Cloning and characterization of the 5' end (exon 1) of the gene encoding human factor X

Enzymatic amplification technique was used to isolate all eight exons and sequences around the splice junctions, putative promoter, and polyadenylation sites of human factor X DNA from a patient with factor X deficiency. Two genetic changes in factor X have been observed in this patient. The patient is most likely a compound heterozygote since there is only 14% activity associated with factor X. A point mutation that resulted in the substitution of cysteine (TGC) for arginine (CGC) at amino acid 366 was found in exon VIII of one allele of the factor X gene. This mutation, which occurs in the catalytic domain, can affect the formation of a disulfide bridge and thus could result in a reduction in factor X activity. Sequencing all the regions revealed a second mutation: a deletion of one nucleotide (TCCT to TCT) in exon VII that would cause a frame shift at amino acid 272 followed by termination. We have also shown that the point mutation in exon VIII creates an ApaL1 restriction site and destroys the HinP1 site. Enzymatic DNA amplification followed by restriction digestion provides a quick, reliable, and sensitive method for carrier detection and antenatal diagnosis in affected kindreds. This is the first characterization of factor X deficiency at the molecular level. We propose to name this mutation Factor XSan Antonio.

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Nucleotide Structure and Characterization of the Murine Gene Encoding Anticoagulant Protein C

Thrombosis and Haemostasis ◽

10.1055/s-0037-1615006 ◽

1998 ◽

Vol 79 (02) ◽

pp. 310-316 ◽

Cited By ~ 6

Author(s):

Louise Jalbert ◽

Elliot Rosen ◽

Ann Lissens ◽

Peter Carmeliet ◽

Désiré Collen ◽

...

Keyword(s):

Protein C ◽

Stop Codon ◽

Transcriptional Start Site ◽

Consensus Sequence ◽

Start Site ◽

Gene Encoding ◽

Exon 2 ◽

Exon 1 ◽

Translation Stop Codon

SummaryThe 15,160 bp murine gene encoding anticoagulation protein C (PC) was cloned and sequenced, including 414 bp upstream of exon 1 and 80 bp downstream of the translation stop codon. Nine exons and eight introns were identified. The first exon was untranslated and contained the major transcriptional start site, the surrounding nucleotide sequence of which matched reasonably well with the consensus eukaryotic Cap element sequence. The translational initiator methio-nine residue was located in exon 2. The other introns were positioned as splices between the major domain units of the protein. The 5’ untranslated region contained two possible CCAAT sequences and GC boxes, but no TATA box was obvious within the optimal range of distances from the transcription start site. The 3’-flanking nucleotides included a probable polyadenylation site (ATTAAA), beginning 80 nucleotides downstream of the translation stop codon, and a downstream consensus sequence (AGTGTTTC) required for the efficient formation of a 3’ terminus of mRNA. Several high probability transcription factor recognition sequences, including proteins that are enriched in, or specific to, the liver, such as C/EBP, C/EBP, HNF1, and HNF3, have been located in the 5’ region of the gene. These results indicate that all elements are present for liver-based transcription of the gene for murine PC.

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Molecular characterization of human factor XSan Antonio

Blood ◽

10.1182/blood.v74.5.1486.1486 ◽

1989 ◽

Vol 74 (5) ◽

pp. 1486-1490 ◽

Cited By ~ 21

Author(s):

SV Reddy ◽

ZQ Zhou ◽

KJ Rao ◽

JP Scott ◽

H Watzke ◽

...

Keyword(s):

Amino Acid ◽

Point Mutation ◽

Human Factor ◽

Catalytic Domain ◽

Dna Amplification ◽

Disulfide Bridge ◽

Factor X ◽

Genetic Changes ◽

Factor X Deficiency

Abstract Enzymatic amplification technique was used to isolate all eight exons and sequences around the splice junctions, putative promoter, and polyadenylation sites of human factor X DNA from a patient with factor X deficiency. Two genetic changes in factor X have been observed in this patient. The patient is most likely a compound heterozygote since there is only 14% activity associated with factor X. A point mutation that resulted in the substitution of cysteine (TGC) for arginine (CGC) at amino acid 366 was found in exon VIII of one allele of the factor X gene. This mutation, which occurs in the catalytic domain, can affect the formation of a disulfide bridge and thus could result in a reduction in factor X activity. Sequencing all the regions revealed a second mutation: a deletion of one nucleotide (TCCT to TCT) in exon VII that would cause a frame shift at amino acid 272 followed by termination. We have also shown that the point mutation in exon VIII creates an ApaL1 restriction site and destroys the HinP1 site. Enzymatic DNA amplification followed by restriction digestion provides a quick, reliable, and sensitive method for carrier detection and antenatal diagnosis in affected kindreds. This is the first characterization of factor X deficiency at the molecular level. We propose to name this mutation Factor XSan Antonio.

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Characterization of the gene encoding the human LW blood group protein in LW+ and LW- phenotypes

Blood ◽

10.1182/blood.v87.7.2962.bloodjournal8772962 ◽

1996 ◽

Vol 87 (7) ◽

pp. 2962-2967 ◽

Cited By ~ 24

Author(s):

P Hermand ◽

PY Le Pennec ◽

P Rouger ◽

JP Cartron ◽

P Bailly

Keyword(s):

Blood Group ◽

Stop Codon ◽

Premature Stop Codon ◽

K562 Cells ◽

Gene Encoding ◽

Potential Binding ◽

Exon 1 ◽

Intercellular Adhesion Molecules ◽

Group Protein

The LW blood group is carried by a 42-kD glycoprotein that belongs to the family of intercellular adhesion molecules. The LW gene is organized into three exons spanning an HindIII fragment of approximately 2.65 kb. The exon/intron architecture correlates to the structural domains of the protein and resembles that of other Ig superfamily members except that the signal peptide and the first Ig- like domain are encoded by the first exon. The 5′UT region (nucleotides -289 to +9) includes potential binding sites for various transcription factors (Ets, CACC, SP1, GATA-1, AP2) and exhibited a significant transcriptional activity after transfection in the erythroleukemic K562 cells. No obvious abnormality of the LW gene, including the 5′UT region, has been detected by sequencing polymerase chain reaction- amplified genomic DNA from RhD+ or RhD- donors and from an Rhnull variant that lacks the Rh and LW proteins on red blood cells. However, a deletion of 10 bp in exon 1 of the LW gene was identified in the genome of an LW (a- b-) individual (Big) deficient for LW antigens but carrying a normal Rh phenotype. The 10-bp deletion generates a premature stop codon and encodes a truncated protein without transmembrane and cytoplasmic domain. No detectable abnormality of the LW gene or transcript could be detected in another LW(a- b-) individual (Nic), suggesting the heterogeneity of these phenotypes.

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Expression, Purification, and Characterization of Recombinant Human Factor X

Protein Expression and Purification ◽

10.1006/prep.1997.0752 ◽

1997 ◽

Vol 10 (3) ◽

pp. 373-378 ◽

Cited By ~ 19

Author(s):

Amy E. Rudolph ◽

Michael P. Mullane ◽

Rhonda Porche-Sorbet ◽

Joseph P. Miletich

Keyword(s):

Human Factor ◽

Factor X ◽

Purification And Characterization

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The Contribution Of Factor VIII In The Intrinsic Activation Of Human Factor X

10.1055/s-0038-1652221 ◽

1981 ◽

Author(s):

K Mertens ◽

A van Wijngaarden ◽

N H van Tilburg ◽

R M Bertina

Keyword(s):

Factor Viii ◽

Calcium Ions ◽

Human Factor ◽

Initial Rate ◽

Factor Xa ◽

Coagulation Factors ◽

Factor Ix ◽

Factor X ◽

Intrinsic Pathway

In the intrinsic pathway, factor X is activated by a complex consisting of activated factor IX, factor VIII, phospholipid and calcium ions. The contribution of each of this components has been estimated by recombination of purified human coagulation factors, and measurement of the initial rate of factor Xa formation using a spectrophoto- metric assay.In the absence of phospholipid and factor VIII, factor X is activated at a low, but significant rate. Addition of either phospholipid or activated factor VIII stimulates the initial rate of this reaction 250 and 500 fold, respectively; addition of both cofactors stimulates more than 100,000 fold. The activation of factor VIII by factor Xa or by thrombin is essential for the stimulating effect of factor VIII. Factor VIII complex, containing both VIIIR:WF and VIII:C, and purified factor VIII:C behave identically in this system; purified factor VIIIR:WF does not influence the rate of factor Xa formation.The same kinetic approach was used for the characterization of preparations of abnormal factor VIII, obtained from plasma of haemophilia A patients.

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Characterization of a cDNA coding for human factor X.

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.81.12.3699 ◽

1984 ◽

Vol 81 (12) ◽

pp. 3699-3702 ◽

Cited By ~ 75

Author(s):

S. P. Leytus ◽

D. W. Chung ◽

W. Kisiel ◽

K. Kurachi ◽

E. W. Davie

Keyword(s):

Human Factor ◽

Factor X

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Characterization of Monoclonal Anti-human Factor VII Antibody (B101/B1) that Recognized Three-dimensional Structures near Arg at Position 79 in the First EGF-like Domain

Thrombosis and Haemostasis ◽

10.1055/s-0037-1614229 ◽

1998 ◽

Vol 79 (01) ◽

pp. 104-109 ◽

Cited By ~ 6

Author(s):

Osamu Takamiya

Keyword(s):

Monoclonal Antibodies ◽

Tissue Factor ◽

Human Factor ◽

Solid Phase ◽

Three Dimensional ◽

Coagulation Factors ◽

Factor Vii ◽

Dimensional Structure ◽

Epidermal Growth

SummaryMurine monoclonal antibodies (designated hVII-B101/B1, hVIIDC2/D4 and hVII-DC6/3D8) directed against human factor VII (FVII) were prepared and characterized, with more extensive characterization of hVII-B101/B1 that did not bind reduced FVIIa. The immunoglobulin of the three monoclonal antibodies consisted of IgG1. These antibodies did not inhibit procoagulant activities of other vitamin K-dependent coagulation factors except FVII and did not cross-react with proteins in the immunoblotting test. hVII-DC2/D4 recognized the light chain after reduction of FVIIa with 2-mercaptoethanol, and hVIIDC6/3D8 the heavy chain. hVII-B101/B1 bound FVII without Ca2+, and possessed stronger affinity for FVII in the presence of Ca2+. The Kd for hVII-B101/B1 to FVII was 1.75 x 10–10 M in the presence of 5 mM CaCl2. The antibody inhibited the binding of FVII to tissue factor in the presence of Ca2+. hVII-B101/B1 also inhibited the activation of FX by the complex of FVIIa and tissue factor in the presence of Ca2+. Furthermore, immunoblotting revealed that hVII-B101/B1 reacted with non-reduced γ-carboxyglutaminic acid (Gla)-domainless-FVII and/or FVIIa. hVII-B101/B1 showed a similar pattern to that of non-reduced proteolytic fragments of FVII by trypsin with hVII-DC2/D4 on immunoblotting test. hVII-B101/B1 reacted differently with the FVII from the dysfunctional FVII variant, FVII Shinjo, which has a substitution of Gln for Arg at residue 79 in the first epidermal growth factor (1st EGF)-like domain (Takamiya O, et al. Haemosta 25, 89-97,1995) compared with normal FVII, when used as a solid phase-antibody for ELISA by the sandwich method. hVII-B101/B1 did not react with a series of short peptide sequences near position 79 in the first EGF-like domain on the solid-phase support for epitope scanning. These results suggested that the specific epitope of the antibody, hVII-B101/B1, was located in the three-dimensional structure near position 79 in the first EGF-like domain of human FVII.

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The Activation of Human Factor X in Sodium Citrate: the Role of Factor VII

Thrombosis and Haemostasis ◽

10.1055/s-0038-1648014 ◽

1976 ◽

Vol 36 (01) ◽

pp. 104-114 ◽

Cited By ~ 3

Author(s):

D. L Aronson ◽

A. J Mustafa

Keyword(s):

Sodium Citrate ◽

Human Factor ◽

Deae Cellulose ◽

Factor Ix ◽

Factor Vii ◽

Factor X

SummaryHuman factor X was purified by several different procedures yielding products which had varying amounts of factor VII and factor IX. Treatment with CHC13 during the fractionation of the factor X removed 95% of the factor VII and factor IX activity and the resulting factor X activated more slowly when incubated in 25% sodium citrate. Removal of residual factor VII by DEAE cellulose chromatography yielded a factor X which activated still more slowly and less completely. When the factor VII, removed by chromatography, was added to the chromatographed factor X, the ability to be activated in 25% sodium citrate was restored. Confirmatory evidence for the role of factor VII in this reaction was the inhibition of the conversion of the factor X by both DFP and SBTI.

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