scholarly journals A Structure Based Study of Selective Inhibition of Factor IXa over Factor Xa

Molecules ◽  
2021 ◽  
Vol 26 (17) ◽  
pp. 5372
Author(s):  
Sibsankar Kundu ◽  
Sangwook Wu
Keyword(s):  
Structural Alignment ◽  
Factor Xa ◽  
Coagulation Factor ◽  
Coagulation Factors ◽  
Selective Inhibition ◽  
Binding Mode ◽  
Pharmacophore Modeling ◽  
Coagulation Cascade ◽  
Modern World ◽  
Factor Ixa

Blood coagulation is an essential physiological process for hemostasis; however, abnormal coagulation can lead to various potentially fatal disorders, generally known as thromboembolic disorders, which are a major cause of mortality in the modern world. Recently, the FDA has approved several anticoagulant drugs for Factor Xa (FXa) which work via the common pathway of the coagulation cascade. A main side effect of these drugs is the potential risk for bleeding in patients. Coagulation Factor IXa (FIXa) has recently emerged as the strategic target to ease these risks as it selectively regulates the intrinsic pathway. These aforementioned coagulation factors are highly similar in structure, functional architecture, and inhibitor binding mode. Therefore, it remains a challenge to design a selective inhibitor which may affect only FIXa. With the availability of a number of X-ray co-crystal structures of these two coagulation factors as protein–ligand complexes, structural alignment, molecular docking, and pharmacophore modeling were employed to derive the relevant criteria for selective inhibition of FIXa over FXa. In this study, six ligands (three potent, two selective, and one inactive) were selected for FIXa inhibition and six potent ligands (four FDA approved drugs) were considered for FXa. The pharmacophore hypotheses provide the distribution patterns for the principal interactions that take place in the binding site. None of the pharmacophoric patterns of the FXa inhibitors matched with any of the patterns of FIXa inhibitors. Based on pharmacophore analysis, a selectivity of a ligand for FIXa over FXa may be defined quantitatively as a docking score of lower than −8.0 kcal/mol in the FIXa-grids and higher than −7.5 kcal/mol in the FXa-grids.

scholarly journals Cryo-EM structures of human coagulation factors V and Va

Blood ◽  
2021 ◽  
Author(s):  
Eliza A Ruben ◽  
Michael J Rau ◽  
James Fitzpatrick ◽  
Enrico Di Cera
Keyword(s):  
Protein C ◽  
Activated Protein C ◽  
Factor Xa ◽  
Coagulation Factor ◽  
Coagulation Factors ◽  
Proteolytic Processing ◽  
Coagulation Cascade ◽  
Factor V ◽  
Prothrombinase Complex ◽  
A2 Domain

Coagulation factor V is the precursor of factor Va that, together with factor Xa, Ca2+ and phospholipids, defines the prothrombinase complex and activates prothrombin in the penultimate step of the coagulation cascade. Here we present cryo-EM structures of human factors V and Va at atomic (3.3 Å) and near-atomic (4.4 Å) resolution, respectively. The structure of fV reveals the entire A1-A2-B-A3-C1-C2 assembly but with a surprisingly disordered B domain. The C1 and C2 domains provide a platform for interaction with phospholipid membranes and support the A1 and A3 domains, with the A2 domain sitting on top of them. The B domain is highly dynamic and visible only for short segments connecting to the A2 and A3 domains. The A2 domain reveals all sites of proteolytic processing by thrombin and activated protein C, a partially buried epitope for binding factor Xa and fully exposed epitopes for binding activated protein C and prothrombin. Removal of the B domain and activation to fVa exposes the sites of cleavage by activated protein C at R306 and R506 and produces increased disorder in the A1-A2-A3-C1-C2 assembly, especially in the C-terminal acidic portion of the A2 domain responsible for prothrombin binding. Ordering of this region and full exposure of the factor Xa epitope emerge as a necessary step for the assembly of the prothrombin-prothrombinase complex. These structures offer molecular context for the function of factors V and Va and pioneer the analysis of coagulation factors by cryo-EM.

scholarly journals Functional assembly of intrinsic coagulation proteases on monocytes and platelets. Comparison between cofactor activities induced by thrombin and factor Xa.

1992 ◽  
Vol 176 (1) ◽  
pp. 27-35 ◽  
Author(s):  
M P McGee ◽  
L C Li ◽  
M Hensler
Keyword(s):  
Factor Viii ◽  
Factor Xa ◽  
Coagulation Factor ◽  
Coagulation Cascade ◽  
Factor X ◽  
Regulatory Pathways ◽  
Factor Ixa ◽  
Factor Viiia ◽  
Human Factor Viii

Generation of coagulation factor Xa by the intrinsic pathway protease complex is essential for normal activation of the coagulation cascade in vivo. Monocytes and platelets provide membrane sites for assembly of components of this protease complex, factors IXa and VIII. Under biologically relevant conditions, expression of functional activity by this complex is associated with activation of factor VIII to VIIIa. In the present studies, autocatalytic regulatory pathways operating on monocyte and platelet membranes were investigated by comparing the cofactor function of thrombin-activated factor VIII to that of factor Xa-activated factor VIII. Reciprocal functional titrations with purified human factor VIII and factor IXa were performed at fixed concentrations of human monocytes, CaCl2, factor X, and either factor IXa or factor VIII. Factor VIII was preactivated with either thrombin or factor Xa, and reactions were initiated by addition of factor X. Rates of factor X activation were measured using chromogenic substrate specific for factor Xa. The K1/2 values, i.e., concentration of factor VIIIa at which rates were half maximal, were 0.96 nM with thrombin-activated factor VIII and 1.1 nM with factor Xa-activated factor VIII. These values are close to factor VIII concentration in plasma. The Vsat, i.e., rates at saturating concentrations of factor VIII, were 33.3 and 13.6 nM factor Xa/min, respectively. The K1/2 and Vsat values obtained in titrations with factor IXa were not significantly different from those obtained with factor VIII. In titrations with factor X, the values of Michaelis-Menten coefficients (Km) were 31.7 nM with thrombin-activated factor VIII, and 14.2 nM with factor Xa-activated factor VIII. Maximal rates were 23.4 and 4.9 nM factor Xa/min, respectively. The apparent catalytic efficiency was similar with either form of factor VIIIa. Kinetic profiles obtained with platelets as a source of membrane were comparable to those obtained with monocytes. These kinetic profiles are consistent with a 1:1 stoichiometry for the functional interaction between cofactor and enzyme on the surface of monocytes and platelets. Taken together, these results indicate that autocatalytic pathways connecting the extrinsic, intrinsic, and common coagulation pathways can operate efficiently on the monocyte membrane.

The Effect of Antithrombin III on the Activity of the Coagulation Factors VII, IX and X

1976 ◽  
Vol 35 (02) ◽  
pp. 295-304 ◽  
Author(s):  
B Østerud ◽  
M Miller-Andersson ◽  
U Abildgaard ◽  
H Prydz
Keyword(s):  
Antithrombin Iii ◽  
Factor Xa ◽  
Coagulation Factors ◽  
Disc Electrophoresis ◽  
Polyacrylamide Gel ◽  
Factor Vii ◽  
Native Form ◽  
Factor Ixa ◽  
Plasma Factor

SummaryAntithrombin III, purified to homogeneity according to Polyacrylamide gel disc electrophoresis and immunoelectrophoresis, inhibited the activity of purified factor IXa and Xa, whereas factor VII was not inhibited either in the active or in the native form.Antithrombin III is the single most important inhibitor of factor Xa in plasma. Factor Xa does not, however, reduce the activity of antithrombin III against thrombin.

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.

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 Direct Determination of Coagulation Factor IIa and Plasmin Activities for Monitoring of Thrombotic State

2020 ◽  
Vol 5 (6) ◽  
pp. 1265-1276
Author(s):  
Junhua Zhang ◽  
Lihui Zou ◽  
Chengyang Liu ◽  
Chuanbao Li ◽  
Meng Wang ◽  
...  
Keyword(s):  
Direct Detection ◽  
Oral Anticoagulants ◽  
Direct Determination ◽  
Coagulation Factor ◽  
High Specificity ◽  
Coagulation Factors ◽  
Coagulation Cascade ◽  
Proteolytic Activation ◽  
Activated State

Abstract Background Current laboratory examinations for hypercoagulable diseases focus on the biomarker content of the activated coagulation cascade and fibrinolytic system. Direct detection of physiologically important protease activities in blood remains a challenge. This study aims to develop a general approach that enables the determination of activities of crucial coagulation factors and plasmin in blood. Methods This assay is based on the proteolytic activation of an engineered zymogen of l-phenylalanine oxidase (proPAO), for which the specific blood protease cleavage sites were engineered between the inhibitory and activity domains of proPAO. Specific cleavage of the recombinant proenzyme leads to the activation of proPAO, followed by oxidation and oxygenation of l-phenylalanine, resulting in an increase of chromogenic production when coupled with the Trinder reaction. Results We applied this method to determine the activities of both coagulation factor IIa and plasmin in their physiologically relevant basal state and fully activated state in sodium citrate–anticoagulated plasma respectively. Factor IIa and plasmin activities could be dynamically monitored in patients with thrombotic disease who were taking oral anticoagulants and used for assessing the hypercoagulable state in pregnant women. Conclusions The high specificity, sensitivity, and stability of this novel assay not only makes it useful for determining clinically important protease activities in human blood and diagnosing thrombotic diseases but also provides a new way to monitor the effectiveness and safety of anticoagulant drugs.

scholarly journals The anticoagulant mechanism of action of heparin in contact-activated plasma: inhibition of factor X activation

Blood ◽  
1985 ◽  
Vol 65 (5) ◽  
pp. 1226-1231 ◽  
Author(s):  
TB McNeely ◽  
MJ Griffith
Keyword(s):  
Antithrombin Iii ◽  
Factor Xa ◽  
Coagulation Factors ◽  
Factor Ix ◽  
Factor X ◽  
Clotting Time ◽  
Intrinsic Pathway ◽  
Blood Coagulation Factors ◽  
Factor Ixa ◽  
Factor X Activation

Abstract The effects of heparin on the activation of blood coagulation factors IX and X in contact-activated plasma were determined in the present study. In the presence and absence of 0.5 U/mL heparin, the amounts of factor IX that were cleaved 30 minutes after the addition of calcium and phospholipid to plasma exposed to glass (ie, contact activated) were essentially identical. In the absence of heparin, however, the plasma clotting time was between three and four minutes, while in the presence of heparin, the clotting time was approximately 40 minutes. More factor IXa was inhibited by antithrombin III in the presence of heparin than in its absence, but factor IXa levels sufficient for factor X activation appeared to be present in the heparinized plasma. Neither an increase in factor Xa nor a decrease in factor X was detected, however, in heparinized plasma. We conclude that the step in the intrinsic pathway of coagulation that is inhibited in the presence of heparin is at the level of factor X activation.

scholarly journals Role of the blood coagulation cascade in hepatic fibrosis

2018 ◽  
Vol 315 (2) ◽  
pp. G171-G176 ◽  
Author(s):  
Asmita Pant ◽  
Anna K. Kopec ◽  
James P. Luyendyk
Keyword(s):  
Blood Coagulation ◽  
Hepatic Fibrosis ◽  
Animal Studies ◽  
Factor Xa ◽  
Coagulation Factor ◽  
Primary Source ◽  
Bleeding Risk ◽  
Normal Function ◽  
Coagulation Cascade ◽  
Rapid Progression

Liver is the primary source of numerous proteins that are critical for normal function of the blood coagulation cascade. Because of this, diseases of the liver, particularly when affiliated with severe complications like cirrhosis, are associated with abnormalities of blood clotting. Although conventional interpretation has inferred cirrhosis as a disorder of uniform bleeding risk, it is now increasingly appreciated as a disease wherein the coagulation cascade is precariously rebalanced. Moreover, prothrombotic risk factors are also associated with a more rapid progression of fibrosis in humans, suggesting that coagulation proteases participate in disease pathogenesis. Indeed, strong evidence drawn from experimental animal studies indicates that components of the coagulation cascade, particularly coagulation factor Xa and thrombin, drive profibrogenic events, leading to hepatic fibrosis. Here, we concisely review the evidence supporting a pathologic role for coagulation in the development of liver fibrosis and the potential mechanisms involved. Further, we highlight how studies in experimental animals may shed light on emerging clinical evidence, suggesting that beneficial effects of anticoagulation could extend beyond preventing thrombotic complications to include reducing pathologies like fibrosis.

Implications of Heparinoid-Induced Inactivation of Coagulation Cascade Factors and In Vivo Proenzyme Concentration.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1621-1621
Author(s):  
Sanjay Patel ◽  
Leslie R. Berry ◽  
Mark W.C. Hatton ◽  
Anthony Chan
Keyword(s):  
Plasma Concentration ◽  
Factor Xa ◽  
Coagulation Factors ◽  
Coagulation Cascade ◽  
Factor Xii ◽  
Initiation Factors ◽  
Inhibition Studies ◽  
The Impact ◽  
Covalent Complex

Abstract Heparin is a commonly used anticoagulant in the treatment of thrombosis. We have compared heparinoid-stimulated inhibition rates with the in vivo plasma concentration of antithrombin (AT)-inhibitable coagulation factors. Second order rate constants (k2) for inhibition of activated factors by either AT + unfractionated heparin (AT+UFH) mixtures or a novel covalent complex of AT and heparin (ATH) (Chan et al, J Biol Chem, 272:22111, 1997) were determined by discontinuous assay. A plot of k2 values (mean ± SEM; n ≥5) versus the respective human plasma concentration of coagulation factors revealed a linear correlation (with R2 values of 0.93 for AT+UFH and 0.90 for ATH, excluding factor XII), in which neutralization efficiency was proportional to in vivo factor level (see Figure). Anticoagulant actions of AT+UFH and ATH were more oriented towards treatment than prophylaxis since inhibition of cascade end point enzymes (thrombin and factor Xa) was more rapid than factors involved in coagulation initiation (factors VIIa and XIa). However, ATH exhibited more enhanced inhibition rates against factors VIIa, IXa and XIa than against factor Xa and thrombin, suggestive of an improved prophylactic profile compared to AT+UFH. Intriguingly, factor XII did not follow this trend, further challenging its role in the coagulation cascade. The impact of these assertions requires confirmation by in vivo inhibition studies. Figure Figure

scholarly journals Autoimmune Factor V Deficiency That Took 16 Years to Diagnose due to Pseudodeficiency of Multiple Coagulation Factors

2021 ◽  
Vol 2021 ◽  
pp. 1-5
Author(s):  
Takaaki Kato ◽  
Takaya Hanawa ◽  
Mea Asou ◽  
Tomohiko Asakawa ◽  
Hisashi Sakamaki ◽  
...  
Keyword(s):  
High Titer ◽  
Coagulation Factor ◽  
Coagulation Factors ◽  
International Normalized Ratio ◽  
University Hospital ◽  
Coagulation Cascade ◽  
Factor V ◽  
Normal Limits ◽  
Factor V Deficiency ◽  
The Right

A 70-year-old man presented to our hospital with intramuscular hemorrhage in the right thigh. He had exhibited a tendency to bleed for the last 16 years and had visited several medical institutions, but no diagnosis had been made. Since the risk of sudden bleeding was assumed to be high due to his age, we decided to examine him in our department. A coagulation abnormality with prothrombin time-international normalized ratio (PT-INR) of 4.5 and activated partial thromboplastin time (aPTT) of 99.6 seconds was observed, but the platelet count, fibrinogen, and PIVKAII were within normal limits. Coagulation activities of factor V, VII, VIII, IX, X, XI, XII, and XIII were all reduced. Anti-factor VIII and IX antibodies which were measured by the Bethesda method, lupus anti-coagulant (diluted Russell snake venom time method) and anti-cardiolipin antibody were also positive. The results of these tests were comparable to those undertaken 15 years ago when they were scrutinized at the university hospital. We suspected the presence of anti-factor V antibodies because there was a dissociation between the thrombotest values measured and those calculated from the PT-INR. Moreover, cross-mixing test showed an immediate inhibitor pattern. Subsequently, factor V antibodies were confirmed by the immunoblot method and the diagnosis of autoimmune factor V deficiency was made. When factor V, which is downstream of the coagulation cascade, is inhibited, coagulation test using the one-stage clotting method shows a pseudolow value. Therefore, extensive abnormalities of coagulation factor activity and inhibitor assay should be interpreted with caution, and the presence of a high titer of factor V inhibitor should be considered.

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