AbstractClinical studies have reported different diagnostic/predictive values of antibodies to domain 1 or 4/5 of β2glycoproteinI in terms of risk of thrombosis and pregnancy complications in patients with antiphospholipid syndrome. To obtain direct evidence for the pathogenic role of anti-domain 1 or anti-domain 4/5 antibodies, we analyzed the in vivo pro-coagulant effect of two groups of 5 sera IgG each reacting selectively with domain 1 or domain 5 in lipopolysaccharide (LPS)-treated rats. Antibody-induced thrombus formation in mesenteric vessels was followed by intravital microscopy, and vascular deposition of β2glycoproteinI, human IgG and C3 was analyzed by immunofluorescence. Five serum IgG with undetectable anti-β2glycoproteinI antibodies served as controls. All the anti-domain 1-positive IgG exhibited potent pro-coagulant activity while the anti-domain 5-positive and the negative control IgG failed to promote blood clot and vessel occlusion. A stronger granular deposit of IgG/C3 was found on the mesenteric endothelium of rats treated with anti-domain 1 antibodies, as opposed to a mild linear IgG staining and absence of C3 observed in rats receiving anti-domain 5 antibodies. Purified anti-domain 5 IgG, unlike anti-domain 1 IgG, did not recognize cardiolipin-bound β2glycoproteinI while being able to interact with fluid-phase β2glycoproteinI. These findings may explain the failure of anti-domain 5 antibodies to exhibit a thrombogenic effect in vivo, and the interaction of these antibodies with circulating β2glycoproteinI suggests their potential competitive role with the pro-coagulant activity of anti-domain 1 antibodies. These data aim at better defining “really at risk” patients for more appropriate treatments to avoid recurrences and disability.
Antiphospholipid syndrome (APS) is a chronic autoimmune disorder characterized by recurrent episodes of vascular thrombosis and adverse pregnancy outcomes in the presence of antibodies to phospholipid-binding proteins (aPL). It occurs either as a primary disease or concomitantly to other connective tissue diseases, particularly systemic lupus erythematosus (SLE).1 Although thrombotic occlusion may affect the vessels of all organs and tissues, common presentations of the syndrome are: a) deep vein thrombosis in the legs often complicated by pulmonary embolism; and b) thrombotic occlusion of cerebral and coronary arteries leading to stroke and myocardial infarction.2 This clinical condition is also associated with pregnancy morbidity, including fetal loss, pre-eclampsia, pre-term delivery, and ‘small for gestational age’ babies.3 These are serious complications that particularly affect young people, and have both social and economic impacts. The disease may sometimes present as catastrophic syndrome, a more severe form of APS characterized by microthrombosis of small vessels in various organs resulting in multiple organ failure.4 Anti-cardiolipin (aCL) and anti-β2glycoprotein I (β2GPI) antibodies and lupus anticoagulant (LA) activity are considered markers of APS and are included among the criteria currently proposed to classify the syndrome.1 Clinical studies have revealed an increased risk of thrombosis and pregnancy complications in patients with medium to high levels of these antibodies and LA present in their plasma.5 The triple positivity of these laboratory markers has also been shown to be associated with more severe forms of APS.5 Conversely, the positivity for a single marker is often associated with a much lower risk of the clinical manifestations of APS.95 It has been widely demonstrated that β2GPI is the main antigen recognized by aPL, and the reactivity against the protein has been shown to be responsible for the positivity for aCL and anti-β2GPI assays, and, in part, for the LA phenomenon strongly associated with the clinical manifestations of APS.10 β2GPI mainly circulates in blood in a circular form and is organized into four domains (D1-D4) composed of 60 amino acids with two disulfide bonds and a fifth domain (D5) containing an extra 24 amino acids that interact with anionic phospholipids on the target cells/tissues.11 Besides the classical diagnostic assays measuring antibodies against whole molecule β2GPI, new tests have recently been developed to detect anti-β2GPI antibody subpopulations reacting with different domains of the protein, particularly the combined domains D4/5 and domain 1 (D1).1412975
In APS patients, a large proportion of anti-β2GPI antibodies react with D1 and recognize a cryptic epitope (Arg39–Arg43) in the native molecule exposed after its interaction with anionic phospholipids1513 or oxidation.1816 Antibodies directed against D1 of β2GPI with or without anti-D4/5 antibodies have frequently been found in APS patients associated with an increased risk of thrombosis and pregnancy complications.241997 In contrast, isolated high levels of anti-D4/5 antibodies have been reported in non-APS patients with leprosy, atopic dermatitis, atherosclerosis and in children born to mothers with systemic autoimmune diseases;6 high levels have also been found in asymptomatic aPL carriers although these antibodies are not associated with either vascular or obstetric manifestations of the APS syndrome.97 This finding prompted some authors to suggest that the ratio between anti-D1 and anti-D4/5 may be a useful parameter for identifying autoimmune APS and for ranking the patients according to their risk of developing the syndrome.7
An isolated positivity for anti-D4/5 is a rare condition and is usually associated with the absence of aCL and/or LA. In the majority of cases, there is some doubt as to the APS clinical profile and classification/diagnostic criteria are not fulfilled.25 The finding that antibodies with this isolated specificity are observed mainly in the absence of clinical manifestations of hypercoagulable states has suggested that they may not be involved in thrombus formation.
The in vivo pathogenic role of aPL has been demonstrated for those directed against the whole molecule and against D1 of β2GPI using animal models of thrombosis developed in rats and mice.2826 However, at present, there is no direct evidence that antibodies to D4/5 do not play an in vivo pathogenic role in blood clotting, nor is it clear whether they are able to interact with soluble or surface-bound β2GPI. Data indicating that the antibodies are ineffective in causing blood clot due to their failure to recognize bound β2GPI will be reported.
Two groups of anti-β2GPI positive sera277 containing isolated antibodies to either D1 or D4/5 domains76 and control sera with undetectable anti-β2GPI antibodies were analyzed. All samples were also tested for aCL antibodies7 and LA activity.29 The anti-D1-positive sera were obtained from APS patients.1 The sera were collected after obtaining informed consent and the IgG were purified by a Protein G column (HiTrap Protein G HP, GE Healthcare) as described.27 The local Istituto Auxologico Italiano ethical committee approved the study.
Purification of β2 glycoprotein I and generation of recombinant domains D4 and D5
Methods of purification of human β2GPI from pooled normal sera and the generation of D4 and D5 domains have been published previously.31302712 Sequence analysis was performed as described32 and compared to the published sequence of β2 GPI.33 The fine specificity against D4 or D5 was investigated by ELISA.27
An in vivo model of antibody-induced thrombus formation was established in male Wistar rats (270-300 g) kept under standard conditions in the Animal House of the University of Trieste, Italy, as previously reported in detail.26 The in vivo procedures were performed in compliance with the guidelines of European (86/609/EEC) and Italian (Legislative Decree 116/92) laws and were approved by the Italian Ministry of University and Research and the Administration of the University Animal House. This study was conducted in accordance with the Declaration of Helsinki. Further details are available in the Online Supplementary Methods.
The mesenteric tissue was collected from rats at the end of the in vivo experiment.26 Deposits of β2GPI were analyzed using the biotinylated monoclonal antibody MBB2 and FITC-labeled streptavidin (Sigma-Aldrich).27 IgG and C3 were detected using FITC-labeled goat anti-human IgG (Sigma-Aldrich) and goat anti-rat C3 (Cappel/MP Biomedicals) followed by FITC-labeled rabbit anti-goat IgG (Dako), respectively. The slides were examined using a DM2000 fluorescence microscope equipped with a DFC 490 photo camera and Application Suite software (Leica).
Antibody binding assays
Different concentrations of β2GPI were added to CL-coated plates and the reactivity of IgG with CL-bound β2GPI was measured.7 The interaction of IgG with soluble β2GPI was evaluated by incubating IgG with increasing concentrations of β2GPI or bovine serum albumin (BSA) as unrelated antigen for one hour (h) at 37°C followed by overnight incubation at 4°C in a rotator. The samples were centrifuged at 3000 g for 5 minutes (min) at room temperature and the residual un-complexed antibodies were tested using β2GPI-coated plates (Combiplate EB, Labsystems) as described.7 Further details are available in the Online Supplementary Methods.
Statistical analysis was performed using GraphPad Prism 6.0 for Windows. The domain reactivity of the anti-β2GPI D4/5 positive sera was expressed as mean+Standard Deviation (SD) and analyzed with the paired Student t-test. Data from in vivo thrombus formation were compared by Dunnett test. The interaction between IgG and β2GPI bound to CL was analyzed with the Kruskall-Wallis with Dunn post-hoc test. The interaction between IgG and soluble β2GPI was expressed as median and interquartile range and analyzed with the two-way repeated measure ANOVA with Sidak post-hoc test. Probabilities of <0.05 were considered statistically significant.
Antibody to phospholipid-binding protein profile of the serum samples
Anti-β2GPI IgG titers were comparable in the anti-D4/5-and anti-D1-positive samples [1.04±0.26 Optical Density (OD) and 1.46±0.48 OD, mean+SD, respectively]. The isolated anti-D4/5-positive samples displayed anti-D4/5 levels of 50.67±9.86 arbitrary units (AU) (mean±SD) while they were negative for aCL (<10 GPL) and LA. The isolated anti-D1-positive samples showed anti-D1 levels of 75.36±17.15 AU (mean±SD), high titers of IgG aCL (124.4±46.9 GPL, mean±SD), and displayed LA activity. Control samples were negative in all the assays. The purified IgG fractions maintained the antigen specificity shown in the whole serum. Clinical and serological data of all the subjects/patients included in the study are reported in Online Supplementary Table S1.
Fine epitope-specificity of antibodies to domains 4/5
The IgG against D4/5 used in this study were selected for their ability to react with the combined domains obtained from INOVA Diagnostics, but it was unclear whether they recognized one or the other domain or both. To clarify this point, we assessed the reactivity of serum IgG towards recombinant D4 and D5. The amino acid sequences of the two domains are reported in Online Supplementary Figure S1. The results presented in Figure 1 clearly show that all the anti-D4/5 reacted with D5 and did not recognize D4. The difference in the reactivity of the various sera IgG towards D4/5 is essentially similar to that observed in their reaction with D5.
Antibodies to domain 5 fail to cause thrombus formation in vivo
To evaluate the pro-coagulant activity of sera containing antibodies to different domains of β2GPI, two groups of serum IgG positive for either D1 or D5 domains were analyzed for their ability to induce thrombus formation followed in vivo by intravital microscopy. IgG from sera negative for antibodies to β2GPI served as a control group. All anti-D1-positive IgG induced blood clots that could be seen from 15 min after serum infusion (Figure 2). Their number progressively increased to reach the highest value after 1 h and was maintained thereafter for up to 90 min. Thrombus formation was associated with vascular occlusion that resulted in a marked decrease, and, in some vessels, in a complete blockage of blood flow. Conversely, the anti-D5-positive IgG did not exhibit pro-coagulant activity and failed to cause reduced blood flow. The latter results were not statistically different from those of anti-β2GPI-negative blood donors at each time point. On the contrary, the data of anti-D1 IgG were statistically different from those of anti-β2GPI-negative samples at all times starting from 15 min of analysis (P<0.05).
Antibodies to domain 5 fail to interact with surface-bound β2glycoprotein I
Having observed an absence of intravascular coagulation in rats that had received anti-D5-positive IgG, we decided to investigate whether this was due to the inability of the antibodies to interact with endothelium-bound β2GPI. To this end, samples of ileal mesentery were analyzed for the presence of β2GPI, human IgG and C3. As expected from our previous findings,30 β2GPI was detected on the vessel endothelium of rats primed with LPS (Figure 3), while it was totally absent in unprimed animals (data not shown). A search for IgG and C3 revealed marked granular deposits of both proteins on endothelial cells of rats treated with anti-D1 IgG, while a milder linear staining for IgG and absence of C3 were observed in rats receiving anti-D5 IgG (Figure 3). The animals treated with anti-β2GPI-negative sera showed negligible staining for IgG and undetectable C3 (Figure 3). Since several molecules other than β2GPI are expressed on the endothelial cell surface and represent potential targets for human IgG, we set out to determine whether the fluorescence was due to the IgG specifically directed against β2GPI. To do this, we set up a β2GPI-dependent CL assay in which the β2GPI supplementation was carried out by adding human purified β2GPI at increasing concentrations instead of fetal calf serum. The system allowed us to test the IgG reactivity with β2GPI added at different concentrations to the CL-plates. The anti-D1 IgG reacted with the β2GPI molecule most likely by recognizing the D1 epitope exposed on the β2GPI molecule following its binding to CL (Figure 4). The IgG level detected in the assay varied in different patients and was related to the concentration of β2GPI used to coat CL. In contrast, anti-D5 IgG failed to interact with CL-bound β2GPI even at the highest concentration of β2GPI, suggesting that D5 domains were not accessible to the antibodies under these experimental conditions. Like the anti-D5 antibodies, in the assay, the IgG from control sera were negative.
Antibodies to domain 5 interact with soluble β2glycoprotein I
Electron microscopy studies have revealed that β2GPI adopts a circular form in plasma and that this is maintained by the interaction of D1 with D5.34 This special conformation prevents the access of autoantibodies to hidden epitopes on D119 and predicts the presence of cryptic epitopes on D5, though this has not been formally proven.35 We first decided to examine the in vivo interaction of the antibodies with circulating β2GPI and the effect of this interaction on β2GPI bound to vascular endothelium. To this purpose, the in vivo model was slightly modified administering IgG intraperitoneally followed 15 h later by LPS given by the same route; this approach would allow sufficient time for the antibodies to react with the target antigen prior to the binding of β2GPI to vascular endothelium promoted by LPS. The IgG from two sera with relatively high levels of antibodies to D1 and D5, respectively, and from an anti-β2GPI-negative serum were tested and the amount of vascular deposits of β2GPI and IgG was evaluated. As expected, the rat treated with anti-D1 developed endovascular thrombi associated with deposition of IgG, both of which were undetectable in animals that received anti-D5-positive or anti-β2GPI-negative IgG (Figure 5). Analysis of the ileal mesentery showed that β2GPI was present on the vascular endothelium of the animals that received the three IgG fractions with no clear difference in the staining intensity observed in the rats treated with anti-D5 and anti-D1 IgG (Figure 5).
Since the in vivo data did not provide convincing evidence of the ability of anti-D5 to prevent binding of circulating β2GPI to vascular endothelium, we decided to further investigate this issue using an in vitro inhibition assay. IgG purified from anti-D5-positive, anti-D1-positive or anti-β2GPI-negative sera were incubated with increasing concentrations of soluble β2GPI and the residual IgG interacting with β2GPI directly bound to the plate wells were measured. The amount of IgG anti-D5 free to bind to solid-phase β2GPI after incubation with the soluble molecule decreased compared to that of the IgG incubated with BSA, particularly at a higher concentration of soluble β2GPI (Figure 6). In contrast, the level of IgG anti-D1 bound to solid-phase β2GPI following incubation with soluble β2GPI was slightly lower, but not significantly different from that of the IgG incubated with BSA.
Antiphospholipid syndrome is now recognized as an antibody-dependent and complement-mediated syndrome and antibodies to β2GPI have been identified as important players in thrombus formation in APS patients.10 Efforts are being made to determine the clinical relevance of antibodies to D1 and D4/5 domains of the molecule detected in these patients. Clinical studies have suggested that antibodies to D4/5, unlike those directed against D1, do not represent a risk factor for thrombosis and pregnancy complications.1497 The in vivo data presented here focused on the thrombotic aspect of the syndrome and support the clinical observation that the anti-D4/5 antibodies are pathologically irrelevant.
The animal model used in this and in previous studies proved to be an invaluable tool to investigate the ability of the anti-β2GPI antibodies to induce blood clots in rats primed with LPS that provides the first hit, followed by the infusion of the antibodies acting as a second hit.10 As expected, all anti-D1 IgG promoted thrombus formation and vascular occlusion, confirming the pathogenicity of these antibodies suggested by clinical observations. It is possible that LA detected in the plasma of these patients may have also contributed to anti-β2GPI-induced blood clots. However, although β2GPI antibody-dependent LA has been shown to correlate with the increased risk of thrombosis,361413 evidence supporting the in vivo prothrombotic activity of LA independently of anti-β2GPI antibody has not yet been provided. Instead, there is good evidence that the antibodies recognizing the D1 domain of β2GPI are directly involved in thrombus formation and vessel occlusion. We have previously shown that a human monoclonal antibody that recognizes D1 induces blood clots and that a CH2-deleted non-complement fixing variant molecule competes with anti-β2GPI antibodies from APS patients and prevents their pro-coagulant activity.27 A similar inhibitory effect was obtained using recombinant D1 to control the thrombus enhancement activity of aPL in mice.37
The in vivo experiments showed that none of the anti-D5 IgG exhibited a prothrombotic activity supporting the observations made in clinical studies that these antibodies are pathologically irrelevant.147 A possible explanation for this finding is the inability of these antibodies to interact with cell-bound β2GPI. In line with this hypothesis, we showed that anti-D5-positive IgG fractions were unable to react with β2GPI bound to CL-coated plates in vitro because of the shielding of D5 in the β2GPI molecule bound to the CL-coated plate. However, in rats treated with LPS (used to promote binding of β2GPI) and anti-D5 IgG, the mild staining for IgG observed on the endothelium of mesenteric vessels did not allow any definite conclusions to be drawn on this issue. It must be emphasized, however, that the staining intensity varied among different sera and was not related to the level of antibodies. The linear deposition of IgG on the mesenteric endothelium from rats treated with anti-D5-positive IgG suggests their interaction with antigens constitutively expressed on endothelial cells. This distribution pattern differs from the irregular staining for IgG seen with the anti-D1-positive IgG most likely explained by their reaction with a plasma-derived molecule, such as β2GPI, bound to the endothelial cell surface. The different distribution of anti-D1 and anti-D5 IgG resembles the well-known difference in the granular and linear distribution patterns of IgG observed in the kidney of patients with SLE and Goodpasture syndrome, respectively. The linear pattern of IgG in Goodpasture is the result of the interaction of the antibodies with their target antigen constitutively expressed on the glomerular basement membrane. In contrast, the granular distribution of IgG in SLE is caused by irregular deposition of circulating immune complexes.3938 The finding that C3 deposition was undetectable on the vascular endothelium of rats treated with anti-D5 IgG is consistent with the failure of these antibodies to induce thrombus formation. We and others have provided convincing evidence that complement activation is critically involved in the coagulation process induced by anti-β2GPI IgG and in this study by antibodies to the D1 domain.43402726
The anti-D4/D5 antibodies present in the sera analyzed in this study selectively recognized the recombinant D5 domain and are likely to inhibit deposition of β2GPI on the endothelium by shielding its binding site for the anionic phospholipid on endothelial cells.44 Our attempt to document ex vivo reduced binding of circulating β2GPI to vascular endothelium of the anti-D5-treated rats was unsatisfactory; this was most likely due to a much higher level of serum β2GPI compared to that of injected antibodies in vivo. The in vitro data obtained under more controlled conditions of IgG and β2GPI concentrations showed a fluid phase interaction between anti-D5 IgG and soluble β2GPI, resulting in a significantly reduced reactivity of these antibodies against surface-bound β2GPI (when the molecule was bound to a plate).
The finding that anti-D5 IgG have no pro-coagulant effect in our in vivo model has important clinical implications suggesting that individuals with isolated presence of these antibodies should not be considered to be at risk of thrombosis. It should be pointed out, however, that anti-D1 and anti-D5 IgG often co-exist in a large proportion of APS patients, and that they are likely to be susceptible to anti-D1-dependent thrombus formation. In view of the ability of the anti-D5 IgG to interact with soluble β2GPI, thus preventing its binding to the target cells, it is tempting to speculate that the anti-D5 IgG may antagonize the pro-coagulant activity of anti-D1 antibodies, according to antibody levels. In accordance with this, we recently published data indicating that patients positive for anti-D1 and anti-D4/5 antibodies have a lower risk of thrombosis if the levels of anti-D4/5 are higher than those of anti-D1 antibodies.97 Overall, our experimental findings fit with the clinical observation and offer new tools for stratifying patients into different risk categories. This would help in better preventing recurrences of the clinical manifestations and avoiding overtreatment, thus ultimately improving the patients’ quality of life and sparing them treatment side-effects.
In conclusion, the data presented in this work indicate that, unlike the anti-D1 positive sera, those containing antibodies against D5 are unable to induce clot formation and vascular occlusion. The failure of the anti-D5 antibodies to promote coagulation is due mainly to their inability to interact with the target epitopes hidden on the surface-bound molecule, and possibly to the recognition of native β2GPI in plasma that may, to some extent, potentially prevent its binding to the surface of activated endothelial cells. The detection of anti-D5 antibodies in patients with a doubtful APS clinical profile and a single positivity for anti-β2GPI in the absence of a positive aCL assay may offer a valuable tool for ruling out a definite APS diagnosis and for identifying subjects at lower risk of clinical manifestations.
The authors would like to thank Linda Vuch, Luca De Maso and Paola A. Lonati for their valuable technical contribution; Michael Mahler, Gary Norman and Filippo Sarra (INOVA Diagnostics and Werfen Italia) for their support.
- ↵PD and CG contributed equally to this work. PLM and FT contributed equally to this work.
- Check the online version for the most updated information on this article, online supplements, and information on authorship & disclosures: www.haematologica.org/content/104/4/819
- FundingThis work was partially supported by Istituto Auxologico Italiano, Ricerca Corrente 2016 (PLM).
- Received May 22, 2018.
- Accepted November 14, 2018.
- Miyakis S, Lockshin MD, Atsumi T. International consensus statement on an update of the classification criteria for definite antiphospholipid syndrome (APS). J Thromb Haemost. 2006; 4(2):295-306. PubMedhttps://doi.org/10.1111/j.1538-7836.2006.01753.xGoogle Scholar
- Gerosa M, Meroni PL, Erkan D. Recognition and management of antiphospholipid syndrome. Curr Opin Rheumatol. 2016; 28(1):51-59. Google Scholar
- Chighizola CB, Andreoli L, de Jesus GR. The association between antiphospholipid antibodies and pregnancy morbidity, stroke, myocardial infarction, and deep vein thrombosis: a critical review of the literature. Lupus. 2015; 24(9):980-984. PubMedhttps://doi.org/10.1177/0961203315572714Google Scholar
- Rodriguez-Pinto I, Moitinho M, Santacreu I. Catastrophic antiphospholipid syndrome (CAPS): Descriptive analysis of 500 patients from the International CAPS Registry. Autoimmun Rev. 2016; 15(12):1120-1124. Google Scholar
- Pengo V, Bison E, Denas G, Jose SP, Zoppellaro G, Banzato A. Laboratory Diagnostics of Antiphospholipid Syndrome. Semin Thromb Hemost. 2018; 44(5):439-444. Google Scholar
- Andreoli L, Nalli C, Motta M. Anti-beta(2)-glycoprotein I IgG antibodies from 1-year-old healthy children born to mothers with systemic autoimmune diseases preferentially target domain 4/5: might it be the reason for their ‘innocent’ profile?. Ann Rheum Dis. 2011; 70(2):380-383. PubMedhttps://doi.org/10.1136/ard.2010.137281Google Scholar
- Andreoli L, Chighizola CB, Nalli C. Clinical characterization of antiphospholipid syndrome by detection of IgG antibodies against beta2-glycoprotein I domain 1 and domain 4/5: ratio of anti-domain 1 to anti-domain 4/5 as a useful new biomarker for antiphospholipid syndrome. Arthritis Rheumatol. 2015; 67(8):2196-2204. Google Scholar
- Bertolaccini ML, Sanna G. The Clinical Relevance of Noncriteria Antiphospholipid Antibodies. Semin Thromb Hemost. 2018; 44(5):453-457. Google Scholar
- Chighizola CB, Pregnolato F, Andreoli L. Beyond thrombosis: Anti-beta2GPI domain 1 antibodies identify late pregnancy morbidity in anti-phospholipid syndrome. J Autoimmun. 2018; 90:76-83. Google Scholar
- Meroni PL, Borghi MO, Raschi E, Tedesco F. Pathogenesis of antiphospholipid syndrome: understanding the antibodies. Nat Rev Rheumatol. 2011; 7(6):330-339. PubMedhttps://doi.org/10.1038/nrrheum.2011.52Google Scholar
- de Laat B, Mertens K, de Groot PG. Mechanisms of disease: antiphospholipid antibodies-from clinical association to pathologic mechanism. Nat Clin Pract Rheumatol. 2008; 4(4):192-199. PubMedhttps://doi.org/10.1038/ncprheum0740Google Scholar
- Iverson GM, Victoria EJ, Marquis DM. Anti-beta2 glycoprotein I (beta2GPI) autoantibodies recognize an epitope on the first domain of beta2GPI. Proc Natl Acad Sci U S A. 1998; 95(26):15542-15546. PubMedhttps://doi.org/10.1073/pnas.95.26.15542Google Scholar
- de Laat B, Derksen RH, Urbanus RT, de Groot PG. IgG antibodies that recognize epitope Gly40-Arg43 in domain I of beta 2-glycoprotein I cause LAC, and their presence correlates strongly with thrombosis. Blood. 2005; 105(4):1540-1545. PubMedhttps://doi.org/10.1182/blood-2004-09-3387Google Scholar
- Pengo V, Ruffatti A, Tonello M. Antibodies to Domain 4/5 (Dm4/5) of beta2-Glycoprotein 1 (beta2GP1) in different antiphospholipid (aPL) antibody profiles. Thromb Res. 2015; 136(1):161-163. PubMedhttps://doi.org/10.1016/j.thromres.2015.04.031Google Scholar
- de Groot PG, Urbanus RT. The significance of autoantibodies against beta2-glycoprotein I. Blood. 2012; 120(2):266-274. PubMedhttps://doi.org/10.1182/blood-2012-03-378646Google Scholar
- Ioannou Y, Zhang JY, Passam FH. Naturally occurring free thiols within beta 2-glycoprotein I in vivo: nitrosylation, redox modification by endothelial cells, and regulation of oxidative stress-induced cell injury. Blood. 2010; 116(11):1961-1970. PubMedhttps://doi.org/10.1182/blood-2009-04-215335Google Scholar
- Passam FH, Rahgozar S, Qi M. Beta 2 glycoprotein I is a substrate of thiol oxidoreductases. Blood. 2010; 116(11):1995-1997. PubMedhttps://doi.org/10.1182/blood-2010-02-271494Google Scholar
- Ioannou Y. The Michael Mason Prize: Pathogenic antiphospholipid antibodies, stressed out antigens and the deployment of decoys. Rheumatology (Oxford). 2012; 51(1):32-36. PubMedhttps://doi.org/10.1093/rheumatology/ker353Google Scholar
- de Laat B, Derksen RH, van Lummel M, Pennings MT, de Groot PG. Pathogenic anti-beta2-glycoprotein I antibodies recognize domain I of beta2-glycoprotein I only after a conformational change. Blood. 2006; 107(5):1916-1924. PubMedhttps://doi.org/10.1182/blood-2005-05-1943Google Scholar
- de Laat B, Pengo V, Pabinger I. The association between circulating antibodies against domain I of beta2-glycoprotein I and thrombosis: an international multicenter study. J Thromb Haemost. 2009; 7(11):1767-1773. PubMedhttps://doi.org/10.1111/j.1538-7836.2009.03588.xGoogle Scholar
- Mahler M, Norman GL, Meroni PL, Khamashta M. Autoantibodies to domain 1 of beta 2 glycoprotein 1: a promising candidate biomarker for risk management in antiphospholipid syndrome. Autoimmun Rev. 2012; 12(2):313-317. PubMedhttps://doi.org/10.1016/j.autrev.2012.05.006Google Scholar
- Pengo V, Ruffatti A, Tonello M. Antiphospholipid syndrome: antibodies to Domain 1 of beta2-glycoprotein 1 correctly classify patients at risk. J Thromb Haemost. 2015; 13(5):782-787. PubMedhttps://doi.org/10.1111/jth.12865Google Scholar
- Chaturvedi S, McCrae KR. Clinical Risk Assessment in the Antiphospholipid Syndrome: Current Landscape and Emerging Biomarkers. Curr Rheumatol Rep. 2017; 19(7):43. Google Scholar
- Ioannou Y, Pericleous C, Giles I, Latchman DS, Isenberg DA, Rahman A. Binding of antiphospholipid antibodies to discontinuous epitopes on domain I of human beta(2)-glycoprotein I: mutation studies including residues R39 to R43. Arthritis Rheum. 2007; 56(1):280-290. PubMedhttps://doi.org/10.1002/art.22306Google Scholar
- Roggenbuck D, Borghi MO, Somma V. Antiphospholipid antibodies detected by line immunoassay differentiate among patients with antiphospholipid syndrome, with infections and asymptomatic carriers. Arthritis Res Ther. 2016; 18(1):111. Google Scholar
- Fischetti F, Durigutto P, Pellis V. Thrombus formation induced by antibodies to beta2-glycoprotein I is complement dependent and requires a priming factor. Blood. 2005; 106(7):2340-2346. PubMedhttps://doi.org/10.1182/blood-2005-03-1319Google Scholar
- Agostinis C, Durigutto P, Sblattero D. A non-complement-fixing antibody to beta2 glycoprotein I as a novel therapy for antiphospholipid syndrome. Blood. 2014; 123(22):3478-3487. PubMedhttps://doi.org/10.1182/blood-2013-11-537704Google Scholar
- Pierangeli SS, Vega-Ostertag ME, Raschi E. Toll-like receptor and antiphospholipid mediated thrombosis: in vivo studies. Ann Rheum Dis. 2007; 66(10):1327-1333. PubMedhttps://doi.org/10.1136/ard.2006.065037Google Scholar
- Pengo V. ISTH guidelines on lupus anticoagulant testing. Thromb Res. 2012; 130(Suppl 1):S76-77. PubMedhttps://doi.org/10.1016/j.thromres.2012.08.283Google Scholar
- Agostinis C, Biffi S, Garrovo C. In vivo distribution of beta2 glycoprotein I under various pathophysiologic conditions. Blood. 2011; 118(15):4231-4238. PubMedhttps://doi.org/10.1182/blood-2011-01-333617Google Scholar
- van Os GM, Meijers JC, Agar C. Induction of anti-beta2-glycoprotein I autoantibodies in mice by protein H of Streptococcus pyogenes. J Thromb Haemost. 2011; 9(12):2447-2456. PubMedhttps://doi.org/10.1111/j.1538-7836.2011.04532.xGoogle Scholar
- Tomaic V, Gardiol D, Massimi P, Ozbun M, Myers M, Banks L. Human and primate tumour viruses use PDZ binding as an evolutionarily conserved mechanism of targeting cell polarity regulators. Oncogene. 2009; 28(1):1-8. PubMedhttps://doi.org/10.1038/onc.2008.365Google Scholar
- Steinkasserer A, Estaller C, Weiss EH, Sim RB, Day AJ. Complete nucleotide and deduced amino acid sequence of human beta 2-glycoprotein I. Biochem J. 1991; 277(Pt 2):387-391. PubMedhttps://doi.org/10.1042/bj2770387Google Scholar
- Agar C, van Os GM, Morgelin M. Beta2-glycoprotein I can exist in 2 conformations: implications for our understanding of the antiphospholipid syndrome. Blood. 2010; 116(8):1336-1343. PubMedhttps://doi.org/10.1182/blood-2009-12-260976Google Scholar
- de Groot PG, Meijers JC. beta(2) -Glycoprotein I: evolution, structure and function. J Thromb Haemost. 2011; 9(7):1275-1284. PubMedhttps://doi.org/10.1111/j.1538-7836.2011.04327.xGoogle Scholar
- Pengo V, Testa S, Martinelli I. Incidence of a first thromboembolic event in carriers of isolated lupus anticoagulant. Thromb Res. 2015; 135(1):46-49. PubMedhttps://doi.org/10.1016/j.thromres.2014.10.013Google Scholar
- Ioannou Y, Romay-Penabad Z, Pericleous C. In vivo inhibition of antiphospholipid antibody-induced pathogenicity utilizing the antigenic target peptide domain I of beta2-glycoprotein I: proof of concept. J Thromb Haemost. 2009; 7(5):833-842. PubMedhttps://doi.org/10.1111/j.1538-7836.2009.03316.xGoogle Scholar
- Agnello V, Koffler D, Kunkel HG. Immune complex systems in the nephritis of systemic lupus erythematosus. Kidney Int. 1973; 3(2):90-99. PubMedGoogle Scholar
- McPhaul JJ, Mullins JD. Glomerulonephritis mediated by antibody to glomerular basement membrane. Immunological, clinical, and histopathological characteristics. J Clin Invest. 1976; 57(2):351-361. PubMedhttps://doi.org/10.1172/JCI108286Google Scholar
- Salmon JE, Girardi G. Theodore E. Woodward Award: antiphospholipid syndrome revisited: a disorder initiated by inflammation. Trans Am Clin Climatol Assoc. 2007; 118:99-114. PubMedGoogle Scholar
- Erkan D, Salmon JE. The Role of Complement Inhibition in Thrombotic Angiopathies and Antiphospholipid Syndrome. Turk J Haematol. 2016; 33(1):1-7. Google Scholar
- Oku K, Nakamura H, Kono M. Complement and thrombosis in the antiphospholipid syndrome. Autoimmun Rev. 2016; 15(10):1001-1004. Google Scholar
- Meroni PL, Macor P, Durigutto P. Complement activation in antiphospholipid syndrome and its inhibition to prevent rethrombosis after arterial surgery. Blood. 2016; 127(3):365-367. PubMedhttps://doi.org/10.1182/blood-2015-09-672139Google Scholar
- Del Papa N, Sheng YH, Raschi E. Human beta 2-glycoprotein I binds to endothelial cells through a cluster of lysine residues that are critical for anionic phospholipid binding and offers epitopes for anti-beta 2-glycoprotein I antibodies. J Immunol. 1998; 160(11):5572-5578. PubMedGoogle Scholar