Anti-ß2-glycoprotein I/HLA-DR Antibody and Adverse Obstetric Outcomes.

Anti-ß2-glycoprotein I/HLA-DR (anti-ß2GPI/HLA-DR) antibody has been reported to be associated with antiphospholipid syndrome and recurrent pregnancy loss (RPL). We conducted a prospective multicenter cross-sectional study aimed at evaluating whether the anti-ß2GPI/HLA-DR antibody is associated with adverse obstetric outcomes and RPL. From 2019 to 2021, serum anti-ß2GPI/HLA-DR antibody levels (normal, <73.3 U) were measured in 462 women with RPL, 124 with fetal growth restriction (FGR), 138 with hypertensive disorders of pregnancy (HDP), 71 with preterm delivery before 34 gestational weeks (preterm delivery (PD) ≤ 34 GWs), and 488 control women who experienced normal delivery, by flow cytometry analysis. The adjusted odds ratios (aORs) of anti-ß2GPI/HLA-DR antibody positivity for adverse obstetric outcomes and RPL were evaluated on the basis of comparisons between the control and each patient group, using multivariable logistic regression analysis. The following were the positivity rates for the anti-ß2GPI/HLA-DR antibody in the patient and control groups: RPL, 16.9%; FGR, 15.3%; HDP, 17.4%; PD ≤ 34 GWs, 11.3%; and the control, 5.5%. It was demonstrated that anti-ß2GPI/HLA-DR antibody positivity was a significant risk factor for RPL (aOR, 3.3 [95% confidence interval {CI} 1.9-5.6], p < 0.001), FGR (2.7 [1.3-5.3], p < 0.01), and HDP (2.7 [1.4-5.3], p < 0.01) although not for PD ≤ 34 GWs. For the first time, our study demonstrated that the anti-ß2GPI/HLA-DR antibody is involved in the pathophysiology underlying FGR and HDP, as well as RPL.

Close link between antiphosphatidylserine/prothrombin antibodies, lupus anticoagulant, and activated protein C resistance in tetra antiphospholipid antibody-positive subjects.

BACKGROUND: Most of the carriers/patients triple-positive for antiphospholipid antibodies (lupus anticoagulant [LAC], immunoglobulin G [IgG]/immunoglobulin M [IgM] anticardiolipin, and anti-ß2-glycoprotein I antibodies) are tetra-positive, being positive for antiphosphatidylserine/prothrombin (aPS/PT) antibodies. The relationship between aPS/PT titer, LAC potency, and resistance to activated protein C (aPC-R) has not been investigated. OBJECTIVES: The aim of this study was to clarify the mutual interdependence of these parameters in tetra-positive subjects. METHODS: Twenty-three carriers and 30 patients with antiphospholipid syndrome, none of whom were being treated with anticoagulants, and 30 age- and sex-matched controls were studied. Detection of aPS/PT, LAC, and aPC-R in each individual was performed with established methods in our laboratory. Carriers and patients were positive for IgG or IgM aPS/PT or for both isotypes without significant difference. Since both IgG and IgM aPS/PT have anticoagulant activity, we used the sum of their titers (total aPS/PT) for the correlation studies. RESULTS: Total aPS/PT in all individuals studied exceeded that in controls. There was no difference in total aPS/PT titers (P = .72), LAC potency (P = .56), and aPC-R (P = .82) between antiphospholipid antibody-carriers and patients with antiphospholipid syndrome. There was a significant correlation between total aPS/PT and LAC potency (r = 0.78; P < .0001) and between total aPS/PT titers and aPC-R (r = 0.80; P < .0001). LAC potency also was correlated significantly with aPC-R (r = 0.72; P < .0001). CONCLUSION: This study shows that there is interdependence between aPS/PT, LAC potency, and aPC-R.

Anti-ß2-glycoprotein I and anti-phosphatidylserine/prothrombin antibodies interfere with cleavage of factor V(a) by activated protein C.

BACKGROUND: The acquired thrombotic risk factor known as lupus anticoagulant (LA) interferes with laboratory clotting assays and can be caused by autoantibodies against ß2-glycoprotein I (ß2GPI) and prothrombin. LA is associated with activated protein C (APC) resistance, which might contribute to thrombotic risk in patients with antiphospholipid syndrome. How antibodies against ß2GPI and prothrombin cause APC resistance is currently unclear. OBJECTIVES: To investigate how anti-ß2GPI and antiphosphatidylserine/prothrombin (PS/PT) antibodies induce APC resistance. METHODS: The effects of anti-ß2GPI and anti-PS/PT antibodies on APC resistance were studied in plasma (of patients with antiphospholipid syndrome) and with purified coagulation factors and antibodies. RESULTS: APC resistance was observed in LA-positive patients with anti-ß2GPI or anti-PS/PT antibodies and in normal plasma spiked with monoclonal anti-ß2GPI or anti-PS/PT antibodies with LA activity. Analysis of factor (F)V cleavage patterns after APC incubation indicated that anti-ß2GPI antibodies attenuated APC-mediated FV cleavage at R506 and R306. APC-mediated cleavage at R506 is required for FV cofactor activity during inactivation of FVIIIa. Assays with purified coagulation factors confirmed that anti-ß2GPI antibodies interfered with the cofactor function of FV during FVIIIa inactivation but not with FVa inactivation. Anti-PS/PT antibodies attenuated APC-mediated FVa and FVIIIa inactivation. Analysis of FV(a) cleavage patterns after APC incubation indicated that anti-PS/PT antibodies interfere with APC-mediated cleavage of FV at positions R506 and R306. CONCLUSION: Anti-ß2GPI antibodies with LA activity contribute to a procoagulant state by causing APC resistance via interference with the cofactor function of FV during FVIIIa inactivation. LA-causing anti-PS/PT antibodies interfere with the anticoagulant function of APC by preventing FV(a) cleavage.

Anti-ß2-glycoprotein I/HLA-DR antibody in infertility.

To evaluate whether anti-ß2-Glycoprotein I/HLA-DR (anti-ß2GPI/HLA-DR) antibody is associated with pathophysiology of infertility, 224 women with infertility were enrolled from July 2020 to December 2021 in this prospective study. The serum levels of anti-ß2GPI/HLA-DR antibody (normal < 73.3 U) were determined in 224 women with infertility. Backgrounds, causes and clinical factors were compared between women with and without anti-ß2GPI/HLA-DR antibody. Forty (17.9 %) of the 224 women tested positive for anti-ß2GPI/HLA-DR antibody. The prevalence of endometriosis was higher in women with anti-ß2GPI/HLA-DR antibody than in women without the antibody (32.5 %, 13/40 vs. 17.4 %, 32/184; P = 0.048). Logistic regression analyses revealed that, among clinical factors and diseases, endometriosis was associated with anti-ß2GPI/HLA-DR antibody positivity in infertile women (adjusted-odds ratio [OR] 3.01, 95 % confidence interval [CI] 1.30-6.99; P = 0.010). Twenty-three (15.5 %) of 148 women who underwent assisted reproductive technology (ART) tested positive for anti-ß2GPI/HLA-DR antibody. The prevalence of recurrent implantation failure (RIF) defined as three or more implantation failures following in vitro fertilization and embryo transfers was higher in women with ART who tested positive for the antibody (43.5 %, 10/23) than in women with ART who tested negative (20.8 %, 26/125; P = 0.032). Logistic regression analyses revealed that RIF was associated with anti-ß2GPI/HLA-DR antibody positivity in women with ART (adjusted-OR 2.92, 95 % CI 1.05-8.11; P = 0.040). Anti-ß2GPI/HLA-DR antibody may be associated with the pathophysiology of infertility, endometriosis and RIF; and can be a potential therapeutic target in infertility.

Antiphospholipid Antibody Assays in 2021: Looking for a Predictive Value in Addition to a Diagnostic One.

Antiphospholipid antibodies (aPL) are mandatory for the diagnosis but are also a risk factor for the antiphospholipid syndrome (APS) clinical manifestations. Lupus anticoagulant (LA), anticardiolipin (aCL), and anti-beta2 glycoprotein I (ß2GPI) assays are the formal laboratory classification/diagnostic criteria. Additional nonclassification assays have been suggested; among them, antiphosphatidylserine-prothrombin (aPS/PT) and antidomain 1 ß2GPI antibodies are the most promising ones although not yet formally accepted. aPL represent the example of a laboratory test that moved from dichotomous to quantitative results consistent with the idea that reporting quantitative data offers more diagnostic/prognostic information for both vascular and obstetric manifestations. Although the general rule is that the higher the aPL titer, the higher the test likelihood ratio, there is growing evidence that this is not the case for persistent low titers and obstetric events. LA displays the highest diagnostic/prognostic power, although some isolated LAs are apparently not associated with APS manifestations. Moreover, isotype characterization is also critical since IgG aPL are more diagnostic/prognostic than IgA or IgM. aPL are directed against two main autoantigens: ß2GPI and PT. However, anti-ß2GPI antibodies are more associated with the APS clinical spectrum. In addition, there is evidence that anti-ß2GPI domain 1 antibodies display a stronger diagnostic/prognostic value. This finding supports the view that antigen and even epitope characterization represents a further step for improving the assay value. The strategy to improve aPL laboratory characterization is a lesson that can be translated to other autoantibody assays in order to improve our diagnostic and prognostic power.

Dendritic Cells and Antiphospholipid Syndrome: An Updated Systematic Review.

Antiphospholipid syndrome (APS) is an autoimmune disease characterized by autoreactive B and T cells against ß2-glycoprotein I (B2GPI), with vascular thrombosis or obstetrical complications. Dendritic cells (DCs) are crucial in the generation of autoimmunity. Here, we conducted a comprehensive systematic review on the relationship between DC and APS. We performed a literature search of PubMed as of 26 March 2021. A total of 33 articles were extracted. DCs are pivotal in inducing inflammatory responses and orchestrating adaptive immunity. DCs contribute to the local inflammation regarding vascular thrombosis or obstetrical complications. Both B2GPI and antiphospholipid antibodies (aPL) can promote antigen presentation by DCs and the generation or maintenance of autoimmunity. In addition, plasmacytoid DC activation is enhanced by aPL, thereby augmenting the inflammatory response. In line with these findings, DC modulation appears promising as a future treatment for APS. In conclusion, our review indicated the crucial role of DCs in the pathogenesis of APS. Deeper understanding of the complex relationship would help in developing new treatment strategies.

Crassolide Suppresses Dendritic Cell Maturation and Attenuates Experimental Antiphospholipid Syndrome.

Antiphospholipid syndrome (APS) is an autoimmune disease characterized by the production of ß2-glycoprotein I (ß2GPI)-dependent autoantibodies, with vascular thrombosis or obstetrical complications. Around 20% of APS patients are refractory to current treatments. Crassolide, a cembranoid diterpene extracted from soft corals, is a potential therapeutic candidate. Here, to examine the anti-inflammatory properties of crassolide, we first determined its effects on bone marrow-derived and splenic dendritic cells (DC). Specifically, we applied lipopolysaccharide (LPS) or ß2GPI stimulation and measured the expressions of CD80 and CD86, and secretions of cytokines. We also determined in the OT-II mice, if bone marrow-derived DC was able to stimulate antigen-specific T cells. Moreover, we examined the therapeutic potential of crassolide postimmunization in a murine model of APS that depended on active immunization with ß2GPI. The vascular manifestations were evaluated in terms of fluorescein-induced thrombi in mesenteric microvessels, whereas the obstetric manifestations were evaluated based on the proportion of fetal loss after pregnancy. We also measured blood titers of anti-ß2GPI antibody, splenic cell proliferative responses and cytokine secretions after ß2GPI stimulation ex vivo. Finally, we determined in these mice, hematological, hepatic and renal toxicities of crassolide. Crassolide after LPS stimulation suppressed DC maturation and secretion of tumor necrosis factor (TNF)-α, interleukin (IL)-6, IL-12 and IL-23, and downstream T cell activation. Crassolide could partially ameliorate both the vascular and obstetric manifestations of APS in BALB/c mice. Both blood titers of anti-ß2GPI antibody and splenic cell proliferation after ß2GPI stimulation were reduced. Splenic Th1 and Th17 responses were also lowered after ß2GPI stimulation. Finally, within therapeutic doses of crassolide, we found no evidence of its toxicity. In conclusion, we showed the ability of crassolide to suppress DC and downstream T cell responses. Crassolide is therefore a potential candidate for adjunctive therapy in APS.

Double positivity of anti-ß2-glycoprotein I domain I and anti-phosphatidylserine/prothrombin antibodies enhances both thrombosis and positivity of anti-ADAMTS13 antibody.

Although a few antiphospholipid syndrome (APS) occurs with acquired thrombotic thrombocytopenic purpura (TTP), the relationship between antiphospholipid antibodies (aPL) and anti-ADAMTS13 (anti-a disintegrin and metalloprotease with thrombospondin type 1 motif, member 13) antibody remains uncertain. We investigated the relationship between high-risk thrombotic aPL and anti-ADAMTS13 antibody. Two hundred and thirty-seven patients with positive lupus anticoagulant and/or anticardiolipin antibody were included. Anti-ß2GPI (anti-ß2-glycoprotein I), anti-ß2GPIdI (anti-ß2-glycoprotein I domain I), anti-PS/PT (anti-phosphatidylserine and prothrombin), ADAMTS13 activity, and anti-ADAMTS13 antibody were measured. Double positivity of anti-ß2GPI and anti-PS/PT increased thrombotic risk more than three-fold and showed increased positivity of anti-ADAMTS13 antibody in comparison with the double negative group. Double positivity of anti-ß2GPIdI and anti-PS/PT presented both effects even more. In the linear regression analysis, double positivity of anti-ß2GPI and anti-PS/PT independently affected the anti-ADAMTS13 antibody level (ß = 1.982, P = 0.042). Our results revealed that double positivity of anti-ß2GPI or anti-ß2GPIdI and anti-PS/PT increased not only thrombotic risk but also the positivity of anti-ADAMTS13 antibody, especially indicating anti-ß2GPIdI showed a higher synergistic effect with anti-PS/PT. We suggest a possible association of anti-ADAMTS13 antibody with a high thrombotic risk of APS. Double positivity of anti-ß2GPI (anti-ß2-glycoprotein I) and anti-PS/PT (anti-phosphatidylserine and prothrombin) antibodies enhanced not only thrombotic risk but also positivity of anti-ADAMTS13 (anti-a disintegrin and metalloprotease with thrombospondin type 1 motif, member 13) antibody. Furthermore, double positivity of anti-ß2GPIdI (anti-ß2-glycoprotein I domain I) combined with anti-PS/PT even more elevated both thrombosis and positivity of anti-ADAMTS13 antibody. Double positivity of ß2GPI and anti-PS/PT was found as an independently significant contributing factor to anti-ADAMTS13 antibody level. We suggest the association between anti-ADAMTS13 antibody and the pathophysiology of antiphospholipid syndrome, which should be further evaluated.

Risk Assessment and Antithrombotic Strategies in Antiphospholipid Antibody Carriers.

Antiphospholipid antibodies (aPL) are a cluster of autoantibodies directed against plasma proteins with affinity for membrane phospholipids. The most frequently tested aPL are lupus anticoagulant (LA), anti-cardiolipin antibodies (aCL), and anti-ß2-glycoprotein I antibodies (anti-ß2GPI). aPL play a key pathogenic role in the development of the antiphospholipid syndrome (APS), a systemic autoimmune disease characterized by recurrent thrombotic and/or pregnancy complications in patients with persistent aPL. However, aPL positivity is occasionally documented in patients with no previous history of thrombotic or pregnancy morbidity. LA activity, multiple aPL positivity, high-titer aPL, and a concomitant systemic autoimmune disease are recognized risk factors for future thrombotic events in asymptomatic carriers. Moreover, an accelerated atherosclerosis with increased cardiovascular (CV) risk has also been associated with aPL positivity, thus exposing aPL carriers to fatal complications and chronic disability requiring cardiac rehabilitation. Overall, an accurate risk stratification is recommended for aPL-positive subjects in order to prevent both venous and arterial thrombotic complications. In this review, we provide an overview of the main antithrombotic and risk assessment strategies in aPL carriers.

Anti-DNA antibodies cross-reactive with ß2-glycoprotein I induce monocyte tissue factor through the TLR9 pathway.

Antibodies specific for cardiolipin (CL)-ß2-glycoprotein I (ß2GPI) are known to induce tissue factor (TF) expression by monocytes and endothelial cells which leads to a prothrombotic state in antiphospholipid syndrome (APS), but the mechanism is not fully elucidated. Previously, we reported that the mouse monoclonal anti-CL-ß2GPI antibody WB-6 cross-reacts with DNA, enters monocytes via binding to cell surface DNA, and induces TF expression. The current study aimed to identify the intracellular signaling pathways involved in this process. The binding of WB-6 to CL-ß2GPI or DNA, and endocytosis was not prevented by chloroquine, but pre-treatment of the cells with chloroquine significantly suppressed TF expression. TLR9 inhibitory oligodeoxynucleotide also suppressed the WB-6-induced TF expression, suggesting a pivotal role of the TLR9 pathway in TF production. Serum antibodies obtained from a patient with APS accompanying systemic lupus erythematosus (SLE) bound to both CL-ß2GPI and DNA, and induced TF in normal monocytes. This effect was suppressed by chloroquine, and abolished by removal of the DNA-binding activity. These results suggest that induction of TF expression results from TLR9 activation by DNA which was internalized together with cross-reactive antibodies produced in secondary APS accompanying SLE.

Peptidic vaccines: The new cure for heart diseases?

Cardiovascular disease continues to be the most common cause of death worldwide. The global burden is so high that numerous organizations are providing counseling recommendations and annual revisions of current pharmacological and non-pharmacological treatments as well as risk prediction for disease prevention and further progression. Although primary preventive interventions targeting risk factors such as obesity, hypertension, smoking, and sedentarism have led to a global decline in hospitalization rates, the aging population has overwhelmed these efforts on a global scale. This review focuses on peptidic vaccines, with the known and not well-known autoantigens in atheroma formation or acquired cardiac diseases, as novel potential immunotherapy approaches to counteract harmful heart disease continuance. We summarize how cancer immunomodulatory strategies started novel approaches to modulate the innate and adaptive immune responses, and how they can be targeted for therapeutic purposes in the cardiovascular system. Brief descriptions focused on the processes that start as either immunologic or non-immunologic, and the ultimate loss of cardiac muscle cell contractility as the outcome, are discussed. We conclude debating how novel strategies with nanoparticles and nanovaccines open a promising therapeutic option to reduce or prevent cardiovascular diseases.

Insight into the hypercoagulable state of high-risk thrombotic APS patients: Contribution of aß2GPI and aPS/PT antibodies.

OBJECTIVE: Most high-risk thrombotic antiphospholipid syndrome (APS) patients test positive for anti-ß2-glycoprotein I (aß2GPI) and anti-phosphatidylserine/prothrombin (aPS/PT) antibodies. Information on the influence of these antibodies on thrombin generation and activated protein C resistance (aPCr) is still sparse and contradictory. METHODS: Plasma of 16 patients poured into a ß2GPI affinity column allowed the perfect separation of aß2GPI and aPS/PT antibodies. aPS/PT antibodies were further purified through a prothrombin affinity column. Obtained material was spiked into normal pooled plasma (NPP) and tested in the thrombin generation assay in the absence or presence of aPC. RESULTS: aPS/PT antibodies showed a marked anticoagulant effect. Affinity purified aPS/PT and aß2GPI antibodies from five patients were compared. aPS/PT antibodies showed significantly prolonged lag time and time to peak (5.0 minutes [interquartile range (IQR)3.5-6.1] versus 2.7 minutes [IQR2.2-3.5], P = .03 and 8.7 minutes [IQR6.7-10.3] versus 5.7 minutes [IQR4.5-6.2], P = .05, respectively) and significantly lower peak and velocity index (143 nmol/L [IQR131-163] versus 171 nmol/L [IQR157-182], P = .03 and 35 nmol/L/min [IQR32-59] versus 72 nmol/L/min [IQR54-77], P = .03, respectively). When aPC was added to the system, aPCr was significantly increased compared to controls for both aß2GPI and aPS/PT antibodies. However, it was significantly stronger using aPS/PT antibodies. Median inhibition of endogenous thrombin potential was 22% (IQR16-33) with aPS/PT compared to 52% (IQR46-56) with aß2GPI antibodies (P = .002). CONCLUSIONS: Aß2GPI antibodies show a mild anticoagulant and moderate procoagulant effect in thrombin generation and moderate aPC resistance. Conversely, aPS/PT antibodies show a strong anticoagulant effect and a strong aPCr.

Autoantibodies specific for C1q, C3b, ß2-glycoprotein 1 and annexins may amplify complement activity and reduce apoptosis-mediated immune suppression.

Neoplastic cells hijack cell death pathways to evade the immune response. Phosphatidylserine, a marker of apoptotic cells, and its highly conserved bridging proteins, annexins and ß2-glycoprotein I, facilitate the efficient removal of apoptotic and necrotic cells via tumor-associated phagocytes in a process called efferocytosis. Efferocytosis results in the clearance of dead and dying cells and local immune suppression. Neoplastic cells also have an increased capacity to activate complement. Complement may facilitate the silent removal of tumor cells and has a dual role in promoting and inhibiting tumor growth. Here I hypothesize that immune response-generating IgG autoantibodies that recognize opsonizing fragments C1q, C3b, and phosphatidylserine-binding proteins (annexins, ß2-glycoprotein I) may reduce tumor growth. I propose that these autoantibodies induce a pro-inflammatory, cytotoxic tumor microenvironment. Further, I predict that autoantibodies can drive neoplastic cell phagocytosis in an Fc receptor-dependent manner and recruit additional complement, resulting in immune-stimulatory effects. Excessive complement activation and antibody-dependent cytotoxicity may stimulate anti-tumor responses, including damage to tumor vasculature. Here I provide insights that may aid the development of more effective therapeutic modalities to control cancer. Such therapeutic approaches should kill neoplastic cells and target their interaction with host immune cells. Thereby the pro-tumorigenic effect of dead cancer cells could be limited while inducing the anti-tumor potential of tumor-associated phagocytes.

Anti-Phospholipid Antibodies in COVID-19 Are Different From Those Detectable in the Anti-Phospholipid Syndrome.

Background: Critically ill patients with coronavirus disease 2019 (COVID-19) have a profound hypercoagulable state and often develop coagulopathy which leads to organ failure and death. Because of a prolonged activated partial-thromboplastin time (aPTT), a relationship with anti-phospholipid antibodies (aPLs) has been proposed, but results are controversial. Functional assays for aPL (i.e., lupus anticoagulant) can be influenced by concomitant anticoagulation and/or high levels of C reactive protein. The presence of anti-cardiolipin (aCL), anti-beta2-glycoprotein I (anti-ß2GPI), and anti-phosphatidylserine/prothrombin (aPS/PT) antibodies was not investigated systematically. Epitope specificity of anti-ß2GPI antibodies was not reported. Objective: To evaluate the prevalence and the clinical association of aPL in a large cohort of COVID-19 patients, and to characterize the epitope specificity of anti-ß2GPI antibodies. Methods: ELISA and chemiluminescence assays were used to test 122 sera of patients suffering from severe COVID-19. Of them, 16 displayed major thrombotic events. Results: Anti-ß2GPI IgG/IgA/IgM was the most frequent in 15.6/6.6/9.0% of patients, while aCL IgG/IgM was detected in 5.7/6.6% by ELISA. Comparable values were found by chemiluminescence. aPS/PT IgG/IgM were detectable in 2.5 and 9.8% by ELISA. No association between thrombosis and aPL was found. Reactivity against domain 1 and 4-5 of ß2GPI was limited to 3/58 (5.2%) tested sera for each domain and did not correlate with aCL/anti-ß2GPI nor with thrombosis. Conclusions: aPL show a low prevalence in COVID-19 patients and are not associated with major thrombotic events. aPL in COVID-19 patients are mainly directed against ß2GPI but display an epitope specificity different from antibodies in antiphospholipid syndrome.

Anti-Domain I ß2-Glycoprotein I Antibodies and Activated Protein C Resistance Predict Thrombosis in Antiphospholipid Syndrome: TAC(I)T Study.

BACKGROUND: Antibodies binding to domain I of ß2-glycoprotein I (aDI) and activated protein C (APC) resistance are associated with an increased risk of thrombosis in cross-sectional studies. The objective of this study was to assess their predictive value for future thromboembolic events in patients with antiphospholipid antibodies (aPL) or antiphospholipid syndrome. METHODS: This prospective multicenter cohort study included consecutive patients with aPL or systemic lupus erythematosus. We followed 137 patients (43.5 ± 15.4 year old; 107 women) for a mean duration of 43.1 ± 20.7 months. RESULTS: We detected aDI IgG antibodies by ELISA in 21 patients. An APC sensitivity ratio (APCsr) was determined using a thrombin generation-based test. The APCsr was higher in patients with anti-domain I antibodies demonstrating APC resistance (0.75 ± 0.13 vs 0.48 ± 0.20, P < 0.0001). In univariate analysis, the hazard ratio (HR) for thrombosis over time was higher in patients with aDI IgG (3.31 [95% CI, 1.15-9.52]; P = 0.03) and patients with higher APC resistance (APCsr >95th percentile; HR, 6.07 [95% CI, 1.69-21.87]; P = 0.006). A sensitivity analysis showed an increased risk of higher aDI IgG levels up to HR 5.61 (95% CI, 1.93-16.31; P = 0.01). In multivariate analysis, aDI IgG (HR, 3.90 [95% CI, 1.33-11.46]; P = 0.01) and APC resistance (HR, 4.98 [95% CI, 1.36-18.28]; P = 0.02) remained significant predictors of thrombosis over time. CONCLUSIONS: Our study shows that novel tests for antibodies recognizing domain I of ß2-glycoprotein I and functional tests identifying APC resistance are significant predictors of thrombosis over time and may be useful for risk stratification.

Rapid and specific detection of oxidized LDL/ß2GPI complexes via facile lateral flow immunoassay.

ß2-Glycoprotein I (ß2GPI) forms indissociable complex with oxidized LDL (oxLDL) into proatherogenic oxLDL/ß2GPI complex through a specific ligand known as 7-ketocholesteryl-9-carboxynonanoate (oxLig-1). Recent discoveries have demonstrated the atherogenicity of these complexes in patients of both systemic and non-systemic autoimmune diseases. Hence, serological level of oxLDL/ß2GPI complexes may represent one crucial clinical parameter for disease prognosis of atherosclerosis-related diseases. Herein, we established a simple, specific and rapid gold nanoparticle (GNP) based lateral flow immunoassay (LFIA) to quantify oxLDL/ß2GPI complexes from test samples. Specificities of hybridoma cell-derived monoclonal antibodies against antigen, optimal conditions for conjugation of antibody with GNP, and sensitivity of oxLDL/ß2GPI LFIA in comparison to an ELISA-based detection method were assessed accordingly. The established oxLDL/ß2GPI LFIA was capable of detecting oxLDL/ß2GPI specifically without interference from autoantibodies and solitary components of oxLDL/ß2GPI present in test samples. A significant correlation (R2 > 0.8) was also obtained with the oxLDL/ß2GPI LFIA when compared to the ELISA-based detection. On the whole, the oxLDL/ß2GPI LFIA remains advantageous over the oxLDL/ß2GPI ELISA. The unnecessary washing step, short developmental and analytical time support facile and rapid detection of oxLDL/ß2GPI as opposed to the laborious ELISA system.

Tetra positive thrombotic antiphospholipid syndrome: Major contribution of anti-phosphatidyl-serine/prothrombin antibodies to lupus anticoagulant activity.

BACKGROUND: The concurrent presence of lupus anticoagulant, anticardiolipin, and anti ß2-glycoprotein I antibodies (triple positive profile) identifies patients at high risk of thromboembolic events. These patients are also positive for anti-phosphatidyl-serine/prothrombin antibodies (tetra-positive profile). OBJECTIVE: Understand which antibody among anti-ß2-glycoprotein I and anti-phosphatidyl-serine/prothrombin is responsible for lupus anticoagulant activity. PATIENTS/METHODS: Affinity purified anti-ß2-glycoprotein I antibodies from plasma of 14 tetra-positive patients spiked into normal pooled plasma were tested. RESULTS AND CONCLUSIONS: Anti-ß2-glycoprotein I antibodies did not prolong the diluted Russell viper venom time and silica clotting time (median ratio 0.98, interquartile ratio [IQR] 0.9-1.06; and 1.0, IQR 0.91-1.03, respectively). Anticoagulant activity remained in the flow-through that was deprived of anti-ß2 glycoprotein I antibodies (median ratio 1.88, IQR 1.58-2.77; and 1.75, IQR 1.17-2.9, respectively). This material was loaded on size-exclusion chromatography Sephacryl S-300 column and showed that anticoagulant activity and anti-phosphatidyl-serine/prothrombin antibodies coeluted in the same fractions. Besides, the flow through was poured into a prothrombin affinity column. Protein yield in three patients ranged from 54 to 91 µg/mL and showed strong positivity in phosphatidyl-serine/prothrombin ELISA. The affinity purified material prolonged the coagulation time of normal pooled plasma: the diluted Russell viper venom ratio in the three patients was 2.09, 1.21, and 1.35; that of silica clotting time was 2.05, 1.5, and 2.13. In conclusion, under the assay conditions used, anticoagulant activity in tetra-positive antiphospholipid syndrome patients may largely be attributable to anti-phosphatidyl-serine/prothrombin antibodies.

Induction of tissue factor expression by anti-ß2-glycoprotein I is mediated by tumor necrosis factor α.

Antiphospholipid antibodies (aPL) are heterogeneous and there is evidence that binding specificity determines which cellular effects they can trigger. We have therefore hypothesised that the induction of tissue factor (TF) in monocytes and endothelial cells by aPL depends on their binding specificity. To further investigate this, we have analyzed the ability of three human monoclonal aPL with distinctly different binding specificities to induce transcription and cell surface expression of TF in monocytes and endothelial cells. Results with human monoclonal aPL were validated with IgG-fractions obtained from patients with APS. We confirmed previous results that a lipid reactive human monoclonal aPL rapidly induced TF transcription and cell surface expression in monocytes and endothelial cells. A monoclonal aPL reactive against ß2 glycoprotein I (ß2GPI) induced TF with a delayed time course. This was fully dependent on the induction of tumor necrosis factor alpha (TNFα) secretion as capture of TNFα by adalimumab prevented TF induction. This pattern was confirmed with patient IgG fractions. Both lipid reactive and anti-ß2GPI induced TF transcription. Unexpectedly, this activity of anti-ß2GPI was mediated fully by TNFα which was secreted in response to incubation with anti-ß2GPI. The role of TNFα in mediating TF induction by anti-ß2GPI may have wider implications for APS pathogenesis.

Detection of anti-domain I antibodies by chemiluminescence enables the identification of high-risk antiphospholipid syndrome patients: A multicenter multiplatform study.

BACKGROUND: Classification of the antiphospholipid syndrome (APS) relies predominantly on detecting antiphospholipid antibodies (aPLs). Antibodies against a domain I (DI) epitope of anti-ß2glycoprotein I (ß2GPI) proved to be pathogenic, but are not included in the current classification criteria. OBJECTIVES: Investigate the clinical value of detecting anti-DI IgG in APS. PATIENTS/METHODS: From eight European centers 1005 patients were enrolled. Anti-cardiolipin (CL) and anti-ß2GPI were detected by four commercially available solid phase assays; anti-DI IgG by the QUANTA Flash® ß2GPI domain I assay. RESULTS: Odds ratios (ORs) of anti-DI IgG for thrombosis and pregnancy morbidity proved to be higher than those of the conventional assays. Upon restriction to patients positive for anti-ß2GPI IgG, anti-DI IgG positivity still resulted in significant ORs. When anti-DI IgG was added to the criteria aPLs or used as a substitute for anti-ß2GPI IgG/anti-CL IgG, ORs for clinical symptoms hardly improved. Upon removing anti-DI positive patients, lupus anticoagulant remained significantly correlated with clinical complications. Anti-DI IgG are mainly present in high-risk triple positive patients, showing higher levels. Combined anti-DI and triple positivity confers a higher risk for clinical symptoms compared to only triple positivity. CONCLUSIONS: Detection of anti-DI IgG resulted in higher ORs for clinical manifestations than the current APS classification criteria. Regardless of the platform used to detect anti-ß2GPI/anti-CL, addition of anti-DI IgG measured by QUANTA Flash® did not improve the clinical associations, possibly due to reduced exposure of the pathogenic epitope of DI. Our results demonstrate that anti-DI IgG potentially helps in identifying high-risk patients.