Discovery and characterization of 2 novel subpopulations of aPS/PT antibodies in patients at high risk of thrombosis

Chinnaraj, Mathivanan; Planer, William; Pengo, Vittorio; Pozzi, Nicola

doi:10.1182/bloodadvances.2019030932

Cited by 24 publications

(32 citation statements)

References 64 publications

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“…In this context, a possible role of the negatively charged surfaces suggested by our SPR binding experiments performed at low ionic strength would be to stimulate the binding of anti-DI antibodies to β 2 GPI by slowing down the dissociation rate, thus resulting in complex stabilization. Such a mechanism is fully consistent with recent and previous data(35, 48), and might be conserved among other aPL.…”

Section: Discussionsupporting

confidence: 92%

“…In addition to properly interacting with phospholipids, the recombinant proteins were also successfully recognized in ELISA assays by aPL isolated from four triple positive APS patients, which contain anti-DI antibodies(22, 35, 40) (Fig. 2E).…”

Section: Resultsmentioning

confidence: 99%

“…Liposomes composed of phosphatidylcholine (PC) or phosphatidylcholine and phosphatidylserine (PS) in a 4:1 molar ratio (PC:PS) were prepared by extrusion using 100 nm polycarbonate membranes (Avanti Polar Lipids, Alabaster, AL), kept a 4°C and used within 7 days. ELISA assays were performed as described before(22, 35, 57). Protein concentrations were determined by reading at 280 nm with molar extinction coefficients adjusted according to the amino acid sequence.…”

Section: Methodsmentioning

confidence: 99%

“…Binding affinities for liposomes were measured as done before(35) using L1 sensor chip in which liposomes were immobilized at 1600 RU. Titrations were performed by injecting increasing concentrations (0-2 μM) of β 2 GPI and its variants in running buffer (20 mM Tris pH 7.4, 150 mM NaCl, 0.1% w/w BSA) at a flow rate of 25 μl/min at 25°C.…”

Section: Methodsmentioning

confidence: 99%

“…Consequently, it remains unclear under what circumstances and how these forms interconvert, what is their physiological role, and how they participate in the mechanism of autoantibody recognition. Encouraged by our recent results with prothrombin(35–37), the second most common antigen of aPL in APS, this work was initiated to investigate the structural and conformational properties of β 2 GPI under conditions relevant to physiology and provide new insights into the mechanism of autoantibody recognition. Our results based on X-ray crystallography, single-molecule FRET (smFRET), SAXS, binding kinetics, and mutational studies unexpectedly reveal that human recombinant β 2 GPI adopts an elongated, flexible conformation, not circular, in which DI is exposed to the solvent and therefore available for autoantibody recognition.…”

Section: Introductionmentioning

confidence: 99%

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X-ray and solution structures of human beta-2 glycoprotein I reveal a new mechanism of autoantibody recognition

Ruben

Planer

Chinnaraj

et al. 2020

Preprint

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31Venous and arterial thromboses in patients suffering from the autoimmune disorder Antiphospholipid 32 Syndrome (APS) are caused by the presence of antiphospholipid antibodies (aPL). Emerging evidence 33 indicates that autoantibodies targeting the epitope R39-R43 in the N-terminal domain, Domain I (DI), of 34 b2-glycoprotein I (b2GPI) are among the most pathogenic aPL in patients with APS. How such 35 autoantibodies engage b2GPI at the molecular level remains incompletely understood. Here, we have 36 used X-ray crystallography, single-molecule FRET, and small-angle X-ray scattering to demonstrate that, 37 in the free form, under physiological pH and salt concentrations, human recombinant b2GPI adopts an 38 elongated, flexible conformation in which DI is exposed to the solvent, thus available for autoantibody 39 recognition. Consistent with this structural model, binding and mutagenesis studies revealed that the 40 elongated form interacts with a pathogenic anti-DI antibody in solution, without the need of phospholipids. 41Furthermore, complex formation was affected neither by the neighboring domains, nor by the presence 42 of the linkers, nor by the glycosylations. Since the pathogenic autoantibody requires residues R39 and 43 R43 for optimal binding, these findings challenge longstanding postulates in the field envisioning b2GPI 44 adopting immunologic inert conformations featuring inaccessibility of the epitope R39-R43 in DI and 45 support an alternative model whereby the preferential binding of anti-DI antibodies towards phospholipid-46 bound b2GPI arises from the ability of the pre-existing elongated form to bind to the membranes and then 47 oligomerize, processes that are likely to be supported by protein conformational changes. Interfering with 48 these steps may limit the pathogenic effects of anti-DI antibodies in APS patients. 503 Significance 51In the autoimmune disorder called Antiphospholipid Syndrome (APS), the presence of autoantibodies 52 targeting the plasma glycoprotein beta-2 glycoprotein I (b2GPI) is associated with arterial and venous 53 thrombosis as well as pregnancy complications. Understanding how b2GPI becomes immunogenic and 54 how autoantibodies in complex with b2GPI cause the blood to clot remains a top priority in the field. By 55 elucidating the structural architecture of b2GPI free in solution, our studies challenge longstanding 56 postulates in the field and shed new light on the pathogenic mechanisms of APS that may help the 57 development of new diagnostics and therapeutic approaches.58 59 4 Introduction 60β2GPI is a 50-kDa multi-domain glycoprotein that circulates in the plasma at a concentration of 0.2 61 mg/ml(1, 2)( Fig 1A). It acquired centerstage in hematology in 1990 when it was recognized by two 62 independent studies as the dominant antigen of antiphospholipid antibodies (aPL) in the Antiphospholipid 63 Syndrome (APS)(3-5), a life-threatening blood clotting disorder characterized by vascular thrombosis and 64 pregnancy morbidity(6). Autoantibodies against β2GPI (anti-β2...

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Section: Discussionsupporting

confidence: 92%