol p

opul

atio

n

[N (

%)

unle

ss s

tate

d ot

herw

ise]

aPL

-neg

ativ

eN

= 3

55aP

L-p

osit

ive

N =

59

Tot

alN

= 4

14^

Cas

esN

=45

Con

trol

sN

=170

Tot

alN

=215

Hom

ocys

tein

e m

ean

(SD

)9.

4 (4

.3)

9.9

(4.5

)9.

5 (4

.4)

9.9

(3.4

)10

.0 (

4.2)

##10

.0 (

4.0)

D-d

imer

(D

D)

mea

n (S

D)

--

-0.

7 (1

.3)

0.6

(1.9

)0.

6 (1

.8)

Fibr

inog

en (

FIB

) m

ean

(SD

)-

--

3.6

(0.9

)3.

8 (0

.8)

3.6

(1.8

).

Fact

or V

III

(FV

III)

mea

n (S

D)

--

-1.

5 (0

.9)

+++

1.3

(0.7

)1.

4 (0

.7)

vWF

mea

n (S

D)

--

-1.

8 (1

.0)

1.4

(0.7

)1.

5 (0

.8)

hsC

RP

mea

n (S

D)

--

-6.

8 (1

0.6)

3.9

(6.7

) #

4.5

(7.8

)

^ One

per

son

was

mis

sing

blo

od s

ampl

es.

* 39 p

artic

ipan

ts in

the

who

le c

ohor

t and

12

in th

e ne

sted

cas

e-co

ntro

l stu

dy p

opul

atio

n w

ere

unce

rtai

n ab

out f

amily

his

tory

: per

cent

ages

wer

e ca

lcul

ated

on

n =

375

and

n =

203

, res

pect

ivel

y;

**A

nti-

plat

elet

age

nts

wer

e A

SA a

nd c

lopi

dogr

el, a

nd a

ntic

oagu

lant

s w

ere

war

fari

n an

d he

pari

n.

*** pe

rcen

tage

cal

cula

ted

on 3

44 f

emal

es;

****

preg

nanc

y m

orbi

dity

bas

ed o

n se

lf-r

epor

t and

acc

ordi

ng to

the

revi

sed

clas

sifi

catio

n fo

r th

e A

PS (

6);

+ APC

R (

acqu

ired

= 4

2, in

heri

ted

= 8

, and

3 in

divi

dual

s di

d no

t hav

e Fa

ctor

V L

eide

n te

stin

g);

+ + A

PCR

mis

sing

in 3

con

trol

s;

+++ Fa

ctor

VII

I (F

VII

I) m

issi

ng in

1 c

ase;

# high

-sen

sitiv

ity C

-rea

ctiv

e pr

otei

n (h

sCR

P) m

issi

ng in

3 c

ontr

ols;

## h

omoc

yste

ine

mis

sing

in 1

con

trol

. SL

E =

sys

tem

ic lu

pus

eryt

hem

atos

us; C

VD

= c

ardi

ovas

cula

r di

seas

e; L

DL

= lo

w-d

ensi

ty-l

ipop

rote

in; e

stro

gen

ther

apy

= h

orm

one

repl

acem

ent t

hera

py o

r or

alco

ntra

cept

ion.


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Table 2

Individuals with new vascular events since entry into the cohort.

Number of individuals with new events

Arterial event Venous event Both

Individuals with previous events (N=24)1

Arterial event 9 2 2

Venous event 2 5 1

Both arterial and venous events 2 1 0

Individuals with no previous event (N=21)* 16 4 1

*The distribution of events in individuals (n = 45) with a new event since entry into the cohort is shown: 24 persons had experienced a previous

event and 21 persons had no previous event. The 45 individuals with new events are from the cohort (n = 414), in which 68 individuals hadprevious events and 346 had no previous event.


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Table 3

New vascular events occurring during follow-up years in the study population (N=45).

Follow-up year N* Arterial events (in N persons) Venous events (in N persons) Total events (in N persons)

1 351 16 (15) 10 (9) 26 (22)**

2 339 8 (6) 1 (1) 9 (7)

3 316 4 (4) 5 (3) 9 (7)

4 308 5 (5) 0 5 (5)

5 300 2 (2) 2 (2) 4 (4)

6 287 3 (3) 0 3 (3)

7 284 5 (5) 2 (2) 7 (7 )

8 204 2 (2) 1 (1) 3 (3)

*N represents the number of individuals in the cohort at each year of follow-up.

**At year 1, 1 person had 2 arterial events, 1 person had 2 venous events, and 2 persons had both arterial and venous events.


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Table 4

Predictors of new arterial and venous events in univariate and multivariate analyses from the nested case-control study population.

Predictor Arterial events Venous events

UnivariateOR (95% CI)

MultivariateOR (95% CI)

UnivariateOR (95% CI)

MultivariateOR (95% CI)

aPL 3.6 (1.4, 8.8) 3.8 (1.0, 14.7) 4.8 (1.4, 17.2) 2.2 (0.3, 15.0)

APCR 1.8 (0.8, 3.9) ND 5.7 (1.7, 18.6) 5.5 (1.1, 26.6)

vWF 1.5 (0.9, 2.4) 1.0 (0.4, 2.6) 6.4 (1.8, 21.8) 5.0 (1.2, 19.8)

hsCRP 1.05 (1.00, 1.07) 1.00 (0.93, 1.07) 1.02 (0.97, 1.07) ND

Smoking 4.2 (1.7, 10.3) 4.4 (1.3, 14.4) 1.9 (0.6, 6.5) ND

Diabetes 7.8 (2.0, 30.5) 9.9 (1.5, 64.6) * ND

Previous AE 7.2 (2.7, 19.1) 10.4 (2.8, 39.0) ND ND

Previous VE ND ND 11.1 (2.3, 54.1) 1.8 (0.2, 16.4)

*Uninterpretable data: one person had diabetes and a new venous event. ND (not done) indicates where variables were not used in the respective

models. OR (95%CI), odds ratio with 95% confidence intervals; aPL, antiphospholipid antibodies; APCR, activated protein C resistance; vWF, vonWillebrand factor; hsCRP, high-sensitivity C-reactive protein; AE, arterial event; VE, venous event. Variables found to predict arterial or venousevents by univariate or multivariate analysis are shown in bold.


antiphospholipid antibodies predict imminent vascular events independently from other risk factors...

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