The IgA Isotype of Anti-ß2 Glycoprotein I Antibodies Recognizes Epitopes in Domains 3, 4, and 5 That Are Located in a Lateral Zone of the Molecule (L-Shaped).

Background: Antiphospholipid syndrome (APS) is characterized by thrombosis and/or pregnancy morbidity with presence of anti-phospholipid antibodies (aPL). The APS classification criteria only consider the aPL of IgG/IgM isotype, however testing of aPL of IgA isotype is recommended when APS is suspected and consensus aPL are negative. IgA anti-ßeta-2 glycoprotein-I (B2GP1) has been clearly related with occurrence of thrombotic events. Antibodies anti-B2GP1 of IgG/M isotypes recognize an epitope in Domain 1 (R39-G43), the epitopes that recognize IgA anti-B2GP1 antibodies are not well-identified. Aim: To determine the zones of B2GP1 recognized by antibodies of IgA isotype from patients with APS symptomatology and positive for IgA anti-B2GP1. Methods: IgA antibodies to Domain-1(D1) and Domain-4/5(D4/5) of B2GP1 (ELISA) and epitope mapping on oligopeptide arrays of B2GP1 were evaluated in sera from a group of 93 patients with at least one thrombotic and with isolated positivity for IgA anti-B2GP1 antibodies (negative for other aPL). Results: A total of 47 patients (50.5%) were positive for anti-D4/5 and 23(25%) were positive for anti-D1. When peptide arrays were analyzed, three zones of B2GP1 reactivity were identified for more than 50% of patients. The center of these zones corresponds to amino acids 140(D3), 204(D4), and 264(D5). The peptides recognized on D3 and D4 contain amino acid sequences sharing high homology with proteins of microorganism that were previously related with a possible APS infectious etiology. In the three-dimensional structure of B2GP1, the three peptides, as the R39-G43 epitope, are located on the right side of the molecule (L-shape). The left side (J-shape) does not bind the antibodies. Conclusions: Patients with thrombotic APS clinical-criteria, and isolated IgA anti-B2GP1 positivity appear to preferentially bind, not to the D1 or D4/5 domains of B2GP1, but rather to three sites in D3, D4, and D5. The sites on D3 and D4 were previously described as the target identified by human monoclonal antibodies derived from patients that were capable of inducing APS in animal models. The localization of these epitopes opens a new route to explore to increase understanding of the patholophysiology of the APS and to propose new alternatives and therapeutic targets.

Antiphospholipid Syndrome and Renal Allograft Thrombosis.

Renal allograft thrombosis is the most frequent and devastating complication in the early postrenal transplantation period. Several risk factors to develop graft thrombosis depending on donors and recipients are well known. Antiphospholipid syndrome (APS) is well recognized as an important cause of kidney injury, with specific clinical and histological features that may lead to renal injury caused by thrombosis at any location within the renal vasculature. There are 3 forms of APS, primary (the most common form), associated to other systemic autoimmune diseases (SAD-APS), and catastrophic. Nevertheless, patients with SAD-APS and renal failure only represent 2% to 5% in hemodialysis or transplantation. The presence of pretransplant antiphospholipid antibodies increases risk of graft thrombosis. A new form of APS based on IgA anti-ß-2-glycoprotein-I (B2GPI) antibodies, representing up to 30% of patients in end-stage renal disease and renal transplantation, is the main independent risk factor for graft thrombosis and early graft loss after renal transplantation. In addition, B2GP1 bound to IgA aB2GP1 immunocomplexes have been described as a marker to predict thrombosis after renal transplantation in patients with antiphospholipid antibodies. Anticoagulation remains the main treatment to prevent renal allograft thrombosis, although new preventive strategies are coming. Future studies may help to identify better therapeutic targets.

Immune Complexes of Beta-2-Glycoprotein I and IgA Antiphospholipid Antibodies Identify Patients With Elevated Risk of Thrombosis and Early Mortality After Heart Transplantation.

Background: The presence of anti-Beta 2 glycoprotein antibodies (aB2GP1) of IgA isotype is common in patients with functional impairment of the organs in which B2GP1 is elaborated. Pretransplant IgA aB2GP1 has been associated with increased risk of thrombosis in kidney and heart transplanted patients and has also been related with early mortality after heart transplantation. Circulating immune complexes between IgA and B2GP1 (B2A-CIC) have been described in the blood of patients positive for IgA aB2GP1 with thrombotic clinical symptoms. In kidney transplanted patients, B2A-CIC is a biomarker that predicts which patients IgA aB2GP1 positive are at risk of thrombosis events following kidney transplantation and may lead to early prophylactic treatment. The prevalence of B2A-CIC and its relation with outcomes after heart transplantation is not known. Methods: Follow-up study based on 151 consecutive patients who received a heart transplant. Autoantibodies and B2A-CIC were quantified in pre-transplant serum samples. Three groups of patients were followed-up for 2 years: Group-1, positive for IgA aB2GP1 and B2A-CIC (N = 19). Group-2, only positive for IgA aB2GP1 (N = 28). Group-0 (control group): IgA aB2GP1 negative (N = 104). Results: Kaplan-Meir survival analysis showed that mortality in B2A-CIC positive was higher than group-0 at 3 months (HR:5.08; 95%CI: 1.36-19.01) and at 2 years (HR:3.82; 95%CI: 1.54-12.66). No significant differences were observed between group-2 and group-0. Multivariate analysis identified B2A-CIC as the most important independent risk factor for early mortality (OR = 6.12; 95% CI: 1.93-19.4). Post-transplant incidence of thrombosis was significantly higher in B2A-CIC positive patients than in the control group (OR: 6.42; 95%CI: 2.1-19.63). Multivariate analysis identified the presence of B2A-CIC (OR: 6.13; 95%CI: 2.1-19.63) and the pre-transplant habit of smoking actively (OR: 4.18; 95%CI: 1.35-12.94) as independent risk factor for thrombosis. The proportion of patients who had thrombotic events or died in the first trimester was significantly higher in group-1 (73.7%) than in group-0 (16.3%; p < 0.001) and in group-2 (39.3%; p = 0.02). Multivariate analysis identified B2A-CIC as the main independent risk factor for early outcomes (mortality or thrombosis) in the first 3 months after heart transplant (OR = 11.42, 95% CI: 1.69-9.68). Conclusion: B2A-CIC are a predictor of early mortality and thrombosis after heart transplant.

Pretransplant IgA-Anti-Beta 2 Glycoprotein I Antibodies As a Predictor of Early Graft Thrombosis after Renal Transplantation in the Clinical Practice: A Multicenter and Prospective Study.

Background: Graft thrombosis is a devastating complication after renal transplantation. We recently described the association of anti-beta-2-glycoprotein-I (IgA-ab2GP1) antibodies with early graft loss mainly caused by thrombosis in a monocenter study. Methods: Multicenter prospective observational cohort study. Setting and participants: Seven hundred forty patients from five hospitals of the Spanish Forum Renal Group transplanted from 2000 to 2002 were prospectively followed-up for 10 years. Outcomes: Early graft loss and graft loss by thrombosis. Measurements: The presence of IgA anti-B2GP1 antibodies in pretransplant serum was examined using the same methodology in all the patients. Results: At transplantation, 288 patients were positive for IgA-B2GP1 (39%, Group-1) and the remaining were negative (Group-2). Graft loss at 6 months was higher in Group-1 (12.5 vs. 4.2% p < 0.001), vessel thrombosis being the most frequent cause of early graft loss, especially in Group-1 (6.9 vs. 0.4% p < 0.001). IgA-aB2GP1 was the most important independent risk factor for graft thrombosis (hazard ratio: 13.83; 95% CI: 3.17-60.27, p < 0.001). Furthermore, the, presence of IgA-aB2GP1 was associated with early graft loss and delayed graft function. At 10 years, survival figures were also lower in Group-1: graft survival was lower compared with Group-2 (60.4 vs. 76.8%, p < 0.001). Mortality was significantly higher in Group-1 (19.8 vs. 12.2%, p = 0.005). Limitations: Patients were obtained during a 3-year period (1 January 2000-31 December 2002) and kidneys were only transplanted from brain-dead donors. Nowadays, the patients are older and the percentage of sensitized and retransplants is high. Conclusion: In a prospective observational multicenter study, we were able to corroborate that pretransplant presence of IgA-aB2GP1 was the main risk factor for graft thrombosis and early graft loss. Therefore, a prospective study is needed to evaluate the efficacy and safety of prophylactic anticoagulation to avoid this severe complication.

Early mortality after heart transplantation related to IgA anti-ß2-glycoprotein I antibodies.

BACKGROUND: The presence of pre-formed IgA anti-ß2-glycoprotein I antibodies (IgA-aB2GP1ab) has been related to early graft loss after kidney transplant. Because ß2-glycoprotein I is produced in both the kidney and heart, we aimed to assess whether the presence of these antibodies may also be associated with poor outcomes after heart transplantation (HT). METHODS: A 2-year follow-up retrospective analysis of 151 consecutive patients who underwent HT between 2004 and 2012 was performed to assess the role of this pre-formed antibody type in HT. The population was divided into 2 groups according to the presence of IgA: Group 1 was positive for IgA-aB2GP1ab (47 patients, 31.1%), and Group 2 was negative for IgA-Ab2GP1ab (104 patients, 68.9%). RESULTS: Early mortality rates within the first 3 months were higher in Group 1 (27.7%) than in Group 2 (9.6%). No differences in donor and recipient characteristics or in causes of death were observed between groups. Multivariate analysis identified the presence of IgA-aB2GP1ab, female gender and blood type A as independent risks factors for early mortality after HT. A greater incidence of thrombotic events during the first 3 months post-HT in Group 1 (23.4% vs 5.8%) and a greater presence of risk factors for thrombotic events, which may have exacerbated them, were observed. After this period, no increase in mortality or in thrombotic events was found when the 2 groups were compared. CONCLUSION: Pre-transplant presence of IgA-aB2GP1ab is associated with both increased early mortality rates and higher thrombotic events after HT.

The Presence of Pretransplant Antiphospholipid Antibodies IgA Anti-ß-2-Glycoprotein I as a Predictor of Graft Thrombosis After Renal Transplantation.

BACKGROUND: Vessel thrombosis is a severe complication after renal transplantation. Antibodies anti-ß-2 glycoprotein-I of IgA isotype (IgA-aB2GP1) have been linked to thrombotic events and mortality in hemodialysis patients. METHODS: All kidney transplanted patients from 2000 to 2011 (n = 1375) in our hospital were followed up for 2 years, evaluating 3 time periods. RESULTS: At transplantation, 401 patients were positive for IgA-aB2GPI (29.2%, group 1), and the remaining patients were negative (group 2). Graft loss at 6 months posttransplantation was higher in group 1 (18% vs 7.2%; P < 0.001). The most frequent cause of early graft loss was vessel thrombosis, especially in group 1 (12.2% vs 2.6% of patients; P < 0.001). In fact, vessel thrombosis was the most important cause of graft loss in the 3 time periods, irrespective of demographic changes and introduction of transplantation with asystolic donors.Notably, IgA-aB2GP1 was an independent risk factor for graft thrombosis (odds ratio, 5.047; P < 0.001). Furthermore, the presence of IgA-aB2GP1 was associated with early graft loss and delayed graft function. Mortality at 24 months was also higher in group 1. CONCLUSIONS: In conclusion, pretransplant IgA-aB2GP1 was the main risk factor for graft thrombosis and early graft loss. Further research should be made on whether anticoagulation in antibody-positive patients could ameliorate this catastrophic complication.

Blockade of cell adhesion molecules enhances cell engraftment in a murine model of liver cell transplantation.

AIM: OLT is the best alternative for patients with end-stage liver diseases. However, as the need for organs surpasses donor availability, alternatives to OLT are required. LCT could be a useful option versus OLT in several patients even though its low cell-engraftment hampers its efficiency. Endothelial cell barrier is the main obstacle for the implantation of cells into the parenchyma. Our study has focused on the modification of the endothelial barrier with monoclonal antibodies against adhesion molecules in order to increase cell engraftment in a mouse model of liver cell transplantation. METHODS: Anti-mouse CD54 and anti-mouse CD61 antibodies were administered intrasplenically to healthy mice within 60 min prior to stem cell transplantation. Animals were sacrificed either short term at 2h or middle term seven days after transplantation. Immunohistochemical techniques to detect alkaline phosphatase activity were used to identify the transplanted cells within the liver parenchyma. RESULTS: Anti-CD54 and anti-CD61 administration increases vascular patency and cell engraftment. This represents a 32% and 45% increase, respectively, of engrafted cells compared to the control (p<0.05). CONCLUSION: Modification of the vascular wall with monoclonal antibodies against endothelial adhesion molecules before cell transplantation enhances cell engraftment into the mouse liver.

Association of early kidney allograft failure with preformed IgA antibodies to ß2-glycoprotein I.

In the current immunosuppressive therapy era, vessel thrombosis is the most common cause of early graft loss after renal transplantation. The prevalence of IgA anti-ß2-glycoprotein I antibodies (IgA-aB2GPI-ab) in patients on dialysis is elevated (>30%), and these antibodies correlate with mortality and cardiovascular morbidity. To evaluate the effect of IgA-aB2GPI-ab in patients with transplants, we followed all patients transplanted from 2000 to 2002 in the Hospital 12 de Octubre prospectively for 10 years. Presence of IgA-aB2GPI-ab in pretransplant serum was examined retrospectively. Of 269 patients, 89 patients were positive for IgA-aB2GPI-ab (33%; group 1), and the remaining patients were negative (67%; group 2). Graft loss at 6 months post-transplant was significantly higher in group 1 (10 of 89 versus 3 of 180 patients in group 2; P=0.002). The most frequent cause of graft loss was thrombosis of the vessels, which was observed only in group 1 (8 of 10 versus 0 of 3 patients in group 2; P=0.04). Multivariate analysis showed that the presence of IgA-aB2GPI-ab was an independent risk factor for early graft loss (P=0.04) and delayed graft function (P=0.04). There were no significant differences regarding patient survival between the two groups. Graft survival was similar in both groups after 6 months. In conclusion, patients with pretransplant IgA-aB2GPI-ab have a high risk of early graft loss caused by thrombosis and a high risk of delayed graft function. Therefore, pretransplant IgA-aB2GPI-ab may have a detrimental effect on early clinical outcomes after renal transplantation.

Renal transplantation dramatically reduces IgA anti-beta-2-glycoprotein I antibodies in patients with endstage renal disease.

IgA anti-beta-2-glycoprotein I (aB2GPI) antibodies have been related to vascular pathology in the general population and mainly in hemodialyzed patients (prevalence 33%) in whom an elevated incidence of thrombosis and mortality is found. In this paper we have studied the presence of IgA aB2GPI antibodies at pretransplant and their evolution after transplantation with a cross-sectional-based follow-up study of a cohort of 288 endstage renal disease (ESRD) patients treated with kidney transplantation. Pretransplant IgA aB2GPI levels were elevated 31.7 ± 4.2 U/mL without differences in age or type of dialysis. Patients with different etiologies of ESRD showed higher levels of IgA aB2GPI than blood donors, except the groups of non-IgA glomerular disease and systemic erythematosus lupus, whose nonsignificant differences were observed. IgA aB2GPI antibodies dropped immediately after transplantation (10.7 ± 1.0 U/mL, P < 0.0001), coinciding with a high degree of immunosuppression, and remained significantly lower than that observed in pretransplant status. Prevalence of patients with elevated antibodies was also less in transplanted patients (8.9% versus 30.4%, P < 0.0001). Among, positivity for IgA aB2GPI was higher than in patients who had received their first transplant that those were retransplanted. This finding could have important clinical implications and can suggest new therapeutic strategies in patients with IgA aB2GPI antibodies.

Isolated IgA anti- ß2 glycoprotein I antibodies in patients with clinical criteria for antiphospholipid syndrome.

Seronegative antiphospholipid syndrome (SNAPS) is an autoimmune disease present in patients with clinical manifestations highly suggestive of Antiphospholipid Syndrome (APS) but with persistently negative consensus antiphospholipid antibodies (a-PL). IgA anti-ß 2 Glycoprotein I (aB2-GPI) antibodies are associated with APS. However, they are not currently considered to be laboratory criteria due to the heterogeneity of published works and the use of poor standardized diagnostic systems. We have aimed to assess aPL antibodies in a group of patients with clinical manifestations of APS (C-APS) to evaluate the importance of the presence of IgA aB2GPI antibodies in APS and its relation with other aPL antibodies. Only 14% of patients with C-APS were positive for any consensus antibody, whereas the presence of isolated IgA aB2GPI antibodies was found in 22% of C-APS patients. In patients with arterial thrombosis IgA aB2GPI, antibodies were the only aPL antibodies present. Serologic profile in primary APS (PAPS) is different from systemic autoimmune disorders associated APS (SAD-APS). IgA aB2GPI antibodies are more prevalent in PAPS and IgG aB2GPI antibodies are predominant in SAD-APS. The analysis of IgA aB2GPI antibodies in patients with clinical manifestations of PAPS might avoid underdiagnosed patients and provide a better diagnosis in patients with SAD-APS. Laboratory consensus criteria might consider including analysis of IgA aB2GPI for APS diagnosis.