scholarly journals Synthesis, Docking, and In Vitro Anticoagulant Activity Assay of Hybrid Derivatives of Pyrrolo[3,2,1-ij]Quinolin-2(1H)-one as New Inhibitors of Factor Xa and Factor XIa

Molecules ◽  
2020 ◽  
Vol 25 (8) ◽  
pp. 1889 ◽  
Author(s):  
Nadezhda Novichikhina ◽  
Ivan Ilin ◽  
Anna Tashchilova ◽  
Alexey Sulimov ◽  
Danil Kutov ◽  
...  
Keyword(s):  
Anticoagulant Activity ◽  
Factor Xa ◽  
Coagulation Factor ◽  
Coagulation Factors ◽  
Protein Targets ◽  
Ic50 Value ◽  
Anticoagulant Drugs ◽  
Factor Xia ◽  
Derivatives Of

Coagulation factor Xa and factor XIa are proven to be convenient and crucial protein targets for treatment for thrombotic disorders and thereby their inhibitors can serve as effective anticoagulant drugs. In the present work, we focused on the structure–activity relationships of derivatives of pyrrolo[3,2,1-ij]quinolin-2(1H)-one and an evaluation of their activity against factor Xa and factor XIa. For this, docking-guided synthesis of nine compounds based on pyrrolo[3,2,1-ij]quinolin-2(1H)-one was carried out. For the synthesis of new hybrid hydropyrrolo[3,2,1-ij]quinolin-2(1H)-one derivatives, we used convenient structural modification of both the tetrahydro- and dihydroquinoline moiety by varying the substituents at the C6,8,9 positions. In vitro testing revealed that four derivatives were able to inhibit both coagulation factors and three compounds were selective factor XIa inhibitors. An IC50 value of 3.68 μM for was found for the best factor Xa inhibitor and 2 μM for the best factor XIa inhibitor.

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

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 217-217 ◽  
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.

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

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 4130-4130 ◽  
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.

scholarly journals Protein Z–dependent protease inhibitor deficiency produces a more severe murine phenotype than protein Z deficiency

Blood ◽  
2008 ◽  
Vol 111 (10) ◽  
pp. 4973-4978 ◽  
Author(s):  
Jing Zhang ◽  
Yizheng Tu ◽  
Lan Lu ◽  
Nina Lasky ◽  
George J. Broze
Keyword(s):  
Protease Inhibitor ◽  
Factor Xa ◽  
Coagulation Factor ◽  
Plasma Vitamin ◽  
Severe Phenotype ◽  
Epinephrine Infusion ◽  
Protein Z ◽  
Thrombotic Risk ◽  
Factor Xia

Abstract Protein Z (PZ) is a plasma vitamin K–dependent protein that functions as a cofactor to dramatically enhance the inhibition of coagulation factor Xa by the serpin, protein Z–dependent protease inhibitor (ZPI). In vitro, ZPI not only inhibits factor Xa in a calcium ion–, phospholipid-, and PZ-dependent fashion, but also directly inhibits coagulation factor XIa. In murine gene-deletion models, PZ and ZPI deficiency enhances thrombosis following arterial injury and increases mortality from pulmonary thromboembolism following collagen/epinephrine infusion. On a factor VLeiden genetic background, ZPI deficiency produces a significantly more severe phenotype than PZ deficiency, implying that factor XIa inhibition by ZPI is physiologically relevant. The studies in mice suggest that human PZ and ZPI deficiency would be associated with a modest thrombotic risk with ZPI deficiency producing a more severe phenotype.

scholarly journals SULFATED DERIVATIVES OF ARABINOGALACTAN AND THEIR ANTICOAGULANT ACTIVITY

2019 ◽  
pp. 47-56
Author(s):  
Svetlana Alekseyevna Kuznetsova ◽  
Natal'ya Yur'yevna Vasilyeva ◽  
Natal'ya Nikolayevna Drozd ◽  
Mikhail Aleksandrovich Mikhailenko ◽  
Tat'yana Petrovna Shakhtshneider ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Sulfur Content ◽  
Anticoagulant Activity ◽  
Factor Xa ◽  
Sulfamic Acid ◽  
In Vitro Study ◽  
Acid Complex ◽  
Derivatives Of ◽  
Salt Forms

The IR spectra and molecular mass distribution of arabinogalactan sulfates in the form of sodium and ammonium salts, obtained using various sulfating reagents, were compared. According to the obtained data, the sulfated derivatives of arabinogalactan differ from each other by the nature of the hydrogen bonds and the molecular weight distribution. Using coagulological tests at the activation of coagulation of platelet-poor human plasma, in vitro study of the anticoagulant properties of arabinogalactan sulfated derivatives in various salt forms differing in methods of  preparation, degree of sulfation and molecular weight was conducted. It was established that the sample in the form of arabinogalactan sodium salt (SAG 1) with a sulfur content of 13.2 wt.% аnd a polydispersity degree of 1.52 showed 2 times more anticoagulant activity than the sample in the form of ammonium salt of arabinogalactan (SAG 2) with the sulfur content of 6.6 wt.% and the degree of polydispersity of 1.30. Antithrombin (aIIa) activity of samples obtained by sulfation with pyridine and sulfuric anhydride complex (SAG 1) and sulfamic acid complex (SAG 2) was, respectively, 23.42±1.86 and 10.20±1.50 U/mg; the anti-factor Xa activity of SAG 1 and SAG 2 was 2.13±0.42 and 0.37±0.08 U/mg; and the ratio aIIa/aXa for SAG1 and SAG 2 was 11 and 28, respectively. The less activity of antifactor Xa(aXA) of SAG as compared to unfractionated heparin (UFG) and higher rations of activities aIIa/aXa may contribute the less provocation of bleeding by SAG samples in the comparition with UFG.

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.

Assays for Phospholipid-Dependent Formation of Thrombin and Xa: A Potential Method for Quantifying Lupus Anticoagulant Activity

1991 ◽  
Vol 66 (04) ◽  
pp. 453-458 ◽  
Author(s):  
John T Brandt
Keyword(s):  
Specific Activity ◽  
Anticoagulant Activity ◽  
Factor Xa ◽  
Clinical Importance ◽  
Potential Method ◽  
Quantitative Expression ◽  
Increased Risk ◽  
Apparent Association

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.

The Anticoagulant Properties of a Modified Form of Protein S

1988 ◽  
Vol 60 (02) ◽  
pp. 298-304 ◽  
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.

Amidolytic Detection of Prothrombin Activation Products after SDS-Gel Electrophoresis

1989 ◽  
Vol 61 (03) ◽  
pp. 386-391 ◽  
Author(s):  
Guido Tans ◽  
Truus Janssen-Claessen ◽  
Jan Rosing
Keyword(s):  
Gel Electrophoresis ◽  
Activated Protein C ◽  
Factor Xa ◽  
Coagulation Factors ◽  
Product Formation ◽  
Chromogenic Substrate ◽  
Nitrocellulose Membrane ◽  
Activation Products ◽  
Factor Xia ◽  
Prothrombin Activation

SummaryIn this paper we report a method via which enzymatically active products formed during prothrombin activation can be detected by simple photographic means after SDS-gel electrophoresis, blotting onto a nitrocellulose membrane and visualization with the chromogenic substrate, S2238. After amidolytic detection the same nitrocellulose membrane can also be used for immunologic detection of prothrombin activation products, thus allowing a complete description of product formation during prothrombin activation.The detection limit of the so-called “amidoblot” is approximately 3 ng thrombin per gel sample which is comparable to the sensitivity of immunoblotting.It is further shown that the amidoblot technique can also be applied to other coagulation factors for which a suitable chromogenic substrate is available (factor XIIa, kallikrein, factor XIa, factor Xa, plasmin and activated protein C).

scholarly journals Inhibition of human coagulation factor VIII by monoclonal antibodies. Mapping of functional epitopes with the use of recombinant factor VIII fragments

1989 ◽  
Vol 263 (1) ◽  
pp. 187-194 ◽  
Author(s):  
A Leyte ◽  
K Mertens ◽  
B Distel ◽  
R F Evers ◽  
M J M De Keyzer-Nellen ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Factor Viii ◽  
Factor Xa ◽  
Coagulation Factor ◽  
Procoagulant Activity ◽  
Coagulation Factor Viii ◽  
Coagulation Cascade ◽  
Restriction Enzyme Digestion ◽  
Proteolytic Activation

The epitopes of four monoclonal antibodies against coagulation Factor VIII were mapped with the use of recombinant DNA techniques. Full-length Factor VIII cDNA and parts thereof were inserted into the vector pSP64, permitting transcription in vitro with the use of a promoter specific for SP6 RNA polymerase. Factor VIII DNA inserts were truncated from their 3′-ends by selective restriction-enzyme digestion and used as templates for ‘run-off’ mRNA synthesis. Translation in vitro with rabbit reticulocyte lysate provided defined radiolabelled Factor VIII fragments for immunoprecipitation studies. Two antibodies are shown to be directed against epitopes on the 90 kDa chain of Factor VIII, between residues 712 and 741. The 80 kDa chain appeared to contain the epitopes of the other two antibodies, within the sequences 1649-1778 and 1779-1840 respectively. The effect of antibody binding to these sequences was evaluated at two distinct levels within the coagulation cascade. Both Factor VIII procoagulant activity and Factor VIII cofactor function in Factor Xa generation were neutralized upon binding to the region 1779-1840. The antibodies recognizing the region 713-740 or 1649-1778, though interfering with Factor VIII procoagulant activity, did not inhibit in Factor Xa generation. These findings demonstrate that antibodies that virtually inhibit Factor VIII in coagulation in vitro are not necessarily directed against epitopes involved in Factor VIII cofactor function. Inhibition of procoagulant activity rather than of cofactor function itself may be explained by interference in proteolytic activation of Factor VIII. This hypothesis is in agreement with the localization of the epitopes in the proximity of thrombin-cleavage or Factor Xa-cleavage sites.

scholarly journals Heparin is more effective than apixaban in inhibiting in vitro contact activated coagulation

2020 ◽  
Vol 41 (Supplement_2) ◽  
Author(s):  
M.M Engelen ◽  
C Van Laer ◽  
M Jacquemin ◽  
C Vandenbriele ◽  
K Peerlinck ◽  
...  
Keyword(s):  
Thrombin Generation ◽  
Heart Valves ◽  
Thrombus Formation ◽  
Oral Anticoagulants ◽  
Direct Oral Anticoagulants ◽  
Factor Xa ◽  
Coagulation Factor ◽  
Mechanical Heart Valves ◽  
Contact Activation

Abstract Introduction Contact of blood with artificial surfaces such as mechanical support devices, catheters, and mechanical heart valves activates the contact activation (CA) pathway of coagulation. Furthermore, recent animal data and clinical studies suggest a more important contribution of CA in pathological thrombus formation in other cardiovascular diseases. Direct oral anticoagulants (DOACs) are recommended as first-line treatment in most patients who require long-term anticoagulation. However, because DOACs directly inhibit a single downstream coagulation factor (thrombin (fXIIa) or factor Xa (fXa)), it has been suggested that their efficacy could be reduced in the presence of strong activation of the CA pathway as compared to anticoagulants that target multiple, more upstream located coagulation factors. Purpose To compare the efficacy of a DOAC (apixaban) and heparin to suppress thrombin generation in the presence of strong CA pathway activation. Methods Pooled platelet-poor plasma was spiked with either apixaban (dissolved in DMSO and PBS) or unfractionated heparin to achieve therapeutic plasma levels. SynthASil, a commercially available mixture of phospholipids and silica, was used to stimulate the CA pathway in two different dilutions (1–80 and 5–80). Downstream coagulation was accessed by Thrombin Generation Test using Thrombinoscope by Stago and associated Thrombin Calibrator (activity 640 nM). The endogenous thrombin potential (area under the thrombin generation curve; ETP), peak thrombin generation (PTG), time to peak (ttPeak) and time to start (ttStart) were accessed. Results With decreasing concentrations of apixaban, stimulation with the lower dose SynthASil reveals an increasing ETP and PTG. As expected, ttPeak and ttStart decreased. Even supratherapeutic levels of apixaban (i.e. 1120 ng/mL) could not inhibit thrombin from being generated, in striking contrast with UFH where no thrombin was formed. Using a five times higher dose of SynthASil showed comparable ETP for all concentrations of apixaban, allocated around the control value. PTG, however, slightly increased with decreasing concentrations of apixaban. ttPeak and ttStart slightly decreased. Except for the subtherapeutic UFH concentration of 0,114 IU/mL, no thrombin was generated with UFH. Conclusion UFH is more effective in inhibiting downstream thrombin generation compared to apixaban as a response to activation of the CA pathway in vitro. These findings could help explain why direct inhibitors were not able to show non-inferiority in patients with mechanical heart valves and support the development of specific CA pathway inhibitors for patients with conditions that activate the CA pathway. Thrombin generation curves Funding Acknowledgement Type of funding source: None

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