Pathogenesis and management of antiphospholipid syndrome · pathogenesis of APS has significant implications for treatment: agents acting outside the coagulation system such as hydroxychloroquine

1

Pathogenesis and management of antiphospholipid syndrome

Deepa Jayakody Arachchillage, Mike Laffan

Department of Haematology, Imperial College Healthcare NHS Trust and Imperial College

London, Hammersmith Hospital, 4th Floor, Commonwealth Building, Du Cane Road, London

W12 0NN,

Address for correspondence:

Dr Deepa RJ Arachchillage, Department of haematology, Imperial College Healthcare NHS

Trust and Imperial College London,, Hammersmith Hospital, 4th Floor, Commonwealth

Building, Du Cane Road, London W12 0NN

Tel: +44 (0) 20 7351 8403, FAX: +44 (0) 2073518402

E-mail: [email protected]

Running head: Antiphospholipid syndrome

Key Words:

Antiphospholipid antibodies, thrombosis, warfarin, pregnancy Complications, complement,

Immunosuppression

Abstract word count: 199

Text word count: 5260

Number of figures and tables: 3 figures and 3 tables

2

Abstract

Antiphospholipid antibodies are a heterogeneous group of autoantibodies which have clear

associations with thrombosis and pregnancy morbidity, and which together constitute the

‘antiphospholipid syndrome’ (APS). However, the pathophysiology of these complications is

not well understood and their heterogeneity suggests that more than one pathogenic

process may be involved. Diagnosis remains a combination of laboratory analysis and clinical

observation but there have been significant advances in identifying specific pathogenic

features such as domain I-specific anti-beta 2GPI antibodies. This in turn has pointed to

endothelial and complement activation as important factors in the pathogenesis of APS.

Consequently, although anticoagulation remains the standard treatment for thrombotic APS

and during pregnancy, the realisation that these additional pathways are involved in the

pathogenesis of APS has significant implications for treatment: agents acting outside the

coagulation system such as hydroxychloroquine for pregnancy complications and sirolimus

as an inhibitor of the mammalian target of rapamycin (mTOR) pathway are now under

evaluation and represent a radical change in thinking for haematologists. Conventional

anticoagulation is also under challenge from new, direct acting anticoagulants. This review

will provide a comprehensive overview of the evolving understanding of APS pathogenesis

and how this and novel therapeutics will alter diagnosis and management.

3

Introduction

Antiphospholipid syndrome (APS) is characterized by vascular thrombosis and/or pregnancy

loss or morbidity in association with persistent positivity of autoantibodies known as

antiphospholipid antibodies (aPL) (Miyakis et al, 2006) (Table 1). Vascular thrombosis could

be arterial, venous, or small vessel, in any tissue or organ and must be confirmed by

objectively (i.e. unequivocal findings of appropriate imaging studies or histopathology).

Thrombosis in APS should show no signs of inflammation in the vessel wall (Miyakis et al,

2006). Although many patients with APS have an associated autoimmune disorder,

thrombotic events in APS are not accompanied by histological evidence of vessel wall

inflammation. Nonetheless, inflammatory mediators and inflammatory responses in

endothelial cells, monocytes and neutrophils are implicated in APS pathogenesis. The

International Consensus Criteria (ICC: Sydney; updated Sapporo) (Miyakis et al, 2006) for

APS were designed for scientific clinical studies but have been adapted for the diagnosis of

APS in routine clinical practice. The British Committee for Standards in Haematology (BCSH)

guidelines (Keeling et al, 2012) adopt similar criteria for the diagnosis of both thrombotic

and obstetric APS. Non-criteria features of APS such as heart valve disease, livedo reticularis,

thrombocytopenia and nephropathy may present in association with thrombosis and/or

pregnancy morbidity or as isolated features. The APS Clinical Features Task Force of the 14th

International Congress on aPL analysed the relevance of the most frequent non-criteria

manifestations and concluded that available data support the inclusion of

thrombocytopenia, heart valve disease, renal microangiopathy (aPL nephropathy), chorea,

and longitudinal myelitis as part of APS Classification Criteria (Abreu et al,2015 ). However,

4

these features are not yet included in the ICC. APS can occur in isolation or in association

with other autoimmune disorders such as systemic lupus erythematous (SLE) or rheumatoid

arthritis (Miyakis et al, 2006).

aPL are a heterogeneous group of autoantibodies, which include lupus anticoagulant (LA),

IgG and IgM anticardiolipin antibodies (aCL) and anti-β2-glycoprotein-I (anti-β2GPI)

antibodies. Their primary targets are phospholipid-binding proteins, although antibodies

directed against phospholipids and other proteins also occur. As early as 1952 Conley and

Hartmann wrote a brief report about two patients with SLE and a ‘peculiar haemorrhagic

disorder’ with prolonged blood clotting times and clear evidence of an anticoagulant in

plasma mixing studies (Conley & Hartmann et al, 1952). As this inhibitor was predominantly

found in patients with SLE, the in vitro anticoagulant phenomenon was called LA. The

paradoxical correlation between vascular thrombosis and LA was first described in 1963 by

Bowie et al (Bowie et al, 1963) and followed by many others. Originally, it was thought that

these autoantibodies bound to phospholipid; hence ‘antiphospholipid antibodies’ but in the

1990s it was shown that aPL recognized phospholipids indirectly via phospholipid-binding

plasma proteins (Galli et al,1990). Although many plasma proteins have been found to be

antibody ligands in APS, antibodies against β2GPI have the most significant association with

pathogenicity (Bas de et al,2004). Some authors have challenged the primacy of anti-β2GPI

antibodies in the pathogenesis of APS (Lackner & Muller-Calleja et al, 2016) and shown that

some co-factor independent antibodies can induce thrombus formation in a mouse model

(Manukyan et al, 2016).

5

Laboratory diagnosis of APS requires presence of LA in plasma or aCL of IgG and/or IgM

isotype in serum or plasma, present in medium or high titre (i.e. >40 GPL or MPL, or >the

99th centile) or anti-β2GPI of IgG and/or IgM isotype in serum or plasma (in titre >the 99th

centile). The positive results must be obtained on two or more occasions, at least 12 weeks

apart (Miyakis et al, 2006). Inter-laboratory variability remains a problem in the

performance of the aCL and anti-β2GPI ELISAs due to the lack of uniformity in reference

materials for calibration.

Nonetheless, identification of these antibodies has allowed development of diagnostic

testing and investigation of pathogenic mechanisms. These in turn offer additional

therapeutic options which are currently under investigation. Even conventional

anticoagulant therapy is under review while the role of direct acting oral anticoagulants is

investigated. The key events in APS are summarised in figure 1. The aim of this review is to

examine the evolving understanding of APS pathogenesis and how this and other

therapeutic options will change the way we manage this potentially devastating disorder.

Pathogenesis of Antiphospholipid syndrome

Despite clear associations between aPL and thrombosis and with pregnancy morbidity, the

pathophysiology of these complications is not well understood, with their heterogeneity

suggesting that more than one pathogenic process is involved. Moreover, even though aPL

are persistently present in the systemic circulation, thrombotic events occur only

occasionally, suggesting that the development of aPL is a necessary, but not sufficient step

in the development of APS and that other factors play a role. Such ‘second hits’ or ‘triggers’

6

likely push the thrombotic/haemostatic balance in favour of thrombosis and may include

infection, endothelial injury, inflammation, immunological and other non-immunological

procoagulant factors such as oestrogen containing contraceptive pills, surgery and

immobility (Pengo et al, 2011). The patient’s genetic constitution, in relation to genes for

inflammatory mediators, may also be a critical variable in the development of clinical APS

manifestations.

Antibody specificities and evidence for pathogenicity of anti-β2-GPI

In 1990, McNeil et al. identified that the binding of aPL to cardiolipin requires the presence

of β2GPI as a cofactor (Mcneil et al,1990). β2GPI is an apolipoprotein; a member of the

complement control family and is considered to be a natural inhibitor of coagulation. In

addition, in vitro studies have shown that it has anti-angiogenic (Yu et al, 2008) and anti-

apoptotic (Maiti et al, 2008) activities. It consists of 326 amino acids, arranged in 5 highly

homologous complement-control protein domains, designated I to V from the N- to the C-

terminus. β2GPI can adopt two different conformations: a circular conformation in plasma

maintained by interaction between the first and fifth domain and an “activated” open

conformation (Agar et al,2010). In the presence of anionic phospholipids, the circular

conformation of the protein unfolds, exposing antigenic determinants that are normally

shielded from the circulation (van Os et al,2011)[Figure 2]. One of these “cryptic” antigenic

determinants, within domain I (DI), is the epitope for the pathologic autoantibodies (de Laat

et al2006, ;Ioannou et al,2007); which will not bind to the closed circulating form. Unfolding

of β2GPI on the endothelial surface may be facilitated by release of molecules such as

thioredoxin in response to oxidative stress which can reduce surface thiols on β2GPI. Binding

7

of β2GPI on endothelial cells (EC) normally has a protective function against oxidative stress

induced cell injury (Ioannou et al,2010; Ioannou et al,2011) but antibody binding fixes β2GPI

in this open conformation on the phospholipid surface and the antibody-β2GPI complexes

bind to a variety of receptors (e.g., Toll-like receptors 2 and 4, annexin A2, and glycoprotein

1bα) on different cell types, including ECs, monocytes, trophoblasts and platelets (de Groot

& Meijers,2011). This binding may trigger intracellular signalling and inflammatory

responses.

Direct evidence for the pathogenicity of these antibodies comes from infusion of

autoantibodies from APS patients to mice with injured blood vessels showing that they

potentiate thrombus formation (Arad et al,2010). Specifically, purified anti-β2GP1 IgG

autoantibodies, but not anti-β2GPI depleted IgG or normal human IgG potentiated thrombus

size in a dose-dependent manner. Although several clinical studies and systematic reviews

suggested that LA is a stronger risk factor for the development of thrombosis (DE Groot et

al, 2005;Galli et al, 2003), other studies have shown that the presence of LA alone is not

associated with thromboembolic events (Pengo et al, 2005, ;Pengo et al,2007). Similar

results were obtained in the Leiden Thrombophila case–control study (de Groot et al,2005)

where positive LA with negative anti-β2GPI or antiprothrombin ELISAs was not a risk for DVT

(OR 1.3, 95% CI: 0.3–6.0). However, a recent systematic review and meta-analysis found

that LA and aCL antibodies were associated with an increased risk of VTE (OR = 6.14 [CI 2.74;

13.8] and OR = 1.46 [CI 1.06; 2.03] respectively) but there was a non-significant trend for

anti-β2GPI (OR = 1.61 [CI 0.76; 3.43]. For arterial thrombosis, all three antibodies showed a

significant association: ORs for LA, aCL and anti-β2GPI were 3.58 (CI 1.29–9.92), 2.65 (CI

8

1.75–4.00) and 3.12 (CI 1.51–6.44) respectively (Reynaud et al, 2014). de Laat and co-

workers found that anti- β2GPI with LA activity are antibodies that are responsible for the

thromboembolic complications in APS (de Laat et al, 2004). Moreover, they showed that a

subgroup of anti-β2GPI recognizing epitope Gly40-Arg43 (G40-R43) in domain I (DI) of the

molecule cause LA and that their presence correlates strongly with thrombosis (de Laat et

al,2005). This has been confirmed by various other studies (Pengo et al,2015 ;de Laat et al,

2009).

Consequently, these pathogenic antibodies will only bind purified β2GPI when coated onto

an appropriate negatively charged surface and this has important implications for the

design, selection and significance of diagnostic assays (de Laat et al,2005 ;de Laat et al,2006

). It has been shown that exposure of the critical DI epitope G40-R43 on β2GPI is highly

variable between commercial anti-β2GPI assays. As a consequence, false negatives can arise

in assays characterized by a reduced exposure of G40-R43 (Pelkmans et al,2013).

The presence of triple aPL positivity, as defined by the detection of LA and high titers of aCL

and anti-β2GPI antibodies, correlates better with both thrombosis and pregnancy morbidity

than any other aPL profile (Pengo et al,2010): the risk of recurrent thrombosis in triple

positive patients was around 30% over a 6-year follow up period (Pengo et al, 2010). Triple

positive patients also have high titres of autoantibodies that bind the major B-cell epitope

on DI of the β2GPI molecule (Banzato et al,2011). Thus DI anti–β2GPI autoantibodies; confer

LA activity associated with the highest risk of thrombosis (de Laat et al, 2009), frequently

present in triple aPL-positive patients, are closely associated with both thrombosis and

pregnancy loss (de Laat et al, 2009) and increase thrombus size in mouse models (Pericleous

9

et al, 2015). Of note, about 30% of patients with anti-β2GPI antibodies are negative for DI-

anti–β2GPI antibodies (Pengo et al,2015) and the clinical significance of autoantibodies

reacting with epitopes other than DI was investigated in a multicentre study (Artenjak et

al,2015). In this study serum samples from 201 autoimmune patients with IgG anti- β2GPI

activity (87 APS, 67 APS plus SLE and 47 with SLE only) were analysed for their binding to six

β2GPI peptides corresponding to amino acid clusters on domains I-II, II, III and III-IV. Results

showed a diverse clinical association with reactivity to different epitopes on β2GPI,

suggesting all domains were relevant. Therefore, more detailed profiling of domain

specificity and avidity of anti-β2GPI antibodies may be useful as risk stratification for clinical

events.

Studies using cell cultures demonstrated that monocytes, ECs and platelets can also be

activated by aPL with anti-β2GPI activity inducing the expression on EC of intercellular cell

adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1) and E-selectin,

and of tissue factor (TF) on both EC and monocytes (Pierangeli et al,2008). Activated

platelets increase expression of glycoprotein IIbIIIa and synthesis of thromboxane A2

(Pierangeli et al,2008). However, it is possible that cells in culture may not behave as they

do in vivo.

Non- criteria Antibodies

The role of so called non-criteria antibodies such as anti-phosphatidylserine/prothrombin,

anti-phosphatidic acid, anti-vimentin/cardiolipin complex anti-protein C/S and variety of

other autoantibodies with specificities such as factor XII, factor X, Annexin A5 and Annexin

10

A2 in the pathogenesis and diagnosis APS remains to be established. The role of DI-anti–

β2GPI antibodies in pathogenesis of APS is discussed above. In a small cross-sectional study

we demonstrated that anti-protein C antibodies are associated with resistance to

endogenous protein C activation and a severe thrombotic phenotype in APS (Arachchillage

et al,2014). Several studies have analysed the role of the IgA isotype of aPL. In particular,

the pathogenicity of IgA aPL was established by demonstrating that mice, injected with IgA

aPL from patients with APS, developed thrombosis (Pierangeli et al,1995). In a recent

systematic review to establish the prevalence of non-criteria antibodies and of resistance to

Annexin A5 anticoagulant activity (AnxA5R) in APS and control populations, 16 retrospective

studies of 1404 APS patients, (1839 disease control and 797 healthy controls) were

examined. It was found that IgA anti-β2GPI antibodies (129/229, 56.3%) were most

prevalent, followed by AnxA5R (87/163, 53.4%) and IgG anti-Domain I (241/548, 44.0%)

(Rodriguez-Garcia et al, 2015). However, in addition to the retrospective data collection, the

results were affected by wide variation in the sample size, discrepancy in assay

methodology and the different cut off levels used for assay positivity. Prospective

multicentre studies with adequate sample size using more uniform methodology are

essential to determine the implications of these antibodies for the management of APS.

Pathogenic mechanisms involving other pathways

There is evidence from several animal models that complement activation, with excess C3a

and C5a generation, plays a role in thrombotic manifestations in APS. Complement

activation contributes to vascular inflammation and complement inhibition may ameliorate

aPL‐induced thrombosis, offering a potential new approach to therapy (Girardi et al, 2003a).

11

Mice treated with IgG from APS patients with high levels of aPL and subjected to a femoral

vein pinch model of thrombosis, developed larger thrombi and higher soluble TF activity

than controls (Romay-Penabad et al,2014). The co-administration of rEV576 (coversin), a

recombinant protein inhibitor of C5 activation, resulted in significantly smaller thrombi and

reduced TF activity (Romay-Penabad et al,2014). Consistent with this hypothesis, low

complement levels (C3, C5) have been demonstrated in patients with APS (Oku et al,2009).

In a study of 186 patients with aPL Breen et al, found significantly increased levels of

fragment Bb and C3a compared to normal controls [NC] (Breen et al, 2012). In the RAPS

(Rivaroxaban in Antiphospholipid Syndrome) trial of 111 APS patients; we demonstrated

that APS patients with previous VTE had significantly increased complement activation

compared to NC which was decreased by rivaroxaban compared to warfarin therapy

(Arachchillage et al, 2016).

Catastrophic APS (CAPS) is a rare but potentially fatal variant of APS characterized by sudden

onset of extensive microvascular thrombosis at multiple sites leading to multi-organ failure

(Cervera et al,2014). Widespread complement activation may contribute to CAPS and this is

supported by individual case reports of patients unresponsive to anticoagulation and

immunosuppression, but successfully treated with C5 complement inhibitor eculizumab

(Shapira et al,2012). As discussed below, complement activation may also be a central

mechanism in aPL‐induced pregnancy loss and intrauterine fetal growth restriction (Salmon

et al, 2003). Complement pathway activation offers several potential targets for therapeutic

complement inhibition as shown in figure 3.

12

Microvascular thrombosis is one of the major pathologic manifestations of APS and may

occur alone or in combination with large-vessel thrombosis. Kidneys represent the most

important site of microvascular thrombotic/microangiopathic APS and is associated with

renal failure, thrombotic microangiopathy, and hypertension (Griffiths et al, 2000). In

patients who undergo renal transplantation for APS, the microvascular thrombotic lesions

often recur. In cultured vascular ECs, IgG antibodies from patients with APS stimulated the

mammalian target of rapamycin (mTOR) through the phosphatidylinositol 3-kinase (PI3K)–

AKT pathway (Canaud et al,2014) leading to cell proliferation. It has been shown that mTOR

pathway activation plays a role in endothelial proliferation and intimal hyperplasia in aPL

positive patients, which leads to microthrombosis, peripheral ischemia, skin ulcers, diffuse

alveolar haemorrhage or aPL nephropathy (Manning & Cantley, 2007). The vascular

endothelium of proliferating intrarenal vessels from patients with APS nephropathy showed

evidence of mTORC pathway activation.

Another proposed mechanism of aPL initiated thrombosis is through interference with the

anticoagulant activity of activated protein C (APC), resulting in acquired activated protein C

resistance (APCr) (Nojima et al, 2005): aPL inhibition of the thrombomodulin mediated

activation of protein C (PC), as well as the anticoagulant activity of APC (Malia et al, 1990),

have been observed. Upregulation of the TF pathway, (Adams et al, 2001) by down

regulation of its principal inhibitor, TF pathway inhibitor (TFPI) (Liestol et al, 2007) may also

increase thrombotic risk and studies have reported the presence of auto-antibodies against

TFPI (αTFPI) in APS patients (Adams et al, 2001). Many in vitro studies have shown aPL

mediated upregulation of TF expression on monocytes (Lambrianides et al, 2010) and

13

endothelial cells associated with inflammatory cytokines and adhesion molecules (Willis et

al, 2015); aβ2GPI has also been shown to suppress TFPI dependent inhibition of the TF

pathway of coagulation (Boles & Mackman et al, 2010).

Pathogenesis of pregnancy complications in APS

The presence of aPL probably constitutes the single most recognisable risk factor in the

majority of cases of recurrent pregnancy loss and late placenta-mediated obstetric

complications. Animal studies have demonstrated that the passive transfer of aPL promotes

fetal loss and placental thrombosis and also inhibits trophoblast and decidual cell functions

in vitro (Branch et al,1990). Cross-species investigations have shown that the exposure of

pregnant rats to a purified IgG fraction from women with APS directly inhibits embryo

growth (Ornoy et al,2003). Immunopathology of obstetric APS differs from that of

thrombotic APS, especially in the case of recurrent early miscarriages, where thrombosis is

neither a universal nor a specific feature (Out et al,1991). Nonetheless, LA may be

associated with extensive placental necrosis, infarction and thrombosis in women with

recurrent pregnancy loss and extensive villous infarction following first trimester

miscarriage in a patient with SLE and positive LA and aCL was first described in 1996 (Nayar

& Lage,1996).

Annexin V is a cationic protein which normally serves an anticoagulant function by

aggregating on trophoblast membranes and blocking FXa and prothrombin binding.

Thrombosis during the development of the normal materno-placental circulation may arise

via interference with (Rand et al, 1997;Rand et al, 2010) this function and women with APS

have disrupted and reduced annexin V binding to the surface of trophoblastic cells of the

14

intervillous space compared with controls (Rand et al, 1997). During differentiation to

syncytium, trophoblast membrane anionic phospholipids also bind β2GPI and hence β2GPI-

dependent aPL which may then lead to defective placentation via complement activation,

inflammatory damage and placental apoptosis (Girardi et al, 2010).

Studies of animal and human placenta have demonstrated that complement activation by

aPL may play a major role in the pathogenesis of recurrent pregnancy loss (Salmon et al,

2003). Appropriate complement inhibition is an essential requirement for normal pregnancy

as evidenced by the finding that deficiency of Crry (a membrane-bound complement

regulatory protein that blocks C3 and C4 activation) leads to progressive embryonic loss in

mice (Xu et al, 2000). It has been hypothesised that aPL bound to trophoblasts activate

complement via the classical pathway, generating split products that mediate placental

injury causing fetal loss and growth restriction. Passive transfer of IgG from women with

recurrent miscarriage and aPL results in a significant increase in the frequency of fetal

resorption and reduced average weight of the surviving fetuses, compared to mice treated

with IgG from healthy individuals (Holers et al, 2002). These effects can be blocked by

inhibition of the complement cascade using a C3 convertase inhibitor or by antibodies or

peptides that block C5a-C5a receptor interactions (Girardi et al, 2003;Pierangeli et al, 1999).

Furthermore, mice deficient in C3 are resistant to the fetal injury induced by aPL (Girardi et

al, 2003; Holers et al, 2002) and studies in factor B-deficient mice indicate that the

alternative complement pathway is also required. Inflammatory tissue injury is likely

mediated by tumour necrosis factor (TNF) alpha, which increases in murine decidua after

exposure to aPL (Berman et al,2005) and antibody blocking of TNF-α reduces fetal

resorption (Blank et al,2003). Finally, the therapeutic effect of heparin has been shown to

15

operate via inhibition of complement rather than inhibition of coagulation (Girardi et al,

2004). The PROMISE study (Predictors of pregnancy outcome: biomarkers In APS and SLE) is

a prospective multicentre observational study which will further evaluate the role of

complement in SLE and aPL-associated pregnancy complications

(https://clinicaltrials.gov/ct2/show/NCT00198068).).

Animal and human cell culture studies have demonstrated that binding of aPL (in particular

β2GPI-dependent antibodies) to human trophoblasts affects other critical cell functions;

inhibition of proliferation and syncitia formation, decreased production of human chorionic

gonadotrophin and defective secretion of growth factors, as well as inducing apoptosis

(Pierangeli et al,2008). Moreover aPL may impair the sequential expression of cell adhesion

molecules, such as integrins and cadherins, in trophoblastic and decidual cells essential for

normal placentation (Di Simone et al, 2002). Mechanisms for aPL-mediated fetal loss and

complications are summarised in Table 2.

Management of thrombosis in antiphospholipid syndrome

Despite the implication of other mechanisms as described above, anticoagulation rather

than immunosuppression remains the mainstay of management in patients with thrombotic

APS. Two randomised controlled trials have demonstrated that high-intensity warfarin is not

superior to moderate-intensity warfarin (INR 2.0-3.0) for the prevention of recurrent VTE

(Crowther et al, 2003;Finazzi et al, 2005). Current recommendation is that patients with APS

and otherwise unprecipitated VTE should remain on anticoagulants indefinitely with a target

INR of 2.5, which reduces recurrent VTE by 80-90% compared to no treatment (Danowski et

16

al,2013 ;Ruiz-Irastorza et al,2011). Indefinite anticoagulation for APS patients is supported

by evidence that APS patients carry a higher risk of recurrent thrombosis compared to aPL

negative subjects and that the thrombotic risk may increase with time (Ruiz-Irastorza et

al,2002). However, in a systematic review, the strength of this association was uncertain

because the available evidence was of very low quality (Garcia et al,2013). Thrombosis after

a clear precipitant should not prompt testing for APS and requires only three months of

anticoagulation (Keeling et al, 2012).

With regards to the optimal intensity of anticoagulation following arterial thrombosis, an

earlier prospective cohort study, the Antiphospholipid Antibodies and Stroke Study (APASS),

found no benefit of low intensity warfarin anticoagulation (target INR 1.4-2.8) over aspirin

(325mg/day) in stroke prevention (Levine et al, 2004). However, the study had important

limitations such as low target INR and aPL testing being performed only on entry to the

study raising the possibility that some participants may have had transient antibody

positivity. Crowther and Finazzi’s studies demonstrated that moderate-intensity warfarin

(INR 2.0-3.0) is effective for arterial thrombosis and APS, although patients with arterial

thrombosis were poorly represented in these trials (only, 24% and 32% respectively)

(Crowther et al, 2003;Finazzi et al, 2005).). Ruiz-Irastorza and colleagues recommended,

following a systematic review of cohort studies, that APS with arterial thrombosis and/or

recurrent venous events should be treated with warfarin at an INR >3.0 (Ruiz-Irastorza et al,

2007). Although the 13th International Congress on Antiphospholipid Antibodies task force

also recommended an INR >3.0 or combined platelet inhibitor-anticoagulant (INR 2.0–3.0)

therapy for arterial thrombosis (Ruiz-Irastorza et al, 2011), this was a non-graded

17

recommendation due to lack of consensus within the panel members. Standard practice

currently is for a target INR of 2.5, with escalation to a higher target INR should thrombosis

recur (Keeling et al, 2012).

Warfarin’s narrow therapeutic range and multiple interactions are further complicated in

APS by the variable responsiveness of thromboplastins to LA, leading to misleading INR

results. However, in the majority of patients with APS monitoring of INR is not a major issue

and the problem is limited to specific thromboplastins (Tripodi et al,2001).

Direct acting oral anticoagulants

Direct acting oral anticoagulants (DOACs) are fixed dose oral anticoagulants with predictable

anticoagulant effect and consequently no need for routine monitoring. DOACs are

established as therapeutic alternatives to VKAs for treatment and secondary prevention of

VTE in patients without APS. Although aPL are reported in approximately 10% of patients

with VTE (Andreoli et al, 2013), phase III clinical trials have not compared warfarin vs DOAC

for this group and experience is limited to case reports. A total of 96 APS patients are

reported to be treated with DOACs (82/96 [85.5%] rivaroxaban, 13/96 [13.5%] dabigatran

and 1/96 [1%] apixaban) (Bachmeyer & Elalamy,2014 ;Schaefer et al,2014 ;Win &

Rodgers,2014 ;Betancur et al,2016 ;Noel et al,2015 ;Sciascia et al,2015 ;Signorelli et al,2016

;Son et al,2015 ). Only 8/96 (8.3%) of the patients received a DOAC as acute treatment of

VTE. 17 patients had recurrent thrombosis. However, those who had recurrent arterial

thrombosis had a severe thrombotic phenotype at the time of starting a DOAC and standard

18

risk APS patients who were on warfarin with target INR of 2.0-3.0 had no recurrent events.

Only 2 patients (2/96 [2.1%]) had bleeding events.

Rivaroxaban has been shown to effectively reduce thrombin generation in plasma from

patients with thrombotic APS compared to warfarin in a recently published randomised

clinical trial (Cohen et al,2016), but there are no data from intervention trials designed to

assess clinical endpoints. Several other clinical trials assessing the use of DOAC is thrombotic

APS are still recruiting participants ([https://clinicaltrials.gov/NCT02157272],

[https://clinicaltrials.gov/NCT02295475],[https://clinicaltrials.gov/NCT02116036]).

However, given their general efficacy, DOAC could be considered as an alternative

anticoagulant in patients with APS and VTE that are usually treated with standard-intensity

warfarin, when there is known VKA allergy or poor anticoagulant control. DOACs have not

been studied in patients with arterial thrombosis even without APS and the comparison has

been always with standard intensity warfarin anticoagulation. Therefore, DOACs are not

recommended in APS patients with arterial thrombosis or those with recurrent VTE whilst

achieving therapeutic anticoagulation with warfarin.

For the small proportion of patients with APS who develop recurrent thrombosis despite

apparently adequate anticoagulation an increase in warfarin intensity is recommended (ie:

target INR of 3.5). There are limited therapeutic options for patients who have recurrent

thrombosis despite high-intensity warfarin. These include addition of an antiplatelet agent

or LMWH, including high-intensity LMWH (maintaining peak anti Xa levels 1.6 – 2.0IU/ml for

once daily dosing and peak 0.8 – 1.0IU/ml for twice daily dosing) or combined factor Xa and

factor IIa inhibitors (Arachchillage et al, 2016). When anticoagulation has failed, other

19

available therapeutic options include combining anticoagulation with immunosuppression

and/or immunomodulation with modalities including rituximab, hydroxychloroquine, statins

rituximab complement inhibitors and mTOR inhibitors such as sirolimus.

Hydroxychloroquine

Hydroxychloroquine (HCQ) is a well-established treatment for patients with SLE and

rheumatoid arthritis due to its anti-inflammatory effects; inhibiting cytosolic phospholipase

A2 and reducing the release of the cytokines interleukin (IL)-6 and TNF (Sperber et al,1993).

It is currently recommended as a baseline therapy in all patients with SLE who have no

contraindications (Ruiz-Irastorza et al, 2010). HCQ also inhibits platelet aggregation and

arachidonic acid release, reducing thrombus size in mice models of APS (Espinola et al,

2002;Rand et al,2010) and protects the Annexin V anticoagulant shield from disruption by

aPL (Rand et al,2010). Its antithrombotic activity is demonstrated by its efficacy as

thromboprophylaxis after hip surgery (Johansson et al, 1981) and in patients with SLE,

without increased bleeding (Schmidt-Tanguy et al,2013). Recent APS consensus guidelines

support its use as an adjuvant to anticoagulation in patients with recurrent thrombosis

despite anticoagulation (Ruiz-Irastorza et al, 2011). A phase III multicentre trial exploring the

effect of HCQ as primary thrombosis prophylaxis in individuals with persistently positive

antiphospholipid antibodies https://clinicaltrials.gov//NCT01784523) has been now

completed and findings are awaited.

https://clinicaltrials.gov/NCT01784523

20

Statins

Statins inhibit the enzyme HMG-CoA reductase which has a central role in hepatic

cholesterol production but also have pleiotropic effects including anti-inflammatory and

antithrombotic actions on EC and monocytes (Ferrara et al, 2003). Furthermore, there is

evidence they increase fibrinolysis and decrease tissue factor mRNA expression; opposite

effects to aPL (Ferrara et al,2004). These observations and the demonstrated VTE reduction

in patients receiving statins (Ridker et al,2009) suggest a possible benefit from their use in

APS. In a prospective open-label pilot study of fluvastatin in aPL positive patients, Erkan et

al, demonstrated that fluvastatin can reduce pro-inflammatory and pro-thrombotic

biomarkers such as interleukin (IL)-6, IL1β, vascular endothelial growth factor, TNF-α and

interferon–α (Erkan et al,2014). Based on available evidence, the 14th International

Congress on Antiphospholipid Antibodies Task Force Report on APS Treatment Trends

stated that statins cannot be recommended in APS patients in the absence of

hyperlipidemia, but may be a useful treatment adjunct in APS patients with recurrent

thrombosis despite adequate anticoagulation (Erkan et al,2014).

Rituximab

Rituximab appears to be an effective treatment for thrombocytopenia and haemolytic

anaemia associated with aPL (Erre et al, 2008) and may improve some other manifestations

such as skin ulcers (Erkan et al,2013). Rituximab has been used for the treatment of difficult

cases such as refractory thrombocytopenia or other non-criteria aPL manifestations, CAPS,

and recurrent VTE in over 50 case reports. In a review by Kumar et al, it was reported that in

19 of 21 published cases; rituximab had a beneficial clinical effect. aPL levels were

21

significantly decreased in ten of 12 cases (Kumar & Roubey,2010). Review of the CAPS

registry of 20 patients treated with rituximab reported a recovery rate of 75% of acute

episodes in combination with multiple treatment strategies including anticoagulation,

steroids, plasma exchange and cyclophosphamide (Berman et al,2013); consequently, the

responses cannot be clearly attributed to rituximab alone. Furthermore, there was no

consistency in the reduction in aPL level in these patients.

Complement inhibition

Experimental evidence discussed above has led to speculation that inhibition of

complement may be a useful therapeutic strategy in APS. So far only preliminary and

anecdotal data are available. A phase II clinical is evaluating the use of eculizumab to enable

renal transplantation and prevent post-transplant thrombotic microangiopathy (TMA) in

patients with a history of CAPS (http://clinicaltrials.gov/show/NCT01029587) is ingoing, but

not recruiting participants. Preliminary data of this study from 80 patients showed that

eculizumab is effective in reducing the incidence of acute antibody-mediated rejection in

sensitized deceased donor kidney transplant recipients (KTR). Patient and graft survival and

kidney function at 1yr were similar to those expected for non-sensitized KTR (Glotz et al,

2015). Another phase II multicentre clinical trial evaluating the safety and tolerability of

intravenous ALXN1007 (C5a inhibitor) in persistently aPL-positive patients with at least one

of the non-criteria manifestations of APS is underway

https://clinicaltrials.gov/NCT02128269.

https://clinicaltrials.gov/NCT02128269

22

Sirolimus

Sirolimus is an mTOR inhibitor which is currently used to prevent reactive arterial stenosis

after coronary stenting. A cohort of patients with APS nephropathy who required

transplantation and were receiving sirolimus had no recurrence of vascular lesions and had

decreased vascular proliferation on biopsy as compared to APS patients not receiving

sirolimus (Canaud et al,2014). Graft survival at 144 months was 7 /10 (70%) versus 3/27

(11%) in the two groups. Further larger studies are required to establish the routine use of

mTOR inhibitor in APS patients.

Potential future therapeutic options

TIFI is a 20-amino acid peptide from human cytomegalovirus, which shares similarities with

the PL-binding site in the β2GPI molecule, DV. In vitro evidence suggests that TIFI inhibits the

binding of labelled β2GPI to human ECs and mouse monocytes (Ostertag et al,2006). In a

mouse model of APS, infusion of TIFI reduced the binding of fluoresceinated β2GPI to ECs

and reduced thrombosis size after aPL infusion (de la Torre et al,2012). A dimeric peptide of

β2-glycoprotein-DI (DI dimer) has a similar effect (Agostinis et al,2014). These peptides are

not yet in clinical trials but may offer a new paradigm for APS treatment in the future.

Management of CAPS

Anticoagulation, intravenous immunoglobulin (IVIG), plasma exchange, immunosuppressive

therapy, prostacyclin, fibrinolytics and defibrotide have all been used in the management of

CAPS. Case reports described above suggest eculizumab may be effective in CAPS

23

unresponsive to other treatments (Shapira et al,2012) and in preventing relapse after kidney

transplantation. There are reported cases of use of rituximab in patients with CAPS with

variable responses (Erre et al, 2008). Espinosa et al reviewed 9 such patients: two patients

died, but notably in three of the surviving patients tests for aPL became negative after

rituximab treatment (Espinosa et al, 2011).

Management of asymptomatic carriers of aPL

Another unanswered question is whether primary thromboprophylaxis is indicated in

subjects with aPL and how risk of a first thrombosis can be stratified in order to personalise

treatment. A prospective evaluation of the incidence and risk factors for a first vascular

event in 258 asymptomatic individuals with aPL determined that the annual incidence rate

was 1.86% compared to 0.1% in the general population with a median follow up of 35

months (range 1-48) (Ruffatti et al, 2011). Hypertension and the presence of LA were

identified as independent risk factors for development of thrombosis (Ruffatti et al, 2011).

In a study of 104 subjects with triple positivity, there were 25 first thrombotic events (5.3%

per year) with a cumulative incidence of 37.1% (95% confidence interval [CI]: 19.9%-54.3%)

after 10 years. Aspirin did not significantly reduce the incidence of thromboembolic events

(Pengo et al, 2011). The Antiphospholipid Antibody Acetylsalicyclic Acid (APLASA) study

randomised individuals with asymptomatic, persistently positive aPL to receive 81mg of

aspirin or placebo daily (Erkan et al, 2007). There was no significant difference in outcome

between the two arms. On current evidence, thromboprophylaxis with aspirin and/or

vitamin K antagonist is not recommended for asymptomatic, aPL positive individuals.

24

Whether subjects who are triple positive aPL should be treated differently remains to be

established.

Treatment of obstetric APS

Heparin and low dose aspirin (LDA) is the treatment of choice for RPL associated with APS.

This is based on several important studies (Kutteh,1996 ;Rai et al,1997). In a meta-analysis

of data from five trials that randomly assigned patients to either heparin and aspirin or

aspirin alone for the management of APS-related pregnancies (Mak et al, 2010), the overall

live birth rates in 334 patients were 74.27 and 55.85% respectively (RR 1.301; 95% CI 1.040,

1.629). The American College of Chest Physicians (ACCP) guidelines (Bates et al, 2012)

recommend treating women with obstetric APS, who meet revised Sapporo criteria with

heparin and LDA in the antepartum period as soon as pregnancy is confirmed. LMWH is

generally favoured due to a lower incidence of heparin induced thrombocytopenia and low

risk of osteopenia compared to unfractionated heparin. The key observation made by

Girardi et al in 2004 that heparin can inhibit complement mediated apoptosis provides a

possible explanation for its efficacy independent of its anticoagulant activity (Girardi et al,

2004). Heparin may also act by inhibiting aPL binding to trophoblastic cell membranes,

modulating trophoblast apoptosis, promoting trophoblast cell invasiveness, and reducing

complement activation with the ensuing inflammatory response at the decidual-placental

interface (Franklin & Kutteh,2003).

Women with obstetric APS but no prior history of VTE, Post-partum thromboprophylaxis

should be considered offered for 6 weeks following delivery based on risk assessment.

25

However, there is variation in guidelines as well as clinical practice regarding this. The Nimes

Obstetricians and Haematologists Antiphospholipid Syndrome (NOH-APS) observational

study reported that the annual rates of deep vein thrombosis (1.46%; range 1.15%-1.82%),

pulmonary embolism (0.43%; range 0.26%-0.66%), superficial vein thrombosis (0.44%; range

0.28%-0.68%), and cerebrovascular events (0.32%; range 0.18%-0.53%) were significantly

higher in women with pure obstetric APS compared with aPL-negative women with

obstetric morbidity, despite low dose aspirin primary prophylaxis (Gris et al, 2012). BCSH

(Keeling et al, 2012) and RCOG guidelines are in keeping with this observation. The RCOG

Green-top Guideline (https://www.rcog.org.uk/globalassets/documents/guidelines/gtg-

37a.pdf), states ‘’Persistent antiphospholipid antibodies (LA and/or anticardiolipin and/or

anti-β2-GPI 1 antibodies) in women without previous VTE should be considered as a risk

factor for thrombosis such that if she has other risk factors she may be considered for

antenatal or postnatal thromboprophylaxis (either 10day or 6 weeks based on risk

assessment). If VTE develops during pregnancy, treatment with therapeutic doses of LMWH

should be employed during the remainder of the pregnancy and for at least 6 weeks

postnatally and until at least 3 months of treatment has been given in total

(https://www.rcog.org.uk/globalassets/documents/guidelines/gtg-37b.pdf). However,

women with thrombosis during pregnancy and positive aPL should be reassessed after

pregnancy in case long-term anticoagulation is indicated.

With standard treatment, approximately 70% of pregnant women with APS have successful

pregnancy outcome (Bramham et al,2010). There are limited therapeutic options and no

guidelines for those who do not respond to heparin and LDA. Addition of low-dose

https://www.rcog.org.uk/globalassets/documents/guidelines/gtg-37a.pdf

https://www.rcog.org.uk/globalassets/documents/guidelines/gtg-37a.pdf

26

prednisolone to standard treatment in the first-trimester improved the live birth rate in

refractory aPL-related first trimester pregnancy loss (Bramham et al,2011): nearly two-thirds

of pregnancies (61%) resulted in live births, of which 8 (57%) were uncomplicated term

pregnancies. However, the frequency of some complications remained elevated, mainly

preterm delivery (21%). Data on the usefulness of IVIG in obstetric APS are conflicting (Parke

et al,1989 ;Branch et al,2001).

In a mouse model of obstetric APS HCQ prevented fetal death and the placental metabolic

changes as measured by proton magnetic resonance spectroscopy (Bertolaccini et al,2016).

In the same study, they showed that HCQ prevented complement activation in vivo and in

vitro. Complement C5a levels in serum samples from APS patients and APS-mice were lower

after treatment with HCQ while the antibodies titres remained unchanged. HCQ prevented

not only placental insufficiency but also abnormal fetal brain development in APS

(Bertolaccini et al,2016). A European multicentre retrospective study of 30 patients with APS

and 35 pregnancies showed a better outcome of pregnancies that were treated by the

addition of HCQ when compared with previous pregnancies under the conventional

treatment (Mekinian et al,2015). Sciascia et al reported HCQ-treatment was associated with

a higher rate of live births (67% vs 57%; P = .05) and a lower prevalence of aPL–related

pregnancy morbidity (47% vs 63%; P = .004) (Sciascia et al,2016).

Conclusion

Despite the clear association between aPL and the thrombotic and obstetric manifestations

of APS, the exact mechanisms by which aPL cause these problems is yet to be fully

27

established. However, the activation of complement and endothelial cells by DI specific anti-

β2GPI antibodies has emerged as an important component. Incorporation of DI specific anti-

β2GPI antibodies into diagnostic panels may allow better identification of those at high risk

for clinical events. Related but distinct pathways also link aPL to the complications of

pregnancy. Warfarin, heparin, and/or antiplatelet drugs are still the standard of care for

APS-thrombosis and although attractive, DOAC can at present only be considered as an

alternative in patients with APS and VTE that are usually treated with standard-intensity

warfarin when there is known VKA allergy or poor anticoagulant control. The realisation that

additional pathways are involved in the pathogenesis of APS has significant implications for

treatment. Agents acting outside the coagulation system such as hydroxychloroquine for

pregnancy complications and sirolimus as an inhibitor of the mammalian target of

rapamycin (mTOR) pathway are now under evaluation and represent a potential radical

change in thinking for haematologists.

Authorship

DRJ Arachchillage performed the literature search and wrote the first draft. M Laffan

critically reviewed the manuscript and both authors approved the final manuscript.

Disclosure of conflict of interest

Authors state that they have no relevant conflict of interest

28

References

Abreu,M.M., Danowski,A., Wahl,D.G., Amigo,M.C., Tektonidou,M., Pacheco,M.S.,

Fleming,N., Domingues,V., Sciascia,S., Lyra,J.O., Petri,M., Khamashta,M., &

Levy,R.A. (2015) The relevance of "non-criteria" clinical manifestations of

antiphospholipid syndrome:14th International Congress on Antiphospholipid

Antibodies Technical Task Force Report on Antiphospholipid Syndrome Clinical

Features. Autoimmun.Rev,14:401-14.

Adams,M.J., Donohoe,S., Mackie,I.J., & Machin,S.J. (2001) Anti-tissue factor pathway

inhibitor activity in patients with primary antiphospholipid syndrome. Br.J.Haematol,

114, 375-379.

Agar,C., van Os,G.M., Morgelin,M., Sprenger,R.R., Marquart,J.A., Urbanus,R.T.,

Derksen,R.H., Meijers,J.C., & de Groot,P.G (2010). Beta2-glycoprotein I can exist in

2 conformations: implications for our understanding of the antiphospholipid

syndrome. Blood, 116,1336.-43.

Agostinis,C., Durigutto,P., Sblattero,D., Borghi,M.O., Grossi,C., Guida,F., Bulla,R.,

Macor,P., Pregnolato,F., Meroni,P.L., & Tedesco,F (2014). A non-complement-fixing

antibody to beta2 glycoprotein I as a novel therapy for antiphospholipid syndrome.

Blood.;123:3478.-87.

Andreoli,L., Chighizola,C.B., Banzato,A., Pons-Estel,G.J., Ramire de,J.G., & Erkan,D.

(2013) Estimated frequency of antiphospholipid antibodies in patients with pregnancy

morbidity, stroke, myocardial infarction, and deep vein thrombosis: a critical review

of the literature. Arthritis Care Res (Hoboken.).,65,1869.-73.

Arachchillage,D.R., Besser,M., Maclean,R., Baglin,T., & van Veen,J.J. (2016) Combined factor IIa and Xa inhibitor therapy for thrombosis whilst on therapeutic anticoagulant. Thromb Res., 143:137-40.

Arachchillage,D.R., Efthymiou,M., Mackie,I.J., Lawrie,A.S., Machin,S.J., & Cohen,H.(2014)

Anti-protein C antibodies are associated with resistance to endogenous protein C

activation and a severe thrombotic phenotype in antiphospholipid syndrome. J

Thromb Haemost, 12:1801.-9.

Arachchillage DR, Mackie IJ, Efthymiou M, Chitolie A, Hunt BJ, Isenberg DA, Khamashta

M, Machin SJ, Cohen H (2016)Rivaroxaban limits complement activation compared

with warfarin in antiphospholipid syndrome patients with venous thromboembolism. J

Thromb Haemost, 14:2177-2186.

Arad,A., Proulle,V., Furie,R.A., Furie,B.C., & Furie,B.(2011) beta(2)-Glycoprotein-1

autoantibodies from patients with antiphospholipid syndrome are sufficient to

potentiate arterial thrombus formation in a mouse model. Blood, 117,3453.-9.

Artenjak,A., Locatelli,I., Brelih,H., Simonic,D.M., Ulcova-Gallova,Z., Swadzba,J.,

Musial,J., Iwaniec,T., Stojanovich,L., Conti,F., Valesini,G., Avcin,T., Cohen

29

Tervaert,J.W., Shoenfeld,Y., Blank,M., Ambrozic,A., Sodin-Semrl,S., Bozic,B., &

Cucnik,S.(2015) Immunoreactivity and avidity of IgG anti-beta2-glycoprotein I

antibodies from patients with autoimmune diseases to different peptide clusters of

beta2-glycoprotein I. Immunol.Res, 61,35.-44.

Bachmeyer,C. & Elalamy,I.(2014) Rivaroxaban as an effective treatment for recurrent

superficial thrombophlebitis related to primary antiphospholipid syndrome. Clin

Exp.Dermatol, 39,840-1.

Banzato,A., Pozzi,N., Frasson,R., De,F., V, Ruffatti,A., Bison,E., Padayattil,S.J., Denas,G.,

& Pengo,V.(2011) Antibodies to Domain I of beta(2)Glycoprotein I are in close

relation to patients risk categories in Antiphospholipid Syndrome (APS). Thromb

Res,.128,583.-6.

Bas de Laat.H., Derksen,R.H., & de Groot,P.G.(2004) beta2-glycoprotein I, the playmaker of

the antiphospholipid syndrome. Clin Immunol,112,161-8.

Bates,S.M., Greer,I.A., Middeldorp,S., Veenstra,D.L., Prabulos,A.M., & Vandvik,P.O.

(2012) VTE, thrombophilia, antithrombotic therapy, and pregnancy: Antithrombotic

Therapy and Prevention of Thrombosis, 9th ed: American College of Chest

Physicians Evidence-Based Clinical Practice Guidelines. Chest, 141, e691S-e736S.

Berman,H., Rodriguez-Pinto,I., Cervera,R., Morel,N., Costedoat-Chalumeau,N., Erkan,D.,

Shoenfeld,Y., & Espinosa,G.(2013) Rituximab use in the catastrophic

antiphospholipid syndrome: descriptive analysis of the CAPS registry patients

receiving rituximab. Autoimmun.Rev,12,1085-90.

Berman,J., Girardi,G., & Salmon,J.E.(2005) TNF-alpha is a critical effector and a target for

therapy in antiphospholipid antibody-induced pregnancy loss. J Immunol, 174,485-90.

Bertolaccini,M.L., Contento,G., Lennen,R., Sanna,G., Blower,P.J., Ma,M.T., Sunassee,K., &

Girardi,G.(2016) Complement inhibition by hydroxychloroquine prevents placental

and fetal brain abnormalities in antiphospholipid syndrome.J Autoimmun, pii: S0896-

8411(16)30040-3. doi: 10.1016/j.jaut.2016.04.008. [Epub ahead of print]

Betancur,J.F., Bonilla-Abadia,F., Hormaza,A.A., Jaramillo,F.J., Canas,C.A., &

Tobon,G.J.(2016) Direct oral anticoagulants in antiphospholipid syndrome: a real life

case series. Lupus,25,658.-62.

Blank,M., Krause,I., Wildbaum,G., Karin,N., & Shoenfeld,Y.(2003) TNFalpha DNA

vaccination prevents clinical manifestations of experimental antiphospholipid

syndrome. Lupus,12,546-9.

Boles J, Mackman N (2010). Role of tissue factor in thrombosis in antiphospholipid antibody

syndrome. Lupus, 19 :370-8.

Bowie,E.J., Thompson,J.H., Jr., Pascuzzi,C.A., & Owen,C.A., Jr. (1963) Thrombosis in

systemic lupus erythematosus despite circulating anticoagulants. J Lab Clin

Med,62,416-30.

30

Bramham,K., Hunt,B.J., Germain,S., Calatayud,I., Khamashta,M., Bewley,S., & Nelson-

Piercy,C.(2010) Pregnancy outcome in different clinical phenotypes of

antiphospholipid syndrome. Lupus,.19,58-64.

Bramham,K., Thomas,M., Nelson-Piercy,C., Khamashta,M., & Hunt,B.J.(2011) First-

trimester low-dose prednisolone in refractory antiphospholipid antibody-related

pregnancy loss. Blood, 117,6948-51.

Branch,D.W., Dudley,D.J., Mitchell,M.D., Creighton,K.A., Abbott,T.M., Hammond,E.H., &

Daynes,R.A.(1990) Immunoglobulin G fractions from patients with antiphospholipid

antibodies cause fetal death in BALB/c mice: a model for autoimmune fetal loss.

Am.J Obstet.Gynecol,163, 210-6.

Branch,D.W., Peaceman,A.M., Druzin,M., Silver,R.K., El-Sayed,Y., Silver,R.M.,

Esplin,M.S., Spinnato,J., & Harger,J.(2000) A multicenter, placebo-controlled pilot

study of intravenous immune globulin treatment of antiphospholipid syndrome during

pregnancy. The Pregnancy Loss Study Group. Am.J Obstet.Gynecol, 182,122-7.

Breen,K.A., Seed,P., Parmar,K., Moore,G.W., Stuart-Smith,S.E., & Hunt,B.J. (2012)

Complement activation in patients with isolated antiphospholipid antibodies or

primary antiphospholipid syndrome. Thromb.Haemost., 107, 423-429.

Canaud,G., Bienaime,F., Tabarin,F., Bataillon,G., Seilhean,D., Noel,L.H., Dragon-

Durey,M.A., Snanoudj,R., Friedlander,G., Halbwachs-Mecarelli,L., Legendre,C., &

Terzi,F.(2014) Inhibition of the mTORC pathway in the antiphospholipid syndrome.

N.Engl.J Med, 371,303-12.

Cervera,R., Rodriguez-Pinto,I., Colafrancesco,S., Conti,F., Valesini,G., Rosario,C., Agmon-

Levin,N., Shoenfeld,Y., Ferrao,C., Faria,R., Vasconcelos,C., Signorelli,F., &

Espinosa,G.(2014) 14th International Congress on Antiphospholipid Antibodies Task

Force Report on Catastrophic Antiphospholipid Syndrome. Autoimmun.Rev, 13,699-

707.

Clemens N, Frauenknecht K, Katzav A, Sommer C, von LP (2009). In vitro effects of

antiphospholipid syndrome-IgG fractions and human monoclonal antiphospholipid

IgG antibody on human umbilical vein endothelial cells and monocytes. Ann N Y

Acad Sci;1173:805-13.

Cohen,H., Hunt,B.J., Efthymiou,M., Arachchillage,D.R., Mackie,I.J., Clawson,S.,

Sylvestre,Y., Machin,S.J., Bertolaccini,M.L., Ruiz-Castellano,M., Muirhead,N.,

Dore,C.J., Khamashta,M., & Isenberg,D.A. (2016) Rivaroxaban versus warfarin to

treat patients with thrombotic antiphospholipid syndrome, with or without systemic

lupus erythematosus (RAPS): a randomised, controlled, open-label, phase 2/3, non-

inferiority trial. Lancet Haematol,3,e426.-36.

Conley C & Hartmann RC (1952) A hemorrhagic disorder caused by circulating anticoagulant in patients with disseminated lupus erythematosus. J Clin Invest, 31,

621.2.

31

Crowther,M.A., Ginsberg,J.S., Julian,J., Denburg,J., Hirsh,J., Douketis,J., Laskin,C.,

Fortin,P., Anderson,D., Kearon,C., Clarke,A., Geerts,W., Forgie,M., Green,D.,

Costantini,L., Yacura,W., Wilson,S., Gent,M., & Kovacs,M.J. (2003) A comparison

of two intensities of warfarin for the prevention of recurrent thrombosis in patients

with the antiphospholipid antibody syndrome. N.Engl.J.Med., 349, 1133-1138.

Danowski,A., Rego,J., Kakehasi,A.M., Funke,A., Carvalho,J.F., Lima,I.V., Souza,A.W., &

Levy,R.A.(2013) Guidelines for the treatment of antiphospholipid syndrome.

Rev.Bras.Reumatol, 53,184-92.

de Groot,P.G., Lutters,B., Derksen,R.H., Lisman,T., Meijers,J.C., & Rosendaal,F.R.(2005)

Lupus anticoagulants and the risk of a first episode of deep venous thrombosis. J

Thromb Haemost,3,1993-7.

de Groot,P.G. & Meijers,J.C.(2011) beta(2) -Glycoprotein I: evolution, structure and

function. J Thromb Haemost, 9,1275-84.

de la Torre,Y.M., Pregnolato,F., D'Amelio,F., Grossi,C., Di,S.N., Pasqualini,F., Nebuloni,M.,

Chen,P., Pierangeli,S., Bassani,N., Ambrogi,F., Borghi,M.O., Vecchi,A., Locati,M.,

& Meroni,P.L.(2012) Anti-phospholipid induced murine fetal loss: novel protective

effect of a peptide targeting the beta2 glycoprotein I phospholipid-binding site.

Implications for human fetal loss. J Autoimmun, 38,J209-15.

de Laat,H.B., Derksen,R.H., Urbanus,R.T., Roest,M., de Groot,P.G (2004). beta2-

glycoprotein I-dependent lupus anticoagulant highly correlates with thrombosis in the

antiphospholipid syndrome. Blood,104:3598.-602.

de Laat.B., Derksen,R.H., Urbanus,R.T., & de Groot,P.G.(2005) IgG antibodies that

recognize epitope Gly40-Arg43 in domain I of beta 2-glycoprotein I cause LAC, and

their presence correlates strongly with thrombosis. Blood,105,1540-5.

de Laat.B., Derksen,R.H., van,L.M., Pennings,M.T., & de Groot,P.G.(2006) Pathogenic anti-

beta2-glycoprotein I antibodies recognize domain I of beta2-glycoprotein I only after

a conformational change. Blood,. 107,1916-24.

de Laat.B., Pengo,V., Pabinger,I., Musial,J., Voskuyl,A.E., Bultink,I.E., Ruffatti,A.,

Rozman,B., Kveder,T., de,M.P., Boehlen,F., Rand,J., Ulcova-Gallova,Z., Mertens,K.,

& de Groot,P.G. (2009) The association between circulating antibodies against

domain I of beta2-glycoprotein I and thrombosis: an international multicenter study. J

Thromb Haemost, 7,1767-73.

Di,S.N., Castellani,R., Caliandro,D., & Caruso,A.(2002) Antiphospholid antibodies regulate

the expression of trophoblast cell adhesion molecules. Fertil.Steril, 77,805-11.

Erkan,D., Aguiar,C.L., Andrade,D., Cohen,H., Cuadrado,M.J., Danowski,A., Levy,R.A.,

Ortel,T.L., Rahman,A., Salmon,J.E., Tektonidou,M.G., Willis,R., &

Lockshin,M.D.(2014) 14th International Congress on Antiphospholipid Antibodies:

task force report on antiphospholipid syndrome treatment trends. Autoimmun.Rev,

13,685-96.

32

Erkan,D., Harrison,M.J., Levy,R., Peterson,M., Petri,M., Sammaritano,L., Unalp-Arida,A.,

Vilela,V., Yazici,Y., & Lockshin,M.D. (2007) Aspirin for primary thrombosis

prevention in the antiphospholipid syndrome: a randomized, double-blind, placebo-

controlled trial in asymptomatic antiphospholipid antibody-positive individuals.

Arthritis Rheum., 56, 2382-2391.

Erkan,D., Vega,J., Ramon,G., Kozora,E., & Lockshin,M.D.(2013) A pilot open-label phase II

trial of rituximab for non-criteria manifestations of antiphospholipid syndrome.

Arthritis Rheum, 65,464-71.

Erkan,D., Willis,R., Murthy,V.L., Basra,G., Vega,J., Ruiz-Limon,P., Carrera,A.L.,

Papalardo,E., Martinez-Martinez,L.A., Gonzalez,E.B., & Pierangeli,S.S.(2014) A

prospective open-label pilot study of fluvastatin on proinflammatory and

prothrombotic biomarkers in antiphospholipid antibody positive patients.

Ann.Rheum.Dis, 73,1176-80.

Erre,G.L., Pardini,S., Faedda,R., & Passiu,G. (2008) Effect of rituximab on clinical and

laboratory features of antiphospholipid syndrome: a case report and a review of

literature. Lupus, 17, 50-55.

Espinola,R.G., Pierangeli,S.S., Gharavi,A.E., & Harris,E.N.(2002) Hydroxychloroquine

reverses platelet activation induced by human IgG antiphospholipid antibodies.


Espinosa,G., Berman,H., & Cervera,R. (2011) Management of refractory cases of

catastrophic antiphospholipid syndrome. Autoimmun.Rev., 10, 664-668.

Ferrara,D.E., Liu,X., Espinola,R.G., Meroni,P.L., Abukhalaf,I., Harris,E.N., &

Pierangeli,S.S. (2003) Inhibition of the thrombogenic and inflammatory properties of

antiphospholipid antibodies by fluvastatin in an in vivo animal model. Arthritis

Rheum., 48, 3272-3279.

Ferrara,D.E., Swerlick,R., Casper,K., Meroni,P.L., Vega-Ostertag,M.E., Harris,E.N., &

Pierangeli,S.S.(2004) Fluvastatin inhibits up-regulation of tissue factor expression by

antiphospholipid antibodies on endothelial cells. J Thromb Haemost, 2,1558-63.

Finazzi,G., Marchioli,R., Brancaccio,V., Schinco,P., Wisloff,F., Musial,J., Baudo,F.,

Berrettini,M., Testa,S., D'Angelo,A., Tognoni,G., & Barbui,T. (2005) A randomized

clinical trial of high-intensity warfarin vs. conventional antithrombotic therapy for the

prevention of recurrent thrombosis in patients with the antiphospholipid syndrome

(WAPS). J.Thromb Haemost., 3, 848-853.

Franklin,R.D. & Kutteh,W.H. (2003) Effects of unfractionated and low molecular weight

heparin on antiphospholipid antibody binding in vitro. Obstet.Gynecol,101,455-62.

Galli,M., Comfurius,P., Maassen,C., Hemker,H.C., de Baets,M.H., van Breda-Vriesman,P.J.,

Barbui,T., Zwaal,R.F., & Bevers,E.M.(1990) Anticardiolipin antibodies (ACA)

directed not to cardiolipin but to a plasma protein cofactor. Lancet,335,1544-7.

33

Galli,M., Luciani,D., Bertolini,G., & Barbui,T. (2003) Lupus anticoagulants are stronger risk

factors for thrombosis than anticardiolipin antibodies in the antiphospholipid

syndrome: a systematic review of the literature. Blood, 101, 1827-1832.

Garcia,D., Akl,E.A., Carr,R., & Kearon,C.(2013) Antiphospholipid antibodies and the risk of

recurrence after a first episode of venous thromboembolism: a systematic review.

Blood, 122,817-24.

Girardi,G.(2010) Role of tissue factor in the maternal immunological attack of the embryo in

the antiphospholipid syndrome. Clin Rev.Allergy Immunol, 39,160-5.

Girardi,G., Berman,J., Redecha,P., Spruce,L., Thurman,J.M., Kraus,D., Hollmann,T.J.,

Casali,P., Caroll,M.C., Wetsel,R.A., Lambris,J.D., Holers,V.M., & Salmon,J.E.

(2003) Complement C5a receptors and neutrophils mediate fetal injury in the

antiphospholipid syndrome. J.Clin.Invest., 112, 1644-1654.

Girardi,G., Redecha,P., & Salmon,J.E. (2004) Heparin prevents antiphospholipid antibody-

induced fetal loss by inhibiting complement activation. Nat.Med., 10, 1222-1226.

Glotz,D., Russ,G., Rostaing,L., Legendre,C., S.Chadban,S., Grinyo,J., Mamode,N., Cozzi,E.,

Lebranchu,Y., Sandrini,S., Couzi,L., & Marks,W. (2015) Eculizumab in Prevention of Acute Antibody-Mediated Rejection in Sensitized Deceased-Donor Kidney Transplant Recipients: 1-Year Outcomes. American Transplant Congress, Abstract number:

3039.

Griffiths MH, Papadaki L, Neild GH (2000) The renal pathology of primary antiphospholipid

syndrome: a distinctive form ofendothelial injury. Q J Med, 93:457–467.

Gris JC, Bouvier S, Molinari N, Galanaud JP, Cochery-Nouvellon E, Mercier E, Fabbro-

Peray P, Balducchi JP, Marès P, Quéré I, Dauzat M (2012). Comparative incidence of

a first thrombotic event in purely obstetric antiphospholipid syndrome with pregnancy

loss: the NOH-APS observational study. Blood ,119:2624-32

Holers,V.M., Girardi,G., Mo,L., Guthridge,J.M., Molina,H., Pierangeli,S.S., Espinola,R.,

Xiaowei,L.E., Mao,D., Vialpando,C.G., & Salmon,J.E. (2002) Complement C3

activation is required for antiphospholipid antibody-induced fetal loss. J.Exp.Med.,

195, 211-220.

Ioannou,Y., Pericleous,C., Giles,I., Latchman,D.S., Isenberg,D.A., & Rahman,A.(2007)

Binding of antiphospholipid antibodies to discontinuous epitopes on domain I of

human beta(2)-glycoprotein I: mutation studies including residues R39 to R43.

Arthritis Rheum,56,280-90.

Ioannou Y, Zhang JY, Passam FH, Rahgozar S, Qi JC, GiannakopoulosB, Qi M, Yu P, Yu

DM, Hogg PJ, Krilis SA (2010). Naturally occurring free thiols within β2-

glycoprotein I in vivo: nitrosylation, redox modification by endothelial cells and

regulation of oxidative stress induced cell injury.Blood,116:1961–70

34

Ioannou,Y., Zhang,J.Y., Qi,M., Gao,L., Qi,J.C., Yu,D.M., Lau,H., Sturgess,A.D.,

Vlachoyiannopoulos,P.G., Moutsopoulos,H.M., Rahman,A., Pericleous,C., Atsumi,T.,

Koike,T., Heritier,S., Giannakopoulos,B., & Krilis,S.A.(2011) Novel assays of

thrombogenic pathogenicity in the antiphospholipid syndrome based on the detection

of molecular oxidative modification of the major autoantigen beta2-glycoprotein I.

Arthritis Rheum,632774-82.

Johansson E, Forsberg K, Johnsson H (1981). Clinical and experimental evaluation ofthe

thromboprophylactic effect of hydroxychloroquine sulfate after total hip replacement.

Haemostasis,10:89–96.

Keeling,D., Mackie,I., Moore,G.W., Greer,I.A., & Greaves,M. (2012) Guidelines on the

investigation and management of antiphospholipid syndrome. Br.J.Haematol., 157,

47-58.

Kumar,D. & Roubey,R.A.(2010) Use of rituximab in the antiphospholipid syndrome.

Curr.Rheumatol.Rep,1240-4.

Kutteh,W.H.(1996) Antiphospholipid antibody-associated recurrent pregnancy loss:

treatment with heparin and low-dose aspirin is superior to low-dose aspirin alone.

Am.J Obstet.Gynecol,174,1584-9.

Lambrianides A, Carroll CJ, Pierangeli SS, Pericleous C, Branch W, Rice J, Latchman DS,

Townsend P, Isenberg DA, Rahman A, Giles IP (2010).Effects of polyclonal IgG

derived from patients with different clinical types of the antiphospholipid syndrome

on monocyte signaling pathways. J Immunol, 184:6622-8.

Levine,S.R., Brey,R.L., Tilley,B.C., Thompson,J.L., Sacco,R.L., Sciacca,R.R., Murphy,A.,

Lu,Y., Costigan,T.M., Rhine,C., Levin,B., Triplett,D.A., & Mohr,J.P. (2004)

Antiphospholipid antibodies and subsequent thrombo-occlusive events in patients

with ischemic stroke. JAMA, 291, 576-584.

Liestol,S., Sandset,P.M., Jacobsen,E.M., Mowinckel,M.C., & Wisloff,F. (2007) Decreased

anticoagulant response to tissue factor pathway inhibitor type 1 in plasmas from

patients with lupus anticoagulants. Br.J.Haematol., 136, 131-137.

Maiti SN, Balasubramanian K, Ramoth JA, Schroit AJ (2008) β-2-glycoprotein 1-dependent

macrophage uptake of apoptotic cells:binding to lipoprotein receptor-related protein

receptor family members. J Biol Chem,283:3761–6

Mak,A., Cheung,M.W., Cheak,A.A., & Ho,R.C. (2010) Combination of heparin and aspirin

is superior to aspirin alone in enhancing live births in patients with recurrent

pregnancy loss and positive anti-phospholipid antibodies: a meta-analysis of

randomized controlled trials and meta-regression. Rheumatology.(Oxford.), 49, 281-

288.

Malia,R.G., Kitchen,S., Greaves,M., & Preston,F.E. (1990) Inhibition of activated protein C

and its cofactor protein S by antiphospholipid antibodies. Br.J.Haematol., 76, 101-

107.

35

Manning,B.D. & Cantley,L.C.(2007) AKT/PKB signaling: navigating downstream.

Cell,129,1261-74.

Manukyan D, Müller-Calleja N1, Jäckel S, Luchmann K, Mönnikes R, Kiouptsi K, Reinhardt

C, Jurk K, Walter U, Lackner KJ (2016). Cofactor-independent human

antiphospholipid antibodies induce venous thrombosis in mice. J Thromb Haemost,

14 :1011-20.

Mcneil,H.P., Simpson,R.J., Chesterman,C.N., & Krilis,S.A.(1990) Anti-phospholipid

antibodies are directed against a complex antigen that includes a lipid-binding

inhibitor of coagulation: beta 2-glycoprotein I (apolipoprotein H).

Proc.Natl.Acad.Sci.U.S.A,87,4120-4.

Mekinian,A., Lazzaroni,M.G., Kuzenko,A., Alijotas-Reig,J., Ruffatti,A., Levy,P., Canti,V.,

Bremme,K., Bezanahary,H., Bertero,T., Dhote,R., Maurier,F., Andreoli,L.,

Benbara,A., Tigazin,A., Carbillon,L., Nicaise-Roland,P., Tincani,A., & Fain,O.(2015)

The efficacy of hydroxychloroquine for obstetrical outcome in anti-phospholipid

syndrome: Data from a European multicenter retrospective study.

Autoimmun.Rev,14,498-502.

Miyakis,S., Lockshin,M.D., Atsumi,T., Branch,D.W., Brey,R.L., Cervera,R., Derksen,R.H.,

DE Groot,P.G., Koike,T., Meroni,P.L., Reber,G., Shoenfeld,Y., Tincani,A.,

Vlachoyiannopoulos,P.G., & Krilis,S.A. (2006) International consensus statement on

an update of the classification criteria for definite antiphospholipid syndrome (APS).

J.Thromb Haemost., 4, 295-306.

Nayar,R. & Lage,J.M.(1996) Placental changes in a first trimester missed abortion in

maternal systemic lupus erythematosus with antiphospholipid syndrome; a case report

and review of the literature. Hum.Pathol, 27,201-6.

Noel,N., Dutasta,F., Costedoat-Chalumeau,N., Bienvenu,B., Mariette,X., Geffray,L.,

Sene,D., Chaidi,R.B., Michot,J.M., Fain,O., Darnige,L., Ankri,A., Cacoub,P.,

Piette,J.C., & Saadoun,D.(2015) Safety and efficacy of oral direct inhibitors of

thrombin and factor Xa in antiphospholipid syndrome. Autoimmun.Rev, 14,680-5.

Nojima,J., Kuratsune,H., Suehisa,E., Iwatani,Y., & Kanakura,Y. (2005) Acquired activated

protein C resistance associated with IgG antibodies against beta2-glycoprotein I and

prothrombin as a strong risk factor for venous thromboembolism. Clin.Chem., 51,

545-552.

Ostertag,M.V., Liu,X., Henderson,V., & Pierangeli,S.S.(2006) A peptide that mimics the Vth

region of beta-2-glycoprotein I reverses antiphospholipid-mediated thrombosis in

mice. Lupus,.15,358-65.

Out,H.J., Kooijman CD,F.A.U., Bruinse HW,F.A.U., & Derksen,R.H.(1991)

Histopathological findings in placentae from patients with intra-uterine fetal death and

anti-phospholipid antibodies. Eur J Obstet Gynecol Reprod Biol,41,179-86.

36

Parke,A., Maier,D., Wilson,D., Andreoli,J., & Ballow,M.(1989) Intravenous gamma-

globulin, antiphospholipid antibodies, and pregnancy. Ann.Intern.Med,110,495-6.

Pelkmans,L., Kelchtermans,H., de Groot,P.G., Zuily,S., Regnault,V., Wahl,D., Pengo,V., &

de Laat B.(2013)Variability in exposure of epitope G40-R43 of domain i in

commercial anti-beta2-glycoprotein I IgG ELISAs. PLoS.One, 8.(8.):e71402.

Pengo,V., Biasiolo,A., Gresele,P., Marongiu,F., Erba,N., Veschi,F., Ghirarduzzi,A.,

Barcellona,D., & Tripodi,A.(2007) A comparison of lupus anticoagulant-positive

patients with clinical picture of antiphospholipid syndrome and those without.

Arterioscler.Thromb Vasc.Biol, 27,e309-10.

Pengo,V., Biasiolo,A., Pegoraro,C., Cucchini,U., Noventa,F., & Iliceto,S.(2005) Antibody

profiles for the diagnosis of antiphospholipid syndrome. Thromb Haemost, 93,1147-

52.

Pengo,V., Ruffatti,A., Legnani,C., Gresele,P., Barcellona,D., Erba,N., Testa,S., Marongiu,F.,

Bison,E., Denas,G., Banzato,A., Padayattil,J.S., & Iliceto,S. (2010) Clinical course of

high-risk patients diagnosed with antiphospholipid syndrome. J Thromb.Haemost., 8,

237-242.

Pengo,V., Ruffatti,A., Legnani,C., Testa,S., Fierro,T., Marongiu,F., De,M., V, Gresele,P.,

Tonello,M., Ghirarduzzi,A., Bison,E., Denas,G., Banzato,A., Padayattil,J.S., &

Iliceto,S. (2011) Incidence of a first thromboembolic event in asymptomatic carriers

of high-risk antiphospholipid antibody profile: a multicenter prospective study.

Blood., 118, 4714-4718.

Pengo,V., Ruffatti,A., Tonello,M., Cuffaro,S., Banzato,A., Bison,E., Denas,G., &

Padayattil,J.S.(2015) Antiphospholipid syndrome: antibodies to Domain 1 of beta2-

glycoprotein 1 correctly classify patients at risk. J Thromb Haemost,13,782-7.

Pericleous,C., Ruiz-Limon,P., Romay-Penabad,Z., Marin,A.C., Garza-Garcia,A., Murfitt,L.,

Driscoll,P.C., Latchman,D.S., Isenberg,D.A., Giles,I., Ioannou,Y., Rahman,A., &

Pierangeli,S.S. (2015) Proof-of-concept study demonstrating the pathogenicity of

affinity-purified IgG antibodies directed to domain I of beta2-glycoprotein I in a

mouse model of anti-phospholipid antibody-induced thrombosis.

Rheumatology.(Oxford), 54,722-7.

Pierangeli,S.S., Chen,P.P., Raschi,E., Scurati,S., Grossi,C., Borghi,M.O., Palomo,I.,

Harris,E.N., & Meroni,P.L.(2008) Antiphospholipid antibodies and the

antiphospholipid syndrome: pathogenic mechanisms. Semin.Thromb Hemost,34,236-

50.

Rai,R., Cohen,H., Dave,M., & Regan,L.(1997) Randomised controlled trial of aspirin and

aspirin plus heparin in pregnant women with recurrent miscarriage associated with

phospholipid antibodies (or antiphospholipid antibodies). Bmj, 314,253-7.

37

Rand,J.H., Wu,X.X., Andree,H.A., Lockwood,C.J., Guller,S., Scher,J., & Harpel,P.C. (1997)

Pregnancy loss in the antiphospholipid-antibody syndrome--a possible thrombogenic

mechanism. N.Engl.J.Med., 337, 154-160.

Rand,J.H., Wu,X.X., Quinn,A.S., Ashton,A.W., Chen,P.P., Hathcock,J.J., Andree,H.A., &

Taatjes,D.J.(2010) Hydroxychloroquine protects the annexin A5 anticoagulant shield

from disruption by antiphospholipid antibodies: evidence for a novel effect for an old

antimalarial drug. Blood, 115,2292-9.

Rand,J.H., Wu,X.X., Quinn,A.S., & Taatjes,D.J.(2010) The annexin A5-mediated

pathogenic mechanism in the antiphospholipid syndrome: role in pregnancy losses

and thrombosis. Lupus, 19,460-9.

Reynaud Q, Lega JC, Mismetti P, Chapelle C, Wahl D, Cathébras P, Laporte S (2014). Risk

of venous and arterial thrombosis according to type of antiphospholipid antibodies in

adults without systemic lupus erythematosus: a systematic review and meta-analysis.

Autoimmun Rev, 13 595-608.

Ridker,P.M.(2009) The JUPITER trial: results, controversies, and implications for

prevention. Circ.Cardiovasc.Qual.Outcomes, 2,279-85.

Rodriguez-Garcia,V., Ioannou,Y., Fernandez-Nebro,A., Isenberg,D.A., & Giles,I.P. (2015)

Examining the prevalence of non-criteria anti-phospholipid antibodies in patients with

anti-phospholipid syndrome: a systematic review. Rheumatology.(Oxford), 54,2042-

50.

Romay-Penabad,Z., Carrera Marin,A.L., Willis,R., Weston-Davies,W., Machin,S.,

Cohen,H., Brasier,A., & Gonzalez,E.B.(2014) Complement C5-inhibitor rEV576

(coversin) ameliorates in-vivo effects of antiphospholipid antibodies. Lupus, 23,1324-

6.

Ruffatti,A., Del,R.T., Ciprian,M., Bertero,M.T., Sciascia,S., Scarpato,S., Montecucco,C.,

Rossi,S., Caramaschi,P., Biasi,D., Doria,A., Rampudda,M., Monica,N., Fischetti,F.,

Picillo,U., Brucato,A., Salvan,E., Vittorio,P., Meroni,P., & Tincani,A. (2011) Risk

factors for a first thrombotic event in antiphospholipid antibody carriers: a prospective

multicentre follow-up study. Ann.Rheum.Dis., 70, 1083-1086.

Ruiz-Irastorza,G., Cuadrado,M.J., Ruiz-Arruza,I., Brey,R., Crowther,M., Derksen,R.,

Erkan,D., Krilis,S., Machin,S., Pengo,V., Pierangeli,S., Tektonidou,M., &

Khamashta,M.(2011) Evidence-based recommendations for the prevention and long-

term management of thrombosis in antiphospholipid antibody-positive patients: report

of a task force at the 13th International Congress on antiphospholipid antibodies.

Lupus,20,206-18.

Ruiz-Irastorza,G., Hunt,B.J., & Khamashta,M.A. (2007) A systematic review of secondary

thromboprophylaxis in patients with antiphospholipid antibodies. Arthritis.Rheum.,

57, 1487-1495.

38

Ruiz-Irastorza,G., Khamashta,M.A., Hunt,B.J., Escudero,A., Cuadrado,M.J., &

Hughes,G.R.(2002) Bleeding and recurrent thrombosis in definite antiphospholipid

syndrome: analysis of a series of 66 patients treated with oral anticoagulation to a

target international normalized ratio of 3.5. Arch.Intern.Med, 162,1164-9.

Ruiz-Irastorza,G., Ramos-Casals,M., Brito-Zeron,P., & Khamashta,M.A. (2010) Clinical

efficacy and side effects of antimalarials in systemic lupus erythematosus: a

systematic review. Ann.Rheum.Dis., 69, 20-28.

Salmon,J.E., Girardi,G., & Holers,V.M. (2003) Activation of complement mediates

antiphospholipid antibody-induced pregnancy loss. Lupus, 12, 535-538.

Schaefer,J.K., Mcbane,R.D., Black,D.F., Williams,L.N., Moder,K.G., &

Wysokinski,W.E.(2014) Failure of dabigatran and rivaroxaban to prevent

thromboembolism in antiphospholipid syndrome: a case series of three patients.


Schmidt-Tanguy,A., Voswinkel,J., Henrion,D., Subra,J.F., Loufrani,L., Rohmer,V., Ifrah,N.,

& Belizna,C.(2013) Antithrombotic effects of hydroxychloroquine in primary

antiphospholipid syndrome patients. J Thromb Haemost, 11,1927-9.

Sciascia,S., Breen,K., & Hunt,B.J.(2015) Rivaroxaban use in patients with antiphospholipid

syndrome and previous venous thromboembolism. Blood Coagul.Fibrinolysis,

26,476-7.

Sciascia,S., Hunt,B.J., Talavera-Garcia,E., Lliso,G., Khamashta,M.A., &

Cuadrado,M.J.(2016) The impact of hydroxychloroquine treatment on pregnancy

outcome in women with antiphospholipid antibodies. Am.J Obstet.Gynecol,

214,273.e1-8.

Shapira,I., Andrade,D., Allen,S.L., & Salmon,J.E.(2012) Brief report: induction of sustained

remission in recurrent catastrophic antiphospholipid syndrome via inhibition of

terminal complement with eculizumab. Arthritis Rheum, 64,2719-23.

Signorelli,F., Nogueira,F., Domingues,V., Mariz,H.A., & Levy,R.A.(2016) Thrombotic

events in patients with antiphospholipid syndrome treated with rivaroxaban: a series

of eight cases. Clin Rheumatol, 35,801-5.

Son,M., Wypasek,E., Celinska-Lowenhoff,M., & Undas,A.(2015) The use of rivaroxaban in

patients with antiphospholipid syndrome: A series of 12 cases. Thromb Res,

135,1035-6.

Sperber,K., Quraishi,H., Kalb,T.H., Panja,A., Stecher,V., & Mayer,L.(1993) Selective

regulation of cytokine secretion by hydroxychloroquine: inhibition of interleukin 1

alpha (IL-1-alpha) and IL-6 in human monocytes and T cells. J Rheumatol, 20,803-8.

The PROMISE study (Predictors of pRegnancy outcome: biomarkers In APS and Systemic

lupus Erythematosus). https://clinicaltrials.gov/ct2/show/NCT00198068. Accessed

20 June 2016.

39

Tripodi,A., Chantarangkul,V., Clerici,M., Negri,B., Galli,M., & Mannucci,P.M.(2001)

Laboratory control of oral anticoagulant treatment by the INR system in patients with

the antiphospholipid syndrome and lupus anticoagulant. Results of a collaborative

study involving nine commercial thromboplastins. Br.J Haematol, 115,672-8.

van Os,G.M., Meijers,J.C., Agar,C., Seron,M.V., Marquart,J.A., Akesson,P., Urbanus,R.T.,

Derksen,R.H., Herwald,H., Morgelin,M., & Groot Pg,D.E.(2011) Induction of anti-

beta2 -glycoprotein I autoantibodies in mice by protein H of Streptococcus pyogenes.

J Thromb Haemost, 9,2447-56.

Willis R, Gonzalez EB, Brasier AR (2015). The journey of antiphospholipid antibodies from

cellular activation to antiphospholipid syndrome. Curr Rheumatol Rep,17:16-0485.

Win,K. & Rodgers,G.M.(2014) New oral anticoagulants may not be effective to prevent

venous thromboembolism in patients with antiphospholipid syndrome. Am.J Hematol,

89,1017.Xu,C., Mao,D., Holers,V.M., Palanca,B., Cheng,A.M., & Molina,H. (2000)

A critical role for murine complement regulator crry in fetomaternal tolerance.

Science, 287, 498-501.

Yu P, Passam FH, Yu DM, Denyer G, Krilis S (2008). β2-glycoprotein I inhibits vascular

endothelial growth factor and basic fibroblast growth factor induced angiogenesis

through its amino terminaldomain. J Thromb Haemost ;6:1215–23.

Zhang,J. & Mccrae,K.R.(2005) Annexin A2 mediates endothelial cell activation by

antiphospholipid/anti-beta2 glycoprotein I antibodies. Blood,105,1964-9.

40

Table 1: The international consensus (revised Sapporo) criteria for diagnosis of obstetric

antiphospholipid syndrome

Clinical criteria Laboratory criteria

1. One or more unexplained deaths of a

morphologically normal fetus at or beyond the

10th week of gestation

2. One or more pre-term births of a

morphologically normal neonate before the 34th

week of gestation because of:

(i) eclampsia or severe pre-eclampsia or

(ii) recognized features of placental insufficiency

3. Three or more unexplained consecutive

spontaneous miscarriages before the 10th week

of gestation, with maternal anatomic or

hormonal abnormalities and paternal and

maternal chromosomal causes excluded

1. LA present in plasma, on two or more

occasions at least 12 weeks apart

2. aCL of immunoglobulin (Ig)G and/or IgM

isotype in serum or plasma, present in medium

or high titre (i.e. >40GPL units or MPL units, or >

the 99th centile), on two or more occasions, at

least 12 weeks apart

3. aβ2GPI of IgG and/or IgM isotype in serum or

plasma (in titre >the 99th centile), present on

two or more occasions at least 12 weeks apart

OAPS is diagnosed if at least one of the clinical criteria and one of the laboratory criteria are met

OAPS: Obstetric antiphospholipid syndrome; LA: lupus anticoagulants; aCL: anticardiolipin

antibodies; aβ2GPI: antiβ2glycoprotein-I antibodies

41

Table 2. Mechanisms for aPL-mediated fetal loss or complications

Mechanism Supporting evidence from studies in humans or animals/comments

Intraplacental thrombosis Animal studies have demonstrated that the passive transfer of aPL

promotes fetal loss and placental thrombosis and also inhibits

trophoblast and decidual cell functions in vitro (Branch et al,1990)

Placental thrombosis and infarction shown in APS patients with

intra-uterine fetal death (Out et al,1991 ;Nayar & Lage,1996)

aPL binding to monocytes, endothelial cells, platelets and plasma

components of the coagulation cascade (Zhang & Mccrae,2005)

may lead to placental thrombosis.

aPL might induce a procoagulant state at the placental level by

disrupting the anticoagulant annexin A5 shield on trophoblast and

endothelial cell monolayers (Rand et al, 1997)

Inflammation β2GPI-dependent aPL are able to react with human stromal

decidual cells in vitro, inducing a proinflammatory

phenotype(Zhang & Mccrae,2005)

Interference with Annexin V function

Thrombosis during the development of the normal materno-

placental circulation may arise via interference with function of

Annexin V (Rand et al, 1997;Rand et al,2010) and women with APS

have diminished annexin V binding to the surface of trophoblastic

cells of the intervillous space compared with controls. (Rand et al,

1997)

Inhibition of syncytium-trophoblast differentiation and defective placentation/placental apoptosis

aPL (in particular β2GPI-dependent antibodies) bind to human

trophoblasts and affect several cell functions (Agostinis et al, 2011;

;Girardi,2003a), inducing cell injury and apoptosis, inhibition of

proliferation and syncitia formation, decreased production of

human chorionic gonadotrophin, defective secretion of growth

factors and impaired invasiveness which lead to defective

placentation (Pierangeli et,1999).

42

Complement activation Passive transfer of IgG from women with recurrent miscarriage and

aPL results in a significant increase in the frequency of fetal

resorption and IUGR, compared to mice treated with IgG from

healthy individuals [(Holers et al, 2002). These effects can be

blocked by inhibition of the complement cascade using a C3

convertase inhibitor or by antibodies or peptides that block C5a-

C5a receptor interactions (Girardi et al, 2003;Pierangeli et al, 1999).

Mice deficient in complement C3 are resistant to fetal injury

induced by aPL (Girardi et al, 2003;Holers et al, 2002) .

Heparin prevents obstetric complications caused by aPL, because it

blocks complement activation rather than through its

antithrombotic properties (Girardi et al, 2004).

aPL = antiphospholipid antibodies; IUGR = intrauterine growth restriction

43

Legends to figures

Figure 1. Key events in the history of Antiphospholipid syndrome (Adapted from figure

originally done by Prof. Mike Greaves)

44

Figure 2. Schematic representation of folded (circular) and unfolded conformation of beta2

glycoprotein I and subsequent binding to anti- beta2 glycoprotein I antibodies (Adapted

from van Os et al, 2011)

In the presence of anionic phospholipids, the circular conformation of the protein unfolds,

exposing antigenic determinants that are normally shielded from the circulation (A-C)_

which facilitate the binding to antibodies (D).

45

Figure 3. Complement pathway activation. aPL induced activation of classical pathway

leading to thrombosis and pregnancy complications and potential targets for therapeutic

complement inhibition (Adapted from Arachchillage & Hillmen, 2015)

The complement cascade is activated by one of the three pathways. Activation leads to the

formation of C3 convertase, resulting in the formation of C5 convertase and membrane

attack complex (MAC). aPL recognize domain I of β2GPI and stabilizes its open configuration

on the cell surface. This is followed by binding of complement factor C1q resulting in the

activation of the complement system. The formed anaphylatoxins cause cell damage and

induce a prothrombotic phenotype, leading to both thrombosis and pregnancy failure.

Potential therapeutic targets of complement inhibition also shown

Documents

Pathogenesis and management of antiphospholipid syndrome · pathogenesis of APS has significant implications for treatment: agents acting outside the coagulation system such as hydroxychloroquine