Bill Giannakopoulos

The Pathogenesis of the Antiphospholipid Syndrome

The antiphospholipid syndrome is a prothrombotic disorder associated with autoantibodies. It is associated with obstetrical complications (mainly spontaneous abortion) as well as venous and arterial thrombotic risks. Insights into disease mechanisms have led to new therapies.

How we diagnose the antiphospholipid syndrome

The antiphospholipid syndrome (APS) is an acquired thrombophilia, characterized by the occurrence of venous and arterial events. This article examines the laboratory and key clinical aspects of APS. Particular focus is given to anti-beta 2-glycoprotein I (beta(2)GPI) antibodies in view of their recent inclusion in the APS classification criteria. The clinical utility of using the beta(2)GPI enzyme-linked immunosorbent assay, in conjunction with the established lupus anticoagulant assays and cardiolipin enzyme-linked immunosorbent assay, for diagnosing and risk stratifying patients suspected of having APS is discussed. The relative importance of the various assays in diagnosing obstetric APS (early and late gestation miscarriages) is explored. The implications of recent epidemiologic findings for possibly understanding the underlying pathophysiologic mechanisms of obstetric APS are highlighted. Insights into which patients with obstetric APS may be at most risk of thrombotic complications are presented.

Naturally occurring free thiols within β2-glycoprotein I in vivo: nitrosylation, redox modification by endothelial cells, and regulation of oxidative stress-induced cell injury

β2-Glycoprotein I (β2GPI) is an evolutionary conserved, abundant circulating protein. Although its function remains uncertain, accumulated evidence points toward interactions with endothelial cells and components of the coagulation system, suggesting a regulatory role in vascular biology. Our group has shown that thioredoxin 1 (TRX-1) generates free thiols in β2GPI, a process that may have a regulatory role in platelet adhesion. This report extends these studies and shows for the first time evidence of β2GPI with free thiols in vivo in both multiple human and murine serum samples. To explore how the vascular surface may modulate the redox status of β2GPI, unstimulated human endothelial cells and EAhy926 cells are shown to be capable of amplifying the effect of free thiol generation within β2GPI. Multiple oxidoreductase enzymes, such as endoplasmic reticulum protein 46 (ERp 46) and TRX-1 reductase, in addition to protein disulfide isomerase are secreted on the surface of endothelial cells. Furthermore, one or more of these generated free thiols within β2GPI are also shown to be nitrosylated. Finally, the functional significance of these findings is explored, by showing that free thiol-containing β2GPI has a powerful effect in protecting endothelial cells and EAhy926 cells from oxidative stress-induced cell death.

How I treat the antiphospholipid syndrome

This article discusses how we approach medical decision making in the treatment of the various facets of the antiphospholipid syndrome (APS), including secondary prophylaxis in the setting of venous and arterial thrombosis, as well as treatment for the prevention of recurrent miscarriages and fetal death. The role of primary thromboprophylaxis is also discussed in depth. Great emphasis is given to incorporating the most up-to-date and relevant evidence base both from the APS literature, and from large, recent, randomized controlled trials (RCTs) of primary and secondary thrombotic prophylaxis in the general population setting (ie, the population that has not been specifically investigated for APS).

Novel Assays of Thrombogenic Pathogenicity in the Antiphospholipid Syndrome Based on the Detection of Molecular Oxidative Modification of the Major Autoantigen β2-Glycoprotein I

Objective Beta-2-glycoprotein I (β2GPI) constitutes the major autoantigen in the antiphospholipid syndrome (APS), a common acquired cause of arterial and venous thrombosis. We recently described the novel observation that β2GPI may exist in healthy individuals in a free thiol (biochemically reduced) form. The present study was undertaken to quantify the levels of total, reduced, and posttranslationally modified oxidized β2GPI in APS patients compared to various control groups. Methods In a retrospective multicenter analysis, the proportion of β2GPI with free thiols in serum from healthy volunteers was quantified. Assays for measurement of reduced as well as total circulating β2GPI were developed and tested in the following groups: APS (with thrombosis) (n = 139), autoimmune disease with or without persistent antiphospholipid antibodies (aPL) but without APS (n = 188), vascular thrombosis without APS or aPL (n = 38), and healthy volunteers (n = 91). Results Total β2GPI was significantly elevated in patients with APS (median 216.2 μg/ml [interquartile range 173.3–263.8]) as compared to healthy subjects (median 178.4 μg/ml [interquartile range 149.4–227.5] [P < 0.0002]) or control patients with autoimmune disease or vascular thrombosis (both P < 0.0001). The proportion of total β2GPI in an oxidized form (i.e., lacking free thiols) was significantly greater in the APS group than in each of the 3 control groups (all P < 0.0001). Conclusion This large retrospective multicenter study shows that posttranslational modification of β2GPI via thiol-exchange reactions is a highly specific phenomenon in the setting of APS thrombosis. Quantification of posttranslational modifications of β2GPI in conjunction with standard laboratory tests for APS may offer the potential to more accurately predict the risk of occurrence of a thrombotic event in the setting of APS.

Ro 60 functions as a receptor for β2-glycoprotein I on apoptotic cells

OBJECTIVE The autoantigens 60-kd Ro/SSA (Ro 60) and beta(2)-glycoprotein I (beta(2)GPI) are both displayed on the surface membrane of apoptotic cells. Epitope-spreading experiments have suggested that these autoantigens may be present as a complex on the apoptotic cell surface. This study was undertaken to investigate whether beta(2)GPI interacts with Ro 60 on apoptotic cells and alters the binding of anti-Ro 60 IgG. METHODS The interaction between soluble recombinant Ro 60 fragments and beta(2)GPI was investigated in vitro by direct and saturation binding assays using native human beta(2)GPI and recombinant domain deletion mutants. Binding of beta(2)GPI to early and late apoptotic cells was assessed by multiparameter flow cytometry, and specificity of binding was determined by competitive inhibition with soluble recombinant Ro 60 and anti-Ro 60 IgG. RESULTS The Ro 60 fragment expressing a surface-exposed epitope (apotope) bound with high affinity (K(d) = approximately 15 nM) to domain V of beta(2)GPI in vitro. Beta(2)-glycoprotein I bound to the surface of apoptotic cells in a dose-dependent manner and was blocked by the Ro 60 apotope fragment. In reciprocal competitive inhibition studies, beta(2)GPI blocked the binding of anti-Ro 60 autoantibodies to apoptotic cells in a dose-dependent manner, and anti-Ro 60 IgG inhibited the binding of beta(2)GPI. Moreover, beta(2)GPI showed a 2-fold increase in binding to apoptotic cells that overexpress Ro 60 on the surface. CONCLUSION These results demonstrate that Ro 60 functions as a novel receptor for beta(2)GPI on the surface of apoptotic cells. The formation of Ro 60-beta(2)GPI complexes may protect against anti-Ro 60 autoantibody-mediated tissue injury.

Beta 2 glycoprotein I is a substrate of thiol oxidoreductases

To the editor: Beta 2 glycoprotein I (β2GPI) is an abundant plasma protein recognized as the major autoantigen in the antiphospholipid syndrome. Although the crystal structure of β2GPI has been resolved,[1][1],[2][2] its normal function remains unknown. We have been intrigued by the presence of a

Redox control of β2-glycoprotein I-von Willebrand factor interaction by thioredoxin-1.

Summary.  Background: β2‐Glycoprotein I (β2GPI) is an abundant plasma protein that is closely linked to blood clotting, as it interacts with various protein and cellular components of the coagulation system. However, the role of β2GPI in thrombus formation is unknown. We have recently shown that β2GPI is susceptible to reduction by the thiol oxidoreductases thioredoxin‐1 and protein disulfide isomerase, and that reduction of β2GPI can take place on the platelet surface. Methods: β2GPI, reduced by thioredoxin‐1, was labeled with the selective sulfhydryl probe Na‐(3‐maleimidylpropionyl)biocytin and subjected to mass spectrometry to identify the specific cysteines involved in the thiol exchange reaction. Binding assays were used to examine the affinity of reduced β2GPI for von Willebrand factor (VWF) and the effect of reduced β2GPI on glycoprotein (GP)Ibα binding to VWF. Platelet adhesion to ristocetin‐activated VWF was studied in the presence of reduced β2GPI. Results:  We demonstrate that the Cys288–Cys326 disulfide in domain V of β2GPI is the predominant disulfide reduced by thioredoxin‐1. Reduced β2GPI in vitro displays increased binding to VWF that is dependent on disulfide bond formation. β2GPI reduced by thioredoxin‐1, in comparison with non‐reduced β2GPI, leads to increased binding of GPIbα to VWF and increased platelet adhesion to activated VWF. Conclusions:  Given the importance of thiol oxidoreductases in thrombus formation, we provide preliminary evidence that the thiol‐dependent interaction of β2GPI with VWF may contribute to the redox regulation of platelet adhesion.

Molecular pathophysiology of the antiphospholipid syndrome: the role of oxidative post‐translational modification of beta 2 glycoprotein I

Summary.  It has been well established that antiphospholipid antibodies and specifically those directed against beta 2 glycoprotein I (β2GPI) are pathogenic for the development of thrombosis in the antiphospholipid syndrome (APS). Several groups have shown that anti‐β2GPI antibodies, in complex with β2GPI, elicit effects on blood cells and coagulation‐fibrinolysis proteins, which prime the arterial and venous vasculature for the development of thrombosis. However, much less is known about the mechanism initiating the production of autoantibodies against β2GPI, a physiological abundant protein of blood. In the current review, novel findings are presented regarding the structure and oxidative post‐translational modifications of β2GPI, which trigger the immune response. The majority of circulating β2GPI exists in a form containing unpaired cysteines (free thiols), which constitutes the reduced form of β2GPI. The free thiols exposed on β2GPI are involved in the interaction with platelets and endothelial cells. We propose that this abundant pool of free thiols may serve as an antioxidant reservoir protecting cells or critical molecules from oxidative stress. Oxidation of β2GPI confers an increase in its immunogenicity through a Th1 immunological mechanism. The clinical significance of these observations is that serum from patients with APS, assessed by a novel ELISA assay, have a significant increase in oxidised β2GPI. These findings hold promise, not only for the delineation of the role of β2GPI as an immunological target, but also for the development of improved diagnostic and prognostic assays for APS.

Domain V of β2-Glycoprotein I Binds Factor XI/XIa and Is Cleaved at Lys317-Thr318

The fifth domain (DV) of β2-glycoprotein I (β2GPI) is important for binding a number of ligands including phospholipids and factor XI (FXI). β2GPI is proteolytically cleaved in DV by plasmin but not by thrombin, VIIa, tissue plasminogen activator, or uPA. Following proteolytic cleavage of DV by plasmin, β2GPI retains binding to FXI but not to phospholipids. Native β2GPI, but not cleaved β2GPI, inhibits activation of FXI by thrombin and factor XIIa, attenuating a positive feedback mechanism for additional thrombin generation. In this report, we have defined the FXI/FXIa binding site on β2GPI using site-directed mutagenesis. We show that the positively charged residues Lys284, Lys286, and Lys287 in DV are essential for the interaction of β2GPI with FXI/FXIa. We also demonstrate that FXIa proteolytically cleaves β2GPI at Lys317-Thr318 in DV. Thus, FXIa cleavage of β2GPI in vivo during thrombus formation may accelerate FXI activation by decreasing the inhibitory effect of β2GPI.