Region of Factor IXa Protease Domain that Interacts with Factor VIIIa: Analysis of Select Hemophilia B Mutants

1999 ◽  
Vol 82 (08) ◽  
pp. 218-225 ◽  
Author(s):  
S. Paul Bajaj
Keyword(s):  
X Chromosome ◽  
Coagulation Factor ◽  
Factor Ix ◽  
Hemophilia B ◽  
Coagulation Cascade ◽  
Factor Vii ◽  
Factor X ◽  
Homologous Proteins ◽  
Dependent Protein ◽  
Factor Ix Deficiency

IntroductionThe identification of coagulation factor IX as a substance required for blood coagulation was first established by Pavlovsky, who reported that a mixture of blood from two hemophiliacs clotted normally.1 Based on this discovery and subsequent observations,2 hemophilia was divided into two conditions-hemophilia A or factor VIII deficiency, the most prevalent condition, and hemophilia B or factor IX deficiency, a less common condition. Since then, much has been learned about the molecular and structural biology of factor IX. It is a vitamin K-dependent protein that participates in the middle phase of the intrinsic as well as the extrinsic coagulation cascade.3 The gene for factor IX consists of eight exons and seven introns, is approximately 34 kb long, and located on the long arm of the X-chromosome at band Xq27.1.4,5 The positions of the introns in the factor IX gene are essentially identical to those of the other three homologous proteins, namely, factor VII, factor X, and protein C;3 the genes for the latter three proteins, however, are not located on the X-chromosome.

scholarly journals Bridging the Missing Link with Emicizumab: A Bispecific Antibody for Treatment of Hemophilia A

2020 ◽  
Vol 120 (10) ◽  
pp. 1357-1370
Author(s):  
Georg Gelbenegger ◽  
Christian Schoergenhofer ◽  
Paul Knoebl ◽  
Bernd Jilma
Keyword(s):  
Clinical Trials ◽  
Hemophilia A ◽  
Coagulation Factor ◽  
Factor Ix ◽  
Coagulation Cascade ◽  
Factor Vii ◽  
Factor X ◽  
Current Standard ◽  
Bleeding Events ◽  
Recombinant Activated Factor Vii

AbstractHemophilia A, characterized by absent or ineffective coagulation factor VIII (FVIII), is a serious bleeding disorder that entails severe and potentially life-threatening bleeding events. Current standard therapy still involves replacement of FVIII, but is often complicated by the occurrence of neutralizing alloantibodies (inhibitors). Management of patients with inhibitors is challenging and necessitates immune tolerance induction for inhibitor eradication and the use of bypassing agents (activated prothrombin complex concentrates or recombinant activated factor VII), which are expensive and not always effective. Emicizumab is the first humanized bispecific monoclonal therapeutic antibody designed to replace the hemostatic function of activated FVIII by bridging activated factor IX and factor X (FX) to activate FX and allow the coagulation cascade to continue. In the majority of hemophilic patients with and without inhibitors, emicizumab reduced the annualized bleeding rate to almost zero in several clinical trials and demonstrated a good safety profile. However, the concurrent use of emicizumab and activated prothrombin complex concentrate imposes a high risk of thrombotic microangiopathy and thromboembolic events on patients and should be avoided. Yet, the management of breakthrough bleeds and surgery remains challenging with only limited evidence-based recommendations being available. This review summarizes published clinical trials and preliminary reports of emicizumab and discusses the clinical implications of emicizumab in treatment of hemophilia A.

Model of a Ternary Complex between Activated Factor VII, Tissue Factor and Factor IX

2002 ◽  
Vol 88 (07) ◽  
pp. 74-82 ◽  
Author(s):  
Shu-wen Chen ◽  
Jean-François Schved ◽  
Jean-Luc Pellequer ◽  
Muriel Giansily-Blaizot
Keyword(s):  
Tissue Factor ◽  
Ternary Complex ◽  
Search Algorithm ◽  
Factor Ix ◽  
Site Directed Mutagenesis ◽  
Coagulation Cascade ◽  
Factor Vii ◽  
Factor X ◽  
Three Dimensional Model ◽  
Inhibitory Peptide

SummaryUpon binding to tissue factor, FVIIa triggers coagulation by activating vitamin K-dependent zymogens, factor IX (FIX) and factor X (FX). To understand recognition mechanisms in the initiation step of the coagulation cascade, we present a three-dimensional model of the ternary complex between FVIIa:TF:FIX. This model was built using a full-space search algorithm in combination with computational graphics. With the known crystallographic complex FVIIa:TF kept fixed, the FIX docking was performed first with FIX Gla-EGF1 domains, followed by the FIX protease/EGF2 domains. Because the FIXa crystal structure lacks electron density for the Gla domain, we constructed a chimeric FIX molecule that contains the Gla-EGF1 domains of FVIIa and the EGF2-protease domains of FIXa. The FVIIa:TF:FIX complex has been extensively challenged against experimental data including site-directed mutagenesis, inhibitory peptide data, haemophilia B database mutations, inhibitor antibodies and a novel exosite binding inhibitor peptide. This FVIIa:TF:FIX complex provides a powerful tool to study the regulation of FVIIa production and presents new avenues for developing therapeutic inhibitory compounds of FVIIa:TF:substrate complex.

Factor VII Deficiency Remains Post-Puberty Due to Point Mutation in the HNF4 Binding Site in the Coagulant Factor VII Gene.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1947-1947
Author(s):  
XingWu Zheng ◽  
Theresa M. Tidd ◽  
Donna DiMichele ◽  
Eleanor S. Pollak
Keyword(s):  
Binding Site ◽  
Point Mutation ◽  
Mutant Allele ◽  
Factor Ix ◽  
Hemophilia B ◽  
Factor Vii ◽  
Start Site ◽  
Expression Plasmid ◽  
Fvii Deficiency ◽  
Factor Ix Deficiency

Abstract A novel T to C point mutation at −60 in the gene for coagulant Factor VII results in life-long severe coagulant Factor VII deficiency in post-pubertal twin males. The clinical course of these patients provides an informative in vivo example of the regulation of expression of vitamin K-dependent clotting protein Factor VII. An analogous point mutation in the HNF4 binding site in the Factor IX gene results in the clinical phenotype Hemophilia B Leyden, a sex-linked antigen-negative Factor IX deficiency that resolves post-puberty. The affected Factor VII deficient patients have prolonged prothrombin times (46 and 52 secs), normal aPTTs and decreased FVII levels of FVII:Coagulant activity: < 1% and FVII:Antigen: < 3%. The −60 mutation, ACTTTG → ACTCTG occurs 9 base pairs before the start site of transcription and 59 bps before the before the start site of translation. The twins are compound heterozygotes and also possess a mutation in exon 8 at amino acid 348, a mutation that has previously been reported to cause FVII deficiency. Both affected individuals have recurrent target joint hemorrhage (shoulder, elbow, ankle) requiring replacement therapy 6–12 times/year. Results: Gel mobility shift assays using a radio-labeled probe spanning from −76 to −46 in the FVII promoter region demonstrate the loss of binding of transcription factor HNF-4. Transient transfection assays in HepG2 cells using 186 bps of the mutant and the wildtype promoters (−185 to +1) revealed a loss of expression with the mutant allele. Co-transfection with an HNF4 expression plasmid resulted in an increase in expression of the wildetype construct in HeLa cells, a non-hepatic cell line. However, co-transfection of the HNF4 expression plasmid failed to increase expression with the construct containing the mutant allele sequence. Conclusion: The lack of phenotypic change of the FVII:C in 19 yo twin boys provides dynamic support of the necessity of an overlapping androgen binding site in the homologous Factor IX gene as responsible for the phenotypic resolution of Factor IX deficiency (Hemophilia B Leyden) post-puberty. It is of interest that an increase in FVII:C did not occur with advancing age in FVII deficiency due to this HNF4 binding site mutation.

scholarly journals A candidate activation pathway for coagulation factor VII

2019 ◽  
Vol 476 (19) ◽  
pp. 2909-2926
Author(s):  
Tina M. Misenheimer ◽  
Kraig T. Kumfer ◽  
Barbara E. Bates ◽  
Emily R. Nettesheim ◽  
Bradford S. Schwartz
Keyword(s):  
Tissue Factor ◽  
Blood Coagulation ◽  
Factor Viia ◽  
Coagulation Factor ◽  
Factor Ix ◽  
Factor Vii ◽  
Factor X ◽  
Contact Activation ◽  
Coagulation Factor Vii ◽  
Factor Viiia

Abstract The mechanism of generation of factor VIIa, considered the initiating protease in the tissue factor-initiated extrinsic limb of blood coagulation, is obscure. Decreased levels of plasma VIIa in individuals with congenital factor IX deficiency suggest that generation of VIIa is dependent on an activation product of factor IX. Factor VIIa activates IX to IXa by a two-step removal of the activation peptide with cleavages occurring after R191 and R226. Factor IXaα, however, is IX cleaved only after R226, and not after R191. We tested the hypothesis that IXaα activates VII with mutant IX that could be cleaved only at R226 and thus generate only IXaα upon activation. Factor IXaα demonstrated 1.6% the coagulant activity of IXa in a contact activation-based assay of the intrinsic activation limb and was less efficient than IXa at activating factor X in the presence of factor VIIIa. However, IXaα and IXa had indistinguishable amidolytic activity, and, strikingly, both catalyzed the cleavage required to convert VII to VIIa with indistinguishable kinetic parameters that were augmented by phospholipids, but not by factor VIIIa or tissue factor. We propose that IXa and IXaα participate in a pathway of reciprocal activation of VII and IX that does not require a protein cofactor. Since both VIIa and activated IX are equally plausible as the initiating protease for the extrinsic limb of blood coagulation, it might be appropriate to illustrate this key step of hemostasis as currently being unknown.

scholarly journals Coagulation Factor IX concentrate: method of preparation and assessment of potential in vivo thrombogenicity in animal models

Blood ◽  
1984 ◽  
Vol 64 (6) ◽  
pp. 1220-1227
Author(s):  
D Menache ◽  
HE Behre ◽  
CL Orthner ◽  
H Nunez ◽  
HD Anderson ◽  
...  
Keyword(s):  
Animal Models ◽  
Specific Activity ◽  
Rabbit Model ◽  
Coagulation Factor ◽  
Factor Ix ◽  
Factor Vii ◽  
Factor X ◽  
Clotting Factors ◽  
Coagulation Factor Ix

Thrombosis and/or disseminated intravascular coagulation (DIC) are complications specifically associated with the use of factor IX complex in some patients. Assuming that these complications might result from zymogen overload, we have produced, using diethylaminoethyl (DEAE)- Sephadex (Pharmacia, Piscataway, NJ) and sulfated dextran chromatography, a factor IX concentrate (coagulation factor IX) that is essentially free of prothrombin, factor VII, and factor X. Factor IX specific activity is at least 5 U/mg protein, a 250-fold purification compared to plasma. Amounts of factors II, VII, and X are less than 5 units each per 100 units of factor IX. The concentrate is essentially free of activated clotting factors and contains no added heparin. In the rabbit stasis model, a dose of 200 factor IX U/kg was less thrombogenic than 100 factor IX U/kg of the DEAE-Sephadex eluate from which the concentrate was derived. Infusion of 200 factor IX U/kg did not induce DIC in the nonstasis rabbit model, whereas 100 factor IX U/kg of the DEAE-Sephadex eluate resulted in DIC in this model. Several factor IX lots were found to have shortened nonactivated partial thromboplastin times (PTTs), but were nonthrombogenic in both animal models. These data indicate that coagulation factor IX concentrate is less thrombogenic than factor IX complex.

A frequent human coagulation Factor VII mutation (A294V, c152) in loop 140s affects the interaction with activators, tissue factor and substrates

2002 ◽  
Vol 363 (2) ◽  
pp. 411-416 ◽  
Author(s):  
Raffaella TOSO ◽  
Mirko PINOTTI ◽  
Katherine A. HIGH ◽  
Eleanor S. POLLAK ◽  
Francesco BERNARDI
Keyword(s):  
Tissue Factor ◽  
Coagulation Factor ◽  
Catalytic Efficiency ◽  
Fibrin Clot ◽  
Enzymic Activity ◽  
Factor Ix ◽  
Site Directed Mutagenesis ◽  
Factor Vii ◽  
Factor X ◽  
Coagulation Factor Vii

Activated Factor VII (FVIIa) is a vitamin-K-dependent serine protease that initiates blood clotting after interacting with its cofactor tissue factor (TF). The complex FVIIa—TF is responsible for the activation of Factor IX (FIX) and Factor X (FX), leading ultimately to the formation of a stable fibrin clot. Activated FX (FXa), a product of FVIIa enzymic activity, is also the most efficient activator of zymogen FVII. Interactions of FVII/FVIIa with its activators, cofactor and substrates have been investigated extensively to define contact regions and residues involved in the formation of the complexes. Site-directed mutagenesis and inhibition assays led to the identification of sites removed from the FVIIa active site that influence binding specificity and affinity of the enzyme. In this study we report the characterization of a frequent naturally occurring human FVII mutant, A294V (residue 152 in the chymotrypsin numbering system), located in loop 140s. This region undergoes major rearrangements after FVII activation and is relevant to the development of substrate specificity. FVII A294V shows delayed activation by FXa as well as reduced activity towards peptidyl and macromolecular substrates without impairing the catalytic efficiency of the triad. Also, the interaction of this FVII variant with TF was altered, suggesting that this residue, and more likely loop 140s, plays a pivotal role not only in the recognition of FX by the FVIIa—TF complex, but also in the interaction of FVII with both its activators and cofactor TF.

scholarly journals Coagulation Factor IX concentrate: method of preparation and assessment of potential in vivo thrombogenicity in animal models

Blood ◽  
1984 ◽  
Vol 64 (6) ◽  
pp. 1220-1227 ◽  
Author(s):  
D Menache ◽  
HE Behre ◽  
CL Orthner ◽  
H Nunez ◽  
HD Anderson ◽  
...  
Keyword(s):  
Animal Models ◽  
Specific Activity ◽  
Rabbit Model ◽  
Coagulation Factor ◽  
Factor Ix ◽  
Factor Vii ◽  
Factor X ◽  
Clotting Factors ◽  
Coagulation Factor Ix

Abstract Thrombosis and/or disseminated intravascular coagulation (DIC) are complications specifically associated with the use of factor IX complex in some patients. Assuming that these complications might result from zymogen overload, we have produced, using diethylaminoethyl (DEAE)- Sephadex (Pharmacia, Piscataway, NJ) and sulfated dextran chromatography, a factor IX concentrate (coagulation factor IX) that is essentially free of prothrombin, factor VII, and factor X. Factor IX specific activity is at least 5 U/mg protein, a 250-fold purification compared to plasma. Amounts of factors II, VII, and X are less than 5 units each per 100 units of factor IX. The concentrate is essentially free of activated clotting factors and contains no added heparin. In the rabbit stasis model, a dose of 200 factor IX U/kg was less thrombogenic than 100 factor IX U/kg of the DEAE-Sephadex eluate from which the concentrate was derived. Infusion of 200 factor IX U/kg did not induce DIC in the nonstasis rabbit model, whereas 100 factor IX U/kg of the DEAE-Sephadex eluate resulted in DIC in this model. Several factor IX lots were found to have shortened nonactivated partial thromboplastin times (PTTs), but were nonthrombogenic in both animal models. These data indicate that coagulation factor IX concentrate is less thrombogenic than factor IX complex.

scholarly journals Coagulation Factor IX for Hemophilia B Therapy

Acta Naturae ◽  
2012 ◽  
Vol 4 (2) ◽  
pp. 62-73
Author(s):  
N. A. Orlova ◽  
S. V. Kovnir ◽  
I. I. Vorobiev ◽  
A. G. Gabibov
Keyword(s):  
Gene Therapy ◽  
Recombinant Protein ◽  
Coagulation Factor ◽  
Factor Ix ◽  
Hemophilia B ◽  
Coagulation Cascade ◽  
Transgenic Organisms ◽  
Current State ◽  
Coagulation Factor Ix ◽  
Functional Factor

Factor IX is a zymogen enzyme of the blood coagulation cascade. Inherited absence or deficit of the IX functional factor causes bleeding disorder hemophilia B, which requires constant protein replacement therapy. Reviewed herein are the current state in the manufacturing of FIX, improved variants of the recombinant protein for therapy, transgenic organisms for obtaining FIX, and the advances in the gene therapy of hemophilia B.

Purification and Properties of an Abnormal Blood Coagulation Factor IX (Factor IXBm)/Kinetics of Its Inhibition of Factor X Activation by Factor VII and Bovine Tissue Factor

1981 ◽  
Vol 45 (01) ◽  
pp. 055-059 ◽  
Author(s):  
B Østerud ◽  
C K Kasper ◽  
K K Lavine ◽  
C Prodanos ◽  
S I Rapaport
Keyword(s):  
Tissue Factor ◽  
Blood Coagulation ◽  
Limited Proteolysis ◽  
Coagulation Factor ◽  
Factor Ix ◽  
Factor Vii ◽  
Factor X ◽  
Factor Xi ◽  
Coagulation Factor Ix ◽  
Bovine Tissue

SummaryAn abnormal blood coagulation factor IX has been isolated from the blood of a hemophilia B patient with a variant of the disease (hemophilia Bm) characterized by a normal concentration of factor IX antigen, negligible factor IX coagulant activity, and a prolonged prothrombin time with bovine tissue factor. The isolated protein (factor IXBm) had the same apparent molecular weight as normal factor IX (55,000) and the same mobility on two dimensional immunoelectrophoresis as normal factor IX. Factor IXBm underwent limited proteolysis induced by activated factor XI, in the presence of Ca2+ ions, or induced by the reaction product of tissue factor, factor VII and Ca2+ ions. A timecourse study showed that activated factor XI cleaved factor IXBm and factor IX at similar rates. However, in contrast to normal factor IX, the limited protelysis of factor IXBm did not generate procoagulant activity.In kinetic experiments purified factor IXBm behaved like a competitive inhibitor (Ki of 0.017 μM) of the activation of factor X by bovine tissue factor and factor VII. Normal factor IX was also found to inhibit the reaction but required a four-fold higher concentration to achieve the same inhibitory effects as factor IXBm.

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