Antiphospholipid Antibodies and the Antiphospholipid Syndrome: From Coagulation to the Clinic

Antiphospholipid syndrome (APS) is a systemic, autoimmune disorder characterized by the presence of antiphospholipid antibodies (APLA) directed to various plasma proteins bound to anionic phospholipids. The clinical syndrome of APS is variable, resulting commonly in venous or arterial thrombosis and/or pregnancy morbidity and is defined by clinical and laboratory consensus criteria (1). “Noncriteria” clinical presentations and severe presentations such as catastrophic APS (a syndrome of multiorgan failure) are less common. Both criteria and noncriteria presentations are described in Table 1. APS patients may have underlying systemic lupus erythematosus, but approximately half of the patients do not and are diagnosed as primary APS (2). Although asymptomatic carriers of APLA are identified, the true prevalence in the general population is unknown, due to the absence of large population-based studies. In symptomatic patients, it is estimated that APLA may be detected in up to 10% of cases of stroke, venous thrombosis, and pregnancy morbidity (3) and in 17% of patients less than 50 years with stroke (4), with an estimated incidence of 1 to 2 persons per 100 000 population (5). Criteria and Non-criteria manifestations of APS. 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 eclampsia or severe preeclampsia or 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. a) Lupus anticoagulant 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). b) Anticardiolipin antibody of IgG and/or IgM isotype in serum or plasma, present in medium or high titer (i.e. >40 GPL or MPL, or >the 99th percentile), on two or more occasions, at least 12 weeks apart, measured according to recommended procedures. c) Anti- β2 glycoprotein-I antibody of IgG and/or IgM isotype in serum or plasma (>the 99th percentile in titer), present on two or more occasions, at least 12 weeks measured according to recommended procedures. a) Hematological: Thrombocytopenia, hemolytic anemia. b) Renal: Acute thrombotic microangiopathy, chronic vaso-occlusive lesions. c) Cardiac: Valve vegetations or thickening. d) Dermatologic: livedo reticularis or racemosa, livedoid vasculopathy (recurrent, painful skin ulcerations). e) Neurologic: Cognitive dysfunction (in the absence of stroke), sub-cortical white matter changes. f) Catastrophic APS: Multi organ thromboses (3 or more) in a patient with persistently positive test results for APLA represents a severe form of APS with associated high mortality. 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 eclampsia or severe preeclampsia or 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. a) Lupus anticoagulant 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). b) Anticardiolipin antibody of IgG and/or IgM isotype in serum or plasma, present in medium or high titer (i.e. >40 GPL or MPL, or >the 99th percentile), on two or more occasions, at least 12 weeks apart, measured according to recommended procedures. c) Anti- β2 glycoprotein-I antibody of IgG and/or IgM isotype in serum or plasma (>the 99th percentile in titer), present on two or more occasions, at least 12 weeks measured according to recommended procedures. a) Hematological: Thrombocytopenia, hemolytic anemia. b) Renal: Acute thrombotic microangiopathy, chronic vaso-occlusive lesions. c) Cardiac: Valve vegetations or thickening. d) Dermatologic: livedo reticularis or racemosa, livedoid vasculopathy (recurrent, painful skin ulcerations). e) Neurologic: Cognitive dysfunction (in the absence of stroke), sub-cortical white matter changes. f) Catastrophic APS: Multi organ thromboses (3 or more) in a patient with persistently positive test results for APLA represents a severe form of APS with associated high mortality. Adapted with permission from Miyakis et al. (1). Adapted with permission from Garcia et al. (8). Criteria and Non-criteria manifestations of APS. 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 eclampsia or severe preeclampsia or 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. a) Lupus anticoagulant 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). b) Anticardiolipin antibody of IgG and/or IgM isotype in serum or plasma, present in medium or high titer (i.e. >40 GPL or MPL, or >the 99th percentile), on two or more occasions, at least 12 weeks apart, measured according to recommended procedures. c) Anti- β2 glycoprotein-I antibody of IgG and/or IgM isotype in serum or plasma (>the 99th percentile in titer), present on two or more occasions, at least 12 weeks measured according to recommended procedures. a) Hematological: Thrombocytopenia, hemolytic anemia. b) Renal: Acute thrombotic microangiopathy, chronic vaso-occlusive lesions. c) Cardiac: Valve vegetations or thickening. d) Dermatologic: livedo reticularis or racemosa, livedoid vasculopathy (recurrent, painful skin ulcerations). e) Neurologic: Cognitive dysfunction (in the absence of stroke), sub-cortical white matter changes. f) Catastrophic APS: Multi organ thromboses (3 or more) in a patient with persistently positive test results for APLA represents a severe form of APS with associated high mortality. 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 eclampsia or severe preeclampsia or 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. a) Lupus anticoagulant 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). b) Anticardiolipin antibody of IgG and/or IgM isotype in serum or plasma, present in medium or high titer (i.e. >40 GPL or MPL, or >the 99th percentile), on two or more occasions, at least 12 weeks apart, measured according to recommended procedures. c) Anti- β2 glycoprotein-I antibody of IgG and/or IgM isotype in serum or plasma (>the 99th percentile in titer), present on two or more occasions, at least 12 weeks measured according to recommended procedures. a) Hematological: Thrombocytopenia, hemolytic anemia. b) Renal: Acute thrombotic microangiopathy, chronic vaso-occlusive lesions. c) Cardiac: Valve vegetations or thickening. d) Dermatologic: livedo reticularis or racemosa, livedoid vasculopathy (recurrent, painful skin ulcerations). e) Neurologic: Cognitive dysfunction (in the absence of stroke), sub-cortical white matter changes. f) Catastrophic APS: Multi organ thromboses (3 or more) in a patient with persistently positive test results for APLA represents a severe form of APS with associated high mortality. Adapted with permission from Miyakis et al. (1). Adapted with permission from Garcia et al. (8). Despite many advances in understanding the pathophysiology of APS, the definitive pathways by which APLA result in the clinical sequelae described in Table 1 remain elusive. The major antigenic targets for APLA are β2-glycoprotein I (β2-GPI), prothrombin (PT) and the phosphatidylserine (PS)/PT complex, all plasma proteins that bind avidly to anionic phospholipid surfaces (6). Supporting evidence from animal models of APS and non-APS thrombosis, in vitro mechanistic studies using human tissues, and APS biomarker studies in humans suggests many pathogenetic pathways by which APLA may lead to thrombosis (6, 7). A “2-hit” model of thrombosis has been proposed, where an initiating “first-hit” injury disrupts the endothelium, and a “second hit” such as infection, inflammation, surgery, or pregnancy potentiates thrombus formation; however, this is not readily identifiable in most cases of APS (6). Evidence exists for priming of the endothelium by oxidative stress and impaired endothelium-dependent vascular responses due to reduction in endothelial nitric oxide synthase activity (6). Autoantibodies to β2-GPI have been shown to cause up regulation of cell-surface expression of tissue factor on endothelial cells and monocytes, dysregulated activation of Factor XI, platelet activation, increased platelet adhesiveness, downregulation of inhibitors of coagulation, and disruption of the annexin 5 anticoagulant shield, thereby exposing procoagulant phosphatidylserine (6, 8). Thus far, this antibody-mediated thrombotic phenotype has led to anticoagulants being the mainstay of treatment for this disorder. However, recent animal model and human studies indicate a greater role for complement activation, endothelial progenitor cell deficiency, and activation of neutrophils with release of neutrophil extracellular traps by APLA, suggesting that APS is more an autoimmune thrombo-inflammatory disorder rather than simply a disorder of coagulation. This has opened up a plethora of potential new therapeutic targets as disease-modifying therapy in severe or anticoagulant refractory APS (7–9). Current laboratory criteria for diagnosis of APS include both quantitative immunoassays for detection of IgG and IgM isotypes of anticardiolipin antibodies (ACA) and anti-β2-GPI, as well as phospholipid-based clotting assays that examine the functional interference from these antibodies, also known as lupus anticoagulant (LA) assays (Table 1). Despite the knowledge of other antigen targets for APLA, running a panel of immunoassays to detect additional APLA antigenic targets, is currently not part of the routine clinical laboratory work up for APLA due to a paucity of evidence supporting the clinical relevance of these noncriteria antibodies. The last decade has seen significant progress in the clinical laboratory diagnosis of APS due to the harmonization initiatives undertaken by various international bodies—the International Society on Thrombosis and Haemostasis, the British Committee for Standards in Haematology, and the Clinical and Laboratory Standards Institute (CLSI). These have led to updated guidelines that have recommended specific assays, cutoffs, and diagnostic algorithms for detecting LA and called attention to controlling important preanalytic, patient, and sample factors to allow accurate diagnosis and appropriate interpretation (10–12). Although, there are minor differences between these laboratory guideline recommendations, harmonization of LA detection has been a major step in minimizing the heterogeneity and lack of generalizability that plagued older diagnostic and therapeutic APS studies. There has been recent recognition that patients with APS vary in terms of risk of thrombosis recurrence based on APLA laboratory profiles and clinical presentation (13, 14). Patients who are persistently APLA positive by a single assay only (single positive) have a lower risk of recurrent thrombosis compared to patients who have persistent, detectable APLA by all 3 assays (triple positive) (13). Among the single-positive patients, the detection of a LA defines a higher risk subgroup of patients than the detection of either ACA or anti-β2-GPI alone (15). Clinically, patients with arterial thrombosis, small vessel thrombosis, and organ or heart valve involvement are considered high risk (13). For patients with venous thromboembolism not associated with APLA, the direct oral anticoagulants (DOACs), with their improved convenience, noninferior efficacy, and lower bleeding risk, have become first-line therapy, replacing oral vitamin K antagonists (VKAs) like warfarin (16). Although DOACs represent an attractive therapeutic option for patients with thrombotic APS given their need for long-term, often lifelong anticoagulation, 2 recent randomized controlled trials have shown some concerning results with higher rates of arterial thrombosis with DOACs compared to VKAs (14, 17, 18). Based on these randomized controlled trials, international guidelines have updated their recommendations for management of APS advising VKAs as the anticoagulant of choice in thrombotic APS (13, 15, 19). In the subgroup of non–high-risk thrombotic APS patients (non–triple positive and no prior history of arterial thrombosis), DOACs may be a suitable anticoagulant, but limited data exist to guide management. All clinical guidelines still recommend using VKA as first-line therapy out of caution until further data from dedicated clinical trials are available in these specific subgroups. However, if an APS patient with non–triple-positive serology and without prior arterial thrombosis is already stable on a DOAC with good adherence, DOACs may be continued after a patient discussion (13, 15, 19). Despite these advances in the laboratory and clinical risk-stratified management of APS, many diagnostic challenges and management controversies remain. Immunoassays for ACA and anti-β2-GPI are not standardized, and external proficiency data show significant interlaboratory variability especially when sample results are not clearly negative or strongly positive (20, 21). While medium or high titers of ACA antibodies are required to meet current laboratory criteria of APS, there is no accepted threshold to categorize medium and high from low (1). A significant proportion of individuals meet clinical criteria for thrombotic or obstetric APS but have only persistent low antibody titers; no consensus exists on optimal treatment for this group (22). Since venous thromboembolism is common in the general population (23), patients being investigated for APLA are usually already on DOACs during sample collection. DOACs are strong inhibitors of specific common pathway clotting factors resulting in significantly high rates of false-positive LA assays and inaccurate diagnoses, a concern not yet appreciated by many treating clinicians (12). The most recent International Society on Thrombosis and Haemostasis guideline has included criteria for holding DOACs for at least 48 h before LA testing, as well as screening for DOACs as a prerequisite before interpreting LA assays (12). However, specific screening tests for DOACs are expensive and not available in most laboratories. Hence, concerns about false-positive LA diagnoses are rampant in the DOAC era and something that laboratories and clinicians will have to work together to overcome. There remain a significant population of patients with classic thrombosis or obstetric presentations without traditional risk factors, who are very likely to have APS but in whom testing for criteria antibodies is negative (seronegative APS). This has resulted in a renewed interest in investigating noncriteria APLA such as the IgA subtypes of both ACA and anti-β2-GPI, β2-GPI domain I antibodies, anti-PT and PS/PT complex antibodies, anti-vimentin/cardiolipin complex antibodies, and Annexin A5 antibody, to name a few (24, 25). However, the current evidence supporting routine testing for noncriteria antibodies is of low quality, and the assays lack standardization. Also, although several antibodies are significantly associated with APS symptoms, their addition to the current criteria panel has not been shown to add independent predictive value (24–26). In the absence of robust evidence, investigating noncriteria antibodies should be done in highly selected patients by expert clinicians and only when there is the potential to change management. The management of anticoagulant refractory thrombotic APS, defined as breakthrough thrombosis on VKA despite therapeutic international normalized ratio, remains both controversial and challenging, as data to guide therapy are restricted to observational studies only. In such patients, increasing the intensity of VKA anticoagulation, switching to low molecular weight heparin, or adding antiplatelet agents, vitamin D, statins, or hydroxychloroquine may be considered (27). In patients with breakthrough thrombosis despite high-intensity anticoagulation, options are extremely limited. Adjuvant therapies such as B-cell inhibition, intravenous immune globulin infusion, plasma exchange, complement inhibition, vasodilators, and hyperbaric oxygen have been described (27). To conclude, APS is a complex disorder with inherent challenges in accurate diagnosis and appropriate management. In our opinion, adhering to harmonized laboratory criteria recommended by international societies has been an important first step in minimizing the heterogeneity and increasing the generalizability of therapeutic randomized controlled trials. However, ongoing efforts should target standardization of APLA immunoassays and controlling the preanalytical and analytical factors that continue to confound accurate diagnosis of LA. Routine investigation of noncriteria antibodies needs further study through well-designed diagnostic management studies. Increasingly, thrombotic APS is being recognized as a spectrum of thrombotic risk, which may require differing anticoagulant strategies for APS subgroups. Recent data supporting APS as a thrombo-inflammatory disorder, with a role for complement and other cellular pathways, have opened up avenues for precision medicine–based therapeutic targets in severe and/or treatment refractory APS. Nonstandard Abbreviations: APS, antiphospholipid syndrome; APLA, antiphospholipid antibodies; β2-GPI, β2-glycoprotein I; PT, prothrombin; PS, phosphatidylserine; ACA, anticardiolipin antibodies; LA, lupus anticoagulant; DOAC, direct oral anticoagulant; VKA, vitamin K antagonist. Author Contributions: All authors confirmed they have contributed to the intellectual content of this paper and have met the following 4 requirements: (a) significant contributions to the conception and design, acquisition of data, or analysis and interpretation of data; (b) drafting or revising the article for intellectual content; (c) final approval of the published article; and (d) agreement to be accountable for all aspects of the article thus ensuring that questions related to the accuracy or integrity of any part of the article are appropriately investigated and resolved. Authors’ Disclosures or Potential Conflicts of Interest: Upon manuscript submission, all authors completed the author disclosure form. Disclosures and/or potential conflicts of interest: Employment or Leadership: R. Selby, Co-Chair, Test Review and Utilization Committee, Laboratories and Genetics Branch, Ontario Ministry of Health (unpaid); J. Abdulrehman, Member of Thrombosis Canada Annual Education Conference 2021 Steering Committee (unpaid). Consultant or Advisory Role: None declared. Stock Ownership: None declared. Honoraria: R. Selby, American Society of Hematology—Education session honorarium, Thrombosis Canada honorarium; J. Abdulrehman, Thrombosis Canada Annual Education Conference 2020—honorarium for speaking, Thrombosis and Hemostasis Societies of North America Summit 2020—honorarium for speaking. Research Funding: R. Selby, Physicians Services Incorporated Foundation (Coapplicant), Association of Hemophilia Clinic Directors of Canada (AHCDC), Shire Canadian Hemophilia Epidemiological Research Program (Coapplicant), CanVECTOR (Canadian Venous Thromboembolism Clinical Trials and Outcomes Research) Network. Canadian Institutes of Health Research (CIHR). ICRH Community Development Program Grants (Coapplicant); J. Abdulrehman, Paul B. Helliwell Foundation Fund for Innovative Research in Non-Malignant Hematology—research grant, Yeo Chair Grant in Benign Hematology—research grant. Expert Testimony: None declared. Patents: None declared.

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