Antiphospholipid Syndrome

Activation of Complement

There is convincing evidence that complement activation plays a role in the APS disease process. When incubated with APS plasma, phospholipid vesicles activate more complement than when incubated with control plasma, as evidenced by more C5a and sC5b-9 generation ( Fig. 139.3 ). Complement activation likely reflects the formation of APS macro-immune complexes on these vesicles. The modified Ham test has also been used to demonstrate complement activation; 69% of APS sera collected within 1 year of a thrombotic event and 86% of CAPS sera tested positive, whereas only 6.8% of SLE sera were positive. Complement activation is induced by anti-β2GPI antibodies, primarily through the classical pathway and germline variants in complement regulatory genes have been found in patients with thrombotic APS and CAPS. The role of complement activation is buttressed by findings in a mouse model of APS antibody-induced pregnancy loss where genetic deletion of C3 or treatment of wild-type mice with a C3 convertase inhibitor protected against pregnancy complications.

Inhibition of Anticoagulant and Fibrinolytic Mechanisms

aPL antibodies accelerate coagulation reactions on endothelial cells and trophoblasts by disrupting the antithrombotic shield provided by annexin A5. Annexin A5 forms a two-dimensional crystalline array over phospholipid bilayers, which shields anionic phospholipids on cell membranes and blocks coagulation proteins from binding and assembling on the cell membrane, thereby preventing phospholipid-dependent coagulation enzyme reactions. Annexin A5 is highly expressed by endothelial cells and localizes to the apical membrane of placental syncytiotrophoblasts where maternal blood interfaces with fetal cells. Pregnant mice treated with anti-annexin A5 antibodies develop placental necrosis, fibrosis, and pregnancy loss, while pregnant annexin A5-null mice experience placental infarction and have reduced litter sizes. Anti-β2GPI IgG/β2GPI immune complexes disrupt the annexin A5 antithrombotic shield by competitively displacing the protein from the surfaces of cell membranes ( Fig. 139.4 ). A mechanistic assay has been developed to assess disruption of annexin A5 anticoagulant activity.

aPL antibodies inhibit several steps in the protein C pathway. These include: (1) reducing protein C activation by the thrombomodulin-thrombin complex, (2) inhibiting assembly of the protein C complex, (3) inhibiting activated protein C (APC) directly or via its cofactor protein S, (4) binding to factors Va and VIIIa and protecting them from proteolysis by APC, and (5) reducing levels of protein C and protein S. Some aPL antibodies bind protein C or S. APC resistance has been described in APS plasmas and has been correlated with anti-β2GPI domain I antibodies.

Autoantibodies directed against TFPI have also been reported in APS patients; these antibodies may inhibit the capacity of TFPI to downregulate activation of factor IX and factor X by the factor VIIa-tissue factor complex. TFPI has a thrombogenic role by serving as a cofactor for promoting aPL-mediated expression of tissue factor by monocytes.

aPL antibodies impact fibrinolytic mechanisms in multiple ways including (1) binding to annexin A2 and reducing its capacity to serve as a receptor for tissue plasminogen activator (t-PA) and plasminogen, (2) reducing the catalytic activity of plasmin or t-PA, (3) increasing the levels of plasminogen activator inhibitor-1, (4) inhibiting autoactivation of factor XII, and (5)reducing plasmin generation because antibodies against β2GPI attenuate its capacity to serve as a cofactor for t-PA-mediated activation of plasminogen.

Activation of Platelets, Monocytes, and Neutrophils

Recent evidence from a mouse model suggests that aPL-induced thrombosis results from platelet activation that promotes endothelial activation and fibrin formation. aPL antibodies can induce platelet aggregation, an effect that may be promoted via signaling through apolipoprotein E receptor 2′ (ApoER2′) with the β2GPI binding site for ApoER2′ on platelets localized to its domain V. Since β2GPI has a dampening effect on platelet adhesion by interfering with the platelet–von Willebrand factor interaction, aPL antibodies increase platelet adhesion in flow systems by interfering with this dampening. Additionally, proteomic studies support a role for platelet disulfide isomerase family members in promoting aPL-mediated thrombosis.

aPL antibodies increase the expression of tissue factor and other cytokines by monocytes—a process that occurs through activation of the p38MAPK and MEK-1/ERK pathways. A recent unexpected finding was that TFPI primes the monocytes for the aPL-mediated signaling that results in tissue factor expression. aPL antibodies may also promote mitochondrial dysfunction and oxidative stress in monocytes, thereby inducing a proinflammatory state.

Finally, anti-β2GPI/β2GPI complexes can promote neutrophils to undergo NETosis, because neutrophil extracellular traps (NETs) can initiate clotting (see Chapter 121 ). The activation of the TLR4/MyD88/MAPKs axis was shown to be involved in the formation of NETs, which then promoted coagulation via tissue factor expression on the NETs along with platelet aggregation. This mechanism was further supported by the proteomic studies.

Effects on Vascular Endothelium

aPL antibodies can bind to, injure, and/or activate cultured vascular endothelial cells. Antibody binding to β2GPI on the endothelial surface may trigger signaling cascades that promote the expression of tissue factor and adhesion molecules.

Like its role in activating platelets, ApoER2′ expressed on the endothelial surface may also be a target for anti-β2GPI/β2GPI complexes to trigger tissue factor and adhesion molecule expression. Evidence from ApoER2′ ^−/− knockout mice supports a role for the protein in mediating APS thrombosis.

Annexin A2 is an endothelial surface receptor for t-PA and plasminogen which serves as a receptor for β2GPI on vascular endothelium. In addition to aPL antibody-mediated inhibition described above, annexin A2 may play a role in mediating the effects of aPL antibodies on the signaling steps that promote thrombosis. Annexin A2 is not a transmembrane protein and so the signaling co-receptors, Toll-like receptor-2 (TLR2) and Toll-like receptor-4 (TLR4), have been implicated as triggers of the signaling cascade. Downstream signaling appears to involve tumor necrosis factor receptor-associated factor 6 (TRAF6) and myeloid differentiation factor 88 (MyD88). Tissue factor expression is mediated by p38 mitogen-activated protein kinase (MAPK). This mechanism is supported by the finding that a mutation in murine TLR-4 that disrupts lipopolysaccharide (LPS) binding attenuated the prothrombotic state in mice injected with aPL antibodies.

aPL antibodies may contribute to other vascular lesions by stimulating the mammalian target of the rapamycin complex (mTORC) pathway. Endothelial cells and platelets activated by aPL antibodies release microparticles containing procoagulant proteins and nucleic acids.

Effects on Trophoblasts and Endometrial Cells

In mice, in addition to disruption of the annexin A5 anticoagulant shield at the maternal-fetal interface, aPL-mediated promotion of tissue factor expression induces trophoblast injury and fetal death. Tissue factor contributed to the C5a-induced oxidative burst in neutrophils that promoted trophoblast and fetal injury in APS.

aPL antibodies may induce trophoblast proliferation, migration, and invasiveness, increase trophoblast apoptosis, and reduce the secretion of human chorionic gonadotropin (HCG) and adhesion molecules. Activation of MYD88 by TLR4 has been linked to increased levels of proinflammatory cytokines (IL-8, IL-1β, monocyte chemoattractant proteins) that influence trophoblast survival. Downregulation of signal transducer and activator of transcription 3 (STAT3) phosphorylation decreases IL-6 expression and reduces trophoblast migration. aPL antibodies have been implicated in the disruption of maternal spiral artery transformation and maturation, as well as differentiation of decidual endometrial cells through activation of signaling pathways such as nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB). Defective placentation may result in impaired blastocyte implantation, thereby leading to fetal loss.

Genetic and Proteomic Studies in Antiphospholipid Syndrome

Genetic factors appear to play a role in the development of aPL antibodies even though familial APS is rare. One study of seven families, which included 30 individuals who met consensus criteria for APS, concluded that the inheritance pattern of aPL antibodies appeared to be autosomal dominant, however no specific linkages could be identified. A study done with peripheral blood mononuclear cells from aPL antibody-positive patients found a gene expression pattern that appeared to correlate with a predisposition for thrombosis. Some of the identified genes encoded proteins that are known to be involved in thrombosis, including apolipoprotein E (ApoE), factor X, and thromboxane. Other genes were not connected with the disease process, such as hypoxia-inducible factor-1alpha (HIF-1α), zinc finger proteins, matrix metalloproteinase 19 (MMP19), interleukin 22 (IL22) receptor, and hematopoietic progenitor cell antigen (CD34) precursor. As mentioned above, germline variants of complement proteins have recently been associated with thrombotic APS and CAPS.

Proteomic studies have revealed a series of proteins that are differentially expressed in monocytes of APS patients with a history of thrombosis. These include annexin A1, annexin A2, ubiquitin Nedd8, Rho A protein, protein disulfide isomerase, and Hsp60. Proteomic analysis of platelets and plasmas from LA-positive patients with a history of thromboembolism supports the role of PDI and NETosis in APS-associated thrombosis. PDI family members were upregulated in platelets and plasma of these patients compared with controls. Leukocyte elastase inhibitor (SERPINB1), an antagonist of NET formation, was decreased and citrullinated histone H3, a NET-specific marker, was increased.

Paths to Development

The recognition of APS stemmed from observations of two laboratory anomalies. One of these, the biologic false-positive serologic test for syphilis (STS), caused by antibodies against cardiolipin (diphosphatidylglycerol), formed the basis for an immunoassay that prompted the identification of “anticardiolipin antibody syndrome,” which was then renamed “antiphospholipid antibody syndrome.” The other anomaly was the finding that some patients had a prolonged activated partial thromboplastin time (aPTT) due to a unique inhibitor of blood coagulation. The first patients identified with this anomaly had SLE, which led to the misnomer “LA”. This brief history is helpful for appreciating that the current aPL tests, which were empirically derived from these two anomalies, are limited because they do not directly measure a disease mechanism. Nevertheless, these assays have become the mainstay markers of this disorder and have proven to be useful as surrogate reporters of thrombotic risk.

The current Sydney criteria for the diagnosis of APS identify LA, aCL, IgG and IgM, and anti-β2GPI IgG and IgM as the key diagnostic assays. To qualify for the diagnosis of APS, positive results must be obtained on two or more occasions at least 12 weeks apart (see Table 139.1 ).

“Criteria” Antiphospholipid Syndrome AssaysLupus Anticoagulant Tests

LA tests are performed in a variety of configurations, all of which share the purpose of detecting antibody-mediated inhibition of phospholipid-dependent blood coagulation reactions.

An updated guidance on LA testing was published by the LA Subcommittee of the International Society on Thrombosis and Haemostasis Scientific and Standardization Committee (ISTH/SCC) in 2020.

LA tests include the dilute Russell viper venom time (dRVVT), modifications of the aPTT with LA-sensitive and LA-insensitive reagents, the kaolin clotting time (KCT), the dilute prothrombin time (dPT) which was previously known as the tissue thromboplastin inhibition time, the hexagonal phase array test, and the ecarin-taipan clotting time. The dRVVT and the silica clotting time, a form of the aPTT, are the most widely performed tests. Interlaboratory comparison studies demonstrate agreement on identification of plasmas containing strongly positive LA activity, but disagreement about samples with weaker activity (these are missed in approximately half the cases) and misdiagnosis of coagulation factor-deficient LA-negative plasmas as being LA-positive.

Despite the limitations, LA positivity is more sensitive and specific for the presence of aPL antibodies than aCL and anti-β2GPI assays. In a systemic review of 7000 patients and 25 studies, the presence of an LA was found to have a strong association with thrombosis, both in patients with and without SLE and regardless of site and type of thrombosis. Also, LA had a stronger association than aCL antibodies for thrombosis. Meanwhile, medium to high titer aCL antibodies were associated with cerebral stroke and myocardial infarction and not deep venous thrombosis. The mean odds ratios (ORs) for thrombosis were 11 for LA, 3 for high-titer aCL antibodies, and 1.6 for elevated levels of aCL antibodies. [LRW2] Furthermore, the Predictors of Pregnancy Outcome: Biomarkers in Antiphospholipid Antibody Syndrome and Systemic Lupus Erythematosus (PROMISSE) study demonstrated that of the available aPL markers, a positive LA was the single strongest predictor of adverse pregnancy outcome in patients with APS. However, routine LA screening is unlikely to benefit the general population since the high relative risks reported in these studies should be seen in the context of the low baseline risks of thrombosis and adverse pregnancy outcomes in disease-free individuals. Nevertheless, LA testing and aPL assay testing may be beneficial to ascertain risk in patients with a high baseline thrombotic risk, such as patients with SLE or a previous thrombosis.

Patient Selection for Lupus Anticoagulant Testing

The recommendations for patient selection for LA testing were expanded in the 2020 ISTH/SSC guidance and are summarized in Table 139.4 .

Prior to ordering testing for patients meeting the ISTH/SSC guidance, clinicians should consider that it is preferable for patients not to be tested in the setting of an acute thrombotic event and that any such testing should be interpreted with caution because false positive and negative results can occur. Whenever possible, LA testing should not be performed in patients receiving anticoagulant treatment because false positives and negatives can occur. When there is a need to perform testing, it is best to suspend anticoagulant treatment when feasible. If patients are tested during treatment with low molecular weight heparin (LMWH), it is recommended that samples be taken, when possible, at least 12 hours after the last dose of LMWH and that the anti-Xa activity be checked concurrently. Direct oral anticoagulants (DOACs) are especially problematic in causing false positive LA tests and, whenever possible, should be held prior to testing.

• Choosing LA assays .The ISTH/SCC has proposed specific criteria for standardizing the diagnosis of LA. When APS is suspected on clinical grounds, it is recommended that laboratories perform two different versions of LA tests. The dRVVT is widely used in clinical laboratories and is believed to be specific for detecting LA in patients at high risk for thrombosis who are not receiving anticoagulant therapy. aPTT tests performed with silica as an activator and low PL content were recommended as the second test of choice because of their sensitivity for LA, however, aPTT assays utilizing ellagic acid as the activator may also be used (see Chapter 127 ).

• Dilute Russell Viper Venom Time . The dRVVT is considered one of the most sensitive LA tests. The assay uses Russell viper venom (RVV) in a system containing limiting quantities of diluted rabbit brain phospholipid. RVV activates factor X, leading to clotting. aPL antibodies which prolong the dRVVT by interfering with the assembly of the prothrombinase complex, an effect reversed by the addition of excess phospholipid (sometimes referred to as a “confirmatory test”). To ensure that prolongation of the clotting time is not the result of a factor deficiency, the procedure includes mixing of patient plasma with control plasma. Anticoagulant therapy with heparin, warfarin or DOACs can yield falsely abnormal test results.

• Activated Partial Thromboplastin Time . The finding of a prolonged aPTT in an otherwise healthy individual without a history of bleeding is often attributable to a LA. aPTT reagents vary in their sensitivity to LA, so it is important to know the characteristics of the particular reagent that is being utilized. The LA needs to be differentiated from antibodies directed against specific coagulation factors (see Chapter 127 ) and from anticoagulant medications. In addition to utilizing specific assays to exclude these possibilities, the clinician can check whether the aPTT is normal if a LA-insensitive aPTT reagent is used. Mixing and incubating the test plasma with normal plasma may be helpful in differentiating LAs from coagulation factor inhibitors. aPTTs performed on mixtures of normal plasma and plasma containing a factor VIII inhibitor usually require incubation for 1 to 2 hours at 37°C to exhibit prolongation, whereas LA-containing plasmas typically prolong the aPTT immediately, without requiring incubation. The clinician should be aware that both types of anticoagulants (i.e., LA and specific coagulation factor inhibitors) may coexist and yield a confusing laboratory picture. LAs may also cause artifactual decreases in contact activation pathway coagulation factor levels because those assays are based on the aPTT; these patients are sometimes misdiagnosed as having multiple coagulation factor deficiencies. This problem can be identified by noting whether there is non-parallelism between dilutions of the plasma and the standard curve used for the assay, or by using an aPTT reagent that is insensitive to LA.

Anticardiolipin IgG and IgM Antibody Assays

The development of the quantitative aCL antibody radioimmunoassay and subsequent enzyme-linked immunosorbent assay (ELISA) was the single advance that led to the identification of APS as a new syndrome. Although aCL ELISAs are sensitive, they are not specific for APS, especially when present at low positive levels or in patients with certain infections. Patient with syphilis can have a false positive aCL antibody test. Alternatively, patients with aCL positive antibodies can have a false positive syphilis Venereal Disease Research Laboratory (VDRL) test since cardiolipin is used as an antigen target to detect non-specific antibodies released by spirochetes. Testing guidelines recommend using β2GPI-dependent cardiolipin targets to avoid detection of non-cofactor related aCL produced by infections or drugs.

aCL may be detected by ELISA or newer methods such as chemiluminescence analysis (CLIA) or multiline dot assay (MLDA). The cutoffs for medium to high aCL titers (i.e., 99th percentile) are greater than 40 IgG Phospholipid Units (GPL)/IgM Phospholipid Units (MPL) in ELISA and greater than 20 Chemiluminescent Units (CU) in CLIA.

High levels of aCL antibodies are associated with an increased risk of venous thromboembolism, myocardial infarction, stroke, and recurrent pregnancy loss. In a 10-year follow-up study, 50% of initially asymptomatic patients with persistently elevated aCL antibodies went on to develop APS. The presence of elevated titers of aCL antibodies 6 months after an episode of venous thromboembolism is a predictor of an increased risk of recurrence and of death.

It is very important for the clinician to recognize that the majority of patients with elevated levels of aCL antibodies on routine screening do not have APS . Many individuals have elevated antibody levels in response to infections; these antibodies have not been associated with thrombotic complications. In various studies, the prevalence of positive immunoassays in the asymptomatic “normal” population has range from 3% to nearly 20%. Therefore, it is important to repeat testing after 12 weeks to determine whether there is persistent positivity.

Anti-β2-Glycoprotein I IgG and IgM Antibody Assays

β2GPI is the major protein cofactor recognized by aPL antibodies. The protein is composed of five homologous domains and assumes a closed conformation in solution and an open conformation when bound to phospholipid membranes (see Fig. 139.2 ). Anti-β2GPI antibodies that bind to the circular closed form generally recognize epitopes between domains one and five, while in the open form, anti-β2GPI antibodies interact with the exposed domain one. Anti-β2GPI ELISAs are less sensitive but more specific for APS than aCL antibody assays. Although antibodies against β2GPI are usually found in conjunction with aCL and anti-phosphatidylserine antibodies, some patients with APS only have antibodies against β2GPI. Despite their higher specificity (98%), anti-β2GPI assays cannot be relied upon as a stand-alone test for APS because of their low sensitivity (40% to 50%) and concurrent testing for both antibodies, along with LA is advised. Antibodies against β2GPI can be detected by ELISA, CLIA, or MLDA. The determined cutoffs for medium to high titers (i.e., 99th percentile) are greater than 40 GPL/MPL in ELISA and greater than 20 CU in CLIA.

In a systematic literature review, 34 of 60 studies showed a significant association between anti-β2GPI antibodies and thrombosis. Anti-β2GPI antibodies were more often associated with venous than arterial thrombotic events and were independent risk factors for venous thrombosis.

Considerations and Limitations of Laboratory Immunoassays

Although a variety of assays are used, including more advanced and automated systems, there is no consensus on a “gold standard” for the detection of aCL and anti-β2GPI antibodies. Studies have shown poor agreement between commercially available immunoassays for detecting aCL and anti-β2GPI IgG/IgM antibodies. Therefore, it is recommended to use the same testing system for patient diagnosis and follow-up. In addition, guidelines recommend laboratories determine their own 99th percentile cutoffs for in-house assays using a population of at least 40 healthy volunteers.

The interpretation of immunoassays should also take into consideration the identified isotype. The aCL and anti-β2GPI IgG isotype have a stronger association with thrombosis than IgM. Although aCL and anti-β2GPI IgM isotypes have been associated with pregnancy morbidity and thrombosis in some reports, these associations have not been confirmed due to lack of paired IgG and IgM results. To not “miss” diagnosing APS patients, the guidelines advise continued measurement of IgG and IgM, with the presence of aCL and anti-β2GPI of the same isotype reinforcing the probability of APS.

Multipositivity for Antiphospholipid Tests and Other Scoring Systems for Clinical Risk

Strong positivity for more than one of the APS criteria assays (LA, aCL, and anti-β2GPI) has been correlated with increased risk for developing clinical events in several retrospective studies and in one prospective study. APS patients who are positive for all three tests (LA, aCL, and anti-β2GPI), referred to as “triple-positive” patients, have the highest risk of thrombosis. This risk is progressively lessened in double-positive (LA-negative) and single-positive patients.

In pregnant women with APS, triple APS assay positivity, with or without previous thromboembolism, is associated with a higher probability of adverse neonatal outcome than double or single aPL antibody-positivity and no thrombosis history. Multipositivity, but not single positivity, for aPL antibodies was associated with antenatal and postnatal deep vein thrombosis (DVT).

Both symptomatic and asymptomatic patients with persistent triple-positive laboratory results (LA-positive, aCL and anti-β2GPI of high titer, double-positive for LA, aCL and/or anti-β2GPI of high titer, or single positive for LA or persistently high aPL titers) should be considered high-risk for future manifestations of APS. Finally, single positivity for aCL IgG or IgM in low-medium titers or anti-β2GPI IgG or IgM in low-medium titers should be considered lower risk for APS but treatment may be warranted if such patients develop thrombosis or pregnancy complications or have other high-risk factors.

The global APS score (GAPSS) is a clinical scoring system that assigns points for the presence of six features—arterial hypertension (1 point), hyperlipidemia (3 points), LA (4 points), aCL antibodies (5 points), anti-β2GPI antibodies (4 points), and antiphosphatidylserine/prothrombin (aPS/PT) antibodies (3 points). GAPSS scores ≥ 10 have the best diagnostic accuracy. Higher GAPSS values are also associated with recurrent thrombotic events. A complementary version, the adjusted GAPSS (aGAPSS), which excludes aPS/PT, was also found to be higher in patients with recurrent thrombosis (arterial or venous) than in those without recurrence. Patients with recurrent arterial, but not venous, thrombosis had higher aGAPSS. A meta-analysis of both the GAPSS and aGAPSS scoring systems showed the highest scores in patients with arterial thrombosis and recurrence. Complement markers including platelet bound C4d (measured by flow cytometry) and low C3 have been included in some composite risk scores.