Antiphospholipid Syndrome

C H A P T E R

32 Antiphospholipid Syndrome Ora Shovman1,2 and Yehuda Shoenfeld1,3,4 1

The Zabludowicz Center for Autoimmune Diseases, Sheba Medical Center, Tel-Hashomer, Affiliated to Tel Aviv University, Tel Aviv, Israel 2Department of Medicine ‘B’, Sheba Medical Center, Tel-Hashomer, Israel 3Past Incumbent of the Laura Schwarz-Kipp Chair for Research of Autoimmune Diseases, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel 4Laboratory of the Mosaics of Autoimmunity, Saint Petersburg University, Saint Petersburg, Israel

O U T L I N E General Introduction and Historical Aspects

607

Diagnostic Procedures

621

Epidemiology

608

Clinical Features and Disease Associations Obstetric Antiphospholipid Syndrome Thrombotic Antiphospholipid Syndrome Noncriteria Antiphospholipid Syndrome Manifestations Catastrophic Antiphospholipid Syndrome

609 610 611

The Mechanisms of Antiphospholipid Antibodies-Mediated Disease Expressions: Clinical Trials and Animal Models Thrombotic Manifestations Obstetric Manifestations The Complement System in Antiphospholipid Syndrome

621 623 624

The Antiphospholipid Antibodies Criteria-Relevant Antiphospholipid Antibodies Noncriteria Antiphospholipid Antibodies

615 615 616

Mortality in the Antiphospholipid Syndrome

626

Treatment of Antiphospholipid Syndrome

626

Conclusions and Future Aspects

627

Seronegative Antiphospholipid Syndrome

619

References

628

Risk Assessment in Antiphospholipid Syndrome

620

Further Reading

634

Genetics

620

Classification Criteria Versus Diagnostic Criteria

621

611 614

625

GENERAL INTRODUCTION AND HISTORICAL ASPECTS In 1983, a discrete syndrome was described by Graham Hughes in which lupus patients with antiphospholipid antibodies (aPL) were prone to arterial/venous thrombosis, recurrent abortions, neurological manifestations, and occasional thrombocytopenia (Hughes, 1983). In the following years, the Hughes’ syndrome was delineated as a systemic disease that can affect both children and adults and can present as a primary disorder or as secondary to other autoimmune disease (Shoenfeld et al., 2009). The definition of this classical

The Autoimmune Diseases, 6th. DOI: https://doi.org/10.1016/B978-0-12-812102-3.00032-4

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608 TABLE 32.1

32. ANTIPHOSPHOLIPID SYNDROME

Summary of the Sydney Consensus Statement on Investigational Classification Criteria for APSa

Antiphospholipid antibody syndrome is present if at least one of the clinical criteria and one of the laboratory criteria that follow are met Clinical criteria 1. Vascular thromboses: One or more documented episodes of arterial, venous, or small vessel thrombosis—other than superficial venous thrombosis—in any tissue or organ. Thrombosis must be confirmed by objective validated criteria. For histopathologic confirmation, thrombosis should be present without significant evidence of inflammation in the vessel wall 2. Pregnancy morbidity: a. One or more unexplained deaths of a morphologically normal fetus at or beyond the 10th week of gestation, with normal fetal morphology documented by ultrasound or by direct examination of the fetus, or b. One or more premature births of a morphologically normal neonate before the 34th week of gestation because of (1) eclampsia or severe preeclampsia defined according to standard definitions or (2) recognized features of placental insufficiency, or c. Three or more unexplained consecutive spontaneous abortions before the 10th week of gestation, with maternal anatomic or hormonal abnormalities and paternal and maternal chromosomal causes excluded Laboratory criteria 1. LA present in plasma, on two or more occasions at least 12 weeks apart, detected according to the guidelines of the International Society on Thrombosis and Haemostasis (Scientific Subcommittee on LAs/phospholipid-dependent antibodies) 2. aCL antibody of IgG and/or IgM isotype in serum or plasma, present in medium or high level (i.e., 40 GPL or MPL, or the 99th percentile), on two or more occasions, at least 12 weeks apart, measured by a standardized ELISA 3. Anti-b2-glycoprotein-I antibody of IgG and/or IgM isotype in serum or plasma (in level. the 99th percentile), present on two or more occasions, at least 12 weeks apart, measured by a standardized ELISA, according to recommended procedures a

Miyakis et al. (2006). aCL, anticardiolipin; LA, Lupus anticoagulant.

autoimmune syndrome has greatly advanced from the original reports and classification criteria for antiphospholipid syndrome (APS) formulated in Sapporo, Japan, in 1998 to the current ones published in 2006 (Miyakis et al., 2006; Cervera and Ra, 2008) (Table 32.1). For the determination of APS, at least one classical clinical criterion [i.e., vascular thrombosis or pregnancy morbidity (PM)] and one serological criterion [i.e., the persistent presence of anticardiolipin (aCL) and/or anti-b2-glycoprotein-I antibody (anti-B2GPI) of IgG or IgM isotype at medium-to-high titers or lupus anticoagulant (LAC)] have to be met. Several new aPL specificities were found, including antibodies directed against domains of B2GPI or coagulation cascade proteins such as prothrombin itself or complexes of prothrombin with phospholipids (i.e., phosphatidylserine/prothrombin complex) (Bertolaccini et al., 2014).

EPIDEMIOLOGY aPL may be detected in up to 1% 5% of the general population, but only the minority of aPL-positive individuals develop APS. aPL appearing in healthy subjects are usually detected transiently at low levels and are frequently clinically insignificant (Shoenfeld et al., 2008a; Biggioggero and Meroni, 2010). They are normally found in older individuals and in association with infections, vaccinations, malignancies, and exposure to certain drugs. The prevalence of aPL in systemic lupus erythematosus (SLE) patients ranges between 30% and 40% (Biggioggero and Meroni, 2010). The Antiphospholipid Syndrome Alliance For Clinical Trials and International Networking conducted a literature review and analyzed the frequency of aPL in patients with PM, stroke (ST), myocardial infarction (MI), and deep venous thrombosis (DVT) (Andreoli et al., 2013a). According to this review, aPL were positive in about 6% of the patients with PM, 13.5% of the patients with ST, 11% of the patients with MI, and 9.5% of the patients with DVT (Andreoli et al., 2013a). It has been estimated that the incidence of APS is approximately 5 new cases per 100,000 individuals per year and the prevalence is 40 50 cases per 100,000 individuals (Cervera, 2017). Secondary APS is estimated to appear in 10% 15% of SLE patients and less in other autoimmune diseases. In contract with aPL-positive healthy individuals, patients with APS either primary or secondary typically present with persistent (more than 12 weeks), high-level aPL seropositivity and significant related morbidity and mortality (Cervera et al., 2009). Catastrophic APS (CAPS) is rare and occurs in less than 1% of all APS cases (Asherson et al., 2003).

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CLINICAL FEATURES AND DISEASE ASSOCIATIONS

CLINICAL FEATURES AND DISEASE ASSOCIATIONS APS is usually diagnosed following obstetric or thrombotic morbidity. Nonetheless, the clinical spectrum of APS is considered nowadays to be wider and includes systemic and organ-specific symptoms induced by both thrombotic and immune-mediated mechanisms (Shoenfeld, 2007; Marai et al., 2004). In addition, a wide spectrum of clinical presentations may arise due to occlusions in one or several vessels. The clinical manifestations of 1000 patients with APS that were in the follow up for 10 years within the EuroPhospholipid project are summarized in Table 32.2. TABLE 32.2 The Most Common APS Manifestations, According to the “Euro-Phospholipid Project” (at the Beginning of the Study) Manifestations

%

Peripheral thrombosis Deep vein thrombosis

38.9

Superficial thrombophlebitis in legs

11.7

Arterial thrombosis in legs

4.3

Venous thrombosis in arms

3.4

Arterial thrombosis in arms

2.7

Subclavian vein thrombosis

1.8

Jugular vein thrombosis

0.9

Neurologic manifestations Migraine

20.2

Stroke

19.8

Transient ischemic attack

11.1

Epilepsy

7.0

Multiinfarct dementia

2.5

Chorea

1.3

Acute encephalopathy

1.1

Pulmonary manifestations Pulmonary embolism

14.1

Pulmonary hypertension

2.2

Pulmonary microthrombosis

1.5

Cardiac manifestations Valve thickening/dysfunction

11.6

Myocardial infarction

5.5

Angina

2.7

Myocardiopathy

2.9

Vegetations

2.7

Coronary by-pass rethrombosis

1.1

Intraabdominal manifestations Renal manifestations (glomerular thrombosis, renal infarction, renal artery thrombosis, renal vein thrombosis)

2.7

(Continued)

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TABLE 32.2 (Continued) Manifestations

%

Gastrointestinal manifestations (esophageal or mesenteric ischemia)

1.5

Splenic infraction

1.1

Cutaneous manifestations Livedo reticularis

24.1

Ulcers

5.5

Pseudovasculitic lesions

3.9

Digital gangrene

3.3

Cutaneous necrosis

2.1

Osteo-articular manifestations Arthralgia

38.7

Arthritis

27.1

Avascular necrosis of bone

2.4

Ophthalmologic manifestations Amaurosis fugax

5.4

Retinal artery thrombosis

1.5

E.N.T. manifestations Nasal septum perforation

0.8

Hematological manifestations Thrombocytopenia ( , 100,000 per μL) Hemolytic anemia

29.6 9.7

Obstetric manifestations (pregnant female 5 590) Preeclampsia

9.5

Eclampsia

4.4

Abruptio placentae

2.0

Fetal manifestations (pregnancies 5 1580) Early fetal losses ( , 10 weeks)

35.4

Late fetal losses ($10 weeks)

16.9

Live births

47.7

Prematures

10.6

Adopted from Cervera, R., Piette, J.C., Font, J., Khamashta, M.A., Shoenfeld, Y., Camps, M.T., et al., 2002. Antiphospholipid syndrome: clinical and immunologic manifestations and patterns of disease expression in a cohort of 1,000 patients. Arthritis Rheum. 46, 1019 1027.

Obstetric Antiphospholipid Syndrome Obstetric complications are a hallmark of APS including fetal and maternal complications and are now recognized as a distinct entity from vascular APS. Early recurrent miscarriages (less than 10 weeks of gestation) occur in about 1% of the general obstetric population, and out of them 15% are associated with APS. Fetal complications in obstetric APS (OAPS) patients include prematurity, intrauterine growth restriction due to placental insufficiency, and early and late pregnancy loss. According to the Euro-Phospholipid Project study, during a 10-year period, the prevalence of these events in APS pregnancies was 48%, 26%, 16.5%, and 5%,

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respectively (Cervera et al., 2015). Fetal death is considered to be the most specific feature of OAPS, and strong association between fetal loss and the presence of aPL was found (Abou-Nassar et al., 2011; Silver et al., 2013). The most common maternal manifestation of APS is preeclampsia, followed by eclampsia and abruptio placentae. The assessment of risk for PM in aPL-positive patients is difficult in general because similar clinical manifestations may be associated with different patterns and combinations of aPL. According to the classification criteria, only women with PM and medium-to-high titer aPL should be diagnosed with OAPS. However, patients with low titer aPL can experience poor pregnancy outcomes similar to high titer patients, and therefore current classification criteria do not incorporate all the OAPS cases (Ofer-Shiber and Molad, 2015). The presence of an associated systemic autoimmune disease, in particular SLE, a history of previous thrombotic events, and complement reduction, has been identified as being predictive of a poor pregnancy outcome (Fredi et al., 2015). In addition, there is evidence that LA and triple positivity may serve as markers of worse outcome (Alijotas-Reig et al., 2015; Yelnik et al., 2016). Angiogenic biomarkers measured as early as the 12th week of pregnancy, in combination with clinical criteria, may be useful to identify patients with aPL at risk of severe adverse pregnancy outcomes (Kim et al., 2016). The possible association between aPL and sterility remains unknown. No thrombosis or SLE was found in babies born to mothers with APS included in European aPL Forum despite the transplacentar transfer of maternal aPL autoantibodies (Mekinian et al., 2013). One study has reported high prolactin levels in 12% of APS patients, and this hyperprolactinemia was associated with reproductive failure, including early and late pregnancy loss, and intrauterine growth retardation (Praprotnik et al., 2010).

Thrombotic Antiphospholipid Syndrome Venous and/or arterial thrombosis are distinctive characteristics of APS (Taraborelli et al., 2012), and vessels in any site and of any size may be involved (Saponjski et al., 2011). Such thrombotic events are the main causes of morbidity and mortality in APS, and they tend to recur particularly in untreated patients (Taraborelli et al., 2012). The confirmation of thrombosis by an objective method is a requirement of the Sapporo criteria, nowadays performed by a diversity of invasive and noninvasive methods such as angiography, ultrasound, computed tomography (i.e., 64 multislice computed tomographic angiography), and others (Saponjski et al., 2011). Histopathology may also be utilized to confirm the diagnosis of APS-associated thrombosis once there is no evidence of inflammation in the vessel wall (Taraborelli et al., 2012). DVT, pulmonary embolism (PE), and STs are the most commonly reported. In line with these data, thrombosis was the most common manifestation of APS in the 1000 patients involved in the Euro-Phospholipid project (Cervera et al., 2002). Venous thrombosis in particular DVT of the lower limbs was a presenting symptom in 39% of the patients. Thrombotic events appeared in 166 (16.6%) patients during the first 5-year period of observation and in 118 patients (15.3%) during the second 5-year period. Among these thrombotic events that developed during the 10-year period, the most common were STs (5.3% of the total cohort), transient ischemic attack (TIA) (4.7%), DVT (4.3%), and PE (3.5%). These events were observed in similar proportions during both periods of the study (Cervera et al., 2009, 2015). Furthermore, thrombotic APS manifestations occurring in atypical sites may cause additional clinical syndromes, including acute coronary syndromes; hepatic arterial or venous (Budd Chiari syndrome) thrombosis; portal, mesenteric, or splenic ischemia; and pancreatic and adrenal insufficiency secondary to acute vascular infarction.

Noncriteria Antiphospholipid Syndrome Manifestations The first definitive classification criteria for APS were formulated during the 8th International Congress on aPL held in Sapporo and were published in 1999 (Wilson et al., 1999). A subsequent revision was later completed during the 11th International Congress on aPL held in Sydney (Miyakis et al., 2006). Initially, these criteria were designed for scientific clinical studies and included major manifestations of APS. Subsequently, they have been adapted for the diagnosis of APS in routine clinical practice. According to the APS Clinical Features Task Force of the 14th International Congress on aPL, additional common non-criteria manifestations including thrombocytopenia, heart valve disease, renal microangiopathy

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(aPL nephropathy), chorea, and longitudinal myelitis may be added to the APS classification criteria (Abreu et al., 2015). However, these features are not yet included in the International Consensus Criteria (Sapporo criteria). Neurological Antiphospholipid Syndrome Neurological manifestations were detailed in the original description of APS and have since remained a main cause for morbidity and mortality among patients affected by this syndrome (Hughes, 1983). In APS, ST is the most common and severe complication and is the only manifestation included in APS criteria (Appenzeller et al., 2012). APS-related STs often occur in young patients and have a tendency to recur without an adequate therapy (Andreoli et al., 2013a). In the Euro-Phospholipid Project Group, it has been demonstrated that the prevalence of ST and TIAs in 1000 APS patients at the beginning of the study was 19.8% and 11.1%, respectively (Cervera et al., 2002). During the 10 years follow-up, STs and TIA occurred in 5.3% and 4.7% of the total cohort, correspondingly (Cervera et al., 2015). In addition to local thrombosis mediated by aPL, valvular heart disease may become a source of emboli and related cerebrovascular events (Andreoli et al., 2013a). Yet, in the last 30 years, aPL positivity has been correlated with a wide variety of non-ST neurological expressions including headache, cognitive dysfunction, psychosis, depression, dementia, migraine, convulsions, chorea, transverse myelitis, Guillain Barre´ syndrome, and a multiple sclerosis-like illness (Chapman et al., 2003; Katzav et al., 2003; Shoenfeld et al., 2004; Rodrigues et al., 2010). Some syndromes, such as migraine or cognitive dysfunction, are frequently described in APS whereas other neurological manifestations are rare. In addition, cognitive dysfunction is strongly associated with aPL, more than any other neurological non-ST APS presentation (Coı´n et al., 2015). One study reported that epilepsy was strongly associated with STs and TIA, presence of SLE, valvulopathy, and livedo reticularis (Shoenfeld et al., 2004). A different rare severe manifestation of APS is transverse myelitis, which requires early aggressive immunosuppressive treatment (Sherer et al., 2002). In addition, APS should be considered in all the unexplained cases of retinal arterial and venous thromboses, as well as in the cases of unusual ocular inflammations, particularly in young individuals (Arnson et al., 2010). Hematologic Antiphospholipid Syndrome Thrombocytopenia occurs in 20% 40% of APS patients (Cervera et al., 2002, 2011b) and is associated with systemic involvement (Krause et al., 2005a). According to the Euro-Phospholipid Project Group, thrombocytopenia appeared in 8.7% of the APS patients during the 10 years follow-up (Cervera et al., 2015). It is usually moderate, with platelet counts greater than 50K, rarely associated with major bleeding episodes, and does not require therapeutic intervention. aPL-positive patients who develop thrombocytopenia without fulfilling the clinical criteria for APS have a potential risk of developing thrombosis. Smoking, LAC, and higher titers of aPL were found to be risk factors for the development of thrombocytopenia (Demetrio Pablo et al., 2017). Hyperferritinemia was found to be linked to thrombocytopenia, LAC, and aCL in SLE patients and it may serve as an early marker for secondary APS in this cohort (Zandman-Goddard et al., 2013). Autoimmune hemolytic anemia is less frequent and was previously reported to occur in 6% 10% of APS patients (Taraborelli et al., 2012; Rottem et al., 2006). According to Euro-Phospholipid Project, it has appeared in 4% of the patients within during 10 years follow-up) Cervera et al., 2015). A significant association between the presence of aPL and a positive Coombs test as well as the cooccurrence of both autoimmune thrombocytopenia and hemolytic anemia (named Evans syndrome) has been described (Rottem et al., 2006; Cervera et al., 2011b). Lymphopenia and neutropenia are well-recognized features of SLE and therefore are found more commonly in patients with APS associated with SLE. Dermatologic Antiphospholipid Syndrome There are several different dermatologic features of APS. Although these features are nonspecific and not included in the classification criteria, they are common (49% of APS patients) and may be the first clinical presentations in 30% 40% of the cases (France`s et al., 2005). The most frequent is livedo reticularis, a red or bluish alteration of the skin with a net-like pattern attributed to blood stasis in capillaries and venules (Toubi et al., 2005). The pathophysiology of livedo reticularis is not well characterized, but the relationship with arterial thrombosis such as Sneddon’s syndrome suggests a possible role for an interaction of aPL with the endothelial cell or other cellular element in blood vessels (Taraborelli et al., 2012). Livedo reticularis may be a prognostic marker of a

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more severe disease, which may be possibly used as a criterion for APS (Toubi and Shoenfeld, 2007). It is associated with arterial and venous thrombosis and PM, irrespective of the presence of aPL (France`s et al., 1999, 2005; Toubi et al., 2005). Cutaneous necrosis is observed in 5.5% of the patients with APS and the most commonly involved sites are the upper and lower extremities, helices of ears, cheeks, trunk, and forehead. These lesions may appear in the postphlebitic state (following a thrombosis) or caused by a circumscribed skin necrosis. A rare association has been found between cryoglobulinemia and APS in patients with cutaneous necrosis (Shachaf and Yair, 2016). APS should be in the differential for pyoderma gangrenosum-like lesions unresponsive to the usual treatment. Subungual splinter hemorrhages may be seen along with thrombotic events elsewhere (France`s et al., 2005). Cutaneous digital gangrene, with preceding ischemic symptoms, has also been observed in up to 7.5% of the patients with APS. Many nonspecific skin lesions of which some resemble vasculitis (pseudovasculitis) including red macules, purpura, cyanotic lesions on the hands and feet, ecchymoses, and painful skin nodules as well as primary anetoderma have also been reported. Cardiac Antiphospholipid Syndrome The heart is one of the organs targeted in APS. Cardiac features include asymptomatic valve lesions, cardiac vegetations causing recurrent STs, accelerated atherosclerosis, MI, intracardiac thrombus, pulmonary hypertension, cardiomyopathy, and diastolic dysfunction (Solte´sz et al., 2007). Valve abnormalities, vegetations, and/or thickening, termed Libman Sacks endocarditis, are the most common manifestations described in 30% 50% of the patients (Ziporen et al., 1996; Blank et al., 2004; Cervera et al., 2011a). It should be noted that the valve damage is more frequent in patients with secondary APS and is highly associated with the presence of aPL (Nesher et al., 1997). aPL represent an independent risk factor for valvular heart disease in SLE patients, along with the disease itself (Watad et al., 2017). In addition, aCL IgG/IgM positivity was connected to valvular abnormalities in primary and secondary APS patients, and higher levels of those antibodies were correlated with an increased risk for these manifestations (Djokovic et al., 2014). Valve abnormalities can lead to an increased risk of embolism and may rarely (4% 6%) require replacement. The mitral valve is most commonly affected followed by the aortic valve. The presence of cardiac valve pathology may be a risk factor for several types of CNS involvement in primary APS including epilepsy, migraine, STs, and TIA (Krause et al., 2005b). Pulmonary hypertension is the second common cardiac manifestation in APS and occurred in about 11% of APS patients in one study (Pardos-Gea et al., 2015). It has been previously demonstrated that pulmonary hypertension occurs mostly due to venous thromboembolic disease with PE. Myocardial ischemia may result from coronary thromboembolism, accelerated atherosclerosis of the coronary arteries, or microvascular injury. MI is diagnosed in 5.5% of the patients with APS (Cervera et al., 2002) and is also significantly associated with the presence of aPL (Cervera et al., 2011a). Similarly, clinically silent myocardial ischemia have been found in young patients with primary APS, and it have also been associated with elevated levels of the IgG class of both aCL and anti-B2GPI antibodies (Padjas et al., 2016). Accelerated atherosclerosis has been found in APS patients more frequently than in the general population (Shoenfeld et al., 2005) and may be driven by different immunopathological factors associated with disease itself. Ventricular dysfunction in APS is rare and may result from valvular disorders, myocardial ischemia, or probably from the direct effect of the aPL on the myocardium. Primary APS seems to be related to diastolic dysfunction while SLE and APS appear to be associated with systolic dysfunction. Moreover, the right ventricle seems to be more involved than left ventricle, especially in primary APS (Tektonidou et al., 2001). Owing to the high incidence of cardiac involvement, echocardiographic follow-up is recommended for all APS patients (Cervera et al., 2011a). Cardiovascular magnetic resonance may be able to identify a high prevalence of occult myocardial scarring and endomyocardial fibrosis in APS and may have a great value in the diagnosis and follow-up of both clinically overt and silent cardiac diseases in APS patients (Mavrogeni et al., 2015). Pulmonary Antiphospholipid Syndrome PE and infarction constitute the most frequent pulmonary manifestation of APS. In one study that included 1000 APS patients, PE was the presenting manifestation in 14% of them and additional 3.5% developed this condition during 10 years of follow-up (Cervera et al., 2002, 2015). Leaving aside this classical thrombotic manifestation, other pulmonary expressions including pulmonary hypertension, acute respiratory distress syndrome

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(ARDS), intraalveolar hemorrhage, and fibrosing alveolitis have been described in the clinical spectrum of APS features (Stojanovich et al., 2012a). Notably, the presence of aPL correlated with distinct pulmonary types of involvement, such as the link between aCL and pulmonary arterial thrombosis, adult respiratory distress syndrome, and fibrosing alveolitis (Stojanovich et al., 2012a). In patients, with CAPS, small vessel thrombosis and systemic inflammatory response may lead to ARDS (Erkan et al., 2010). Diffuse alveolar hemorrhage may occasionally present as the initial manifestation of APS and cause cough, dyspnea, and fever with or without hemoptysis and, in some cases, progress to acute respiratory failure (CartinCeba et al., 2014). The aPL positivity may help in the identification of SLE patients who are at risk for developing pulmonary hypertension and pulmonary arterial hypertension (Zuily et al., 2017). Renal Antiphospholipid Syndrome APS-mediated thrombosis can affect different parts of the kidney depending on the type and size of the vessels. These thrombotic events may result in various manifestations that reflect the site and size of the involved vessel. Large vessel involvement is usually in the form of thrombosis and/or stenosis that present as marked hypertension, renal dysfunction, and pain. In the case of occlusive lesions, both in situ thrombosis and embolization from heart lesions can take place (Taraborelli et al., 2012). Renal involvement in APS also includes small vessel vaso-occlusive nephropathy defined as “APS nephropathy” or “aPL-associated nephropathy.” The major clinical presentations of this condition are hypertension, hematuria, proteinuria (ranging from mild to nephrotic level), and renal insufficiency (Tektonidou, 2014). Investigation in such cases should include a renal biopsy (Chaturvedi et al., 2011; Tektonidou, 2014). In a histological view, aPL-associated nephropathy is characterized by acute lesions with thrombotic microangiopathy, and less commonly, chronic lesions with fibrous, intimal hyperplasia, organizing thrombi with or without recanalization, fibrous occlusions of arteries or arterioles, and focal cortical atrophy. Similar clinical and histological characteristics have been found within all the different groups of patients with positive aPL (primary APS, SLE-related APS, CAPS, and SLE/non-APS with positive aPL) (Tektonidou, 2014). In SLE patients undergoing kidney biopsy, aPL-associated nephropathy, isolated or concomitant with SLE nephritis, should be considered. Moreover, it has been demonstrated that the inclusion of renal vascular lesions in the histological classification system of lupus nephritis improves renal outcome predictions. Thus thrombotic microangiopathy was associated with the poorest renal outcome among the other renal vascular lesions (Wu et al., 2013). aPL screening should be performed when aPL-associated nephropathy lesions are found in kidney biopsy, after the exclusion of other reasons for similar histological lesions such as malignant hypertension, thrombotic thrombocytopenic purpura, hemolytic uremic syndrome, scleroderma, or human immunodeficiency (Tektonidou, 2014).

Catastrophic Antiphospholipid Syndrome The term CAPS was first used in 1992 to define an accelerated form of APS resulting in multiorgan failure (Asherson, 1992). CAPS is characterized by widespread intravascular thrombosis within a short period of time resulting in multiorgan failure (Carmi et al., 2017). CAPS usually involves multiple small vessels, but sometimes large vessels may be concomitantly occluded as well (Erkan et al., 2010). The international “CAPS Registry” was created in 2000 by the European Forum on Antiphospholipid Antibodies and included a large cohort of 500 patients with CAPS (Rodrı´guez-Pinto´ et al., 2016). In this cohort, 60% had primary APS and 40% had APS associated with another autoimmune disease, mainly SLE (75%). In 65% of CAPS episodes, the most common precipitating factor was infections (49%), especially in young patients. In this cohort, several organs and systems were involved, including kidneys (73%), lungs (60%), brain (56%), heart (50%), and skin (47%). Among laboratory features, thrombocytopenia was the most common (67%). Schistocytes, the markers of thrombotic microangiopathic hemolytic anemia, were present in 22%. LAC, aCL IgG, and antiB2GPI IgG antibodies were the most frequently observed aPL (83%, 81%, and 78% respectively). Interestingly, in a series of 14 patients with CAPS, high levels of ferritin have been reported in 71% of the cases (Agmon-Levin et al., 2013). In the aforementioned CAPS Registry, the 12-year mortality rate in the general cohort was 37%, while a higher mortality of 48% was found in SLE patients secondary to severe cardiac and brain involvement (Rodrı´guez-Pinto´ et al., 2016).

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THE ANTIPHOSPHOLIPID ANTIBODIES Criteria-Relevant Antiphospholipid Antibodies aPL is a group of over 20 different autoantibodies directed against a variety of antigens including negatively charged phospholipids, phospholipid-binding proteins, and factors related to hemostasis (de Groot et al., 2012). However, the revised laboratory criteria for the classification of APS include only LAC, aCL, and anti-B2GPI antibodies (Miyakis et al., 2006). In recent years, the role of these three aPL in the pathogenesis of APS and their relation to APS manifestations have been challenged. Lupus Anticoagulant LAC defines a heterogeneous group of immunoglobulins that inhibits phospholipid-dependent coagulation reactions in vitro and is detected by prolongation of functional coagulation assay. LAC is associated with severe clinical manifestations and considered the most important acquired risk factor for thrombosis and fetal loss. Thus a high incidence of LAC was found in patients with ST, DVT, and both early and late PM (Andreoli et al., 2013a). These findings have been further demonstrated by a recent metaanalysis showing that LAC is associated with a higher risk for thrombotic events in comparison with aCL and anti-B2GPI antibodies (Reynaud et al., 2014). Finally, LAC positivity has been shown to be the strongest predictor of poor pregnancy outcomes after 12 weeks of pregnancy (Lockshin et al., 2012; Yelnik et al., 2016). A “weak” positive LAC result (a measurement that results in value just above the mean 1 2SD) should be considered positive when making clinical decisions, according to the 14th International Congress on Antiphospholipid Antibodies Task Force (Bertolaccini et al., 2014). Anticardiolipin The thrombotic risk of aCL antibodies, particularly high titer IgG aCL, has been previously demonstrated in patients with primary APS and APS associated with SLE (Long et al., 1991; Danowski et al., 2009). However, the importance of aCL in OAPS has been recently challenged by the several studies, which observed that isolated aCL positivity had no significant influence on adverse pregnancy outcomes in aPL-positive women with and without SLE (Lockshin et al., 2012; Yelnik et al., 2016). aCL antibodies were associated with an increased risk of venous thromboembolism and arterial thrombosis in patients without SLE, but this trend was less statistically significant than a similar association between LAC and thrombotic events (Reynaud et al., 2014). The effects of aCL antibodies’ isotypes and their ability to predict different clinical manifestations of APS remain controversial issues. The IgG aCL isotype has been shown to be an independent risk factor for thrombosis, especially venous, in several previous and recent studies (Danowski et al., 2009; Domingues et al., 2016). Although IgM aCL is included in the Sydney APS Classification Criteria, the clinical importance of isolated IgM aCL is under debate. One study hasn’t shown an increased incidence of venous or arterial thrombosis in patients with IgM aCL positivity (Danowski et al., 2009), and a different study has shown an association between IgM aCL and venous thrombosis (Samarkos et al., 2006). It has been lately shown that the risk for jugular venous thrombosis and cerebral venous sinus thrombosis was correlated with the titer of aCL IgM, and no correlation with aCL IgG was found (Stojanovich et al., 2012b). Anti-b2-Glycoprotein-I Antibody Originally, it was assumed that aPL bind to phospholipids and were thus named “aPL antibodies.” However, in the 1990s it has been shown that aPL recognize phospholipids indirectly via phospholipid-binding plasma proteins (Galli et al., 1990). B2GPI is the most important phospholipid-binding protein, present either in a circulating form or bound to cells (de Groot et al., 2012). The B2GPI molecule has five homologous domains (I V) (Fig. 32.1). B2GPI participates in the innate immune responses [i.e., lipopolysaccharide (LPS) scavenging and clearance of unwanted anionic microparticles] and has antithrombotic properties. It has been proposed previously that B2GPI is the most important antigenic target (Meroni et al., 2011). However, the predominant role of anti-B2GPI in the pathogenesis and clinical manifestations of APS has been recently challenged. According to a critical analysis of published data regarding the pathogenic role of anti-B2GPI, this antibody may not play a central role in the pathogenesis and clinical manifestations of APS (Lackner and Mu¨ller-Calleja, 2016). Thus no association has been

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FIGURE 32.1 The plasma protein B2GPI is a phospholipid-binding protein. It is constructed of five domains (I V) that can present in a circular nonactive form or in an active, open J-shaped conformation, as presented in the figure. Each of the 5 homologous domains of B2GPI includes approximately 60 amino acids, and certain peptides in each domain have been identified as autoantigens (3 of which are detailed in this figure). Anti-B2GPI antibodies can form a complex with B2GPI and activate intracellular signaling only in its open conformation while cryptic epitopes are exposed. With acknowledgment to Prof. Miri Blank (The Zabludovicz Center for Autoimmune Diseases, Israel). B2GPI, B(2)-glycoprotein I.

found between anti-B2GPI antibodies, both IgG and IgM, and adverse pregnancy outcomes in women with aPL and/or SLE (Lockshin et al., 2012; Yelnik et al., 2016). Large systematic literature review and metaanalysis have showed that anti-B2GPI antibodies were associated with an increased risk of arterial thrombosis (OR 3.12, 95% CI 1.51 6.44) but only a nonsignificant trend was identified for venous thromboembolism (Reynaud et al., 2014). In addition, in one recent study, anti-B2GPI antibodies were the most common antibodies in patients with thrombotic APS and had the highest sensitivity and negative predictive value with regard to APS diagnosis during the first visit (Ahluwalia et al., 2016). Higher titers of anti-B2GPI, both IgM and IgG, were observed in patients with SLE and secondary APS compared to negative aPL SLE patients and positive aPL SLE patients without APS clinical features. In this study, anti-B2GPI positivity was accompanied by almost a sixfold increased risk of secondary APS in SLE patients (Dima et al., 2015).

Noncriteria Antiphospholipid Antibodies Only three aPL are regarded as APS-relevant criteria, despite the fact that more than 30 different antibodies have been described in APS patients (the so-called autoantibody explosion in APS) (Shoenfeld et al., 2008b). Antiphospholipid Antibodies of the IgA Isotype These are present in up to 40% of SLE patients and are especially common in Afro-Caribbean individuals. aCL antibodies of the IgA isotype were found to be thrombogenic in mice, and anti-B2GPI IgA was documented in a subgroup of seronegative women with recurrent pregnancy losses. IgA class aPL were also an independent risk factor for cardiovascular mortality and thrombotic events in hemodialysis patients (Serrano et al., 2012) and SLE patients (Sweiss et al., 2010). Interestingly, unlike IgG directed at domains IV (DIV) and V (DV) of B2GPI, IgA antibodies that bind to these domains may contribute to thrombosis atherosclerosis (Iverson et al., 2006) and early pregnancy loss (Staub et al., 2006). Several studies failed to show the usefulness of IgA aCL and IgA anti-B2GPI testing, owning to several reasons, including the low prevalence of these antibodies, the fact that they are found along with other aPL in most cases, and, mainly, the failure to enhance the diagnostic accuracy of routine testing (Meijide et al., 2013). Recent studies suggest that isolated IgA anti-B2GPI may identify additional patients with clinical features of APS and hence recommended testing for these antibodies when other aPL are negative and APS is suspected (Murthy et al., 2013). Based on the published evidence, IgA aPL testing may contribute to the risk assessment of thrombosis or/and PM in selected cases, mainly in SLE patients (Andreoli et al., 2013b). The above-discussed clinical utility of IgA aCL and IgA anti-B2GPI has been a subject of debate in the 14th International Congress on Antiphospholipid Antibodies Task Force (Bertolaccini et al., 2014).

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Low Level Antiphospholipid Antibodies The value and clinical significance of low positive aPL values have been the topic of several publications. According to the 2006 classification criteria update, medium-to-high titers of the IgG and/or IgM isotypes of aCL and/or anti-B2GPI antibodies and/or a positive test for LAC should be present on two consecutive occasions, at least 12 weeks apart. The threshold between low and medium aCL or anti-B2GPI antibody titer is 40 GPL or MPL units, or the 99th percentile of the values obtained from the reference subjects for both aCL and anti-B2GPI antibodies, respectively. It has been previously reported that high levels of aPL are correlated with a worse outcome (Simchen et al., 2011). However, even lower levels should be taken into consideration by the treating physician. Thus a recent study in this field has demonstrated that persistent low titers of aCL and/or anti-B2GPI antibodies were present in the patients with OAPS (Gardiner et al., 2013). In another study, the overall risk for vascular and obstetrical manifestations of APS was found to be similar both in patients with low titer of aCL/anti-B2GPI IgG/IgM antibody and in patients with moderate-to-high titers. This indicates that low titer aCL/B2GPI IgG/ IgM tested positive twice 12 weeks apart may be sufficient for diagnosing APS in routine clinical practice (OferShiber and Molad, 2015). Moreover, technical aspects may play a role while evaluating aPL levels. Given the variability of aPL assays, it is difficult to find a standard numerical cutoff value distinguishing between low- and medium-high titers of antibodies. The definition of medium-positive antibody titers depends on the performance characteristics of the particular assay, the statistical method, and the reference population. The most appropriate approach was summarized in the 14th International Congress on Antiphospholipid Antibodies Task Force, and according to it, the significance of a low positive aPL result depends on the whole risk profile of the patient with respect to clinical manifestations (Bertolaccini et al., 2014). Autoantibodies to Domain 1 of b2-Glycoprotein-I Antibody The role of anti-B2GPI antibodies directed at different domains of B2GPI has become a focus of attention. Antibodies directed against domain I of B2GPI (anti-B2GPI-DI) have been previously found to be associated with an increased risk of thrombosis and pregnancy complications (de Laat et al., 2005, 2009) and represent one of the noncriteria aPL. Recent promising data support this association between anti-B2GPI-DI and APS clinical manifestation (Pengo et al., 2015a). Additional studies have highlighted the strong association between IgG anti-B2GPIDI antibodies and thrombosis. Thus assessment of IgG anti-B2GPI-DI in 35 patients with primary APS and 51 patients with secondary APS indicated that anti-B2GPI-DI antibodies, but not non-DI anti-B2GPI antibodies, were significantly correlated with thrombotic events (OR 3.27; 95% CI 1.59 6.71) (Zhang et al., 2016). In this report, IgG anti-B2GPI-DI antibodies were detected in 48.6% of the patients with primary APS and in 45.1% of the patients with secondary APS, and their levels were significantly increased in APS patients in comparison with controls. IgG anti-B2GPI-DI antibodies were found in 81.4% of APS patients with positive anti-B2GPI antibodies, supporting the idea that DI is the major epitope in B2GPI. Recently, the domain profile of anti-B2GPI antibodies has been evaluated in a large cohort of patients. In this work, no association has been found between anti-B2GPI-DI or anti-B2GPI-DIV/DV antibodies and APS clinical criteria. However, a high ratio ( . 1.5) of anti-B2GPI-DI to anti-B2GPI-DIV/DV may predict systemic autoimmunity and, consequently, may be a useful biomarker for APS (Andreoli et al., 2015). An additional study evaluating aCL and/or aB2GPI-positive patients suggests that the addition of anti-B2GPI-DI positivity increases the likelihood of APS by three to five times. Positivity for IgG, IgA, or IgM anti-B2GPI-DI antibodies increased the strength of association between aCL and/or anti-B2GPI antibodies and thrombotic manifestations in APS (Pericleous et al., 2016). Recent studies have found that patients with multiple aPL have a higher prevalence and higher titers of anti-B2GPI-DI antibodies (Pengo et al., 2015a). Significantly higher levels of IgG aB2GP1-DI were found in patients with triple aPL positivity, compared with patients with double and single aPL positivity (Zhang et al., 2016). Specific autoantibodies directed at other domains of B2GPI were less extensively studied. Nonetheless, the presence of an antibody directed at a peptide derived from domain III of B2GPI was found to be a significant predictor of recurrent spontaneous abortions (Shoenfeld et al., 2003). Antiphosphatidylethanolamine Antibodies Antiphosphatidylethanolamine (aPE) antibodies were found to be associated with fetal loss and venous thrombosis (Pierangeli et al., 2011). In a different cohort, it has been reported that aPE antibodies were found in 19% of

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the patients with unexplained recurrent early or mid-to-late pregnancy losses, suggesting an association between these antibodies and pregnancy losses (Sugi et al., 2004). The pathogenic role of aPE in pregnancy complications was assessed in a mice model, and it was found that passive immunization with aPE antibodies significantly induced thrombosis and hemorrhage in the placenta (Velayuthaprabhu et al., 2011). In contrast, several studies failed to find an association between aPE and PM and thrombosis in SLE patients (Bertolaccini et al., 2012). The role of aPE in patients with seronegative APS remains a field of broad and current interest that requires additional investigation. The diagnostic value of aPE is still not established and aPE assays are not yet standardized. Similarly, there are no definitive therapeutic recommendations for patients with thrombotic or obstetric events and isolated aPE. Antiphosphatidylserine Antibodies Antiphosphatidylserine (aPS) positivity was documented in women with recurrent pregnancy losses who were negative for aCL (Sater et al., 2012). In addition, aPS can inhibit trophoblast development and invasion and impede syncytiotrophoblast formation further supporting their pathogenic role (Blank and Shoenfeld, 2004). The diagnostic value of aPS antibodies in APS has been recently assessed in a prospective observational study consisting of 212 patients with thrombosis and recurrent pregnancy failure. In this study, aPS antibodies were detected in 47.6% of the patients with thrombosis and in 52% of the patients with pregnancy failure. This prevalence of aPS antibodies in these two groups was similar to the prevalence of LAC (57% and 53%), anti-B2GPI antibodies (45.7% and 56%), and aCL (52% and 56%). In this study, 75% of the patients with confirmed primary APS had aPS antibodies. Thus aPS may be used as a diagnostic tool in clinical cases when other aPL are absent (Khogeer et al., 2015). Antiprothrombin Antibodies Antiprothrombin antibodies (aPT) similarly to anti-B2GPI are phospholipid-binding proteins. The coexistence of IgG aPT and LACs was found to be an essential risk factor for venous thromboembolism in patients with SLE (Nojima et al., 2001) and to play a role in the pathogenesis of thrombosis in APS (de Groot et al., 1998; Von Landenberg et al., 2003; Haj-Yahia et al., 2003). Besides, aPT was linked to thrombosis and disease progression in other conditions such as acute ischemic STs in young women (Cojocaru et al., 2008), fetal death in women with previous uneventful pregnancies and seronegative aPL (Marozio et al., 2011), and more advanced primary biliary cirrhosis (Agmon-Levin et al., 2010). A recent systematic review of 38 studies on aPT has shown that the presence of aPT is linked to a higher risk of thrombosis (OR 1.82, 95% CI 1.44 2.75) (Sciascia et al., 2014). aPT may be detected by directly coating prothrombin on irradiated ELISA plates (aPT) (Bertolaccini et al., 2013). Antiphosphatidylserine/Prothrombin Antibodies Antibodies directed at the complex phosphatidylserine/prothrombin (aPS/PT) were reported to be markers of APS with a high degree of concordance with LAC activity (Hoxha et al., 2012; Vlagea et al., 2013; Fabris et al., 2014). In a recent study that included 62 patients with inconclusive LAC analysis, the prevalence of aPS/PT antibodies was found to be 48% and allowed to discover a significant number of previously unrecognized aPLpositive cases with negative aCL and aB2GPI. Therefore, testing for aPS/PT antibodies may enhance the diagnostic performance for APS (Fabris et al., 2014). In a cohort of 295 individuals that included primary and secondary APS and APS-related diseases, aPS/PT correlated with particular expressions of APS, namely venous thrombosis (OR 7.44; 95% CI 3.97 13.92) and obstetric abnormalities (OR 2.37; 95% CI 1.04 5.43), but not with arterial thrombosis (Vlagea et al., 2013). According to the systematic review of the literature, aPS/PT antibodies were found to be a strong risk factor for arterial (more significant) or venous thrombosis (less significant) (Sciascia et al., 2014). In line with this, a recent study has demonstrated that aPS/PT antibodies were found in 63% of 108 APS patients and were significantly associated with thrombosis (OR 3.4), especially arterial thrombosis (OR 4.8), independently of aCL and antiB2GPI (Zhu et al., 2017). Controversial data have been published regarding the connection between aPS/PT and PM. In one earlier study that included 208 women with history of pregnancy complications relevant to APS, the prevalence of ˇ aPS/PT was 13.0%. Moreover, 6.5% of these patients had isolated aPS/PT positivity (Zigon et al., 2015). In contrast, according to another study, no association has been found between aPS/PT positivity and history of PM (Zhu et al., 2017). The presence of aPS/PT antibodies may be a risk factor for more severe APS. Thus

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aPS/PT antibodies, especially in high titers, were correlated with severe thrombosis, severe pregnancy complications inducing prematurity, and vascular microangiopathy (Hoxha et al., 2017). In addition, the inclusion of aPS/PT as a second-level assay to confirm APS classification was recommended in one study (Vlagea et al., 2013). A recent report presented at the 14th International Congress on aPL antibodies concluded that testing for aPS/ PT can contribute to a better identification of patients with APS and to the assessment of the risk of thrombosis (Bertolaccini et al., 2014). Antiannexin A5 Antibodies Annexin A5 is one of the most important proteins in the annexin A group. It has an anticoagulant activity that prevents the binding and activation of clotting factors by the phospholipid shell that covers cells. aPL disturb this shield and expose phospholipids, thereby accelerating blood coagulation reactions. A significantly high correlation has been reported between annexin A5 resistance measured by flow cytometric assay (FCA) and the diagnosis of APS (Rodrı´guez-Garcı´a et al., 2015). In a different recent report, positive FCA results were correlated with the presence of aCL and anti-B2GPI and were associated with APS features among patients with SLE (Avriel et al., 2016). A possible mechanism of annexin A5 resistance is linked to neutralization of annexin A5 by antiannexin A5 antibodies, which exposes the phospholipids on cell membranes for the accessibility of phospholipid-dependent coagulation enzymes. In one recent study conducted on primary and secondary APS patients and disease and healthy controls, the levels of both IgG and IgM antiannexin A5 antibodies were significantly increased in patients with APS, compared with controls. Significant correlations were found between IgG antiannexin A5 antibodies and arterial thrombotic events (OR 2.60; 95% CI 1.44 4.71) and between IgG antiannexin A5 antibodies and venous thrombotic events (OR 2.80; 95% CI 1.55 5.06). In contrast, obstetric complications and IgG or IgM antiannexin A5 antibodies were not correlated. It has been suggested that antiannexin A5 antibodies could serve as a diagnostic biomarker for patients with APS (Zhang et al., 2017). Metaanalysis: Prevalence of Different Noncriteria Antiphospholipid Antibodies Several additional investigations have addressed themselves to identify the prevalence of different noncriteria aPL in patients with APS. A recent metaanalysis of 16 such retrospective studies was conducted, incorporating 1404 APS patients, 1839 disease control patients, and 797 healthy patients. This analysis was based on studies published before June 2014 and reviewed the following noncriteria antibodies: IgA aCL, IgA anti-B2GPI, aPS, aPE, antiphosphatidylinositol, anti-B2GPI-DI, aPT, antiphosphatidylcholine, antivimentin/cardiolipin complex, and antiphoshatidic acid. Studies that evaluated the resistance to annexin A5 in APS patients were also included. It has been shown that the prevalence of all noncriteria aPL was significantly increased in APS patients compared with controls. The most common laboratory characteristics were IgA anti-B2GPI antibodies (56.3%), resistance to annexin A5 (53.4%), and IgG anti-B2GPI-DI antibodies (44.0%) (Rodrı´guez-Garcı´a et al., 2015).

SERONEGATIVE ANTIPHOSPHOLIPID SYNDROME The seronegative APS definition has been suggested for patients with clinical manifestations indicative of APS but with persistently negative results utilizing assays to detect criteria aPL (Rodriguez-Garcia et al., 2012). Apparently these patients exhibit similar frequencies of thrombotic events and obstetric morbidity. Transient or false-negative aPL tests may explain some of these cases, as even today anti-B2GPI is routinely tested in only a small number of laboratories, and standardization of other criteria aPL is yet to be accomplished. In addition, noncriterion aPL are currently tested in only a few research laboratories (Cervera et al., 2012). Therefore, new antigenic targets and different methodological approaches have been investigated in order to discover aPL in seronegative APS. One study analyzed the sera from 24 seronegative APS patients utilizing several methods: thin layer chromatography (TLC immunostaining) for all aPL, ELISA for antivimentin/cardiolipin, antiannexin A5 and aPT antibodies, and dot blot that was also used for antiannexin A5 and aPT antibodies (Conti et al., 2014). This study has found that 54.2% of the cohort were positive for aCL antibodies according to the TLC immunostaining assay. In addition, 45.8% of them had IgG antibodies against vimentin/cardiolipin, 12.5% against prothrombin, and 4.2% against annexin A5. At least one aPL/cofactor antibody was detected using these assays in 79.2% of the patients. By combining TLC immunostaining for aCL and ELISA for antivimentin/cardiolipin

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antibodies, aPL/cofactors were detected in two-thirds of the seronegative APS patients. Furthermore, recent evidence suggests that anti-B2GPI-DI, IgA aCL, or IgA anti-B2GPI may be used to identify seronegative APS patients (Meroni et al., 2014).

RISK ASSESSMENT IN ANTIPHOSPHOLIPID SYNDROME APS manifestations may vary greatly between different patients. Some patients with aPL remain healthy, while others develop PM and/or thrombosis. The value of different aPL profiles in the stratification of risks for thrombosis or obstetric events is under current investigation. Among aPL, LAC, triple positivity, and isolated persistently positive aCL at medium-high titers have been shown to be associated with a high risk for thrombosis (reviewed in Khamashta et al., 2016). In line with this, LAC and triple positivity were also correlated with worse pregnancy outcome (Alijotas-Reig et al., 2015). While the presence of aPL is a risk factor for APS, additional risk factors have been incorporated in the global APS score (GAPSS) that was developed in 2013 (Sciascia et al., 2013). The GAPPS combines independent risk factors for thrombosis and pregnancy loss, including aPL profiles (criteria aPL and noncriteria aPL), and conventional cardiovascular risk factors and autoimmune antibody profiles, including anti-nuclear antibodies (ANA), extractable nuclear antigens antibodies (ENA), and anti-double stranded DNA antibodies (dsDNA), among others. It has been demonstrated that arterial hypertension, hyperlipidemia, LAC, and IgG/IgM isotypes of aCL, antiB2GPI, and aPS/PT were independent risk factors for thrombosis and/or PM. Each risk factor was assigned a number of points within the GAPSS, as follows: IgG/IgM aCL (5 points), IgG/ IgM anti-B2GPI (4 points), LAC (4 points), IgG/IgM aPS/PT (3 points), hyperlipedemia (3 points), and arterial hypertension (1 point). The GAPSS model was initially developed in patients with SLE, and higher GAPSS scores were associated with thrombosis and/or pregnancy loss (Sciascia et al., 2013). Later, the GAPSS scoring system was evaluated in a cohort 62 patients with primary APS (Sciascia et al., 2015). Higher values of GAPSS were found in APS patients who had thrombosis alone, in comparison with patients who had previous pregnancy loss alone. In addition, patients with GAPSS values higher or equal to 11 had a higher risk of recurrent thrombotic events. Furthermore, a different study has shown that GAPSS correlated with a history of APS manifestations, particularly with thrombosis, suggesting that it may be used as a quantitative marker for APS (Oku et al., 2015). Recently, GAPSS above 16 was reported as a predictor of thrombosis in a study that included APS and SLE patients (Zuily et al., 2015).

GENETICS The role of various genetic factors in the pathogenesis of APS was evaluated in several studies, including animal model studies, family studies, and population studies. The current data suggest the existence of a genetic predisposition to APS, both as a primary condition or in association with SLE. This genetic predisposition is partly associated with the HLA system, and several family studies have shown that specific haplotypes, especially those containing DR4 and DRw53, may be correlated with aPL production or APS itself (reviewed in Sebastiani et al., 2016). According to different population studies, the HLA loci relevant to the pathogenesis of primary APS appeared to be DRB1 04, DR7, DRw53, DQB1 0301/4, DQB1 0604/5/6/7/9, DQA1 0102, and DQA1 0301/2. With regard to aPL in SLE patients, the majority of the reports indicate that these antibodies are associated with DR4, DR7, the closely linked antigen DRw53, and DQB1 0302 (Sebastiani et al., 2016). Several other genes unrelated to the HLA system may also increase the susceptibility to APS, including STAT 4 (Yin et al., 2009), 12q24.12 (Ochoa et al., 2013), and PTPN22 (Bottini et al., 2006). In addition, in a recent metaanalysis including 1507 APS patients, a strong association was found between valine/leucine (247) polymorphism of B2GPI and APS, thrombosis, and anti-B2GPI positivity (Lee et al., 2012). In addition to genetic factors, epigenetic processes such as DNA methylation, histone modification, noncoding RNA, and nucleosome remodeling may be the topics of future research and provide additional insights regarding APS (Zhang and Zhang, 2015).

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CLASSIFICATION CRITERIA VERSUS DIAGNOSTIC CRITERIA To date, there are no diagnostic criteria available for APS and therefore physicians should consider this diagnosis in the presence of minor features, even when major manifestations are absent. As mentioned earlier, there are minor clinical features and several noncriteria antibodies that may be potentially included in the disease’s classification criteria. Recently, it has been suggested to classify APS as definite, probable/possible, or uncertain. According to this suggestion, if the patient satisfies the 2006 Sydney APS Classification Criteria and has triple positivity, the diagnosis is considered definite. These patients may not require repeat testing after 12 weeks and have a high risk for recurrence of thrombosis and pregnancy loss. The diagnosis of APS is considered as probable in patients with double positivity (mostly LAC negative but with aCL IgG or IgM .99th percentile and anti-B2GPI IgG or IgM of the same isotype .99th percentile) and proven venous/arterial thrombosis and/or pregnancy loss. The diagnosis is “uncertain” if only one of the tests turns positive. Low titer single test positivity may be significant in obstetric cases only (Pengo et al., 2015b).

DIAGNOSTIC PROCEDURES Great efforts have been made to standardize the tests evaluating the presence of aPL. Several committees published guidelines regarding different coagulation and immunological tests. In 2009, the subcommittee on LAC/ aPL antibodies of the International Society of Thrombosis and Haemostasis (ISTH) initiated the new guidelines for the detection of LAC (Pengo et al., 2009). Generally, two tests are used for the detection of LAC: dilute Russell venom time (dRVVT) and a sensitive activated thromboplastin time (aPTT). The test for the detection of LAC consists of three steps: screening for prolongation in a phospholipid-sensitive test, mixing with normal plasma to discriminate between the presence of an inhibitor and coagulation factor deficiencies, and confirmation through result shortening in a phospholipid-rich test screening, mixing, and confirmatory studies. It has been found that the mixing test is essential to avoid false-positives in patients with prolonged dRVVT (Devreese and de Laat, 2015). In addition, the Scientific and Standardization Committee of the ISTH initiated new recommendations regarding aCL and anti-B2GPI ELISAs. In particular, the aCL ELISA cutoff should be calculated by the 99th percentile based on a population of healthy volunteers, a value that is quite different from the usually adopted cutoff of 40 GPL (Devreese et al., 2014). Novel assay techniques have been proposed for aPL testing, such as chemiluminescence-based methods, fluorescence enzyme immunoassays, or line immunoassays (LIAs). A comparison of the LIA and ELISA techniques in 56 APS patients, 24 aPL carriers, and 73 aPL-positive patients with infectious diseases revealed a good intermethod agreement for the detection of IgG/IgM anti-B2GPI and aCL in APS patients. Unlike ELISA, according to LIA, the prevalence of aCL and anti-B2GPI IgG in aPL carriers and in aPL-positive patients with infectious diseases was significantly reduced in comparison with APS patients. This indicates that LIA discriminates patients with APS from aPL-positive asymptomatic carriers and infectious patients (Roggenbuck et al., 2016). Confirmation of aPL positivity in order to avoid the detection of transient antibodies is a mandatory part of the classification criteria for APS. However, it may delay the diagnosis of APS and influence the treatment decisions. It has been demonstrated that initial aPL profiles may predict aPL persistence after 12 weeks. For example, repeated testing of 161 aPL-positive individuals who initially had one or more aPL showed that in 98% of subjects with triple positivity at initial testing, aPL profile was confirmed after 12 weeks. The aPL profile confirmation rates in double-positive and single-positive groups were 84% and 40%, respectively. These results have raised the possibility that triple positivity at initial screening may help in the early identification of APS patients (Pengo et al., 2013).

THE MECHANISMS OF ANTIPHOSPHOLIPID ANTIBODIES-MEDIATED DISEASE EXPRESSIONS: CLINICAL TRIALS AND ANIMAL MODELS The pathogenic effects of these antibodies are exerted through binding to receptor on target cells, including monocytes, endothelial cells, and trophoblasts, leading to the recruitment of cell surface receptors and subsequent perturbation of intracellular signaling pathways (Blank et al., 1991; Giannakopoulos and Krilis, 2013).

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In the last three decades, much effort has been devoted to clarify the pathogenic potential of aPL (Bakimer et al., 1992). Both vascular thrombosis and pregnancy loss are the classical clinical expressions of the APS, therefore aPL pathogenicity was studied focusing on these clinical aspects. Notably, aPL-related vascular thrombosis can occur both on the arterial and the venous side, making this acquired procoagulant condition very peculiar in human disease. The first evidence of the aPL-related pathogenic potential was obtained through animals that carried aPL as part of their disease (i.e., lupus-like-prone mice), or alternatively naive mice that were infused with antibodies or stimulated to produce their own aPL (Ziporen et al., 1997; Katzav et al., 2010). In a long series of studies, different polyclonal and monoclonal aPL with various specificities were utilized inducing pregnancy losses and enhancing the coagulation process. Probably in no other autoimmune disorders has there been so much evidence produced to show the pathogenicity in vivo of these autoantibodies. This body of evidence was pivotal in proving autoantibody-mediated damage and at the same time prompted further in vitro studies aiming to define the cellular pathogenic effects of aPL. Originally both pregnancy loss and vascular occlusions were ascribed to the thrombogenic effect of aPL. Nowadays, it is clear that placental thrombosis can be responsible for pregnancy failures, but at the same time it is accepted that aPL can exert a direct nonthrombotic effect on trophoblast cells (Di Simone et al., 2000) and other cells (Del Papa et al., 1995). Over the years, the concept of aPL itself has also been redefined. In fact, starting from the hypothesis that these autoantibodies were directed to phospholipids, we now know that in most cases they are directed to phospholipid-binding proteins (Giannakopoulos et al., 2011). Even if these antibodies are detected with different assays, the main target of the so-called aPL was identified as B2GPI, a phospholipidbinding protein. Almost all pathogenic effects of aPL have been shown using highly purified anti-B2GPI antibodies (Meroni et al., 2011). However, at that time, antibodies that bind to phospholipids in a cofactorindependent manner were generally considered irrelevant for the pathogenesis of APS. In recent years, there is direct evidence that cofactor-independent aPL presented in the blood of APS patients may contribute to the pathogenesis of the APS. Thus two human monoclonal cofactor-independent aPL that had been obtained from patients with APS stimulated proinflammatory and procoagulant responses in monocytes and endothelial cells (Mu¨ller-Calleja et al., 2015). Endosomal NADPH-oxidase 2 (NOX2) activation was mediated by cofactor-independent aPL with subsequent induction of cytokines, inflammasome components, and tissue factor (TF). All effects induced by the monoclonal aPL were reproduced with IgG fractions of APS patients suggesting that effects of the monoclonal aPL are relevant for the APS. In a different study, two monoclonal human aPL-induced thrombus formation in vivo mouse model (Manukyan et al., 2016). Rapid induction of thrombus formation within 3 hours after infusion of the aPL was observed, and this thrombogenic effect of aPL was not reintroduced in NOX2-deficient mice. It has been found that thrombus formation depends on TF induction in monocytes. These accumulating data suggest that cofactor-independent aPL play an important role in pathogenesis of APS (Lackner and Mu¨ller-Calleja, 2016). Several noncriteria autoantibodies such as aPS/PT, aPT, and anti-B2GPI-DI have been proposed to be relevant to the pathogenesis of APS. The prothrombotic property of aPS PT antibodies has been demonstrated in vitro (Oku et al., 2013) and in vivo (Yamada et al., 2017). Both purified IgG fractions obtained from the sera of aPS/PT positive patients with APS and murine monoclonal aPS/PT antibodies induced TF expression and shortening of coagulation time in cells in the presence of prothrombin in procoagulant cells via p38 mitogen-activated protein kinase (MAPK) phosphorylation pathway. The pathogenicity of aPS PT antibodies in vivo has also been demonstrated in rat model of thrombosis induced by intravenous injection of aPS PT antibodies (Yamada et al., 2017). A direct demonstration of the pathogenic effect of anti-B2GPI-DI antibodies has been recently observed in animal model, in which infusion of human monoclonal anti-B2GPI-DI antibodies resulted in fetal losses in pregnant mice and blood clots in rat mesenteric microcirculation following priming with LPS. A variant of this antibody, lacking the CH2 domain, is effective in preventing blood clot formation and fetal loss induced by aPL (Agostinis et al., 2014). In a different work, intraperitoneal injection of two polyclonal IgG fractions (anti-B2GPI-DI rich and anti-B2GPI-DI poor IgG) isolated from APS patients in vivo mouse model demonstrated that anti-B2GPI-DI-rich IgG induced significantly larger thrombi compared with anti-B2GPIDI poor IgG. Similarly, anti-B2GPI-DI-rich IgG significantly increased the procoagulant activity of the carotid artery endothelium and peritoneal macrophages isolated from the experimental animals (Pericleous et al., 2015).

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Thrombotic Manifestations Many factors are involved in the pathogenesis of thrombotic manifestations. Several mechanisms have been proposed regarding the development of thrombosis, including the activation of endothelial cells, monocytes, platelets, coagulation, and complement pathways, along with the inhibition of fibrinolytic and anticoagulation pathways (Merashli et al., 2015). Consequently, it has been recently indicated that vasculopathy, related mainly to severe intimal hyperplasia, may also play a role in arterial vascular occlusion secondary to stenotic lesions and in PM. In this regard, the vascular endothelium of proliferating intrarenal vessels from patients with APS nephropathy demonstrated signs of mTOR activation, which is the mammalian target of rapamycin (Canaud et al., 2014). It has been shown that in cultured vascular endothelial cells, IgG antibodies from patients with APS stimulated mTOR through the phosphatidylinositol 3-kinase AKT pathway. Treatment with sirolimus (rapamycin) led to the inhibition of mTOR in APS patients who underwent renal transplantation, which in turn inhibited the recurrence of vascular lesions and decreased vascular proliferation on biopsy, compared with APS patients who were not treated with sirolimus. Therefore, it was concluded that the activation of mTOR stimulates intimal hyperplasia, leading to the formation of the chronic vascular lesions seen in APS. It has also been found that mTOR is able to induce a prothrombogenic phenotype leading to thrombosis (Canaud and Terzi, 2014). Antiphospholipid Antibodies and the Coagulation Cascade Several mechanisms have been described to explain the thrombophilic properties of aPL, generally related to their interaction with the coagulation and fibrinolysis systems or with cells involved in thrombus formation. These include B2GPI anti-B2GPI complexes that were found to be localized to atherosclerotic plaques, mainly to oxidized low-density lipoprotein, to induce autoimmune thombogenesis (Matsuura et al., 2003), and to amplify thrombus size (Arad et al., 2011). Activated protein C (APC) is a natural anticoagulant that interacts with factors Va and VIIIa impairing their procoagulant activity. In the presence of B2GPI anti-B2GPI complexes, APC cannot exert its action possibly because it is unable to bind Va/VIIIa or, alternatively, the formed APC/Va/VIIIa complex is sterically impaired in its binding to a phospholipid surface (Vlachoyiannopoulos and Routsias, 2010). aPL antibodies can also impair the anticoagulant function of antithrombin. This can occur because some of the targets such as thrombin or activated factor IX, when bound by aPL, are no longer available for antithrombin action (Chen et al., 2010). On the other hand, aPL can interfere with the fibrinolytic cascade. Fibrin degradation, which allows thrombus remodeling and dissolution, is mediated by the active enzyme plasmin which is derived from the conversion of plasminogen. This conversion is mediated by tissue-type plasminogen activator (tPA) and urokinase-type plasminogen activator, and both of them are controlled by specific inhibitors -plasminogen activator inhibitors PAI-1 and PAI-2. In the presence of aPL, PAI-1 activity was reported as enhanced resulting in reduction of tPA and plasminogen activation. Apparently B2GPI protects tPA from the action of PAI-1, thereby promoting fibrinolysis; if B2GPI is bound by its specific antibodies, it cannot exert its protective action with the consequent prevalent action of the inhibitory effect of PAI-1 (Vlachoyiannopoulos and Routsias, 2010). Another impairment of fibrinolytic activity seems to be due to a possible direct binding of aPL to plasmin, followed by its inactivation (Yang et al., 2004). The data reported so far represent only some of the plausible mechanisms by which aPL interfere with the coagulation process, and recently another possible explanation has been formulated (Lambrianides et al., 2011). Many of the proteins involved in coagulation/fibrinolysis processes belong to the family of serine protease (SP) enzymes, such as APC, thrombin, plasmin, tPA, and others. These proteins share a high homology region at their catalytic domain. Most aPL recognize a conformational epitope that is shared by B2GPI molecule and the catalytic domain of SP. Therefore the presence of aPL can result in binding of SP molecules and impairment of their enzymatic activity, resulting in enhanced risk of thrombosis. Antiphospholipid Antibodies Cellular Interactions One of the most studied pathogenic effects of aPL deals with the interaction of antibodies with the cells involved in the coagulation process. This basically occurs through the recognition of B2GPI that has adhered to cell membranes. It is known that anti-B2GPI antibodies can upregulate adhesion molecules such as endothelial leukocyte adhesion molecule-1, vascular cell adhesion molecule-1, intracellular adhesion molecule-1, and TF, conditioning their expression on the cell surface. This process was described in vitro on monolayers of human umbilical vein endothelial cells (HUVECs) (Meroni et al., 1996) and its consequences were shown in vivo on CD1 mice

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infused with aPL: in fact in this model, leukocytes were seen to adhere to vascular endothelium favoring clotting (Pierangeli et al., 1995). In the presence of aPL, HUVECs also significantly increase the production of some proinflammatory cytokines such as IL-6 and IL-1B. Therefore the consequence of the aPL effect on endothelium is the shift toward a proadhesive/procoagulant as well as proinflammatory phenotype. These profound modifications are basically due to the presence of B2GPI on the endothelial surface. A number of possible receptors for B2GPI have been described on the endothelial cell surface. Heparin sulfate, annexin 2 receptor, Toll-like receptors (TLR) 2 and 4, and apolipoprotein E receptor 2 were all shown to be involved in the thrombogenic mechanisms related to aPL using in vitro and in vivo models (Poulton et al., 2012). It was also observed that animals lacking one of these receptors are only partially resistant to the pathogenic potential of aPL, suggesting that they play a similar, probably redundant, role (Meroni et al., 2011). The binding of antiB2GPI to its antigen on the endothelial surface should produce intracellular signaling, able to upregulate the cellular expression of adhesion molecules and TF. The signal can start from the receptors that are sensitive to the clustering of B2GPI that follows their specific antibody binding. Among receptors, those having a cytoplasmic tail, TLR 2 and 4 are most likely to be the favorite candidate. Several intracellular pathways have been described as activated, including nuclear factor κB, p38 MAPK, myeloid differentiation primary response protein (MyD88), and tumor necrosis factor receptor-associated factor 6. The above-reported mechanisms are well defined in endothelial cells, but they are also at least partially described in monocytes, platelets, and in other cells serving as a possible target of aPL-mediated damage (Meroni et al., 2011; Poulton et al., 2012). The thrombophilic effect of aPL was also investigated at platelet level. In subjects with aPL, platelet activation was proven by the increase of thromboxane B2 and the decrease in vascular prostacyclin. Receptors of B2GPI at the platelet surface seem to be the apolipoprotein E receptor 2 and the platelet glycoprotein Ib alpha chain (Cognasse et al., 2005; Urbanus et al., 2008). In vitro, the pathogenic effect of affinity purified anti-B2GPI antibodies was shown as an increased aggregation and intracellular signaling activation of platelets in the presence of low doses of thrombin stimulation. In vivo, the B2GP1 anti-B2GP1 complex binds to platelets and activates thrombus-associated platelets. This enhanced platelet activation leads to increased activation of the endothelium and fibrin generation (Proulle et al., 2014). In this respect, a recent study (Vlachoyiannopoulos and Routsias, 2010) has underlined the importance of platelet factor 4, a protein derived from platelet alpha granules and belonging to the chemokine family. Platelet factor 4 is secreted by platelets but can also bind to the platelet surface as well as anionic molecules and B2GPI, both in solid phase and in solution. Platelet factor 4 is able to gather two B2GPI molecules, so favoring an efficient antibody binding. Complexes containing platelet factor 4, dimerized B2GPI, and anti-B2GPI antibodies can induce an activated procoagulant phenotype in platelets. Notably, platelet factor 4 is expressed in different cells such as endothelial cells, monocytes, T cells, and dendritic cells suggesting that its capacity to dimerize B2GPI could favor immune system sensitization and the production of pathogenic anti-B2GPI antibodies. Neutrophil extracellular traps (NETs) represent an important activator of the coagulation cascade and an important component of arterial and venous thrombi. In one study, freshly isolated APS neutrophils demonstrated enhanced NET release in a spontaneous manner. Circulating aPL both purified IgG fractions and antiB2GPI monoclonals can promote NET release from neutrophils. A positive correlation was found between circulating levels of NETs and IgG anti-B2GPI positivity, LAC positivity, and triple positivity in APS patients (Yalavarthi et al., 2015). A decreased degradation of NETs was found in a subgroup of patients with both primary and secondary APS and was associated with antibodies against NETs in patients with secondary APS (Leffler et al., 2014). Cell-released vesicles and exosomes are important systems of intercellular communication. aPL may promote pathogenic effects on vascular cells (endothelial cells, platelets, monocytes) through the release of extracellular vesicles, which include exosomes and microparticles. In this regard, an increased number of monocyte and endothelial microparticles was found in APS patients, in comparison with healthy controls (Vikerfors et al., 2012). Notably, endothelial cells exposed to polyclonal IgG from APS patients produced significantly more endothelial microparticles than those exposed to polyclonal IgG from healthy subjects (Pericleous et al., 2013).

Obstetric Manifestations The pathogenesis of pregnancy failure in APS patients seems to be multifactorial, and it is well known that aPL are associated with reproductive failure (reviewed in Blank and Shoenfeld, 2010).

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The mechanisms responsible for aPL-mediated obstetric manifestations include intraplacental thrombosis, inflammation, interference with annexin A5 function, inhibition of syncytium-trophoblast differentiation, defective placentation/placental apoptosis, and complement activation (reviewed in Arachchillage et al., 2017). Although many different obstetric expressions of APS have been described, the most typical are midpregnancy losses, mainly related to defective placentation and intrauterine growth restriction. Therefore, the first pathogenic mechanism to be investigated was the presence of intraplacental thrombosis (Inbar et al., 1993; Levy et al., 1998). Indeed the above-described thrombophilic properties of aPL favor an increased thrombosis occurrence, particularly during pregnancy, that can work as a “second hit” since pregnancy per se is characterized by an increased thrombosis risk, even in the general population. A potent anticoagulant acting mainly, but not exclusively, on the trophoblast surface is annexin A5 that produces the so-called protective shield, by binding negatively charged phospholipids such as phosphatidylserine. The presence of annexin A5 at the intervillous surface was found to be significantly reduced in patients with APS, thus confirming that B2GPI anti-B2GPI complexes are able to displace in vivo annexin A5 from the cell surface as shown in vitro (Hunt et al., 2011). Furthermore, it is recognized that aPL can also directly interact with trophoblast during syncytium formation (Di Simone et al., 2000) when it expresses phosphatidylserine at its outer surface. As shown by in vitro experiments, B2GPI can bind to negatively charged phospholipids such as phosphatidylserine on the cell surface and become a target of circulating anti-B2GPI antibodies. Direct placental damage induced by aPL may be caused by several mechanisms including inhibition of trophoblast differentiation and syncytialization, induction of trophoblast apoptosis, impairment of trophoblast invasiveness and trophoblast expression of adhesion molecules, and also inhibition of the angiogenic factors production by trophoblasts (Tong et al., 2015). An additional mechanism for preeclampsia is related to the internalization of aPL by trophoblasts with the subsequent acceleration of cell death and release of debris that can activate maternal endothelial cells (Viall et al., 2013). Inflammation has been described as one of the main mechanisms of aPL-induced PM, and it has recently gained additional support from an in vitro study, which showed that aPL can induce trophoblasts to produce interleukin-1β by inflammasome activation (Mu¨ller-Calleja et al., 2015). A novel mechanism of trophoblast inflammation may be related to endothelial microparticles production upon exposure to aPL. MicroRNA released via exosomes may induce the trophoblast to secrete the proinflammatory cytokines IL-8 through activation of TLR 8 (Gysler et al., 2016). Current research focuses on the identification of aPL targets on cell membranes and on the intracellular signaling pathways. In particular, a recent study suggests that apolipoprotein E receptor 2 may be the key molecule mediating trophoblast dysfunction in a mouse model (Ulrich et al., 2016). In a different study, it was shown that TLR 4 mediated the inhibition of trophoblast invasion in vitro by purified aPL IgG from patients with OAPS, but this effect was not observed when aPL IgG from non-OAPS was used (Poulton et al., 2015).

The Complement System in Antiphospholipid Syndrome Complement is apparently involved in APS as antibodies cannot exert their pathogenic effect in animals that lack complement factors or receptors. This effect has been observed in both pregnancy loss (Holers et al., 2002) and in a thrombosis model (Fischetti et al., 2005). If the binding of aPL to their target cells (endothelial cells, trophoblasts) can activate complement, several activation molecules can be released. A recently described damage mechanism focused on C5a as a possible activator of neutrophils via its receptor on the cell surface (Girardi, 2010); according to this model, the consequence of C5a binding to neutrophils is TF production that can exert procoagulant and proinflammatory actions, and these can cause both thrombosis and fetal loss. Direct evidence that activation of the complement system is an important mechanism for aPL-mediated thrombosis was demonstrated in an APS mouse model of induced thrombosis (Romay-Penabad et al., 2014). In this study, the thrombosis was induced in a femoral vein pinch manner, and it was found that mice treated with IgG from APS patients with high levels of aPL developed larger thrombi and higher soluble TF activity than controls. Furthermore, the coadministration of rEV576 (coversin), a recombinant protein inhibitor of C5 activation, resulted in significantly smaller thrombi and reduced TF activity (Romay-Penabad et al., 2014). These data suggest that complement inhibition may significantly reduce aPL-mediated venous thrombosis and TF production. Hypocomplementemia was frequently observed in a cohort of 36 patients with primary APS, reflecting complement activation and consumption, and was also correlated with LAC activity but not with particular

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clinical manifestations (Oku et al., 2009). In contrast, one large study that included 2399 aPL-positive SLE patients found that in aCL-positive patients, the presence of hypocomplementemia (both low C3 and C4) was strongly associated with DVT (Durcan et al., 2016). The assumption that the complement system is involved in pregnancy morbidities in APS patients is supported by histopathological evaluation of the placentae in women with aPL (Viall and Chamley, 2015). While lower complement levels were observed in patients with OAPS, no correlation has been found between hypocomplementemia and obstetric complications in these patients (Reggia et al., 2012). The reason of complement activation in APS has not been fully clarified yet. One recent study that included primary APS patients detected the presence of antibodies directed against C1q which is the initiator of the classical complement pathway. These antibodies bind to C1q on anionic phospholipids and accelerate complement activation. Thus autoantibodies against C1q were detected in 36% of APS patients and their titers were significantly higher compared to normal healthy controls. Neither the prevalence nor the titers of anti-C1q antibodies were correlated with a specific manifestation of APS (arterial thrombosis, venous thrombosis, PM) and no correlation was found between titers of anti-C1q and titers of aPL. In addition, patients with refractory APS tended to have higher serum titers of anti-C1q antibodies than patients with nonrefractory APS (Oku et al., 2016). It has been suggested that the presence of aPL alone is not enough to cause thrombus formation. Thus the activation of the complement pathway due to the existence of anti-C1q antibodies may play an additional role in the pathogenesis of APS.

MORTALITY IN THE ANTIPHOSPHOLIPID SYNDROME The mortality rate in patients with APS is still relatively high despite the current treatment. One of the most prominent studies that investigated mortality in APS patients was performed within the Euro-Phospholipid project (Cervera et al., 2015). In this study, the mortality rate observed during a 10-year period (1999 2009) was 9.3% (5.3% in the first 5-year period and 4% in the second 5-year period), and the mean age of death was 59 years. No differences were detected in the mortality rate or the causes of death between patients with primary APS and patients with APS associated with SLE. Similarly, there were no differences in the causes of death between patients receiving different treatments (immunosuppressive or anticoagulant agents). The causes of death were severe thrombotic events including MI, STs, and PE (36.5%), followed by infections (26.9%) and hemorrhages (10.7%). In addition, among the nine patients who developed CAPS, five patients (55.6%) died. Despite the similar mortality rates in both the first and second 5-year periods of the study, there were some differences in the causes of death: fatal thrombotic events were more frequent in the first period, while malignancies were more frequent during the second period. According to this study, no clinical or immunological parameter with prognostic significance for mortality was identified.

TREATMENT OF ANTIPHOSPHOLIPID SYNDROME In contrast to the large body of evidence on the pathogenesis, mechanisms, and diagnosis of APS, there is still a gap in our knowledge on appropriate therapy for patients affected by this disease. The management of patients with APS is currently directed to antithrombotic medications. Balancing an individual’s risk of thrombosis against the benefits and risks of antithrombotic therapies is crucial for optimizing management and preventing morbidity in patients with APS or aPL-positive-asymptomatic ones. Primary thromboprophylaxis in aPL carriers is mainly based on controlling any additional vascular risk factors that should be treated according to the cardiovascular disease prevention guidelines for the general population. All aPL carriers should receive thromboprophylaxis with usual doses of low molecular weight heparin (LMWH) in high-risk situations including surgery, prolonged immobilization, and puerperium period. Estrogen-containing oral contraceptives are not recommended while progestin-only contraception is considered to be safe. Low-dose aspirin (LDA, 75 100 mg/day) is recommended in subjects with persistent positivity of multiple and/or high titer aPL (Ruiz-Irastorza et al., 2010). In accordance with a recent metaanalysis, a lower rate of first thrombotic events was found in aPL-positive patients receiving aspirin in comparison with nontreated patients (7.8% vs 15.2%, P , .0001). According to the subgroup analysis, aspirin had a significant protective effect in asymptomatic aPL-positive individuals (OR 0.50, 95% CI 0.25 0.99), in SLE patients (OR 0.55, 95% CI 0.31 0.98), and in OAPS patients (OR 0.25, 95% CI 0.10 0.62) (Arnaud et al., 2014, 2015).

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No difference in the frequency of thrombosis was observed in aPL-positive patients treated with lowintensity anticoagulation target INR (1.5 and LDA) in comparison with those treated with LDA only (Cuadrado et al., 2014). Definite APS patients with a first venous thrombosis event should receive oral anticoagulant therapy to a target of INR 2.0 3.0. Patients with definite APS and arterial thrombosis and/or recurrent events should be treated with oral anticoagulant therapy target with an INR of over 3.0, or receive anticoagulation therapy combined with antiaggregant agents, with an INR target between 2.0 and 3.0. In addition, indefinite antithrombotic therapy is recommended in patients with definite APS and thrombosis (Sciascia et al., 2017). It has been recently demonstrated that persistent negative aPL profile is not an indication to stop oral anticoagulant therapy in APS patients. Thus in one study, thrombosis recurrence appeared in 46% of 24 primary APS patients with persistent negative aPL profile (Medina et al., 2017). Oral anticoagulation therapies have been developed during the last years including direct anti-Xa inhibitors, including rivaroxaban, apixaban, and edoxaban, and a direct thrombin inhibitor named dabigatran etexilate. According to the rivaroxaban in antiphospholipid syndrome trial, rivaroxaban might be an effective alternative in patients with APS and previous venous thromboembolism (Cohen et al., 2016). The use of new oral anticoagulants in APS patients with arterial events and/or high-risk aPL profile requires further investigation. With regard to pregnancy management in women with APS, after pregnancy confirmation, the patient should discontinue oral anticoagulants because of teratogenicity and switch to LDA in combination with LMWH. LMWH is commonly prescribed at prophylactic doses in women without previous thrombosis, or therapeutic doses in women with previous thrombotic episodes (Erkan et al., 2014; Levy et al., 2015). Several clinical studies have examined the role of immunosuppressive therapy in APS patients and aPL-positive subjects. It has been previously shown that hydroxychloroquine (HCQ) has a beneficial effect on primary arterial and venous thromboses prevention in aPL-positive individuals and in SLE patients with or without aPL (Tektonidou et al., 2009). The use of HCQ as an additional treatment in refractory APS cases is also recommended (Negrini et al., 2017). Furthermore, an addition of HCQ to the current therapy may be considered in cases of OAPS when a standard treatment with aspirin and a heparin agent has failed, or in selected cases including women with previous thrombosis and/or ischemic placenta-mediated complications (Sciascia et al., 2016). Rituximab, an anti-CD20 chimeric monoclonal antibody, may be considered in difficult-to-treat APS patients, possibly in those with hematologic and microthrombotic/microangiopathic manifestations (Erkan et al., 2014). In cases of CAPS, an aggressive therapy is highly recommended using anticoagulation, glucocorticoids, and plasma exchange and/or intravenous immunoglobulins (Puente et al., 2009). This combination was retrospectively assessed and found to be advantageous (Espinosa et al., 2011), noting that APS has been revealed as a complex syndrome with multiple pathophysiological mechanisms previously unknown. In this context, new therapeutic approaches have been defended and empirically tested, with potentially promising results. For patients with refractory CAPS, rituximab has been reported to be safe and effective (Sukara et al., 2015). In addition, several reports have described the successful use of eculizumab, a humanized monoclonal antibody against complement protein C5, in CAPS and severe cases of APS, including APS and thrombotic microangiopathy (reviewed in Sciascia et al., 2017). Peptide therapy and mTOR inhibition are new potential targets in APS that require additional investigation (Andrade and Tektonidou, 2016).

CONCLUSIONS AND FUTURE ASPECTS APS is a systemic autoimmune disease, mediated by autoantibodies directed at phospholipids and phospholipid-binding proteins. Since its definition some 40 years ago, our understanding of the mechanisms underlining this disease has greatly improved. Nonetheless, much is yet to be accomplished regarding the definition of subgroups of APS patients, the accurate diagnosis and interpretation of criteria and noncriteria aPL, and the appropriate treatments for APS- and aPL-affected patients. These goals may be achieved by conducting welldesigned, large-scale, multicenter clinical trials to explore and address the unmet needs of better, safer, and targeted management of APS.

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Further Reading Agmon-Levin, N., Blank, M., Zandman-Goddard, G., Orbach, H., Meroni, P.L., Tincani, A., et al., 2011. Vitamin D: an instrumental factor in the anti-phospholipid syndrome by inhibition of tissue factor expression. Ann. Rheum. Dis. 70, 145 150. Cimaz, R., Meroni, P.L., Shoenfeld, Y., 2006. Epilepsy as part of systemic lupus erythematosus and systemic antiphospholipid syndrome (Hughes syndrome). Lupus 15, 191 197. de Groot, P.G., Meijers, J.C., 2011. beta(2)-Glycoprotein I: evolution, structure and function. J. Thromb. Haemost. 9, 1275 1284. Di Prima, F.A., Valenti, O., Hyseni, E., Giorgio, E., Faraci, M., Renda, E., et al., 2011. Antiphospholipid syndrome during pregnancy: the state of the art. J. Prenat. Med. 5, 41 53. Erkan, D., Vega, J., Ramo´n, 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 (2), 464 471.

VI. MULTISYSTEM AUTOIMMUNE DISEASES