Steven A. Krilis

Background: βeta-2 glycoprotein I (β2GPI, Apolipoprotein H) is a plasma glycoprotein best known as a major autoantigen in autoimmune disorders such as antiphospholipid syndrome (APS) and systemic lupus erythematosus (SLE), both of which confer elevated cardiovascular risk. Despite its prominence in autoimmunity, its role in lipid metabolism and potential sex-specific effects remain poorly understood. Methods: We investigated β2GPI’s influence on lipoprotein profiles using β2GPI knockout (KO) and wild-type (WT) mice subjected to normal chow (NC) and high-fat (HF) diets, as well as plasma from β2GPI-deficient patients and aged and sex matched controls. Lipoprotein fractions were analyzed for cholesterol and apolipoprotein content, and protein interactions were assessed by co-immunoprecipitation. Results: In animal studies, female β2GPI KO mice—but not males—displayed significantly increased total plasma cholesterol on a HF diet and greater cholesterol content within HDL fractions. Apo E was enriched in HDL fractions from female KO mice under both NC and HF diets, and plasma Apo E was elevated in HF-fed female KOs. In WT females on HF diet, β2GPI was enriched in HDL fractions, and β2GPI co-immunoprecipitated with Apo E. In human studies, the β2GPI-deficient female patient exhibited increased HDL cholesterol, a shift toward larger HDL particles, and enriched Apo E in HDL fractions relative to controls. Co-immunoprecipitation confirmed β2GPI–Apo E interaction in human plasma, with binding requiring Domain V of β2GPI. Conclusions: Our findings identify β2GPI as a sex-specific regulator of HDL metabolism. In females, β2GPI modulates Apo E-containing HDL particles, influencing cholesterol distribution and lipoprotein composition. These results reveal a novel mechanism linking β2GPI to lipid homeostasis, with potential implications for cardiovascular risk in women with autoimmune disease. Targeting β2GPI–Apo E interactions may represent a therapeutic avenue for correcting dysregulated HDL metabolism in female-specific cardiometabolic and autoimmune contexts.

Objective: Atherosclerotic coronary artery disease is well recognised as an inflammatory disorder that is also influenced by oxidative stress. β2-GPI (β-2-glycoprotein-I) is a circulating plasma protein that undergoes post-translational modification and exists in free thiol as well as oxidized forms. The aim of this study was to assess the association between these 2 post-translational redox forms of β2-GPI and atherosclerotic coronary artery disease. Approach and Results: Stable patients presenting for elective coronary angiography or CT coronary angiography were prospectively recruited. A separate group of patients after reperfused ST-segment–elevation myocardial infarction formed an acute coronary syndrome subgroup. All patients had collection of fasting serum and plasma for quantification of total and free thiol β2-GPI. Coronary artery disease extent was quantified by the Syntax and Gensini scores. A total of 552 patients with stable disease and 44 with acute coronary syndrome were recruited. While total β2-GPI was not associated with stable coronary artery disease, a higher free thiol β2-GPI was associated with its presence and extent. This finding remained significant after correcting for confounding variables, and free thiol β2-GPI was a better predictor of stable coronary artery disease than hs-CRP (high-sensitivity C-reactive protein). Paradoxically, there were lower levels of free thiol β2-GPI after ST-segment–elevation myocardial infarction. Conclusions: Free thiol β2-GPI is a predictor of coronary artery disease presence and extent in stable patients. Free thiol β2-GPI was a better predictor than high-sensitivity C-reactive protein.

β₂-Glycoprotein I is the major autoantigen in the antiphospholipid syndrome (APS), a prothrombotic disorder characterized by the occurrence of either venous or arterial thrombosis. In women it is also associated with an increased risk of obstetric complications such as recurrent miscarriages. We have identified that the plasma protein β₂-glycoprotein I in healthy individuals exists in an optimal ratio between two distinct forms, an oxidized and free thiol, reduced form. This ratio is disrupted in pathophysiological conditions associated with increased oxidative stress such as the APS, but also in the setting of age-related macular degeneration and gram-negative sepsis. We have developed assays that quantify plasma/serum levels of total and free thiol β₂-glycoprotein I which can potentially be used for risk stratification and prognostic purposes in the early stages of the aforementioned conditions.

Angiotensinogen mediates an important role in the pathophysiology of preeclampsia, a disorder of pregnancy characterized by hypertension and proteinuria usually after 20 weeks of gestation. Angiotensinogen is found in two distinct posttranslational forms in the plasma, an oxidized and a reduced (free thiol) form. Higher levels of the oxidized form are associated with an increased risk of preeclampsia. We have developed novel ELISA assays to quantitate the levels of total and free thiol angiotensinogen allowing for calculation of the amount of oxidized angiotensinogen species. We describe the methodology for performing these assays.

Reperfusion after a period of ischemia results in reperfusion injury (IRI) which involves activation of the inflammatory cascade. In cardiac IRI, IgM natural antibodies (NAb) play a prominent role through binding to altered neoepitopes expressed on damaged cells. Beta 2 Glycoprotein I (β2GPI) is a plasma protein that binds to neoepitopes on damaged cells including anionic phospholipids through its highly conserved Domain V. Domain I of β2GPI binds circulating IgM NAbs and may provide a link between the innate immune system, IgM NAb binding and cardiac IRI. This study was undertaken to investigate the role of Β2GPI and its Domain V in cardiac IRI using wild-type (WT), Rag-1 ^-/- and β2GPI deficient mice. Compared with control, treatment with Domain V prior to cardiac IRI prevented binding of endogenous β2GPI to post-ischemic myocardium and resulted in smaller myocardial infarction size in both WT and β2GPI deficient mice. Domain V treatment in WT mice also resulted in less neutrophil infiltration, less apoptosis and improved ejection fraction at 24 h. Rag-1 -/- antibody deficient mice reconstituted with IgM NAbs confirmed that Domain V prevented IgM NAb induced cardiac IRI. Domain V remained equally effective when delivered at the time of reperfusion which has therapeutic clinical relevance.Based upon this study Domain V may function as a universal inhibitor of IgM NAb binding in the setting of cardiac IRI, which offers promise as a new therapeutic strategy in the treatment of cardiac IRI.

Ras guanine nucleotide-releasing protein-4 (RasGRP4) is an evolutionarily conserved calcium-regulated, guanine nucleotide exchange factor and diacylglycerol/phorbol ester receptor. While an important intracellular signaling protein for CD117⁺ mast cells (MCs), its roles in other immune cells is less clear. In this study, we identified a subset of in vivo-differentiated splenic CD117⁺ dendritic cells (DCs) in wild-type (WT) C57BL/6 mice that unexpectedly contained RasGRP4 mRNA and protein. In regard to the biologic significance of these data to innate immunity, LPS-treated splenic CD117⁺ DCs from WT mice induced natural killer (NK) cells to produce much more interferon-γ (IFN-γ) than comparable DCs from RasGRP4-null mice. The ability of LPS-responsive MCs to cause NK cells to increase their expression of IFN-γ was also dependent on this intracellular signaling protein. The discovery that RasGRP4 is required for CD117⁺ MCs and DCs to optimally induce acute NK cell-dependent immune responses to LPS helps explain why this signaling protein has been conserved in evolution.

This chapter reviews several important themes pertaining to the antiphospholipid syndrome (APS), including a description of the clinical features, a discussion of the main autoantigen, beta 2-glycoprotein I (β₂GPI), and insights into the characteristics of the pathogenic anti-β₂GPI autoantibodies. Evidence-based considerations for when to test for APS are explored, along with the clinical significance of patients testing positive on multiple APS assays, so-called triple positivity. A detailed review of recently published laboratory guidelines for the detection of lupus anticoagulant and the solid-phase anticardiolipin and anti-β₂GPI ELISAs is undertaken. Finally, a brief review of nonclassification criteria laboratory assays with potential future diagnostic utility is presented.