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At least 73 records · Page 4Linked to original sources
PubMed2002

Ixolaris, a novel recombinant tissue factor pathway inhibitor (TFPI) from the salivary gland of the tick, Ixodes scapularis: identification of factor X and factor Xa as scaffolds for the inhibition of factor VIIa/tissue factor complex.

Saliva of the hard tick and Lyme disease vector, Ixodes scapularis, has a repertoire of compounds that counteract host defenses. Following sequencing of an I scapularis salivary gland complementary DNA (cDNA) library, a clone with sequence homology to tissue factor pathway inhibitor (TFPI) was identified. This cDNA codes for a mature protein, herein called Ixolaris, with 140 amino acids containing 10 cysteines and 2 Kunitz-like domains. Recombinant Ixolaris was expressed in insect cells and shown to inhibit factor VIIa (FVIIa)/tissue factor (TF)-induced factor X (FX) activation with an inhibitory concentration of 50% (IC(50)) in the picomolar range. In nondenaturing gel, Ixolaris interacted stoichiometrically with FX and FXa but not FVIIa. Ixolaris behaves as a fast-and-tight ligand of the exosites of FXa and gamma-carboxyglutamic acid domainless FXa (des-Gla-FXa), increasing its amidolytic activity. At high concentration, Ixolaris attenuates the amidolytic activity of FVIIa/TF; however, in the presence of DEGR-FX or DEGR-FXa (but not des-Gla-DEGR-FXa), Ixolaris becomes a tight inhibitor of FVIIa/TF as assessed by recombinant factor IX (BeneFIX) activation assays. This indicates that FX and FXa are scaffolds for Ixolaris in the inhibition of FVIIa/TF and implies that the Gla domain is necessary for FVIIa/TF/Ixolaris/FX(a) complex formation. Additionally, we show that Ixolaris blocks FXa generation by endothelial cells expressing TF. Ixolaris may be a useful tool to study the structural features of FVIIa, FX, and FXa, and an alternative anticoagulant in cardiovascular diseases.

Amino Acid Sequence↗
PubMed1988

Factor X Roma: a congenital factor X variant defective at different degrees in the intrinsic and the extrinsic activation.

A factor X molecular variant was identified in a 13-year-old girl affected by a bleeding tendency. Factor X antigen levels and activation by Russel's viper venom (tested both by clotting and amidolytic assays) were normal. Factor X crossed immunoelectrophoresis was found to be identical to that of the control plasma. Factor X functional activity was low (3% of the normal) if tested by PTT-derived assays, whereas it was found at intermediate levels (about 30-50% of the normal) if measured by prothrombin time-derived assays. The defect in the extrinsic activation was more clearly disclosed using as activating agent thromboplastin from ox brain. The factor X of the patient was completely adsorbed by aluminum hydroxide. The parents of the propositus (first degree cousins) showed factor X functional levels compatible with a condition of heterozygosity for the abnormality. This factor X molecular variant appears different from the other ones so far described and was named 'Factor X Roma'.

Adolescent↗
PubMed1992

Neutralization of factor X activity by factor X-specific monoclonal antibodies.

Factor X, a vitamin K-dependent protein, is the plasma zymogen for the active serine protease factor Xa. Factor Xa is the proteolytic enzyme for prothrombinase, the multi-protein membrane complex that catalyses the cleavage of prothrombin to thrombin. A panel of 10 monoclonal antibodies (identified by their corresponding clone numbers: 1, 2, 3, 5, 7, 26, 27, 54, 73, and 79) to factor X were produced by immunizing mice with purified factor X. All of the antibodies bound both human factor X and factor Xa in a solid-phase ELISA and binding of the antibodies was not affected by removal of Ca2+ with EDTA. In immunoblot analysis, antibody alpha HFX-54 bound to the light chain and antibodies alpha HFX-1, -5, -7, and -26 bound to the heavy chain of reduced factor X. Antibodies alpha BFX-2b, alpha HFX-27, -54, and -73 prolonged both the factor X-dependent clotting time and activated partial thromboplastin time (APTT) of normal plasma while antibody alpha HFX-1 only prolonged the APTT. None of the antibodies significantly inhibited factor X activation by purified Russell's viper venom factor X activator. In prothrombin activation assays using purified factor Xa, factor Va, prothrombin, Ca2+ and phospholipid vesicles, seven of the antibodies (alpha HFX-1, -3, -26, -27, -54, -73 and alpha BFX2b) showed some inhibition of thrombin generation ranging from 18 to 60% of the control. The decrease in factor X plasma clotting activity was most likely due to inhibition of factor Xa activity in prothrombinase, although some antibody-dependent inhibition of factor X activation may contribute to the observed inhibition of plasma clotting. Prothrombinase activity on platelets was inhibited in an identical manner by the monoclonal antibodies. When prothrombin was activated in the absence of factor Va, only antibody alpha BFX-2b inhibited activation. Calcium-independent determinants on both the heavy chain (determinants 1 and 26) and light chain (determinant 54) of factor X may play a role in prothrombin activation by prothrombinase. Other epitopes (antibodies alpha HFX-3, -27, -73) appeared to be influenced by association of factor Xa with factor Va. Topographic regions on factor X important for factor X activation and factor Xa function may be identified by the use of these monoclonal antibodies.

Antibodies, Monoclonal↗
PubMed2000

The fungal CPCR1 protein, which binds specifically to beta-lactam biosynthesis genes, is related to human regulatory factor X transcription factors.

Here we report the isolation and characterization of a novel transcription factor from the cephalosporin C-producing fungus Acremonium chrysogenum. We have identified a protein binding site in the promoter of the beta-lactam biosynthesis gene pcbC, located 418 nucleotides upstream of the translational start. Using the yeast one-hybrid system, we succeeded in isolating a cDNA clone encoding a polypeptide, which binds specifically to the pcbC promoter. The polypeptid shows significant sequence homology to human transcription factors of the regulatory factor X (RFX) family and was designated CPCR1. A high degree of CPCR1 binding specificity was observed in in vivo and in vitro experiments using mutated versions of the DNA binding site. The A. chrysogenum RFX protein CPCR1 recognizes an imperfect palindrome, which resembles binding sites of human RFX transcription factors. One- and two-hybrid experiments with truncated versions of CPCR1 showed that the protein forms a DNA binding homodimer. Nondenaturing electrophoresis revealed that the CPCR1 protein exists in vitro solely in a multimeric, probably dimeric, state. Finally, we isolated a homologue of the cpcR1 gene from the penicillin-producing fungus Penicillium chrysogenum and determined about 60% identical amino acid residues in the DNA binding domain of both fungal RFX proteins, which show an overall amino acid sequence identity of 29%.

Acremonium↗
PubMed1998

Factor X Nagoya 1 and Nagoya 2: a CRM- factor X deficiency and a dysfunctional CRM+ factor X deficiency characterized by substitution of Arg306 by Cys and of Gly366 by Ser, respectively.

We have identified, in two unrelated patients, factor X deficiency that we have designated factor X Nagoya 1 and Nagoya 2, respectively. The proband with factor X Nagoya 1 showed factor X activity level of 3% and factor X antigen level < 10% of the normal control value. All the exons and intron/exon junctions of the factor X gene were studied using a strategy combining polymerase chain reaction (PCR) amplification and nonradioactive single-strand conformational polymorphism (SSCP) analysis. Exon 8 containing DNA fragment of the proband with factor X Nagoya 1 showed aberrant migration on SSCP analysis. All exon-containing DNA fragments amplified by PCR were sequenced, and we identified a C-to-T substitution in exon 8 in the human factor X gene of the proband, which results in the replacement of Arg306 by Cys. This genetic defect has been transmitted from her father, and her sister also carried the same mutation; both showed almost half the normal levels of both factor X activity and antigen. The coordinates of human factor Xa indicated that Arg306 in the catalytic domain is positioned at the beginning of the alpha-helix near the second EFG-like domain. The substitution for Arg of Cys has been supposed to cause the destruction of local alpha-helix formation, possibly leading to the secretion problem. The proband with dysfunctional factor X Nagoya 2 was characterized by factor X activity level of 34% with normal factor X antigen level of 80%. We identified one substitution of G for A in exon 8 in the human factor X gene of the proband, which results in the replacement of Gly366 by Ser. As the Gly366 is positioned at the primary substrate binding pocket. the replacement of Gly with Ser would cause a defect of substrate binding, leading to the loss of enzymatic activity.

Adult↗
PubMed2002

Gene targeting of tissue factor, factor X, and factor VII in mice: their involvement in embryonic development.

Inactivation of specific genes in mammals by gene targeting has accelerated our ability to determine gene function. Nearly all genes involved in the blood coagulation system have been knocked out in mice. Tissue factor (TF) is the main initiator of the coagulation system and functions as a cell surface receptor for coagulation factor VII (FVII). Knockout studies have shown that TF deficiency results in lethality around embryonic day (E) 8.5-10.5. The results suggest a role for TF in embryonic blood vessel development and maintenance of vascular integrity in the yolk sac. In addition, TF may be involved in the maintenance of the placental labyrinth. Factor X (FX) deficiency causes partial embryonic lethality between E11.5-12.5. FX-/- mice that were born died from fatal neonatal bleeding. In contrast, FVII deficiency is not embryonic lethal, but FVII-/- neonates died from hemorrhage within the first days after birth. The various lethal phenotypes of deficiencies of the different coagulation factors suggest involvement in processes beyond hemostasis. Both TF/FVIIa and FXa can trigger intracellular signaling events in certain cell types. Signaling by coagulation proteases and protease-activated receptors (PARs) may have important roles in embryonic development.

Animals↗
PubMed1996

Activation of factor X by factor VIIa complexed with human-mouse tissue factor chimeras requires human exon 3.

In an attempt to define sequence elements in human and mouse tissue factor (TF) that are responsible for the species specificity observed in their interaction with human factor VIIa (HVIIa), we constructed human-mouse chimeric TF cDNAs, inserted them into plasmid vectors, and induced their expression in E. coli. Assays for procoagulant activity were carried out with the resulting E. coli lysates using (HVIIa) human and mouse (MVIIa). The ratio of the procoagulant activities, HVIIa/MVIIa, revealed that human TF exon 3 was essential for activity when the TF:VIIa complex was formed with HVIIa. By ligating the maltose binding protein (MBP) gene to TF cDNAs it was possible to construct, express and purify MBP-TF chimeras as well as to estimate their specific activities. With selected MBP-TF chimeras and HVIIa we determined kinetic parameters for the activation of human factor X. Replacement of exon 3 in human TF cDNA with the corresponding exon from mouse TF cDNA resulted in both lower affinity for HVIIa and failure to convert bound HVIIa into a potent protease.

Amino Acid Sequence↗
PubMed1994

Human monocytes support factor X activation by factor VIIa, independent of tissue factor: implications for the therapeutic mechanism of high-dose factor VIIa in hemophilia.

High doses of recombinant factor VIIa are useful in managing bleeding in hemophiliacs with inhibitors. Whether this therapeutic effect of factor VIIa is dependent on tissue factor (TF) is a matter of debate. We examined the ability of freshly isolated human monocytes (which lack TF) to support the activation of coagulation-factor X by factor VIIa. The rate of factor-X activation by factor VIIa was accelerated in the presence of monocytes compared with the rate of X activation in solution. This activation of factor X on monocytes was saturable with a K1/2 of about 400 to 600 pmol/L factor VIIa. The rate of activation was not inhibited by an excess of inhibitory anti-TF antibody or a Gla-containing fragment of prothrombin. In contrast to monocytes, an endothelial cell line did not support activation of factor X by factor VIIa. Our findings suggest that at least one cell type can accelerate activation for factor X by factor VIIa in the absence of TF. This activity requires higher concentrations of factor VIIa than does the TF mechanism. The concentrations of VIIa required are of a similar order of magnitude to those required for a therapeutic effect of VIIa in bleeding hemophiliacs with inhibitors.

Cells, Cultured↗
PubMed1984

The contribution of Ca2+ and phospholipids to the activation of human blood-coagulation Factor X by activated Factor IX.

The role of the cofactors Ca2+ and phospholipid in the activation of human Factor X by Factor IXa was investigated. By use of a sensitive spectrophotometric Factor Xa assay, it was demonstrated that human Factor IXa can activate Factor X in the absence of cofactors. The presence of Ca2+ as the only cofactor resulted in a 7-fold stimulation of the Factor Xa formation. Kinetic analysis of the Ca2+-stimulated reaction showed that the apparent Km of Factor X was 4.6 microM, whereas the apparent Vmax. for Factor Xa formation was 0.0088 mol of Xa/min per mol of IXa. The presence of phospholipid as the only cofactor had no effect on the rate of Factor Xa formation. However, a several-hundred-fold stimulation was observed when Ca2+ and phospholipid were present in combination. The activation of Factor X in the presence of Ca2+ and phospholipid was found to be kinetically heterogeneous, involving both phospholipid-bound and free reactants. Quantitative data concerning the phospholipid binding of Factors IXa and X were used to study the relation between the rate of Factor Xa formation and the binding of enzyme and substrate to the phospholipid membrane. The results support the hypothesis that phospholipid-bound Factor X is the substrate in the phospholipid-stimulated reaction; however, phospholipid-bound and free Factor IXa seem to be equally efficient in catalysing the activation of phospholipid-bound Factor X.

Calcium↗
PubMed1985

Factor X Padua: a 'new' congenital factor X abnormality with a defect only in the extrinsic system.

A family with a new factor X defect is reported. The proposita is a 56-year-old female. She is asymptomatic and no consanguinity is present between the parents. The main features of the defect are: prolongation of prothrombin time and derivative tests but normal partial thromboplastin time. Factor X was found to be low (about 25-30% of normal) only if tissue thromboplastins were used in the assay system. Chromogenic substrate S-2222 also yielded decreased factor X levels. However, factor X activity was normal with cephalin and cephalin-RVV mixture. Factor X antigen was normal in three immunological systems (electroimmunoassay, an Elisa method and laser nephelometry). Crossed immunoelectrophoresis and antigen-antibody kinetics recorded in a laser nephelometer failed to show major differences from normal factor X. Both sons of the proposita, the father and other family members showed slightly decreased factor X levels and normal factor X antigen and were considered heterozygous for the abnormality. The toponym factor X Padua is proposed to indicate this peculiar abnormality.

Factor X Deficiency↗
PubMed1991

The gamma-carboxyglutamic acid and epidermal growth factor-like domains of factor X. Effect of isolated domains on prothrombin activation and endothelial cell binding of factor X.

Factor Xa is the enzymatically active constituent of the prothrombinase complex, which catalyzes the conversion of prothrombin to thrombin. We have isolated fragments, from tryptic digests of factor X, that consists of the gamma-carboxyglutamic acid (Gla) region linked to one or two epidermal growth factor (EGF)-like domains. Calcium ion binding measurements indicated that these fragments have a native conformation. The factor X-GlaEGF fragments inhibit factor Xa-induced blood clotting in a manner suggesting that they compete with factor Xa for phospholipid binding sites. The same conclusion was reached when thrombin generation was studied in a system of purified components (factor Xa, factor Va, prothrombin, phospholipid, and Ca2+). There was no evidence for a strong interaction between the EGF-like domains of factor Xa and factor Va in either system. However, experiments in the purified system without phospholipid indicated a direct, albeit weak, interaction between the Gla region of factor Xa and factor Va and between the COOH-terminal EGF-like domain of factor Xa and factor Va. Using domain-specific Fab fragments, we have confirmed that the conformation of the serine protease region alters dramatically upon activation of factor X. Furthermore, we have demonstrated that the conformation of the Gla region is affected by the activation, whereas the EGF-like domains appear to be unaltered. The association constant for factor X binding to endothelial cells was two orders of magnitude lower than that for binding of factor IX to these cells. Binding of the Gla and GlaEGF fragments suggested Gla-mediated binding to phospholipid rather than binding to a specific receptor.

1-Carboxyglutamic Acid↗