Characterization of Recombinant Human Coagulation Factor XFriuli

1996 ◽  
Vol 75 (02) ◽  
pp. 313-317 ◽  
Author(s):  
D J Kim ◽  
A Girolami ◽  
H L James
Keyword(s):  
Catalytic Domain ◽  
Coagulation Factor ◽  
Molecular Size ◽  
Binding Pocket ◽  
Site Directed Mutagenesis ◽  
Bleeding Tendency ◽  
Factor X ◽  
Amino Terminal ◽  
Normal Plasma ◽  
Plasma Factor

SummaryNaturally occurring plasma factor XFriuli (pFXFr) is marginally activated by both the extrinsic and intrinsic coagulation pathways and has impaired catalytic potential. These studies were initiated to obtain confirmation that this molecule is multi-functionally defective due to the substitution of Ser for Pro at position 343 in the catalytic domain. By the Nelson-Long site-directed mutagenesis procedure a construct of cDNA in pRc/CMV was derived for recombinant factor XFriuli (rFXFr) produced in human embryonic (293) kidney cells. The rFXFr was purified and shown to have a molecular size identical to that of normal plasma factor X (pFX) by gel electrophoretic, and amino-terminal sequencing revealed normal processing cleavages. Using recombinant normal plasma factor X (rFXN) as a reference, the post-translational y-carboxy-glutamic acid (Gla) and (β-hydroxy aspartic acid (β-OH-Asp) content of rFXFr was over 85% and close to 100%, respectively, of expected levels. The specific activities of rFXFr in activation and catalytic assays were the same as those of pFXFr. Molecular modeling suggested the involvement of a new H-bond between the side-chains of Ser-343 and Thr-318 as they occur in anti-parallel (3-pleated sheets near the substrate-binding pocket of pFXFr. These results support the conclusion that the observed mutation in pFXFr is responsible for its dysfunctional activation and catalytic potentials, and that it accounts for the moderate bleeding tendency in the homozygous individuals who possess this variant procoagulant.

Heparin and Low Molecular Weight Heparins Inhibit Prothrombinase Formation but not its Activity in Plasma

1994 ◽  
Vol 72 (06) ◽  
pp. 862-868 ◽  
Author(s):  
Frederick A Ofosu ◽  
J C Lormeau ◽  
Sharon Craven ◽  
Lori Dewar ◽  
Noorildan Anvari
Keyword(s):  
Factor Xa ◽  
Catalytic Efficiency ◽  
Thrombin Inhibitor ◽  
Lag Phase ◽  
Factor V ◽  
Factor X ◽  
Normal Plasma ◽  
Plasma Factor ◽  
Prothrombin Activation ◽  
Plasma Heparin

SummaryFactor V activation is a critical step preceding prothrombinase formation. This study determined the contributions of factor Xa and thrombin, which activate purified factor V with similar catalytic efficiency, to plasma factor V activation during coagulation. Prothrombin activation began without a lag phase after a suspension of coagulant phospholipids, CaCl2, and factor Xa was added to factor X-depleted plasma. Hirudin, a potent thrombin inhibitor, abrogated prothrombin activation initiated with 0.5 and 1.0 nM factor Xa, but not with 5 nM factor Xa. In contrast, hirudin did not abrogate prothrombin activation in plasmas pre-incubated with 0.5,1.0 or 5 nM α-thrombin for 10 s followed by the coagulant suspension containing 0.5 nM factor Xa. Thus, thrombin activates plasma factor V more efficiently than factor Xa. At concentrations which doubled the clotting time of contact-activated normal plasma, heparin and three low Mr heparins also abrogated prothrombin activation initiated with 0.5 nM factor Xa, but not with 5 nM factor Xa. If factor V in the factor X-depleted plasma was activated (by pre-incubation with 10 nM a-thrombin for 60 s) before adding 0.5,1.0, or 5 nM factor Xa, neither hirudin nor the heparins altered the rates of prothrombin activation. Thus, none of the five anticoagulants inactivates prothrombinase. When 5 or 10 pM relipidated r-human tissue factor and CaCl2 were added to normal plasma, heparin and the three low Mr heparins delayed the onset of prothrombin activation until the concentration of factor Xa generated exceeded 1 nM, and they subsequently inhibited prothrombin activation to the same extent. Thus, hirudin, heparin and low Mr heparins suppress prothrombin activation solely by inhibiting prothrombinase formation.

scholarly journals Proteolytic inactivation of blood coagulation factor IX by thrombin

Blood ◽  
1985 ◽  
Vol 66 (6) ◽  
pp. 1302-1308 ◽  
Author(s):  
W Kisiel ◽  
KJ Smith ◽  
BA McMullen
Keyword(s):  
Human Factor ◽  
Coagulation Factor ◽  
Factor Ix ◽  
Light Chains ◽  
Factor X ◽  
Amino Terminal ◽  
Coagulation Factor Ix ◽  
Human Factor Ix ◽  
Coagulant Activity

Coagulation factor IX is a vitamin K-dependent glycoprotein that circulates in blood as a precursor of a serine protease. Incubation of human factor IX with human alpha-thrombin resulted in a time and enzyme concentration-dependent cleavage of factor IX yielding a molecule composed of a heavy chain (mol wt 50,000) and a doublet light chain (mol wt 10,000). The proteolysis of factor IX by thrombin was significantly inhibited by physiological levels of calcium ions. Under nondenaturing conditions, the heavy and light chains of thrombin- cleaved factor IX remained strongly associated, but these chains were readily separated by gel filtration in the presence of denaturants. Amino-terminal sequence analyses of the isolated heavy and light chains of thrombin-cleaved human factor IX indicated that thrombin cleaved peptide bonds at Arg327-Val328 and Arg338-Ser339 in this molecule. Comparable cleavages were observed in bovine factor IX by bovine thrombin and occurred at Arg319-Ser320 and Arg339-Ser340. Essentially, a complete loss of factor IX procoagulant activity was associated with its cleavage by thrombin. Furthermore, thrombin-cleaved factor IX neither developed coagulant activity after treatment with factor XIa nor inhibited the coagulant activity of native factor IX. These data indicate that thrombin cleaves factor IX near its active site serine residue, rendering it incapable of activating factor X. Whether or not this reaction occurs in vivo is unknown.

A hitherto undescribed plasma factor acting at the contact phase of blood coagulation (Flaujeac factor): case report and coagulation studies

Blood ◽  
1975 ◽  
Vol 46 (5) ◽  
pp. 761-768 ◽  
Author(s):  
MJ Lacombe ◽  
B Varet ◽  
JP Levy
Keyword(s):  
Blood Coagulation ◽  
Coagulation Factor ◽  
Old French ◽  
Bleeding Tendency ◽  
Contact Phase ◽  
Hageman Factor ◽  
Coagulation Defect ◽  
Plasma Factor ◽  
Fletcher Factor

Abstract This paper reports an asymptomatic coagulation defect responsible for an abnormality at the contact phase of blood coagulation in vitro, distinct from Hageman factor and Fletcher factor deficiencies. Coagulation studies in a 50-yr-old French woman without bleeding tendency revealed the following results: whole-blood clotting time in glass tubes and activated partial thromboplastin time with kaolin and ellagic acid were greatly prolonged; one-stage prothrombin was normal; no circulating anticoagulant was detected, and the infusion of normal plasma corrected the coagulation defect with an estimated half-life of 6.5 days; the levels of factor VIII, IX, XI, and XII were normal; mutual correction was obtained with a Fletcher factor-deficient plasma; the level of whole complement was normal. Studies of the contact phase of blood coagulation and contact-induced fibrinolysis showed the same abnormalities as in Hageman factor- and Fletcher-deficient plasmas. These results indicate that the patient's plasma is deficient in a previously undescribed coagulation factor, which participates in the initial stage of the blood coagulation process in vitro. Family studies revealed consanguinity in the propositus' parents. The assay of this newly described factor in the propositus' children revealed a partial defect, compatible with a heterozygous state, in three of the four tested children. This indicates a recessive inheritance of this new blood coagulation defect.

scholarly journals Modulation of erbB kinase activity and oncogenic potential by single point mutations in the glycine loop of the catalytic domain.

1994 ◽  
Vol 14 (10) ◽  
pp. 6868-6878 ◽  
Author(s):  
H K Shu ◽  
C M Chang ◽  
L Ravi ◽  
L Ling ◽  
C M Castellano ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Conformational Changes ◽  
Kinase Activity ◽  
Catalytic Domain ◽  
Single Point ◽  
Point Mutations ◽  
Full Length ◽  
Site Directed Mutagenesis ◽  
Tyrosine Kinase Domain ◽  
Amino Terminal

Avian c-erbB is activated to a leukemia oncogene following truncation of its amino-terminal ligand-binding domain by retroviral insertion. The insertionally activated transcripts encode protein products which have constitutive tyrosine kinase activity and can induce erythroleukemia but not sarcomas. We have previously found that a valine-to-isoleucine point mutation at position 157 (V157I mutant) within the tyrosine kinase domain of this truncated erbB can dramatically activate the sarcomagenic potential of the oncogene and increase the kinase activity of this oncoprotein. This mutation lies at position 157 of the insertionally activated c-erbB product, affecting a highly conserved valine residue of the glycine loop involved in ATP binding and phosphate transfer. To investigate the functional importance of this residue in the catalytic activity of kinases, we have introduced at this position, by site-directed mutagenesis, codons representing the remaining 18 amino acid residues. Most of the mutants have diminished activity, with six of them completely devoid of kinase activity, indicating the sensitivity of this region to conformational changes. Some of these mutants displayed increased kinase activity and greater transforming potential in comparison with IA c-erbB, but none had levels as high as those of the V157I mutant. In general, the sarcomagenic potential of the various erbB mutants correlated with their autophosphorylation state and their ability to cause phosphorylation of MAP kinase. However, there are important exceptions such as the V157G mutant, which lacks enhanced autophosphorylation but is highly sarcomagenic. Studies of this and other autophosphorylation site mutants point to the existence of an autophosphorylation-independent pathway in sarcomagenesis. The requirement for leukemogenic potential is much less stringent and correlates with positivity of kinase activity. When the valine-to-isoleucine substitution was put in context of the full-length erbB protein, the mutation relaxed the ligand dependence and had a positive effect on the transforming potential of the full-length c-erbB.

scholarly journals Regions 301–303 and 333–339 in the catalytic domain of blood coagulation Factor IX are Factor VIII-interactive sites involved in stimulation of enzyme activity

1999 ◽  
Vol 339 (2) ◽  
pp. 217-221 ◽  
Author(s):  
Joost A. KOLKMAN ◽  
Peter J. LENTING ◽  
Koen MERTENS
Keyword(s):  
Catalytic Domain ◽  
Coagulation Factor ◽  
Factor Ix ◽  
Factor X ◽  
Factor Ixa ◽  
Factor Viiia ◽  
Α Helix ◽  
Factor X Activation ◽  
Affinity Interaction ◽  
Stimulation Of

The contribution of the Factor IX catalytic domain to Factor VIIIa binding has been evaluated by functional analysis of Factor IX variants with substitutions in α-helix region 333–339 and region 301–303. These regions were found to play a prominent role in Factor VIIIa-dependent stimulation of Factor X activation, but do not contribute to the high-affinity interaction with Factor VIIIa light chain. We propose that complex assembly between Factor IXa and Factor VIIIa involves multiple interactive sites that are located on different domains of these proteins.

The Disulfide Bond between Cys22 and Cys27 in the Protease Domain Modulate Clotting Activity of Coagulation Factor X

2019 ◽  
Vol 119 (06) ◽  
pp. 871-881 ◽  
Author(s):  
Fang Li ◽  
Changming Chen ◽  
Si-Ying Qu ◽  
Ming-Zhu Zhao ◽  
Xiaoling Xie ◽  
...  
Keyword(s):  
Disulfide Bond ◽  
Catalytic Domain ◽  
Coagulation Factor ◽  
Major Change ◽  
Coagulation Factors ◽  
Hek293 Cells ◽  
Factor X ◽  
Wild Type ◽  
Protease Domain ◽  
Dynamic Simulations

AbstractThe Cys22-Cys27 disulfide bond of factor X (FX) protease domain is not conserved among coagulation factors and its contribution to the physiological haemostasis and implication in the pathogenesis of haemostatic and thrombotic disorders remain to be elucidated. Mutation p.Cys27Ser was identified in a pedigree of congenital FX deficiency and fluorescence labelling study of transiently transfected HEK293 cells showed accumulation of FX p.Cys27Ser within cell, indicating incompetent secretion partially responsible for the FX deficiency. The clotting activity of FX p.Cys27Ser was decreased to about 90% of wild-type, while amidolytic and pro-thrombinase activities (kcat/Km) determined with recombinant FXa mutant were 1.33- and 4.77-fold lower. Molecular dynamic simulations revealed no major change in global structure between FXa p.Cys27Ser and wild-type FXa; however, without the Cys22-Cys27 disulfide bond, the insertion of newly formed N terminal of catalytic domain after the activation cleavage is hindered, perturbing the conformation transition from zymogen to enzyme. The crystal structure of FXa shows that this disulfide bond is solvent accessible, indicating that its stability might be subject to the oxidation/reduction balance. As demonstrated with FX p.Cys27Ser here, Cys22-Cys27 disulfide bond may modulate FX clotting activity, with reduced FX pertaining less pro-coagulant activity.

scholarly journals A Study of Lymphocyte Transformation as a Mechanism for Spontaneous Factor VIII Inhibitor Formation

1977 ◽  
Author(s):  
J. Katz ◽  
R. Lea ◽  
E.D. Gomperts
Keyword(s):  
Factor Viii ◽  
Mononuclear Cells ◽  
Lymphocyte Transformation ◽  
Factor Viii Inhibitor ◽  
Bleeding Tendency ◽  
Normal Plasma ◽  
Plasma Factor ◽  
Lymphocyte Cultures ◽  
Viii Inhibitor

Two elderly patients with a bleeding tendency due to spontaneous Factor VIII inhibition were investigated. An in vitro study was designed to investigate lymphocyte transformation and the production of Factor VIII inhibitor. Blood samples from patients were separated on a ficoll-hypaque gradient and the mononuclear cells were cultured with haemophiliac plasma. Factor VIII concentrate and normal plasma as sources of Factor VIII antigen. The results showed that the patients lymphocytes were stimulated by some samples of haemophiliac plasma, but not by the Factor VIII concentrates or normal plasma. However, normal control lymphocytes were similarly stimulated by these samples of haemophiliac plasma suggesting that immune complexes were present in the haemophiliac plasma and probably caused the stimulation. Furthermore, the patients lymphocytes were stimulated with phytohaemagglutinin candida and tuberculin and normal responses were obtained with AB serum. However, the sera of both patients inhibited candida and tuberculin stimulated lymphocyte cultures and the inhibition correlated with the presence of Factor VIII inhibitor. In this study a cellular immune mechanism for the production of spontaneous Factor VIII inhibitor could not be demonstrated, however a serum inhibitor of candida and tuberculin stimulated lymphocyte cultures was found.

scholarly journals Processivity of dextransucrases synthesizing very-high-molar-mass dextran is mediated by sugar-binding pockets in domain V

2020 ◽  
Vol 295 (17) ◽  
pp. 5602-5613 ◽  
Author(s):  
Marion Claverie ◽  
Gianluca Cioci ◽  
Marlène Vuillemin ◽  
Pauline Bondy ◽  
Magali Remaud-Simeon ◽  
...  
Keyword(s):  
Molar Mass ◽  
Catalytic Domain ◽  
Binding Pocket ◽  
Site Directed Mutagenesis ◽  
High Molar Mass ◽  
Double Mutation ◽  
Sugar Binding ◽  
Binding Pockets ◽  
Domain V ◽  
Very High

The dextransucrase DSR-OK from the Gram-positive bacterium Oenococcus kitaharae DSM17330 produces a dextran of the highest molar mass reported to date (∼109 g/mol). In this study, we selected a recombinant form, DSR-OKΔ1, to identify molecular determinants involved in the sugar polymerization mechanism and that confer its ability to produce a very-high-molar-mass polymer. In domain V of DSR-OK, we identified seven putative sugar-binding pockets characteristic of glycoside hydrolase 70 (GH70) glucansucrases that are known to be involved in glucan binding. We investigated their role in polymer synthesis through several approaches, including monitoring of dextran synthesis, affinity assays, sugar binding pocket deletions, site-directed mutagenesis, and construction of chimeric enzymes. Substitution of only two stacking aromatic residues in two consecutive sugar-binding pockets (variant DSR-OKΔ1-Y1162A-F1228A) induced quasi-complete loss of very-high-molar-mass dextran synthesis, resulting in production of only 10–13 kg/mol polymers. Moreover, the double mutation completely switched the semiprocessive mode of DSR-OKΔ1 toward a distributive one, highlighting the strong influence of these pockets on enzyme processivity. Finally, the position of each pocket relative to the active site also appeared to be important for polymer elongation. We propose that sugar-binding pockets spatially closer to the catalytic domain play a major role in the control of processivity. A deep structural characterization, if possible with large-molar-mass sugar ligands, would allow confirming this hypothesis.

A Review of Coagulation Abnormalities of Autoimmune Acquired Factor V Deficiency with a Focus on Japan

2021 ◽  
Author(s):  
Akitada Ichinose ◽  
Tsukasa Osaki ◽  
Masayoshi Souri
Keyword(s):  
Coagulation Factor ◽  
Nationwide Survey ◽  
Autoimmune Disorder ◽  
Antibody Detection ◽  
Extensive Literature ◽  
Factor V ◽  
Bleeding Tendency ◽  
Factor X ◽  
Timely Initiation ◽  
Fv Antibody

AbstractCoagulation factor V (or FV for the purpose of medical safety) is an essential cofactor of coagulation factor X in the common pathway of coagulation; severe FV deficiency leads to a bleeding tendency. Although both congenital and acquired FV deficiencies are widely recognized, FV deficiency also presents as an autoimmune disorder. A nationwide survey on autoimmune coagulation factor deficiencies (AiCFDs) conducted in Japan by our Japanese Collaborative Research Group identified 24 new patients with autoimmune FV deficiency (AiFVD) in the past 5 years. Furthermore, our extensive literature search confirmed that 177 AiFVD cases have been reported in previous articles published from Japan. Patients with AiFVD in Japan were predominantly men, with age similar to those with other AiCFDs. AiFVD was confirmed as a relatively mild type of bleeding diathesis, associated with lower mortality rate than that for AiFVD and other AiCFDs reported in previous studies. Patients with AiFVD had variable FV inhibitor titers and both neutralizing anti-FV autoantibodies and nonneutralizing counterparts. Although spontaneous resolution occurs in some patients, timely initiation of hemostatic and immunosuppressive therapies helps arrest the bleeding and eliminate anti-FV antibodies, resulting in a high cumulative recovery rate. Immunological anti-FV antibody detection is recommended to avoid missing AiFVD cases for the presence of nonneutralizing anti-FV autoantibodies. Further investigation is necessary to clarify the long-term prognosis and optimal management of AiFVD.

Murine Model of Suppressed Intrinsic Pathway Using a FX Variant: FX Roma.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 728-728
Author(s):  
Paris Margaritis ◽  
Shing Jen Tai ◽  
Rodney M. Camire ◽  
Danielle Dunn ◽  
Katherine A. High
Keyword(s):  
Factor Viia ◽  
Es Cells ◽  
Site Directed Mutagenesis ◽  
Intrinsic Activity ◽  
Bleeding Tendency ◽  
Factor X ◽  
Intrinsic Pathway ◽  
Hprt Gene ◽  
Extrinsic Pathway ◽  
Coagulation Testing

Abstract The complex interplay between the intrinsic and extrinsic pathways of coagulation is incompletely understood. The existence of Factor X variants that can be asymmetrically activated through one but not both of these pathways affords one strategy for analyzing the relationship of the two pathways. The Factor X Roma (FXRoma) variant, originally described in a patient with mild bleeding tendency (severe following trauma, De Stefano et al., Br J Haematol.1988, 69(3):387–91), is due to a missense mutation (Thr318→Met) in exon 8. Coagulation testing revealed markedly decreased activity (1–3%) in the intrinsic pathway as measured by aPTT, but substantially higher activity (30–50%) in the extrinsic pathway as measured by PT. Using site-directed mutagenesis and transient transfection in HEK 293 cells, we confirmed this differential activity in extrinsic and intrinsic pathways for a variant human Factor X (FX) recombinant protein with a Thr318→Met change. FX antigen was assessed by ELISA as previously described (Larson PJ et al., Biochemistry.1998, 37(14):5029–38) and FX activity by PT or aPTT; analysis of conditioned medium from FX wild-type and FX Thr318→Met showed respectively: antigen 100%, 82%; intrinsic activity 120 U/mg (100%), 1.8 U/mg (1.5%); extrinsic activity 190 U/mg (100%), 81 U/mg (41%). To further study this variant, we constructed a mouse homozygous for the analogous mutation (Thr318→Met) in the murine FX gene. This residue is conserved in human, murine and canine FX. We utilized mouse ES cells in which exon 8 of the murine FX gene had previously been deleted and replaced with a neomycin resistance gene and a partial HPRT gene through a targeted recombination event. These cells were used for a second electroporation event with a targeting construct carrying an exon 8 cassette containing the Thr318→Met substitution, and the missing portion of the HPRT gene. After selection on HAT medium, correctly targeted ES cells were micro-injected into blastocysts and implanted into pseudopregnant mice to generate chimeric mice. Heterozygous offspring of chimeric males were intercrossed to obtain animals homozygous for the FXRoma mutation. Coagulation testing revealed that FXRoma heterozygotes showed the expected 52.1±5.9% intrinsic activity and 142.8±25.1% extrinsic activity. More importantly, FXRoma homozygotes showed 8.5±2.7% intrinsic activity and 107.7±41.1% extrinsic activity. Thus the murine FXRoma mutation demonstrates a coagulation profile similar to that observed with the human variant. Thus the FXRoma mouse can model the effect of impeded intrinsic pathway more accurately than e.g. a FIX knockout mouse, because both FX and FIX can act as substrates for the Factor VIIa-Tissue Factor complex and all the components of the Xase complex will be essentially at normal or near-normal levels. The ability to carry out studies of long-term effects of an impeded pathway, and to analyze the response of these animals to prothrombotic stimuli, will allow us to determine the safety and efficacy of therapeutic approaches based on impeding the intrinsic pathway.

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