Page 192 - 2019_04-Haematologica-web

P. Durigutto et al.
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 var- ious organs resulting in multiple organ failure.4 Anti-cardi- olipin (aCL) and anti-β2glycoprotein I (β2GPI) antibodies and lupus anticoagulant (LA) activity are considered markers of APS and are included among the criteria cur- rently proposed to classify the syndrome.1 Clinical studies have revealed an increased risk of thrombosis and preg- nancy complications in patients with medium to high lev- els 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.5-9 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
10
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 inef- fective in causing blood clot due to their failure to recog- nize bound β2GPI will be reported.
Methods
Serum source
Purification of β glycoprotein I and generation of 2
Animal model
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 per- formed 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.
Immunofluorescence analysis
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 strep- tavidin (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
associated with the clinical manifestations of APS.
β2GPI
mainly circulates in blood in a circular form and is organ-
ized 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
recombinant domains D4 and D5
the classical diagnostic assays measuring antibodies
Methods of purification of human β2GPI from pooled normal sera and the generation of D4 and D5 domains have been pub- lished previously.12,27,30,31 Sequence analysis was performed as described32 and compared to the published sequence of β2GPI.33 The fine specificity against D4 or D5 was investigated by ELISA.27
against whole molecule β2GPI, new tests have recently
been developed to detect anti-β2GPI antibody subpopula-
tions reacting with different domains of the protein, par-
ticularly the combined domains D4/5 and domain 1
(D1).5-7,9,12-14
In APS patients, a large proportion of anti-β2GPI anti- bodies react with D1 and recognize a cryptic epitope (Arg39–Arg43) in the native molecule exposed after its interaction with anionic phospholipids13,15 or oxidation.16- 18 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.7,9,19-24 In contrast, isolated high levels of anti-D4/5 antibodies have been reported in non-APS patients with leprosy, atopic dermatitis, athero- sclerosis 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 mani- festations of the APS syndrome.7,9 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 sug- gested that they may not be involved in thrombus forma- tion.
The in vivo pathogenic role of aPL has been demonstrat- ed for those directed against the whole molecule and against D1 of β2GPI using animal models of thrombosis developed in rats and mice.26-28 However, at present, there
Different concentrations of β2GPI were added to CL-coated plates and the reactivity of IgG with CL-bound β2GPI was meas- ured.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.
Two groups of anti-β2GPI positive sera7,27 containing isolated antibodies to either D1 or D4/5 domains6,7 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 puri- fied by a Protein G column (HiTrap Protein G HP, GE Healthcare) as described.27 The local Istituto Auxologico Italiano ethical com- mittee approved the study.
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haematologica | 2019; 104(4)