Structure of prothrombin in the closed form reveals new details on the mechanism of activation

Chinnaraj, Mathivanan; Chen, Zhiwei; Pelc, Leslie A.; Grese, Zachary R.; Bystranowska, Dominika; Di, Enrico; Pozzi, Nicola

doi:10.1038/s41598-018-21304-1

Cited by 35 publications

(80 citation statements)

References 46 publications

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“…Prothrombin wild-type (proTWT) and mutants were expressed in mammalian cells as previously described. 26 Prethrombin-2 was expressed in Escherichia coli. 27 Prethrombin-1, fragment-1, kringle-1, and kringle-2 were obtained by limited proteolysis using thrombin and factor Xa.…”

Section: Protein Production and Purificationmentioning

confidence: 99%

“…Small unilamellar vesicles composed of phosphatidylcholine (PC) or phosphatidylcholine and phosphatidylserine (PS) in a 3:1 molar ratio (PC:PS) were prepared by extrusion using 100-nm polycarbonate membranes (Avanti Polar Lipids), kept at 4°C, and used within 7 days. 26 Protein concentrations were determined by reading at 280 nm with molar extinction coefficients adjusted based on the amino acid sequence. All other chemicals were purchased from MilliporeSigma.…”

Section: Protein Production and Purificationmentioning

confidence: 99%

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

et al. 2019

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Anti-phosphatidylserine/prothrombin (aPS/PT) antibodies are often detected in patients with antiphospholipid syndrome (APS), but how aPS/PT engage prothrombin at the molecular level remains unknown. Here, the antigenic determinants of immunoglobulin G aPS/PT were investigated in 24 triple-positive APS patients at high risk of thrombosis by using prothrombin mutants biochemically trapped in closed and open conformations, and relevant fragments spanning the entire length of prothrombin. Two novel unexpected findings emerged from these studies. First, we discovered that some aPS/PT are unique among other anti-prothrombin antibodies insofar as they efficiently recognize prothrombin in solution after a conformational change requiring exposure of fragment-1 to the solvent. Second, we identified and characterized 2 previously unknown subpopulations of aPS/PT, namely type I and type II, which engage fragment-1 of prothrombin at different epitopes and with different mechanisms. Type I target a discontinuous density-dependent epitope, whereas type II engage the C-terminal portion of the Gla-domain, which remains available for binding even when prothrombin is bound to the phospholipids. Based on these findings, APS patients positive for aPS/PT were classified into 2 groups, group A and group B, according to their autoantibody profile. Group A contains mostly type I antibodies whereas group B contains both type I and type II antibodies. In conclusion, this study offers a first encouraging step toward unveiling the heterogeneity of anti-prothrombin antibodies in correlation with thrombosis, shedding new light on the mechanisms of antigen–autoantibody recognition in APS.

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Section: Protein Production and Purificationmentioning

confidence: 99%

Section: Protein Production and Purificationmentioning

confidence: 99%

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

et al. 2019

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“…2B). The second version, called ST-β 2 GPI, contains a shorter, non-cleavable purification tag at the N-terminus that, based on our previous work(36), is expected not to affect the conformational properties of the protein (Fig. 2B).…”

Section: Resultsmentioning

confidence: 99%

“…Selective labeling of the unpaired Cys residues with Alexa Fluor 555-C2-maleimide as the donor and Alexa Fluor 647-C2-maleimide as the acceptor was achieved as described recently for prothrombin(36, 37). FRET measurements of freely diffusing single molecules were performed with a confocal microscope MicroTime 200 (PicoQuant, Berlin, Germany), as detailed elsewhere(36, 37).…”

Section: Methodsmentioning

confidence: 99%

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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“…This suggests that the activating cleavage site is shielded from PKa when FXII is in a globular form, restricting FXII activation to the activating surface. Similar mechanisms have already been described for prothrombin and plasminogen [11][12][13].…”

Section: Introductionmentioning

confidence: 99%

Factor XII truncation accelerates activation in solution

Maat

Clark

Boertien

et al. 2019

Journal of Thrombosis and Haemostasis

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Summary During contact system activation, factor XII is progressively cleaved by plasma kallikrein. We investigated the role of factor XII truncation in biochemical studies. Factor XII contains naturally occurring truncating cleavage sites for a variety of enzymes. Truncation of factor XII primes it for activation in solution through exposure of R353. Summary BackgroundThe contact activation system and innate immune system are interlinked in inflammatory pathology. Plasma kallikrein (PKa) is held responsible for the stepwise processing of factor XII (FXII). A first cleavage activates FXII (into FXIIa); subsequent cleavages truncate it. This truncation eliminates its surface‐binding domains, which negatively regulates surface‐dependent coagulation. ObjectivesTo investigate the influence of FXII truncation on its activation and downstream kallikrein‐kinin system activation. MethodsWe study activation of recombinant FXII variants by chromogenic assays, by FXIIa ELISA and western blotting. ResultsWe demonstrate that FXII truncation primes it for activation by PKa in solution. We demonstrate this phenomenon in three settings. (i) Truncation at a naturally occurring PKa‐sensitive cleavage site, R334, accelerates FXIIa formation in solution. A site‐directed mutant FXII‐R334A displays ~50% reduced activity when exposed to PKa. (ii) A pathogenic mutation in FXII that causes hereditary angioedema, introduces an additional plasmin‐sensitive cleavage site. Truncation at this site synergistically accelerates FXII activation in solution. (iii) We identify new, naturally occurring cleavage sites in FXII that have so far not been functionally linked to contact system activation. As examples, we show that non‐activating truncation of FXII by neutrophil elastase and cathepsin K primes it for activation by PKa in solution. ConclusionsFXII truncation, mediated by either pathogenic mutations or naturally occurring cleavage sites, primes FXII for activation in solution. We propose that the surface‐binding domains of FXII shield its activating cleavage site, R353. This may help to explain how the contact system contributes to inflammatory pathology.

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Structure of prothrombin in the closed form reveals new details on the mechanism of activation

Cited by 35 publications

References 46 publications

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

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

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

Factor XII truncation accelerates activation in solution

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