Antiphospholipid Antibody Syndrome. Rare Diseases of the Immune System

2. The Paradox of Lupus Anticoagulant

Alessandra Banzato  and Vittorio Pengo 

(1)

Department of Cardiologic Thoracic and Vascular Sciences, Padua University Hospital, Via Giustiniani, 2, Padua, 35121, Italy

Alessandra Banzato

Email: alessandra.banzato@unipd.it

Vittorio Pengo (Corresponding author)

Email: vittorio.pengo@unipd.it

Keywords

Lupus anticoagulantAPSβ2-glycoprotein IThrombosisPregnancy loss

2.1 History

In the 1940s a report from the University of California Medical School described a young man with a fatal condition manifesting as moderate thrombocytopenia and prolonged whole blood clotting time with a hemorrhagic diathesis and intracranial and peripheral venous thrombosis [1]. Even if the prolonged clotting time was attributed to “hypothromboplastinemia” and the crude tests available did not demonstrate a coagulation inhibitor, it may be speculated whether this was the first description of a lupus inhibitor. Afterward, Ley et al. [2] reported on a young man with a fatal disease with abnormal bleeding, arthralgias, and leg vein thrombosis, with prolonged blood clotting time and prothrombin time attributed to hypoprothrombinemia. At examination, as well as tissue bleeds, there were renal changes indicative of SLE and cerebral infarcts.

In the 1950s it was progressively more acknowledged that some patients with systemic lupus erythematosus (SLE) have a circulating anticoagulant factor. Conley [3], of Johns Hopkins, wrote a brief report about two patients with lupus and a “peculiar hemorrhagic disorder” with prolonged blood clotting and prothrombin times, and clear evidence of an anticoagulant in plasma mixing studies. Especially, both patients had biological false-positive tests for syphilis.

These observations were complemented by Frick and Weimer in the 1955s, who reported three patients with convincing evidence of coagulation inhibitors, including in one subject with confirmed lupus. In each case there were positive serological tests for syphilis, and in only one of the three was there a definite hemorrhagic diathesis. Furthermore, they provided evidence for transplacental transfer of the anticoagulant in one subject and postulated an “immunologic” pathogenesis [4]. The picture of lupus anticoagulant was slowly emerging, with evidence of its immunoglobulin association, and, in retrospect, the link to anticardiolipin was present, identified through the biological false-positive tests for syphilis in these patients. Subsequent case reports demonstrated that on serum electrophoresis the Wasserman reagent and the anticoagulant localized to the same region of gamma globulins [5]. The hypothesis of Frick [4] that the mechanism of action was “directed against thromboplastin” was reasonable at the time and not all that far from the truth.

In the 1960s it was increasingly recognized that the in vitro anticoagulant phenomenon in SLE could be associated with a thrombotic rather than a hemorrhagic diathesis: Bowie et al. [6] reported on the presence of circulating anticoagulant in eight of 11 patients with SLE of whom three suffered from deep vein thrombosis and one from ischemic leg ulcers and livedo reticularis.

The term “lupus anticoagulant” (LAC) was coined only later [7], in 1972, to designate an inhibitor of coagulation that impairs prothrombinase activation of prothrombin (PT), which was recognized in the plasma of patients with systemic SLE. Some arguments favored the hypothesis that the inhibitor was directed against phospholipids (PL) since preincubation with PL reduced its activity and the inhibitory effect was more pronounced when PL were diluted. It seems likely that the excess of descriptions of a hemorrhagic diathesis in the early case reports with what came to be known in the 1970s as lupus coagulation inhibitor [8], and soon after lupus anticoagulant, was simply due to the presentation with bleeding as the principal symptom in these unusual cases and the lack of any reason to explore coagulation in SLE patients without such a history of bleeding. Subsequent case reports highlighted that the lupus anticoagulant-associated prothrombotic condition can arise in the absence of SLE or other connective tissue disorders [9]. A significant diagnostic breakthrough occurred with the development of assays for anticardiolipin (aCL) that were considerably more sensitive than other assays. Harris et al. in 1983 [10] demonstrated that almost two-thirds of serum samples from a cohort of 65 patients with SLE had high levels of anticardiolipin.

Furthermore, over 90 % of those with LAC had raised anticardiolipin antibody levels, confirming the relationship previously suggested by cases with both LAC and biological false-positive tests for syphilis. Finally, there was a correlation between raised aCL titer and history of thrombosis within this patient cohort with SLE [10]. In the ‘80s, observations in four women, only one of whom had SLE, led to the postulation of a possible correlation between LAC and pregnancy morbidity [11]. This postulation was later confirmed [12]. Further case reports around this time suggested additional disease associations, for example, chorea gravidarum [13]. It was in the 1980s that the nomenclature “antiphospholipid antibody syndrome” (APS) began to be generally used [14], abbreviated to “antiphospholipid syndrome” afterward. The term primary antiphospholipid syndrome was introduced to describe those cases in which there was no background disease [1516]. There were also reports of cases with unusually acute and severe multiorgan vasoocclusive episodes associated with positive tests for antiphospholipid antibodies (aPL) [1718]. In 1992 Ronald Asherson of Cape Town, South Africa, coined the term catastrophic antiphospholipid syndrome (CAPS) [19]. It became clear that CAPS is a rare form of microangiopathic thrombosis that has shared features with thrombotic thrombocytopenic purpura, disseminated intravascular coagulation, and systemic inflammatory response syndrome (SIRS) as seen in sepsis. Because of these similarities, there is the possibility of both under- and overdiagnosis, especially in view of the lack of specificity of aPL. Multiorgan involvement is typical with a predilection for the lung, brain, and kidney. It may complicate previously diagnosed more typical APS or be the presenting manifestation, and there is often a trigger for the acute episode, such as infection or anticoagulant withdrawal.

LAC is now considered the most important acquired risk factor for thrombosis and fetal loss. The current definition of LAC is autoantibodies that in vitro inhibit PL-dependent coagulation reactions in the absence of specific coagulation factor inhibition.

2.2 Autoantigen

Individuals with LAC have circulating autoantibodies that inhibit blood coagulation. The autoantibodies are of the IgG or IgM isotype and are mainly directed against two phospholipid (PL)-binding plasma proteins, β2GPI and PT [20]. The presence of such inhibitors represents a well-recognized risk factor for venous and arterial thromboembolism, as well as pregnancy loss. These two proteins are mainly involved in the LAC phenomenon. β2GPI is a 326-amino-acid PL-binding protein with a molecular weight of 50 kDa belonging to the complement control protein superfamily.

Its plasma concentration is approximately 200 μg/ml, and as 40 % is bound to lipoproteins, it is also termed “apolipoprotein H.” Composed of five homologous “sushi” domains of approximately 60 amino acids and binding to anionic PL (aPL) with its fifth positively charged domain, it may have a role in the elimination of apoptotic cells from circulation.

Prothrombin (PT), a 579-amino-acid-long vitamin K-dependent glycoprotein with a molecular weight of 72 kDa, has central role in blood coagulation. Its plasma concentration is approximately 1.5 μmol/l. PT binds to PL through N-terminal γ-carboxyglutamic acid domain in the presence of calcium ions and is activated to thrombin by the prothrombinase complex (activated factor X, factor V, calcium ions, and negatively charged PL).

Both proteins can be purified from human plasma. Human β2GPI is purified from normal plasma by means of perchloric acid precipitation and affinity chromatography on heparin–sepharose followed by cation-exchange chromatography. β2GPI is a very conserved protein among species, and bovine and human β2GPI cannot be distinguished from an antigenic point of view.

PT is purified from normal plasma by precipitation with barium chloride, which is then removed by ammonium sulfate. The supernatant is then applied to an anion-exchange column, and PT is eluted with a gradient of sodium chloride. Human and bovine PT differ as bovine PT may not be recognized by some LACs.

2.3 Autoantibody

The LAC phenomenon is related to the presence of autoantibodies of IgG or IgM isotypes directed against β2GPI, PT, or both. This has been demonstrated by testing the anticoagulant properties of affinity-purified aβ2GPI antibodies on normal plasma. By prolonging PL-dependent coagulation tests (in particular diluted Russell viper venom time (dRVVT)), the action of these antibodies is inhibited by an excess of PL. It has been proposed that the molecular mimicry mechanism between infectious agents and the β2GPI molecule may generate anti-β2GPI antibodies [21].

Moreover, a strong homology exists between β2GPI-related peptides (target epitopes for anti-β2GPI antibodies) and different common pathogens. Furthermore, β2GPI polymorphism (in particular the Val 247 allele) has recently been associated with both a high frequency of anti-β2GPI antibodies and a stronger antibody reactivity than the Leu247 β2GPI allele. It is possible that this type of polymorphism may favor the occurrence of molecular mimicry. With regard to antiprothrombin antibodies, LAC activity was first demonstrated in the purified system consisting of human PT, factor Xa, factor Va, and calcium. The same properties have been shown by murine monoclonal antibodies against human β2GPI and PT. How these immunoglobulins determine thromboembolic events or obstetric complications is still unknown as many theories have been put forward but none are considered totally resounding. One possibility is that antibodies responsible for LAC in vitro also interfere with the in vivo function of PL-dependent anticoagulant pathways. In fact, PL-dependent inactivation of factors Va/VIIIa by the thrombomodulin–protein C–protein S system or by PL-dependent tissue factor (TF) inhibition by tissue factor pathway inhibitor (TFPI) may be impaired in the presence of LAC [22].

Alternatively, β2GPI (or annexin V, another putative autoantigen for LAC) may exert an anticoagulant effect in vivo, whereas autoantibodies to β2GPI (or annexin V) may damage it. Thrombus formation may thus take place on the surface of activated monocytes, platelets, or endothelial cells. While original, these theories are controverted by the fact that congenital β2GPI deficiency is not a risk factor for thrombosis.

Many autoimmune diseases are associated with genes in the major histocompatibility complex (MHC) region. MHC class II alleles (HLA-DR and DQ) may be involved in the genetic predisposition to develop LAC. HLA-DQw7 (DQB1*0301), linked to HLA-DR5 and -DR4 haplotypes, was significantly increased in LAC-positive patients as compared to race-matched normal controls. Moreover, the HLADQB1*0302 (DQ8) allele, typically carried on HLA-DR4 haplotypes, was associated with anti-β2GPI, but there are variations in HLA associations in different ethnic groups. The HLA-DPB1 locus may also contribute to the genetic predisposition to develop anti-β2GPI and clinical manifestations of APS in patients with SLE, but this association is unclear.

Previous criteria for the diagnosis of LAC were those proposed by Brandt in 1995 [23]: prolongation in at least one PL-dependent coagulation test out of two or more screening assays (activated partial thromboplastin time (aPTT), kaolin clotting time (KCT), dRVVT, diluted PT time (dPT)).

2.4 LAC Detection

According to the Brandt guidelines, in our laboratory dRVVT was determined essentially [2425], by using a single batch of reagents and an automatic coagulation analyzer. The screening test was considered positive when a 1:1 mixture of test plasma and normal plasma yielded a clotting time exceeding 42″. This cutoff value was usually calculated by using the 95th percentile of coagulation times obtained in frozen plasma samples from healthy subjects and corresponded to a ratio to normal plasma of 1.19. Results were confirmed in patient plasmas by test normalizing clotting time (confirming test), which has been reported to be diagnostic also in patients on oral anticoagulant treatment [2627]. KCT was determined using 2 % kaolin in distilled water [25] and was considered positive when a 1:1 mixture of normal and patient plasma yielded a coagulation time greater than 159″. This meant a ratio to pooled normal plasma of 1.31 (the cutoff value is calculated as for dRVVT). To exclude factor deficiency, test plasma was mixed with pooled plasma at a ratio of 1:1. A confirmatory test using an excess of PL or activated platelets (platelet neutralization procedure) or exagonal PL will neutralize the anticoagulant effect, demonstrating that the inhibitor is actually sensitive to PL. In any case, the presence of other inhibitors or heparin, which interferes with most LAC assays, should be excluded (Fig. 2.1).

A322602_1_En_2_Fig1_HTML.jpg

Fig. 2.1

Flow chart for diagnosis of LAC

Numerous variables affect screening tests for LAC detection. The low content of PL renders the test more sensitive, and KCT can be considered the most sensitive as no external PL is added to the assay, with the only available PL being those present in the plasma tested. Unfortunately, KCT is hard to automate, as most photo-optical devices cannot be used in the presence of the kaolin reagent, which tends to scatter light. The presence of platelets in this and in other assays greatly affects the results, particularly when plasma is frozen before testing. Centrifuging fresh plasma twice or using a double filter (through a 0.2-μm filter) may get around this problem. aPTT is a general coagulation screening test, KCT and dPT are used less frequently, and dRVVT has become the most commonly used screening assay for detection of LAC. We have modified the original test by diluting both the venom and the PL, thereby greatly increasing its sensitivity. Because Russell viper venom activates factor X directly, the test is normal in patients with some factor deficiency and in those with an inhibitor to factor VIII, the most common coagulation inhibitor associated with a bleeding tendency.

The performance of laboratories across the world to detect LA is a matter of concern. Misclassification of positive or negative LA plasma sample is commonly encountered in external surveys. Recently, in a survey of centralized LA diagnosis, we reported that about 33 % of plasma samples collected by thrombosis centers and labeled locally as LA positive were reported as LA negative in a central laboratory [28].

The poor laboratory performance is due to the lack of standardized tests and/or inappropriate application of the diagnostic criteria. Recently, the Brandt guidelines were updated [29]. Particular emphasis was given to several aspects discussed in this official communication. A new paragraph was dedicated to the patient selection. Testing for LAC should be limited to patients who have a significant probability of having the antiphospholipid syndrome (APS). Appropriateness to search for LAC can be graded according to clinical characteristics into low, moderate, and high.

·               High: unprovoked venous thromboembolism (VTE) and (unexplained) arterial thromboembolism (ATE) in young patients (<50 years of age), thrombosis at unusual sites, late pregnancy loss, any thrombosis, or pregnancy morbidity in patients with autoimmune diseases (systemic lupus erythematosus, rheumatoid arthritis, autoimmune thrombocytopenia, autoimmune hemolytic anemia)

·               Moderate: accidentally found prolonged aPTT in asymptomatic subjects, recurrent spontaneous early pregnancy loss, and provoked VTE in young patients

·               Low: VTE or ATE in elderly patients

Modalities for blood collection and processing are fully delineated, and the choice of tests is limited to dRVVT and a sensitive aPTT. Calculation of cutoff values for each diagnostic step are clearly stated. To avoid misinterpretation, it is recommended to perform laboratory procedures 1–2 weeks after discontinuation of treatment or when the INR is less than 1.5. Bridging VKA discontinuation with LMWH is recommended with the last dose of LMWH administered more than 12 h before the blood is drawn for LAC testing. LAC result should always be considered in the context of a full laboratory aPL profile comprising aCL and aβ2GPI antibodies ELISAs. The presence of medium- to high-titer aCL and aβ2GPI of the same isotype (most often IgG) is in agreement with a positive LAC and identifies patients at high risk for thrombosis and fetal losses [30]. Diagnostic steps are confirmed to be those of previous guidelines: (a) screening step, (b) mixing test, and (c) confirmatory test.

Results of screening tests are potentially suggestive of LA when their clotting times are longer than the local cutoff value. Results should be expressed as follows:

 $$ \mathrm{dRVVT}\;\mathrm{Screen}\;\mathrm{Ratio}=\frac{\mathrm{Patient}\;\mathrm{dRVVT}\;\mathrm{Screen}\;\mathrm{results}\;\left(\mathrm{in}\;\mathrm{seconds}\right)}{\mathrm{PNP}\;\mathrm{dRVVT}\;\mathrm{Screen}\;\left(\mathrm{in}\;\mathrm{seconds}\right)} $$

Perform testing on patient plasmas mixed with the pooled normal plasma (PNP) at 1:1 proportion and express results as follows:

 $$ \mathrm{dRVVT}\;\mathrm{mixing}\;\mathrm{Ratio}=\frac{\mathrm{Patient}\;\mathrm{dRVVT}\;\mathrm{mixing}\;\left(1:1\right)}{\mathrm{PNP}\;\mathrm{dRVVT}\;\left(\mathrm{in}\;\mathrm{seconds}\right)} $$

Confirmatory Test: Results are confirmatory of LA if the % correction of patient’s plasma at low (screen) and high (confirm) phospholipid concentration is above the local cutoff value.

Sensitive aPTT: same procedure as dRVVT should be used.

2.5 Significance of Positive LAC

LAC is an important test in vascular medicine, since its detection on two occasions 12 weeks apart in a patient with venous or arterial thromboembolism allows us to suspect the antiphospholipid syndrome (APS), and this in turn determines a different approach to secondary prevention and treatment. As thrombosis is a common event, laboratory diagnosis of LAC becomes crucial in identifying patients with APS.

When medium- to high-titer beta-2 glycoprotein I-dependent anticardiolipin antibodies and LAC are both present in the same patient’s plasma, a complete positive antiphospholipid profile may render physicians more confident with the diagnosis of APS. However, APS diagnosis becomes problematic, due to the abovementioned laboratory pitfalls, when LAC is the only positive test among those used to study antiphospholipid antibodies. Moreover, if antihuman prothrombin antibodies are responsible for LAC, they are poorly associated with thromboembolic events [31]. Therefore, the development of simple coagulation tests improving the clinical significance of positive LA is important [32].

Among the so-called antiphospholipid antibodies, the presence of LAC is strongly associated with thrombosis and obstetric complications. Thromboembolic complications, venous thromboembolism (deep vein thrombosis and/or pulmonary embolism), and cerebral ischemia (transient ischemic attack (TIA) or stroke) are the most frequent. In a systematic review of the literature, LAC has been shown to be statistically associated with both venous and arterial thromboembolism with an odds ratio ranging from 4.09 to 16.2. Obstetric complications include fetal death, preeclampsia or eclampsia, and multiple abortions. The association of other antiphospholipid antibodies such as anticardiolipin antibodies (aCL) and aβ2GPI with the clinical manifestations described above is less striking. The contemporaneous positivity in coagulation (LAC) and solid-phase assays (aCL and aβ2GPI antibodies) is due to the presence of a common pathogenic group of aβ2GPI antibodies.

As LAC is less sensitive with respect to solid-phase assays, triple positivity suggests that the aβ2GPI antibodies present in plasma are able to express LAC activity. In the absence of standardization and reference material for testing, an antiphospholipid laboratory profile could help to better classify these patients [33]. With this in mind we examined the results of all the three tests obtained in 618 consecutive subjects and compared subjects with previously documented thrombosis-related events with those without, for the most part, normal subjects. When antiphospholipid antibody profiles, instead of individual test positivity, were considered in a multivariate analysis taking into account age, gender, the presence of SLE or other autoimmune diseases, and established risk factors for venous and arterial thromboembolism, triple positivity was found to be a strong independent risk factor (odds ratio 33.3, confidence interval 7.0–157.6). Significance was maintained when an association with venous or arterial thromboembolism was considered. Double positivity with negative LAC was a highly significant risk factor for obstetric complications only (odds ratio 10.8, confidence interval 2.9–40.8). Other combinations were not statistically significant. The analysis of a complete antiphospholipid antibody profile, as compared to a single testing, can thus better identify patients at risk. In fact, it is a common experience that some isolated and often transient episodes of LAC positivity, such as those not infrequently found in children or young adults during a coagulation screening before surgery, are benign and not associated with thromboembolism. In the same way, isolated aCL positivity can easily be found in infectious diseases where no association with thrombosis has been reported. It is important to underline that only some antibodies to a specific domain of β2GPI express LAC activity and correlate strongly with thromboembolic events [34]. Other autoantibodies to β2GPI may not be pathogenic, and this may explain why studies on aβ2GPI antibody detection in solid-phase assays do not produce uniform results as IgG aβ2GPI is associated with thrombosis in only a subset of patients. In those cases (from 2 to 10 %) in which aβ2GPI is the sole antibody detected in patients with clinical manifestations of APS, it may not be pathogenic. In conclusion, a complete pattern of aPL antibodies comprising LAC, aCL, and aβ2GPI is important for identifying pathogenic aβ2GPI antibodies and the associated risk of clinical manifestations.

The prevalence of LAC in the normal population is reported to be 3.6 % according to screening using the KCT, but it may be much less. The prevalence of LAC in the SLE population has been reported to be between 10 and 50 %, depending on the test used [35].

The lupus anticoagulant is detected according to a set of diagnostic criteria recommended by the Scientific and Standardization Committee of the International Society of Thrombosis and Haemostasis. The sensitivity and specificity of these procedures vary, depending on the type of test, reagents, instrumentation, cutoff values, and the expression of results utilized. Many surveys have been carried out to evaluate the performance of clinical laboratories in LAC diagnosis. A recent survey was carried out within the framework of activities of the Italian Federation of Anticoagulation Clinics using affinity-purified lyophilized IgG aβ2GPI from a single patient. Overall, 69, 68, and 59 out of 70 participants were able to detect LAC in plasma with high, intermediate, and low potency (sensitivity 99, 97, and 84 %).

Conversely, 69, 50, and 53 out of 70 were able to rule out LAC when examining normal, heparinized, and deficient plasma (specificity, 99, 71, and 76 %). This survey showed that sensitivity is satisfactory, whereas specificity should be improved.

We have recently demonstrated that LAC with associated aCL and aβ2GPI antibody positivity (triple positivity) is a risk factor for thromboembolic recurrence. Moreover, LAC in these patients constantly remains positive with time. Thromboembolic events may occur anywhere without provocation. It is the experience of authors that appropriate oral anticoagulant treatment is essential as recurrence is more frequent at the subtherapeutic international normalized ratio (i.e., INR <2.0). A first distinction in methods to detect LAC in relation to clinical manifestation of APS considered LAC to be clinically relevant when detected by means of dRVVT. In fact, the dRVVT rather than the KCT profile may better identify β2GPI-dependent LAC and is associated with thrombosis in LAC-positive patients. Coagulation tests assessing LAC can be improved by making them very sensitive to the presence of aβ2GPI antibodies. The mechanism underlying LAC activity of aβ2GPI antibodies is related to their ability to enhance binding of β2GPI to PL, thus impeding binding/activation of clotting factors. Since β2GPI binding to aPL as well as its enhanced binding to PL in the presence of specific autoantibodies might be influenced by calcium and ionic strength, we evaluated the effect of various calcium concentrations in common tests assessing the presence of LAC in patients with functionally active aβ2GPI antibodies. We were thus able to distinguish plasmas from patients with clinically relevant antihuman β2GPI LAC. A reduction in the final calcium concentration (from 10 to 5 mM) increased coagulation times in both dRVVT and dilute dPT when plasmas of patients with aβ2GPI antibodies were used. Instead, all LAC-positive antiβ2GPI antibody-negative patients showed decreased coagulation times. These results were confirmed by running dRVVT of normal plasma spiked with affinity-purified IgG anti-aβ2GPI antibodies. Thus when a PL-dependent coagulation test is run twice at different final calcium concentrations, aβ2GPI LAC can be identified. Another test system to discriminate between LAC caused by anti-β2GPI from those due to antiprothrombin antibodies has recently been described [32]. In an aPTT-based test, the addition of cardiolipin vesicles shortened or prolonged coagulation time in the presence of aβ2GPI or antiprothrombin LAC, respectively.

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