Natural IgM antibodies inhibit microvesicle-driven coagulation and thrombosis

Blood ◽  
2020 ◽  
Author(s):  
Georg Obermayer ◽  
Taras Afonyushkin ◽  
Laura Goederle ◽  
Florian Puhm ◽  
Waltraud C. Schrottmaier ◽  
...  
Keyword(s):  
Therapeutic Potential ◽  
Coagulation Factor ◽  
Mechanistic Explanation ◽  
Factor X ◽  
Thrombotic Risk ◽  
Igm Antibodies ◽  
Plasmatic Coagulation ◽  
Natural Igm

Thrombosis and the complications associated with it are a major cause of morbidity and mortality worldwide. Microvesicles (MVs), a class of extracellular vesicles, are increasingly recognized as mediators of coagulation and biomarkers of thrombotic risk. Thus, identifying factors targeting MV-driven coagulation may help in the development of novel antithrombotic treatments. We have previously identified a subset of circulating MVs that is characterized by the presence of oxidation-specific epitopes and bound by natural IgM antibodies targeting these structures. Here, we investigated whether natural IgM antibodies, which are known to have important anti-inflammatory house-keeping functions, inhibit the procoagulatory properties of MVs. We found that the extent of plasma coagulation is inversely associated with the levels of both free and MV-bound endogenous IgM. Moreover, the oxidation epitope-specific natural IgM antibody LR04, which recognizes malondialdehyde adducts, reduced MV-dependent plasmatic coagulation and whole blood clotting without affecting thrombocyte aggregation. Intravenous injection of LR04 protected mice from MV-induced pulmonary thrombosis. Of note, LR04 competed the binding of coagulation factor X/Xa to MVs, providing a mechanistic explanation for its anticoagulatory effect. Thus, our data identify natural IgM antibodies as hitherto unknown modulators of MV-induced coagulation in vitro and in vivo and their prognostic and therapeutic potential in the management of thrombosis.

scholarly journals Manipulating Adenovirus Hexon Hypervariable Loops Dictates Immune Neutralisation and Coagulation Factor X-dependent Cell Interaction In Vitro and In Vivo

2015 ◽  
Vol 11 (2) ◽  
pp. e1004673 ◽  
Author(s):  
Jiangtao Ma ◽  
Margaret R. Duffy ◽  
Lin Deng ◽  
Rachel S. Dakin ◽  
Taco Uil ◽  
...  
Keyword(s):  
Coagulation Factor ◽  
Cell Interaction ◽  
Factor X ◽  
Dependent Cell

scholarly journals Biodistribution and retargeting of FX-binding ablated adenovirus serotype 5 vectors

Blood ◽  
2010 ◽  
Vol 116 (15) ◽  
pp. 2656-2664 ◽  
Author(s):  
Raul Alba ◽  
Angela C. Bradshaw ◽  
Lynda Coughlan ◽  
Laura Denby ◽  
Robert A. McDonald ◽  
...  
Keyword(s):  
Coagulation Factor ◽  
Adenovirus Type ◽  
Factor X ◽  
High Affinity ◽  
Serotype 5 ◽  
Adenoviral Transduction ◽  
High Doses ◽  
Immunohistochemical Studies

AbstractA major limitation for adenoviral transduction in vivo is the profound liver tropism of adenovirus type 5 (Ad5). Recently, we demonstrated that coagulation factor X (FX) binds to Ad5-hexon protein at high affinity to mediate hepatocyte transduction after intravascular delivery. We developed novel genetically FX-binding ablated Ad5 vectors with lower liver transduction. Here, we demonstrate that FX-binding ablated Ad5 predominantly localize to the liver and spleen 1 hour after injection; however, they had highly reduced liver transduction in both control and macrophage-depleted mice compared with Ad5. At high doses in macrophage-depleted mice, FX-binding ablated vectors transduced the spleen more efficiently than Ad5. Immunohistochemical studies demonstrated transgene colocalization with CD11c+, ER-TR7+, and MAdCAM-1+ cells in the splenic marginal zone. Systemic inflammatory profiles were broadly similar between FX-binding ablated Ad5 and Ad5 at low and intermediate doses, although higher levels of several inflammatory proteins were observed at the highest dose of FX-binding ablated Ad5. Subsequently, we generated a FX-binding ablated virus containing a high affinity Ad35 fiber that mediated a significant improvement in lung/liver ratio in macrophage-depleted CD46+ mice compared with controls. Therefore, this study documents the biodistribution and reports the retargeting capacity of FX binding-ablated Ad5 vectors in vitro and in vivo.

scholarly journals Human Coagulation Factor X-Adenovirus Type 5 Complexes Poorly Stimulate an Innate Immune Response in Human Mononuclear Phagocytes

2014 ◽  
Vol 89 (5) ◽  
pp. 2884-2891 ◽  
Author(s):  
Karsten Eichholz ◽  
Franck J. D. Mennechet ◽  
Eric J. Kremer
Keyword(s):  
Coagulation Factor ◽  
Human Adenovirus ◽  
Adenovirus Type ◽  
Natural Host ◽  
Innate Immune ◽  
Mononuclear Phagocytes ◽  
Factor X ◽  
Dynamic Interactions

ABSTRACTOne of the first lines of host defense against many viruses in vertebrates is the innate immune system, which detects pathogen-associated molecular patterns (PAMPs) using pathogen recognition receptors (PRR). The dynamic interactions between pathogens and hosts create, in some cases, species-specific relationships. Recently, it was shown that murine factor X (mFX)-armored human adenovirus (HAd) stimulated a mFX-Toll-like receptor 4 (TLR4)-associated response in mouse macrophagesin vitroandin vivo. Given the importance of studies using animals to better understand host-pathogen interactions, we asked if human FX (hFX)-armored HAd type 5 (HAd5) was capable of activating innate immune sensors in primary human mononuclear phagocytes. To this end, we assayed human mononuclear phagocytes for their ability to be stimulated by hFX-armored HAd5 via a TLR/NF-κB pathway, in particular, a TLR4 pathway. In our hands, we found no significant interaction, activation, or maturation of human mononuclear phagocytes caused by the presence of hFX-armored HAd5.IMPORTANCEAnimals, and mice in particular, are often used as informative and powerful surrogates for how pathogens interact with natural host systems. When possible, extended and targeted studies in the natural host can then be performed. Our data will help us understand the differences in preclinical testing in mice and clinical use in humans in order to improve treatment for HAd diseases and Ad vector effectiveness.

scholarly journals A mutated factor X activatable by thrombin corrects bleedings in vivo in a rabbit model of antibody-induced hemophilia A

Haematologica ◽  
2019 ◽  
Vol 105 (9) ◽  
pp. 2335-2340
Author(s):  
Toufik Abache ◽  
Alexandre Fontayne ◽  
Dominique Grenier ◽  
Emilie Jacque ◽  
Alain Longue ◽  
...  
Keyword(s):  
Factor Viii ◽  
Hemophilia A ◽  
Factor Viia ◽  
Rabbit Model ◽  
Coagulation Factor ◽  
Factor Ix ◽  
Factor X ◽  
Factor Viiia

Rendering coagulation factor X sensitive to thrombin was proposed as a strategy that can bypass the need for factor VIII. In this paper, this non-replacement strategy was evaluated in vitro and in vivo in its ability to correct factor VIII but also factor IX, X and XI deficiencies. A novel modified factor X, named Actiten, was generated and produced in the HEK293F cell line. The molecule possesses the required post-translational modifications, partially keeps its ability to be activated by RVV-X, factor VIIa/tissue factor, factor VIIIa/factor IXa and acquires the ability to be activated by thrombin. The potency of the molecule was evaluated in respective deficient plasmas or hemophilia A plasmas, for some with inhibitors. Actiten corrects dose dependently all the assayed deficient plasmas. It is able to normalize the thrombin generation at 20 μg/mL showing however an increased lagtime. It was then assayed in a rabbit antibody-induced model of hemophilia A where, in contrast to recombinant factor X wild-type, it normalized the bleeding time and the loss of hemoglobin. No sign of thrombogenicity was observed and the generation of activated factor X was controlled by the anticoagulation pathway in all performed coagulation assays. This data indicates that Actiten may be considered as a possible non replacement factor to treat hemophilia's with the advantage of being a zymogen correcting bleedings only when needed.

scholarly journals Influence of Coagulation Factor X on In Vitro and In Vivo Gene Delivery by Adenovirus (Ad) 5, Ad35, and Chimeric Ad5/Ad35 Vectors

2009 ◽  
Vol 17 (10) ◽  
pp. 1683-1691 ◽  
Author(s):  
Jenny A Greig ◽  
Suzanne MK Buckley ◽  
Simon N Waddington ◽  
Alan L Parker ◽  
David Bhella ◽  
...  
Keyword(s):  
Gene Delivery ◽  
Coagulation Factor ◽  
Factor X ◽  
In Vivo Gene Delivery

scholarly journals Resistance of Platelets in Hypercholesterolemia to Inhibition by Activated Coagulation Factor X

2011 ◽  
Vol 2011 ◽  
pp. 1-6
Author(s):  
Nighat Kahn ◽  
Bilal Khan ◽  
Asru K. Sinha
Keyword(s):  
Platelet Aggregation ◽  
Platelet Activation ◽  
Factor Xa ◽  
Coagulation Factor ◽  
Factor X ◽  
High Cholesterol ◽  
Normal Dispersion ◽  
Normal Controls

Platelet hyperactivity may be involved in the pathogenesis of both thrombogenesis and hypercholesterolemia. The cholesterol-enriched states may contribute to accelerated development of atherosclerosis. The effect of high cholesterol on platelet activation and on inhibition by coagulation factor Xa, was studied in vitro. Incubation of normal platelets (n=20) with cholesterol-rich dispersion resulted in a small increase of platelet aggregation (PA) and thromboxane A2 (TXA2) synthesis when compared with platelets incubated with cholesterol-normal dispersion. In hypercholesterolemic patients (n=20), ADP-induced PA and TXA2 synthesis showed only small increases over normal controls. Addition of factor Xa (1 unit/mL) prevented the ADP-induced PA and markedly inhibited TXA2 synthesis in normal platelets (1.3±0.2 and 8.7±2.0 pmol TXA2/108 platelets, with and without factor Xa, resp.). However, factor Xa failed to significantly suppress TXA2 synthesis in cholesterol-incubated normal platelets (9.5±1.4 and 11.8±1.3 pmol TXA2/108 platelets, with and without factor Xa; resp., P=NS) as well as in platelets from patients with hypercholesterolemia (8.6±4.0 and 10.9±4.9 pmol TXA2/108 platelets, with and without factor Xa; resp., P=NS). Exposure of platelets to high cholesterol concentrations, in vitro and in vivo, marginally increased PA and TXA2 synthesis but resulted in loss of responsiveness to factor Xa, which could significantly contribute to platelet activation in hypercholesterolemic states.

Proteolytic processing of human coagulation factor IX by plasmin

Blood ◽  
2000 ◽  
Vol 95 (3) ◽  
pp. 943-951 ◽  
Author(s):  
John A. Samis ◽  
Gillian D. Ramsey ◽  
John B. Walker ◽  
Michael E. Nesheim ◽  
Alan R. Giles
Keyword(s):  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Coagulation Factor ◽  
Proteolytic Processing ◽  
Factor Ix ◽  
Stable Complex ◽  
Factor X ◽  
Active Site Probe

Previous studies have shown that thrombin generation in vivo caused a 92% decrease in factor IX (F.IX) activity and the appearance of a cleavage product after immunoblotting that comigrated with activated F.IX (F.IXa). Under these conditions, the fibrinolytic system was clearly activated, suggesting plasmin may have altered F.IX. Thus, the effect(s) of plasmin on human F.IX was determined in vitro. Plasmin (50 nM) decreased the 1-stage clotting activity of F.IX (4 μM) by 80% and the activity of F.IXa (4 μM) by 50% after 30 minutes at 37°C. Plasmin hydrolysis of F.IX yields products of 45, 30, 20, and 14 kd on reducing sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and 2 products of 52 and 14 kd under nonreducing conditions. Plasmin-treated F.IX did not bind the active site probe, p-aminobenzamidine, or form an SDS-stable complex with antithrombin. It only marginally activated human factor X in the presence of phospholipid and activated factor VIII. Although dansyl-Glu-Gly-Arg-chloromethyl ketone inactivated–F.IXa inhibited the clotting activity of F.IXa, plasmin-treated F.IX did not. Plasmin cleaves F.IX after Lys43, Arg145, Arg180, Lys316, and Arg318, but F.IXa is not appreciably generated despite cleavage at the 2 normal activation sites (Arg145 and Arg180). Tissue plasminogen activator–catalyzed lysis of fibrin formed in human plasma results in generation of the 45- and 30-kd fragments of F.IX and decreased F.IX clotting activity. Collectively, the results suggest that plasmin is able to down-regulate coagulation by inactivating F.IX.

scholarly journals Identification of coagulation factor (F)X binding sites on the adenovirus serotype 5 hexon: effect of mutagenesis on FX interactions and gene transfer

Blood ◽  
2009 ◽  
Vol 114 (5) ◽  
pp. 965-971 ◽  
Author(s):  
Raul Alba ◽  
Angela C. Bradshaw ◽  
Alan L. Parker ◽  
David Bhella ◽  
Simon N. Waddington ◽  
...  
Keyword(s):  
Amino Acids ◽  
Gene Transfer ◽  
Coagulation Factor ◽  
Single Mutation ◽  
Factor X ◽  
Electron Microscopic ◽  
Contact Points ◽  
Serotype 5

Recent studies have demonstrated the importance of coagulation factor X (FX) in adenovirus (Ad) serotype 5–mediated liver transduction in vivo. FX binds to the adenovirus hexon hypervariable regions (HVRs). Here, we perform a systematic analysis of FX binding to Ad5 HVRs 5 and 7, identifying domains and amino acids critical for this interaction. We constructed a model of the Ad5-FX interaction using crystallographic and cryo-electron microscopic data to identify contact points. Exchanging Ad5 HVR5 or HVR7 from Ad5 to Ad26 (which does not bind FX) diminished FX binding as analyzed by surface plasmon resonance, gene delivery in vitro, and liver transduction in vivo. Exchanging Ad5-HVR5 for Ad26-HVR5 produced deficient virus maturation. Importantly, defined mutagenesis of just 2 amino acids in Ad5-HVR5 circumvented this and was sufficient to block liver gene transfer. In addition, mutation of 4 amino acids in Ad5-HVR7 or a single mutation at position 451 also blocked FX-mediated effects in vitro and in vivo. We therefore define the regions and amino acids on the Ad5 hexon that bind with high affinity to FX thereby better defining adenovirus infectivity pathways. These vectors may be useful for gene therapy applications where evasion of liver transduction is a prerequisite.

Ancylostoma caninum Anticoagulant Peptide Blocks Metastasis In Vivo and Inhibits Factor Xa Binding to Melanoma Cells In Vitro

1998 ◽  
Vol 79 (05) ◽  
pp. 1041-1047 ◽  
Author(s):  
Kathleen M. Donnelly ◽  
Michael E. Bromberg ◽  
Aaron Milstone ◽  
Jennifer Madison McNiff ◽  
Gordon Terwilliger ◽  
...  
Keyword(s):  
Active Site ◽  
Thrombin Generation ◽  
Factor Xa ◽  
Coagulation Factor ◽  
Melanoma Cells ◽  
Factor V ◽  
Ancylostoma Caninum ◽  
Metastatic Activity

SummaryWe evaluated the in vivo anti-metastatic activity of recombinant Ancylostoma caninum Anticoagulant Peptide (rAcAP), a potent (Ki = 265 pM) and specific active site inhibitor of human coagulation factor Xa originally isolated from bloodfeeding hookworms. Subcutaneous injection of SCID mice with rAcAP (0.01-0.2 mg/mouse) prior to tail vein injection of LOX human melanoma cells resulted in a dose dependent reduction in pulmonary metastases. In order to elucidate potential mechanisms of rAcAP’s anti-metastatic activity, experiments were carried out to identify specific interactions between factor Xa and LOX. Binding of biotinylated factor Xa to LOX monolayers was both specific and saturable (Kd = 15 nM). Competition experiments using antibodies to previously identified factor Xa binding proteins, including factor V/Va, effector cell protease receptor-1, and tissue factor pathway inhibitor failed to implicate any of these molecules as significant binding sites for Factor Xa. Functional prothrombinase activity was also supported by LOX, with a half maximal rate of thrombin generation detected at a factor Xa concentration of 2.4 nM. Additional competition experiments using an excess of either rAcAP or active site blocked factor Xa (EGR-Xa) revealed that most of the total factor Xa binding to LOX is mediated via interaction with the enzyme’s active site, predicting that the vast majority of cell-associated factor Xa does not participate directly in thrombin generation. In addition to establishing two distinct mechanisms of factor Xa binding to melanoma, these data raise the possibility that rAcAP’s antimetastatic effect in vivo might involve novel non-coagulant pathways, perhaps via inhibition of active-site mediated interactions between factor Xa and tumor cells.

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