scholarly journals Apixaban, an orally available anticoagulant, inhibits SARS-CoV-2 replication by targeting its major protease in a non-competitive way

2021 ◽  
Author(s):  
Otavio Augusto Chaves ◽  
Carolina Q. Sacramento ◽  
Natalia Fintelman-Rodrigues ◽  
Jairo Ramos Temerozo ◽  
Filipe Pereira-Dutra ◽  
...  
Keyword(s):  
Three Dimensional ◽  
Factor Xa ◽  
Coagulation Factor ◽  
Dependent Manner ◽  
Allosteric Site ◽  
Substrate Complex ◽  
Mechanism Of Inhibition ◽  
Competitive Mechanism ◽  
Viral Particles ◽  
Dual Mechanism

Anticoagulants are associated with clinical benefit against the 2019 coronavirus disease (COVID-19), preventing COVID-19 associated coagulopathy. Blood coagulation factor Xa (FXa) and SARS-CoV-2 major protease (Mpro) share over 80% homology at the three-dimensional protein level. Thus, it is worth interrogating whether there is crosstalk between inhibitors and substrates between these enzymes. Here, we found that the clinically-approved FXa inhibitor apixaban targets SARS-CoV-2 Mpro with a 21-fold higher potency than boceprevir (GC376). Apixaban displayed a non-competitive mechanism of inhibition towards Mpro, since it targets the enzyme/substrate complex and the allosteric site onto the viral protease. Enzymatic assays were further validated in infected Calu-3 cells, which reveal that apixaban decreases the production of infectious viral particles in a dose-dependent manner, with an inhibitory potency in the micromolar range. Our results are in line with the proposed early use of anticoagulants, including FXa inhibitors, to improve clinical outcome of COVID-19 patients. In this context, apixaban may display a dual mechanism of action by targeting FXa to prevent coagulopathy and, at some level, SARS-CoV-2 Mpro.

scholarly journals Crystal Structure of Isoform CBd of the Basic Phospholipase A2 Subunit of Crotoxin: Description of the Structural Framework of CB for Interaction with Protein Targets

Molecules ◽  
2020 ◽  
Vol 25 (22) ◽  
pp. 5290
Author(s):  
Dorota Nemecz ◽  
Maciej Ostrowski ◽  
Marc Ravatin ◽  
Frederick Saul ◽  
Grazyna Faure
Keyword(s):  
Cystic Fibrosis ◽  
Phospholipase A2 ◽  
Three Dimensional ◽  
Factor Xa ◽  
Coagulation Factor ◽  
Presynaptic Receptors ◽  
Dimensional Structure ◽  
Protein Targets ◽  
Structural Framework ◽  
Crotalus Durissus

Crotoxin, from the venom of the South American rattlesnake Crotalus durissus terrificus, is a potent heterodimeric presynaptic β-neurotoxin that exists in individual snake venom as a mixture of isoforms of a basic phospholipase A2 (PLA2) subunit (CBa2, CBb, CBc, and CBd) and acidic subunit (CA1–4). Specific natural mutations in CB isoforms are implicated in functional differences between crotoxin isoforms. The three-dimensional structure of two individual CB isoforms (CBa2, CBc), and one isoform in a crotoxin (CA2CBb) complex, have been previously reported. This study concerns CBd, which by interaction with various protein targets exhibits many physiological or pharmacological functions. It binds with high affinity to presynaptic receptors showing neurotoxicity, but also interacts with human coagulation factor Xa (hFXa), exhibiting anticoagulant effect, and acts as a positive allosteric modulator and corrector of mutated chloride channel, cystic fibrosis transmembrane conductance regulator (CFTR), implicated in cystic fibrosis. Thus, CBd represents a novel family of agents that have potential in identifying new drug leads related to anticoagulant and anti-cystic fibrosis function. We determined here the X-ray structure of CBd and compare it with the three other natural isoforms of CB. The structural role of specific amino acid variations between CB isoforms are analyzed and the structural framework of CB for interaction with protein targets is described.

scholarly journals Activation of coagulation releases endothelial cell mitogens.

1986 ◽  
Vol 103 (2) ◽  
pp. 419-428 ◽  
Author(s):  
C Gajdusek ◽  
S Carbon ◽  
R Ross ◽  
P Nawroth ◽  
D Stern
Keyword(s):  
Growth Factors ◽  
Endothelial Cell ◽  
Cell Function ◽  
Factor Xa ◽  
Coagulation Factor ◽  
Mitogenic Activity ◽  
Dependent Manner ◽  
Factor X ◽  
Binding Studies ◽  
Endothelial Cell Function

Recent studies have indicated that endothelial cell function includes elaboration of growth factors and regulation of coagulation. In this paper we demonstrate that activated coagulation Factor X (Factor Xa), a product of the coagulation mechanism generated before thrombin, induces enhanced release of endothelial cell mitogens, linking these two functions. Mitogenic activity generated by cultured bovine aortic endothelial cells in response to Factor Xa included platelet-derived growth-factor-like molecules based on a radioreceptor assay. Effective induction of mitogens by Factor Xa required the integrity of the enzyme's active center and the presence of the gamma-carboxyglutamic acid-containing domain of the molecule. Factor Xa-induced release of mitogens from endothelium occurred in serum-free medium and was not altered by hirudin or antibody to Factor V, indicating that it was a direct effect of Factor Xa and was not mediated by thrombin. Elaboration of mitogenic activity required only brief contact between Factor Xa and endothelium, and occurred in a time-dependent manner. Generation of enhanced mitogenic activity in response to Factor Xa was unaffected by the presence of actinomycin D and was not associated with increased hybridization of RNA from treated cells to a v-sis probe. Release of mitogenic activity was dependent on the dose of Factor Xa, being half-maximal at 0.5 nM and reaching a maximum by 5 nM. Radioligand binding studies demonstrated a class of endothelial cell sites half-maximally occupied at a Factor Xa concentration of 0.8 nM. The close correspondence between the parameters of Factor Xa-induced mitogen release and Factor Xa binding suggests these sites may be related. When Factor X was activated on the endothelial cell surface by Factors IXa and VIII, the Factor Xa formed resulted in the induction of enhanced release of mitogenic activity. These data suggest a mechanism by which the coagulation system can locally regulate endothelial cell function and vessel wall biology before thrombin-induced release of growth factors from platelets.

Characterization of Antithrombin-Specific RNA Aptamers for Use in Anticoagulant Therapy

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 4236-4236
Author(s):  
Yolanda Fortenberry
Keyword(s):  
Unfractionated Heparin ◽  
Vascular Injury ◽  
Conflicts Of Interest ◽  
Factor Xa ◽  
Coagulation Factor ◽  
Dependent Manner ◽  
Rna Aptamer ◽  
Rna Molecules ◽  
Factor Ixa

Abstract Historically, unfractionated heparin (a complex polysaccharide classified as a glycosaminoglycan) has been used in the treatment and prevention of thromboembolic disorders. However, due to complications associated with heparin (such as bleeding and heparin-induced thrombocytopenia), low molecular weight heparins (LMWHs) have been developed and are being used in place of unfractionated heparin. LMWHs have proven effective as antithrombotic agents; however, the effects of LMWHs cannot be readily reversed, while unfractionated heparin can be reversed by protamine sulfate. The overall goal of this project was to characterize an antithrombin (AT)-specific RNA aptamer that will combine the safety and efficacy of LMWH with the reversibility of unfractionated heparin. I have developed an RNA molecule (aptamer) that binds to AT. Using Systematic Evolution of Ligands by EXponential enrichment (SELEX) we created a combinatorial library consisting of single stranded RNA molecules with 20-40 randomized positions resulting in approximately 1014 different sequences. The RNA libraries after round 12 consisted of several different aptamers, and there was a progressive decrease in library complexity. We isolated four individual RNA molecules that bind in the nanomolar range to AT. Each displayed the same general properties, they accelerated factor Xa inhibition by AT in a dose-dependent manner. It should be noted that as anticipated, the aptamers did not promote the enhanced inhibition of thrombin by AT. Overall, our results show that these molecules are able to enhance the inhibition of factor Xa by AT. We suspect that these aptamers mimic the action of LMWH by binding to the D-helix of AT. Our previous AT specific RNA aptamer, Aptamer 7-4.16 was shown to prolonged bleeding in vivo in a vascular injury model. These data showed that Aptamer 7-4.16 is as effective as heparin in preventing clotting after vascular injury. We suspect that these new aptamers will have a similar effect in vivo. Proof of the concept was established by the work of Rusconi et al. (Nature, 2002), who developed an RNA aptamer and RNA aptamer antidote directed against coagulation factor IXa. The factor IXa aptamer completely inhibits the activation of factor X. Its anticoagulant action is controlled by the antidote, which is able to reverse the effects of the factor IXa aptamer within seconds. This aptamer/antidote pair is currently being tested clinically. Our data suggesting that antidotes to our AT aptamers have the ability to reverse its activity. If successful, this aptamer/antidote pair will have advantages over LMWHs because not only will it be able to control thrombosis in a fashion similar to LMWHs, but the availability of an antidote will allow for better therapeutic regulation and intervention. Disclosures No relevant conflicts of interest to declare.

Meclofenamate inhibits prostaglandin E binding and adenylyl cyclase activation in human myometrium

1991 ◽  
Vol 129 (3) ◽  
pp. 439-445 ◽  
Author(s):  
A. López Bernal ◽  
S. Buckley ◽  
C. M. P. Rees ◽  
J. M. Marshall
Keyword(s):  
Inhibitory Effect ◽  
Human Myometrium ◽  
Prostaglandin E ◽  
Dependent Manner ◽  
Synthesis Inhibitor ◽  
Traditional Role ◽  
Mechanism Of Inhibition ◽  
Receptor Sites ◽  
Competitive Mechanism ◽  
Dose Dependent

ABSTRACT The effect of sodium meclofenamate on the binding of [3H]prostaglandin E2 ([3H]PGE2) to membranes from human myometrium was investigated. Meclofenamate inhibited the binding of [3H]PGE2 to high-affinity (dissociation constant 1·5 nmol/l) sites in a reversible dose-dependent manner (inhibition constant 11 μmol/l). The mechanism of inhibition was mainly competitive, but at high doses of meclofenamate (≥ 100 μmol/l) there was loss of PGE receptor sites. Of several PG synthesis inhibitors tested, only meclofenamate and, to a lesser extent, mefenamic acid had a significant inhibitory effect. PGE2 stimulated cyclic AMP generation in slices of human myometrium and this was inhibited by meclofenamate in a dose-dependent manner (50% inhibition occurred at 9 μmol/l). Again, this effect was specific for meclofenamate and fitted a competitive mechanism at doses in the range 1–10 μmol/l and a non-competitive mechanism at higher doses. The data show that meclofenamate, in addition to its traditional role as a PG synthesis inhibitor, affects directly PGE receptor binding and activation. Journal of Endocrinology (1991) 129, 439–445

Inhibition of Factors Xa and Iia Results in Synergistic Inhibition of Thrombin Generation with Minimal Clot Time Elevation.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1877-1877
Author(s):  
Kathleen M. Welch ◽  
Kamlai Saiya-Cork ◽  
Weston R. Gould ◽  
Robert J. Leadley
Keyword(s):  
Thrombin Generation ◽  
Ex Vivo ◽  
Factor Xa ◽  
Coagulation Factor ◽  
Specific Factor ◽  
Synergistic Activity ◽  
Dependent Manner ◽  
Clotting Time ◽  
Concentration Dependent Manner

Abstract Inhibition of either coagulation factor Xa (FXa) or thrombin (FIIa) alters ex vivo biomarkers of coagulation and decreases thrombus size in animal models of experimental thrombosis. The objective of this study was to determine if combining FXa and FIIa inhibitors would synergistically reduce the magnitude of IIa generated without elevating markers of bleeding. A synergistic combination may translate into lower doses, more effective anticoagulation and better safety. To predict the FXa and FIIa inhibitor combinations with the maximum potential for synergy, PD 0313052, a potent, selective FXa inhibitor, and argatroban, a potent FIIa inhibitor, were each tested independently and in combination using an in vitro thrombin generation assay. Individually, PD 0313052 and argatroban reduced total thrombin generation (TG) in a concentration dependent manner with IC50’s of 497±148 and 882±193 nM, respectively. Subsequently, PD 0313052 and argatroban were combined in 96 well plates at concentrations ranging from 0.125x to 8x their respective IC50 concentrations. Analysis using the Bliss Independence Model identified statistically significant synergistic activity, with the greatest increase (33%) over simple additivity at 249 and 441 nM FXa to FIIa, respectively, both below their respective IC50 concentrations. Furthermore, combinations of PD 0313052 and argatroban were evaluated in an assay measuring activated clotting time (ACT). Although both PD 0313052 and argatroban dose-dependently elongated the ACT, the combination of 0.5x the TG IC50 concentrations, which demonstrated the greatest synergy in the TG assay, showed the smallest increase in the ACT, prolonging clotting time by only 15%. These data demonstrate that the combination of a specific factor Xa inhibitor and a specific IIa inhibitor can synergistically reduce thrombin generation without appreciable elevation of ACT, suggesting that dual inhibition of FXa and FIIa, using relatively low doses of each, may provide efficacious and safe treatment for thromboembolic diseases.

Effects of the Factor Xa Inhibitor Apixaban on Venous Thrombosis and Hemostasis in Rabbits.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 917-917 ◽  
Author(s):  
Pancras C. Wong ◽  
Carol A. Watson ◽  
Earl J. Crain ◽  
Joseph M. Luettgen ◽  
Martin L. Ogletree ◽  
...  
Keyword(s):  
Venous Thrombosis ◽  
Prothrombin Time ◽  
Bleeding Time ◽  
Ex Vivo ◽  
Vena Cava ◽  
Factor Xa ◽  
Coagulation Factor ◽  
Vehicle Group ◽  
Dependent Manner ◽  
Venous Thrombus

Abstract Apixaban is a small-molecule, potent, reversible and direct inhibitor of human coagulation factor Xa (FXa) (Ki = 0.08 nM) with greater than 30000-fold selectivity over other coagulation proteases. It is orally bioavailable in rats, dogs, chimpanzees and humans, and is currently in clinical development for prevention and treatment of thromboembolic diseases. The objective of this study was to compare antithrombotic and bleeding time effects of apixaban and warfarin in rabbits. We selected rabbits as the animal model because apixaban has similar potency in inhibiting human and rabbit FXa, but is less potent in inhibiting rat and dog FXa (Ki (nM): 0.16, rabbit; 1.3, rat; 1.8, dog). We induced the formation of venous thrombus by placing threads in the vena cava, and bleeding by the incision of cuticles in anesthetized rabbits. Apixaban was infused IV 60 min before the initiation of thrombosis. Warfarin was administered orally for 4 days before the study. Thrombus weight averaged 73±5 mg and bleeding time averaged 183±7 s in the vehicle group (n=6 per group). Apixaban and warfarin inhibited the formation of venous thrombus in a dose-dependent manner. The estimated IC50 for apixaban was 0.16±0.04 μM. At antithrombotic doses studied, apixaban did not alter blood pressure and heart rate, suggesting hemodynamic effects are not likely to be involved in its antithrombotic activity. At the antithrombotic ID80, apixaban and warfarin increased bleeding time by 9±4% and 516±24%, respectively (n=6 per group). At this dose, apixaban increased ex vivo activated partial thromboplastin time and prothrombin time to 1.4±0.1 and 1.7±0.1 times control, respectively, and warfarin increased prothrombin time to 4.5±0.1 times control. Apixaban at antithrombotic doses selectively inhibited ex vivo FXa but not thrombin activity. In summary, these findings demonstrate potent activity of apixaban as a selective FXa inhibitor exhibiting potential strong efficacy in prevention of venous thrombosis at doses that preserve hemostasis and produce less increases in systemic anticoagulation than warfarin.

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.

Isolation and Structural Charaderization of a Potent Inhibitor of Coagulation Factor Xa from the Leech Haementeria ghilianii

1989 ◽  
Vol 61 (03) ◽  
pp. 437-441 ◽  
Author(s):  
Cindra Condra ◽  
Elka Nutt ◽  
Christopher J Petroski ◽  
Ellen Simpson ◽  
P A Friedman ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Salivary Gland ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
Western Blot ◽  
Potent Inhibitor ◽  
Factor Xa ◽  
Coagulation Factor ◽  
Sds Page ◽  
Bovine Factor

SummaryThe present work reports the discovery and charactenzation of an anticoagulant protein in the salivary gland of the giant bloodsucking leech, H. ghilianii, which is a specific and potent inhibitor of coagulation factor Xa. The inhibitor, purified to homogeneity, displayed subnanomolar inhibition of bovine factor Xa and had a molecular weight of approximately 15,000 as deduced by denaturing SDS-PAGE. The amino acid sequence of the first 43 residues of the H. ghilianii derived inhibitor displayed a striking homology to antistasin, the recently described subnanomolar inhibitor of factor Xa isolated from the Mexican leech, H. officinalis. Antisera prepared to antistasin cross-reacted with the H. ghilianii protein in Western Blot analysis. These data indicate that the giant Amazonian leech, H. ghilianii, and the smaller Mexican leech, H. officinalrs, have similar proteins which disrupt the normal hemostatic clotting mechanisms in their mammalian host’s blood.

Activation of Human Coagulation Factor VIII by Activated Factor X, the Common Product of the Intrinsic and the Extrinsic Pathway of Blood Coagulation

1982 ◽  
Vol 47 (02) ◽  
pp. 096-100 ◽  
Author(s):  
K Mertens ◽  
R M Bertina
Keyword(s):  
Factor Viii ◽  
Human Factor ◽  
Intrinsic Factor ◽  
Factor Xa ◽  
Coagulation Factor ◽  
Factor X ◽  
Extrinsic Factor ◽  
Extrinsic Pathway ◽  
Factor Ixa ◽  
The Common

SummaryThe intrinsic activation of human factor X has been studied in a system consisting of purified factors and in plasma. In both these systems factor Xa stimulated the activation of factor X by factor IXa plus factor VIII This is due to the activation of factor VIII by factor Xa. When this factor Xa is formed via the extrinsic pathway, the extrinsic factor X activator functions as a stimulator of the intrinsic factor X activator.

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