Anticoagulant activity screening of an in-house database of natural compounds for discovering novel selective factor Xa inhibitors; a combined in silico and in vitro approach

SummaryLupus anticoagulants (LAs) are antibodies which interfere with phospholipid-dependent procoagulant reactions. Their clinical importance is due to their apparent association with an increased risk of thrombo-embolic disease. To date there have been few assays for quantifying the specific activity of these antibodies in vitro and this has hampered attempts to purify and characterize these antibodies. Methods for determining phospholipid-dependent generation of thrombin and factor Xa are described. Isolated IgG fractions from 7 of 9 patients with LAs were found to reproducibly inhibit enzyme generation in these assay systems, permitting quantitative expression of inhibitor activity. Different patterns of inhibitory activity, based on the relative inhibition of thrombin and factor Xa generation, were found, further substantiating the known heterogeneity of these antibodies. These systems may prove helpful in further purification and characterization of LAs.

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The Anticoagulant Properties of a Modified Form of Protein S

Thrombosis and Haemostasis ◽

10.1055/s-0038-1647048 ◽

1988 ◽

Vol 60 (02) ◽

pp. 298-304 ◽

Cited By ~ 17

Author(s):

C A Mitchell ◽

S M Kelemen ◽

H H Salem

Keyword(s):

Monoclonal Antibody ◽

Anticoagulant Activity ◽

Activated Protein C ◽

Factor Xa ◽

Protein S ◽

Human Neutrophil Elastase ◽

Factor X ◽

Sds Page

SummaryProtein S (PS) is a vitamin K-dependent anticoagulant that acts as a cofactor to activated protein C (APC). To date PS has not been shown to possess anticoagulant activity in the absence of APC.In this study, we have developed monoclonal antibody to protein S and used to purify the protein to homogeneity from plasma. Affinity purified protein S (PSM), although identical to the conventionally purified protein as judged by SDS-PAGE, had significant anticoagulant activity in the absence of APC when measured in a factor Xa recalcification time. Using SDS-PAGE we have demonstrated that prothrombin cleavage by factor X awas inhibited in the presence of PSM. Kinetic analysis of the reaction revealed that PSM competitively inhibited factor X amediated cleavage of prothrombin. PS preincubated with the monoclonal antibody, acquired similar anticoagulant properties. These results suggest that the interaction of the monoclonal antibody with PS results in an alteration in the protein exposing sites that mediate the observed anticoagulant effect. Support that the protein was altered was derived from the observation that PSM was eight fold more sensitive to cleavage by thrombin and human neutrophil elastase than conventionally purified protein S.These observations suggest that PS can be modified in vitro to a protein with APC-independent anticoagulant activity and raise the possibility that a similar alteration could occur in vivo through the binding protein S to a cellular or plasma protein.

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Comparison of the Anticoagulant and Antithrombotic Effects of Synthetic Thrombin and Factor Xa Inhibitors

Thrombosis and Haemostasis ◽

10.1055/s-0038-1645198 ◽

1990 ◽

Vol 63 (02) ◽

pp. 220-223 ◽

Cited By ~ 19

Author(s):

J Hauptmann ◽

B Kaiser ◽

G Nowak ◽

J Stürzebecher ◽

F Markwardt

Keyword(s):

Factor Xa ◽

Thrombin Inhibitors ◽

Factor Xa Inhibitors ◽

Venous Stasis ◽

Clotting Time ◽

Thrombosis Model ◽

Activity Factor ◽

Antithrombotic Effects

SummaryThe anticoagulant effect of selected synthetic inhibitors of thrombin and factor Xa was studied in vitro in commonly used clotting assays. The concentrations of the compounds doubling the clotting time in the various assays were mainly dependent on their thrombin inhibitory activity. Factor Xa inhibitors were somewhat more effective in prolonging the prothrombin time compared to the activated partial thromboplastin time, whereas the opposite was true of thrombin inhibitors.In vivo, in a venous stasis thrombosis model and a thromboplastin-induced microthrombosis model in rats the thrombin inhibitors were effective antithrombotically whereas factor Xa inhibitors of numerically similar IQ value for the respective enzyme were not effective at equimolar dosageThe results are discussed in the light of the different prelequisiles and conditions for inhibition of thrombin and factor Xa in the course of blood clotting.

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A Novel Prothrombin Time Assay for Assessing the Anticoagulant Activity of Oral Factor Xa Inhibitors

Clinical and Applied Thrombosis/Hemostasis ◽

10.1177/1076029612441859 ◽

2012 ◽

Vol 19 (5) ◽

pp. 522-528 ◽

Cited By ~ 26

Author(s):

Yu Chen Barrett ◽

Zhaoqing Wang ◽

Robert M. Knabb

Keyword(s):

Prothrombin Time ◽

Anticoagulant Activity ◽

Factor Xa ◽

Factor Xa Inhibitors

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High Throughput in Silico Identification of Novel Phytochemical Inhibitors for the Master Regulator of Inflammation (TNFα)

10.26434/chemrxiv.14178212.v2 ◽

2021 ◽

Author(s):

Pratap Kumar Parida ◽

Dipak Paul ◽

Debamitra Chakravorty

Keyword(s):

Small Molecule ◽

High Throughput ◽

In Silico ◽

Small Molecule Inhibitors ◽

Binding Free Energy ◽

Natural Compounds ◽

Free Energy Calculations ◽

Compound Libraries ◽

Relative Binding

<a>The over expression of Tumor necrosis factor-α (TNFα) has been implicated in a variety of disease and is classified as a therapeutic target for inflammatory diseases (Crohn disease, psoriasis, psoriatic arthritis, rheumatoid arthritis).Commercially available therapeutics are biologics which are associated with several risks and limitations. Small molecule inhibitors and natural compounds (saponins) were identified by researchers as lead molecules against TNFα, however, </a>they were often associated with high IC50 values which can lead to their failure in clinical trials. This warrants research related to identification of better small molecule inhibitors by screening of large compound libraries. Recent developments have demonstrated power of natural compounds as safe therapeutics, hence, in this work, we have identified TNFα phytochemical inhibitors using high throughput in silico screening approaches of 6000 phytochemicals followed by 200 ns molecular dynamics simulations and relative binding free energy calculations. The work yielded potent hits that bind to TNFα at its dimer interface. The mechanism targeted was inhibition of oligomerization of TNFα upon phytochemical binding to restrict its interaction with TNF-R1 receptor. MD simulation analysis resulted in identification of two phytochemicals that showed stable protein-ligand conformations over time. The two compounds were triterpenoids: Momordicilin and Nimbolin A with relative binding energy- calculated by MM/PBSA to be -190.5 kJ/Mol and -188.03 kJ/Mol respectively. Therefore, through this work it is being suggested that these phytochemicals can be used for further in vitro analysis to confirm their inhibitory action against TNFα or can be used as scaffolds to arrive at better drug candidates.

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A Comparison of Anticoagulation Activity of a Potent Heparin Preparation in Different Test

10.1055/s-0039-1687438 ◽

1979 ◽

Author(s):

A.S. Bhargava ◽

J. Heinick ◽

Chr. Schöbel ◽

P. Günzel

Keyword(s):

Blood Clotting ◽

Anticoagulant Activity ◽

Factor Xa ◽

Thrombin Time ◽

Beagle Dogs ◽

Clotting Time ◽

Relative Potencies ◽

Heparin Preparation

The anticoagulant effect of a new potent heparin preparation was compared with a commercially available heparin in vivo after intravenous application in beagle dogs. The anticoagulant activity was determined using thrombin time, activated partial thromboplastin time and whole blood clotting time after 5, 10 and 30 minutes of application. The relative potency of the new heparin preparation (Scherinq) was found to be 1.62 to 2.52 times higher than heparin used for comparison (150 USP units/mg, Dio-synth). The anticoagulant properties of both preparations were also studied in vitro using dog and human plasma. The relative potencies in vitro correlated well with those obtained in vivo. Further characterization with amidolytic method using chromogenic substrate for factor Xa and thrombin (S-2222 and S-2238 from KABI, Stockholm) showed that heparin (Schering) contains 243 to 378 USP units/raq depending upon the test systems used to assay the anticoagulation activity and in addition, proves the validity of the amidolytic method.

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Design, synthesis, and in vitro biological activity of benzimidazole based factor Xa inhibitors

Bioorganic & Medicinal Chemistry Letters ◽

10.1016/s0960-894x(00)00139-6 ◽

2000 ◽

Vol 10 (9) ◽

pp. 963-966 ◽

Cited By ~ 79

Author(s):

Zuchun (Spring) Zhao ◽

Damian O Arnaiz ◽

Brian Griedel ◽

Steven Sakata ◽

Jerry L Dallas ◽

...

Keyword(s):

Biological Activity ◽

Factor Xa ◽

Factor Xa Inhibitors ◽

Design Synthesis

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The Synthetic Pentasaccharide, Fondaparinux, Reduces Inflammation and Neutrophil Accumulation in Kidney Ischemia-Reperfusion Injury.

Blood ◽

10.1182/blood.v104.11.217.217 ◽

2004 ◽

Vol 104 (11) ◽

pp. 217-217 ◽

Cited By ~ 3

Author(s):

Gernot Schabbauer ◽

Rolf D. Frank ◽

Todd Holscher ◽

Yuichiro Sato ◽

Michael Tencati ◽

...

Keyword(s):

Ischemia Reperfusion Injury ◽

Inflammatory Diseases ◽

Anticoagulant Activity ◽

Ischemia Reperfusion ◽

Factor Xa ◽

Coagulation Factor ◽

Inflammatory Cells ◽

Neutrophil Accumulation ◽

Kidney Ischemia

Abstract Acute inflammatory diseases are often accompanied by coagulation activation leading to local thrombotic complications and disseminated intravascular coagulation. Recent studies support the concept of crosstalk between coagulation and inflammation. The synthetic pentasaccharide, fondaparinux, is a selective antithrombin-dependent inhibitor of coagulation factor Xa. In this study, we investigated the effect of fondaparinux in a lethal murine model of kidney ischemia-reperfusion (I/R) injury that is associated with coagulation and inflammation. Fondaparinux treatment of I/R-injured mice significantly reduced serum creatinine levels and increased survival from 0 to 44% compared with saline treated control mice. In contrast, depletion of fibrinogen with ancrod was not protective, suggesting that fondaparinux may have additional properties beyond its anticoagulant activity. Indeed, fondaparinux significantly reduced IL-6 and MIP-2 expression but did not reduce MCP-1 expression. Furthermore, fondaparinux significantly decreased neutrophil accumulation in the injured kidneys. Finally, we showed that fondaparinux reduced recruitment of neutrophils into the peritoneum in a model of acute peritonitis and inhibited the binding of U937 cells to P-selectin in vitro. Our data indicate that fondaparinux has both anticoagulant and anti-inflammatory activity reducing fibrin deposition and blocking the binding of inflammatory cells to activated endothelium. Fondaparinux may be useful in the treatment of acute inflammatory diseases.

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In Vitro Evaluation of Apixaban, a Novel, Potent, Selective and Orally Bioavailable Factor Xa Inhibitor.

Blood ◽

10.1182/blood.v108.11.4130.4130 ◽

2006 ◽

Vol 108 (11) ◽

pp. 4130-4130 ◽

Cited By ~ 13

Author(s):

Joseph M. Luettgen ◽

Tracy A. Bozarth ◽

Jeffrey M. Bozarth ◽

Frank A. Barbera ◽

Patrick Y. Lam ◽

...

Keyword(s):

Human Plasma ◽

Serine Proteases ◽

Anticoagulant Activity ◽

Factor Xa ◽

Coagulation Factor ◽

Competitive Inhibitor ◽

Rat Plasma ◽

Reversible Inhibitor ◽

Antithrombotic Agent

Abstract Apixaban, previously known as BMS-562247, is a high affinity, highly selective, orally-active, reversible inhibitor of coagulation factor Xa (fXa), in clinical studies as a therapeutic agent for prevention and treatment of thromboembolic diseases. The in vitro characteristics of apixaban were evaluated in purified systems and in human blood from healthy volunteers. Detailed kinetic analysis of apixaban inhibition of human fXa showed that it is a readily reversible, potent and competitive inhibitor versus a synthetic tripeptide substrate with a Ki of 0.08 nM, an association rate of 2 × 107 M−1s−1and a dissociation half life of 3.4 min. Weak affinity (Ki ~3 μM) is observed for thrombin, plasma kallikrein, and chymotrypsin. Affinity for trypsin and all other serine proteases tested is negligible with Ki > 15 μM. Apixaban is an effective inhibitor of free fXa and of prothrombinase, in buffer, platelet poor plasma, and whole blood. The anticoagulant activity of apixaban was determined in platelet-poor human plasma. Apixaban causes concentration dependent prolongation of the fXa mediated clotting assays. The human plasma concentration required to produce a doubling of the clotting time is 3.6 μM for prothrombin time, 7.4 μM for activated partial thromboplastin time and 0.4 μM for HepTest. To support preclinical efficacy and safety studies purified fXa from rabbit, dog and rat plasma was also found to be inhibited by apixaban (0.17, 2.6, and 1.3 nM, respectively). In summary the in vitro properties of apixaban show that it is a highly selective and potentially potent antithrombotic agent for venous and arterial thrombotic diseases.

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