Detection of annexin A8 antibodies in serum of patients with antiphospholipid syndrome

Introduction Antibodies specific for annexin A8 (AnxA8) have not been investigated in patients suffering from antiphospholipid syndrome (APS) yet. The aim of this study was to compare the presence of AnxA8 antibodies in serum of APS patients with that of age-matched healthy controls and to investigate whether AnxA8 antibodies are potential biomarkers for APS. Materials and methods We enrolled 22 APS patients and 22 healthy controls in this case-control study. We used sodium dodecyl sulfate polyacrylamide gel electrophoresis and immunoblot to investigate the presence of AnxA8 antibodies, and we applied enzyme-linked immunosorbent assay to investigate the presence of cardiolipin (CL) and beta-2-glycoprotein I (ß2GPI) antibodies. Results The serum of 9/22 APS patients showed AnxA8 IgG isotype antibody reactivity compared to serum of 2/22 healthy controls (P = 0.034). When we also included weak immunoblot signals, 12/22 APS patients exhibited AnxA8 IgG isotype antibody reactivity compared to 3/22 healthy controls (P = 0.005). We also investigated the presence of AnxA8 IgM isotype antibodies in the serum of APS patients but found no statistically significant difference between the APS patient group and healthy control group (P = 0.500). We further investigated the presence of ß2GPI and CL IgG and IgM isotype antibodies. AnxA8 IgG isotype antibodies were present in APS patients in a similar frequency as the APS “criteria” antibody against CL (P = 0.764). Conclusion We demonstrated that AnxA8 IgG isotype antibodies are potential biomarkers for the diagnosis of APS.


Introduction
Antiphospholipid syndrome (APS) is an autoimmune disorder that is clinically characterized by thrombosis and/or obstetric complications (1)(2)(3). Antiphospholipid syndrome can occur alone (primary APS) or with other autoimmune diseases (secondary APS), e.g., systemic lupus erythematosus (SLE) (4). Due to the lack of specificity in clinical manifestations, the diagnosis of APS is based on the occurrence of clinical symptoms and the detection of at least one of the three antiphospholipid antibodies (aPL, "criteria" aPLs), i.e., IgG or IgM isotype antibodies directed against β2glycoprotein I (aß2GPI) and cardiolipin (aCL), or a positive lupus anticoagulant (LA) functional assay. Patients diagnosed with APS are placed on lifelong anticoagulation, which is associated with a risk of bleeding complications. Antiphospholipid antibodies titers are used for diagnosis of APS according to the revised Sapporo criteria (> 40 IgM phospholipid units [MPL] or > 40 IgG phospholipid units [GPL]; here, one unit is defined as one microgram of antibody per milliliter or > 99th percentile Scholz P. et al. Annexin A8 antibodies and APS for aCL and > 99th percentile for anti-β2GPI) (3,5). These criteria also require the presence of aPL on two occasions, 12 weeks apart, to avoid misdiagnosing APS in patients with a low titer or transient aPL (6). Laboratory testing is important not only for the diagnosis of APS, but also for risk assessment. Lupus anticoagulant assay is a stronger predictor of risk for vascular thrombosis compared to aCL or aß2GPI, but the greatest risk of thrombosis is found in people with multiple aPLs (7). Antiphospholipid antibodies are directed against a heterogeneous group of antigens, e.g., negatively charged molecules, proteins, or phospholipid-protein complexes. Besides the well investigated three "criteria" aPLs, a growing number of "noncriteria" antibodies against various biomolecules, such as prothrombin/phosphatidylserine, vimentin/cardiolipin, protein S, protein C, annexin A2 (AnxA2), annexin A5 (AnxA5), oxidized low-density lipoproteins, lysobisphosphatidic acid, and sulfatides, have been linked to the occurrence of APS (7). These "non-criteria" aPLs have been proposed as relevant in APS and useful to subclassify APS with clinical manifestations (8,9). Therefore, identification of "non-criteria" aPLs is important to assess the risk of APS patients and possibly diagnose patients with APS-like symptoms but without clearly defined laboratory criteria for an APS (seronegative APS, SNAPS).
Annexin A8 (AnxA8) was originally described as an anticoagulant and an inhibitor of phospholipase A 2 activity due to the 56% association with vascular anticoagulant-alpha (VAC-α, synonyms: AnxA5, lipocortin V) (10). In contrast to other annexins, AnxA8 has a low affinity to phosphatidylserine and hardly interacts with the cell surface of dying cells (11). Annexin A8 is associated specifically with late endosomes and involved in actin-based late endosome motility (12). It is activated by p53 signalling (13). Furthermore, AnxA8 may regulate epidermal growth factor receptor signalling and trafficking (14). Therefore, prior research speculated that AnxA8 has tumour suppressor effects (15). However, the biological function of AnxA8 remains unclear.
Recently, the presence of high AnxA8 antibody titers was reported in a patient suffering from SNAPS (16). Antibodies specific for AnxA8 have yet not been investigated in patients suffering from APS.
Here, we compared the presence of AnxA8 antibodies in serum of 22 APS patients with that of 22 age-matched healthy controls and investigated whether AnxA8 antibodies are potential biomarkers for APS.  (3). The diagnostic criteria require one clinical event, i.e., thrombosis or obstetric complication, and two positive antibody blood tests for one of the "criteria" aPLs 12 weeks apart. All APS patients suffered exclusively from thromboembolic events, except 4 patients who also suffered obstetric manifestations of APS. Patients were excluded if they had other thrombotic risk factors, e.g., factor V-Leiden and prothrombin mutation, antithrombin, protein C and protein S deficiency, and pathological factor VIII and factor XII activity. Remaining samples from routine laboratory testing were used to identify patients for inclusion in the control group. Patients in the control group had no APS symptoms or other autoimmune diseases and no reported thromboembolic or obstetric events in their medical history. Our study was approved by the Ethical Committee of the University Hospital of Cologne (application number 14-176).

Materials and methods
Blood samples (remaining samples) were collected from July 2014 to March 2017 in 4.7 mL serum monovettes, centrifuged at 2772xg for 10 minutes, and stored in aliquots at -70 °C.
We used AnxA8 for the detection of AnxA8 IgG and IgM isotype antibodies in the serum of APS patients. Recombinant expression of AnxA8, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and immunoblot analysis were performed as described previously (16). Briefly, AnxA8 was recombinantly expressed in bacteria and purified by affinity-and endotoxin-removal gel chromatography.
We further investigated the presence of ß2GPI and CL IgG and IgM isotype antibodies by immunoassay. A functional LA assay was not performed, as APS patients were anticoagulated and anticoagulation could affect the performance of the LA assay. The serum of 10/22 APS patients was positive for aCL IgG isotype antibodies, and the serum of 8/22 APS patients was positive for aCL IgM isotype antibodies. The serum of 9/18 APS patients was positive for ß2GPI IgG isotype antibodies, and the serum of 9/18 APS patients was positive for ß2GPI IgM isotype antibodies. For 4 APS patients, no ß2GPI antibody analysis was available. Frequency of AnxA8 IgG isotype antibodies was similar to frequency of "criteria" CL antibodies (P = 0.764). Interestingly, one APS patient without CL or ß2GPI antibodies was positive for AnxA8 IgG isotype antibodies.

Discussion
In this case-control study, we investigated the presence of AnxA8 antibodies in the serum of 22 APS patients and 22 healthy controls. We found a statistically significant difference in the frequency of AnxA8 IgG isotype antibodies between the APS group and the healthy control group. Recently, we reported the case of a SNAPS patient with a history of six pregnancy losses and a fulminant stroke, with no evidence of the three "criteria" aPLs, but with high antibody titers against AnxA2 and AnxA8 (16).
Antiphospholipid syndrome diagnosis requires meeting the revised Sapporo criteria of 2006 and detection of at least one of three "criteria" aPLs (3). Meanwhile, numerous studies describe the presence of "non-criteria" antibodies in the course of APS. The presence of multiple antibodies is commonly associated with the greatest risk of thrombosis, and increasing interest has been focused on "non-criteria" antibodies (18,19). The precise relevance of those antibodies and the diagnostic value of a positive test result are unclear, as these antibodies were mostly detected by immunoassays and not by a functional test, as it was described for Annexin A5 anticoagulant activity (20). In our study, we used specific immunoblot analysis instead of ELISA analysis to include the molecular size of the detected band as additional selection criteria for the investigation of the serum samples. Several serum samples exhibited antibody reactivity against remaining LPS traces of affinity-and endotoxin-purified AnxA8, which may yield false positive results in ELISA. Analysis by ELISA, therefore, is not suited to discriminate between a specific reactivity against AnxA8 or an unspecific cross-reactivity against remaining traces of LPS.
Prior research on "non-criteria" antibodies was of relatively small sample size, and this is also a limitation of our study (20). However, we found a significant difference in the presence of AnxA8 IgG isotype antibodies between the tested groups. Antibodies can occur transiently, particularly IgM isotype antibodies that are linked to infectious disorders. Therefore, persistently elevated aPL levels are a mandatory laboratory criteria for diagnosis of APS, and aPL tests must be repeated within 12 weeks. We investigated the presence of AnxA8 antibodies only once; however, we found a highly significant correlation between the presence of AnxA8 IgG isotype antibodies and APS and identified AnxA8 IgG isotype antibodies as potential biomarkers for the diagnosis of APS.