ANTIPHOSPHOLIPID SYNDROME (D ERKAN, SECTION EDITOR)

Clinical Relevance of β2-Glycoprotein-I Plasma Levelsin Antiphospholipid Syndrome (APS)

Alessandra Banzato & Vittorio Pengo

Published online: 6 May 2014# Springer Science+Business Media New York 2014

Abstract Antiphospholipid syndrome (APS) is characterizedby the presence of antiphospholipid (aPL) antibodies associat-ed with thrombosis or pregnancy morbidity. The antibodiesmainly involved in this disorder are directed againstβ2-glycoprotein I (β2-GPI). β2-GPI plasma level is usuallynot reported in studies on APS, because it is not regarded asrelevant to the diagnosis and prognosis of APS. Neverthelessits measurement may be important for understanding the path-ophysiology of the syndrome. This review summarizes avail-able data from the literature on plasma concentrations ofβ2-GPI in patients with different antibody profiles.

Keywords Beta-2-glycoprotein I . Antiphospholipidsyndrome . Plasma levels . APS . Antiphospholipidantibodies . Thrombosis

Introduction

Beta-2 glycoprotein I (β2-GPI), an apolipoprotein with anapproximate molecular weight of 50 kD, is the main factorimplicated in the pathophysiology of antiphospholipid syn-drome (APS). Discovered more than 50 years ago [1–3] itconsists of five short consensus repeats, also called sushidomains [4]. The crystal structure of this protein was firstreported in 1999 by two independent groups [5, 6]; it has aJ-shaped conformation, with a vertical ascending part calleddomain I and a positive area in the bottom part of the moleculecalled domain V. Domains I, II, III, and IVare each composed

of approximately 60 amino acids whereas domain V differsfrom the other four domains and consists of 82 amino acidsincluding a C-terminal, hydrophilic, positively charged patchenabling β2-GPI to bind to anionic surfaces, for example asactivated blood cells [6].

The physiological function of β2-GPI is not clear. It isbelieved to be involved in regulation of FXI and in plasmin-ogen activation [7, 8]. It contributes to triglyceride clearance[9] and regulates uptake and proteolytic degradation of oxi-dized low-density lipoprotein by scavenger cells [10]. Itsprocoagulant activity is attributed to reduction of protein C-related activity, thus inhibiting the phospholipid-dependentcoagulation pathway [11]. In APS patients, another antibodyis believed to be involved in protein C activity: anti-cardiolipin antibodies (aCLs) [12, 13].

Recently our group demonstrated an important interactionbetween β2-GPI and thrombin [14]. β2-GPI has unique anti-coagulant properties and selectively inhibits the procoagulantfunctions of thrombin without altering its anticoagulant activ-ity. These findings shed light on a novel physiological antico-agulant function of this protein.

For many years, attention has been focused on anti-β2-GPIantibodies rather than on the β2-GPI itself. However, there isgrowing interest in understanding the physiological effect ofβ2-GPI and determining its plasma levels.

Historical Perspective

In 1993, Vlachoyiannopoulos et al. [15] studied the corre-lation between anti-phosphatidylethanolamine (PE) anti-bodies, aCLs, and β2-GPI plasma levels in patients withAPS and systemic lupus erythematosus (SLE). They foundthat APS patients had higher β2-GPI levels than SLE pa-tients and its concentration was associated with aCL andanti-phosphatidylethanolamine (aPE) activity.

This article is part of the Topical Collection on Antiphospholipid Syndrome

A. Banzato (*) :V. PengoDepartment of Cardiac Thoracic and Vascular Sciences, Universityof Padova, Via Giustiniani, 2, 35128 Padova, Italye-mail: alessandra.banzato@unipd.it

Curr Rheumatol Rep (2014) 16:424DOI 10.1007/s11926-014-0424-9

In 1995, McNally et al. [16] measured plasma levels of β2-GPI antigen and β2-GPI binding antibodies in patients withSLE and primary APS (PAPS). They found both were in-creased in patients with PAPS and a history of thromboem-bolic complications or recurrent fetal loss. In their work, theymeasured β2-GPI antigen (β2-GPI:Ag), β2-GPI aPL cofactoractivity (β2-GPI:Cof), and antibodies to β2-GPI (alpha β2-GPI) in SLE patients with aPL antibodies (SLE-aPL+) and inpatients with primary APS (PAPS). β2-GPI:Ag levels weresignificantly increased in SLE-aPL + patients and PAPS pa-tients compared with SLE-aPL − patients and normal healthycontrols. The β2-GPI:Cof/Ag ratio was significantly reducedin SLE-aPL + patients compared with SLE-aPL − patients,indicating functional modification of β2-GPI in SLE-aPL +patients. These results suggested involvement of β2-GPI inthe pathogenic mechanism of thrombosis associated withaPLs.

The same group [17] investigated levels of β2-GPI inAPS patients. Although the total β2-GPI levels of the SLE-aPL + patients were significantly increased compared withSLE-aPL − patients and normal controls, no significantdifferences between free β2-GPI levels were observed be-tween these groups. These results suggested that levels ofcomplexed β2-GPI were increased in patients with aPL,perhaps as a result of immune complex formation, or al-tered binding to other plasma constituents.

In 1998, Balasubramanian et al. [18] measured plasma β2-GPI levels, by use of a novel competitive ELISA assay, todetermine the concentration of free β2-GPI and the fraction ofantibody-bound β2-GPI. In this way, quantification of totalantigen in individuals with autoimmune antibodies could beeasily achieved. When plasma samples from normal individ-uals (free from blocking antibodies) were assessed with bothcompetitive and standard ELISA the results did not differ.However, in plasma samples from APS patients, β2-GPIlevels were significantly lower, indicating that a substantialfraction of the plasma β2-GPI was bound by antibody.

Mehdi et al. [19], in 1999, reported a correlation between agenetic variation in the apolipoprotein H gene (APOH, gene)and plasma concentrations of β2-GPI (apoH, protein). APOHis polymorphic, with three common alleles (APOH*1,APOH*2, and APOH*3). The APOH*2 allele is the mostfrequent in all populations. The authors sought to determinethe effect of APOH polymorphism on apoH levels innormoglycemic non-Hispanic Whites. Levels of apoH proteinwere positively related to the polymorphism in older women(a 3.4 % phenotypic variance). However, analysis of varianceshowed that APOH polymorphism and apoH protein levelswere positively correlated in both men and women. As inother metabolic pathways, genetic polymorphism might becorrelated with β2-GPI (apoH, protein) levels.

In 2000, Yasuda et al. [20] studied β2-GPI deficiency inhealthy Japanese individuals. Low plasma concentrations of

β2-GPI were not associated with apparent abnormalities inlipoprotein metabolism.

Lower levels of β2-GPI might, in fact, be determined byreduced gene expression as related to a functional polymor-phism at the transcriptional initiation site [21]. Large inter-individual variability in β2-GPI plasma levels, mostly underthe control of genetic factors, is widely accepted but its mo-lecular basis remains largely unknown. When analyzed byDHPLC, the 5’ flanking region of the β2-GPI gene had apoint mutation at the transcriptional initiation site (-1C→A)with a carrier frequency of 12.1 %. The mutation was associ-ated with significantly lower β2-GPI plasma levels(P<0.0001) and low occurrence of aPL in lupus patients(4.8 % antibody-positive group vs. 16.6 % in the antibody-negative group; P=0.019).

In 2004, Song et al. [22] evaluated the correlation betweenthrombotic events in patients with liver cirrhosis and theprevalence of β2-GPI antibodies. Plasma β2-GPI levels wereaffected by the presence of cirrhosis and correlated well withChild classification. Anti-β2-GPI antibodies were not corre-lated either with β2-GPI levels or with thrombosis occurrencein these patients.

Fig. 1 a Patients with a high-risk aPL pattern (triple-positivity) have ahigh plasma β2GPI concentration (0.386±0.19 mgmL−1) compared withother, less risky, profiles (double or single-positivity patients, 0.218±0.09 mg mL−1 and 0.229±0.09 mg mL−1, respectively). b Patients withcatastrophic APS (CAPS) have higher β2GPI levels during the quiescentperiod (0.431±0.094 mg mL−1) than on the day of catastrophic clinicalmanifestations (0.361±0.088 mg mL−1, P=0.3)

424, Page 2 of 5 Curr Rheumatol Rep (2014) 16:424

Table1

Articlesreportingβ2-G

PIlevels

Articlesanalyzed

Results

Author

Year

Journal

Beta2

levels

Groupsevaluated

Rem

arks

VlachoyiannopoulosPG

etal.[15]

1993

Autoimmunity

APS

patientshadhigh

β2-G

PIlevels

SLEandAPS

APS

patientshadhigher

β2-G

PIlevelsthan

SLEpatients

McN

ally

etal.[16]

1995

BrJRheum

atol

Highβ2-G

PIlevelsin

APS

SLEor

PAPS

Resultssuggestthatβ

2-G

PIiscrucialin

thrombosisassociated

with

APS

McN

ally

etal.[17]

1995

Blood

CoagulF

ibrinolysis

Highβ2-G

PIlevelsin

APS

SLE+APS

orSLE

β2-G

PIcomplexed

iselevated

inpatientswith

APS

BalasubramanianKetal.[18]

1998

Throm

bRes

Highβ2-G

PIlevelsin

APS

APS

β2-G

PIplasmafractio

nisboundto

antibodies

inAPS

patients

MehediH

etal.[19]

1999

Hum

Genet

Low

β2-G

PIlevel

Polym

orphism

ornot

Geneticvariations

ofβ2-G

PIdeterm

ine

differentβ

2-G

PIplasmalevels

YasudaSetal.[20]

2000

Atherosclerosis

Low

β2-G

PIlevel

β2-G

PIdeficiency

Abnormallip

oprotein

metabolism

caused

β2-G

PIlevelsvariation

MehediH

etal.[21]

2003

Eur

JBiochem

Low

β2-G

PIlevel

Polym

orphism

Geneticvariations

ofβ2-G

PIdeterm

inedifferent

β2-G

PIplasmalevels

Song

KSetal.[22]

2004

Clin

ApplT

hrom

bHem

ost

Low

β2-G

PIlevel

Liver

cirrhosis

Noβ2-G

PIvariations

with

positiv

ea-β2-G

PIantib

odies

Lin

Fetal.[23]

2006

Lupus

Low

β2-G

PIlevel

Throm

botic

disordersand

inflam

mation

Largestreductionin

β2-G

PIlevelsoccurred

inpatientswith

thehighestC

RPvalues

deLaatB

etal.[24]

2009

Blood

Highβ2-G

PIlevel

Elderly

men

with

myocardial

infarctio

nReduced

β2-G

PIlevelsareassociated

with

alower

risk

ofmyocardialinfarction

Gries

A[25]

2009

Clin

Chim

Acta

Low

erβ2-G

PIlevel

Liver

cirrhosispopulatio

nβ2-G

PIplasmalevelsarereducedin

liver

disorders

Banzato

etal.

2014

CurrRheum

atol

Rep

Highβ2-G

PIplasmalevelin

triple-positive

APS

Triple,double,singleaPLor

CAPS

β2-G

PIishigh

intriplepositiv

itypatientsand

decreasesin

catastrophiccondition

Curr Rheumatol Rep (2014) 16:424 Page 3 of 5, 424

To further investigate the involvement of β2-GPI in throm-bosis and inflammation, in 2006, Lin et al. [23] measuredcirculating levels of β2-GPI in healthy controls, normal preg-nancies, patients with non-hemorrhagic stroke, patients withacute coronary syndrome, and patients with elevated C-reactive protein (CRP). β2-GPI levels were positively corre-lated with age (r=0.274, P<0.001) and dropped significantlyafter the eighth week of pregnancy (P=0.002). Reduced levelsof β2-GPI protein over time were reported for patients withstroke and myocardial infarction. As for inflammation, β2-GPI levels were negatively related to CRP (r=−0.284,P<0.001) and positively related to albumin and transferrin(r=0.372 and 0.453, respectively, with P<0.001 for both).

In 2009, de Laat et al. [24] showed that β2-GPI plasmalevels were related to the risk of myocardial infarction in oldermen. When men with a first myocardial infarction were com-pared with controls there was no important correlation be-tween β2-GPI plasma levels and the incidence of myocardialinfarction in the overall population. However, they found adose-dependent protective effect of increasing β2-GPI plasmalevels in men 60 years and older (OR 0.41, 95 % CI 0.22–0,74). This protective effect persisted despite high levels ofvWF.

Gries, in 2009 [25], studied β2-GPI plasma levels in livercirrhosis. They found thatβ2-GPI plasma levels are reduced inchronic liver disease.

Personal Experience

Our group set up a “sandwich” ELISA using a rabbit poly-clonal anti-human β2-GPI coated on a plate and a mousemonoclonal anti-human β2-GPI to detect the plasma concen-tration of bound β2-GPI. The system was developed by usinga phosphatase-labeled goat anti-mouse antibody. Increasingconcentrations of human purified β2-GPI were used to gener-ate a calibration reference curve. Plasma of 23 patients withAPS and triple positivity (LAC+, IgG aCL+, IgG aβ2-GPI+),eight with double positivity (IgG aCL+, IgG aβ2-GPI+, LACnegative), five with single positivity (IgG aβ2-GPI+, LAC andaCL negative) and 20 controls were evaluated. Moreover,plasma of four catastrophic APS patients (CAPS) were eval-uated on the day of CAPS clinical manifestations and afterrecovery. Median values between groups were compared byuse of the Mann–Whitney test (Fig. 1).

β2GPI concentrations were significantly higher in plasmafrom patients with triple positivity (LAC+, IgG aCL+, IgGaβ2GPI+) than in that from control subjects (0.386±0.19 mg mL−1 vs 0.175±0.06 mg mL−1, P=0.009). For dou-ble positivity (IgG aCL+, IgG aβ2GPI+) and single positivity(IgG aβ2GPI+) patients β2GPI levels were similar to those ofcontrols (0.218±0.09 mg mL−1 and 0.229±0.09 mg mL−1,respectively). Plasma from patients with CAPS contained

higher concentrations of β2GPI during the quiescent period(0.431±0.094 mg mL−1) than on the day of catastrophicclinical manifestations (0.361±0.088 mg mL−1, P=0.3). Ourresults revealed that the aPL profile was an important deter-minant of β2-GPI level.

Clinical Significance of Measuring β2-GPI Levels

On the basis of the results presented in Table 1, it is reasonableto affirm that β2-GPI plasma levels are increased in patientswith APS. The presence of free β2-GPI and β2-GPI–a β2-GPIcomplexes or altered binding to other plasma constituentscould explain the larger amount of circulating protein [17,18, 26]. Moreover, this might account for the higher β2-GPIlevels in APS patients than in SLE patients. Looking at ourown results, it is apparent that levels are increased in patientswith aβ2-GPI antibodies and especially in those with a hightiter of antibodies and those with triple positivity. On the otherhand, the increased amount of β2-GPI may be related tooverproduction by the liver because of its rapid turnover.This second hypothesis is not easy to demonstrate, becausethe protein should be tagged and re-injected in patients withAPS. Interestingly enough, the level of β2-GPI decreases inpatients with CAPS, meaning that its rapid consumption istaking place without immediate compensation by the liver. Tounderstand if this consumption is because of formation ofthrombi in the microcirculation remain to be proved. We donot see, at this time, a need for systematic measurement of theprotein in clinical practice; this is, at the moment, a matter forresearch only.

Conclusions and Future Research Directions

It could be useful and relevant to understand whether β2-GPIis directly involved in micro thrombi formation in CAPS.Immunochemical studies of biopsies of patients with CAPSmight provide insight into this.

Compliance with Ethics Guidelines

Conflict of Interest Alessandra Banzato and Vittorio Pengo declarethat they have no conflict of interest.

Human and Animal Rights and Informed Consent This article doesnot contain any studies with animal subjects performed by any of theauthors.

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