Plasma factor IX: The tip of the iceberg?

The vitamin K-dependent plasma proteins demonstrate remarkable similarities in their structures: all have multiple domains in common and extensive homology is observed within many of these domains. In order to investigate the structure-function relationship of these proteins, we have interchanged domains of one protein (factor IX) with that of another (factor VII) and have compared the expression of these fusion proteins with recombinant and native factors IX and VII. Oligonucleotide-directed mutagenesis was used to generate four fusion proteins: factor IX/VII-1, which contains the factor IX leader and gla domain fused to the growth factor and serine protease of factor VII; factor VII/IX-1, a reciprocal fusion protein of factor IX/VII-1; factor IX/VII-2, which contains the factor IX leader adjoined to the mature factor VII protein sequence; and factor VII/IX-2, the reciprocal fusion protein of factor IX/VII-2. The cDNAs encoding all four proteins were cloned into mammalian expression vectors, and to date three of these (factors IX/VII-1, 2 and VII/IX-1) have been transfected into baby hamster kidney (BHK) cells or 293 cells and characterized. Factors IX/VII-1 and VII/IX-1 were both secreted at levels comparable to recombinant factors IX and VII. The factor IX/VII-1 was identical in molecular weight to native or recombinant factor VII (i.e., 53 K). Factor VII/IX-1 was expressed as two proteins with molecular weights around 68 kd, as observed with recombinant factor IX. The factor IX/VII-1 protein has been purified to homogeneity and has been found to possess factor VII biological activity, but at a specific activity approximately 20% that of plasma factor VII. Thus, the gla domain of one clotting factor is capable of directing the activation of another and of generating biologically active protein. In contrast, no activity was observed with the factor IX/VII-2 fusion protein, indicating that there are limits to the interchanges which can generate functional blood clotting factors.

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ACQUIRED INHIBITORS OF PLASMA FACTOR IX A STUDY OF THEIR INDUCTION, PROPERTIES AND NEUTRALIZATION

The American Journal of the Medical Sciences ◽

10.1097/00000441-196601000-00008 ◽

1966 ◽

Vol 251 (1) ◽

pp. 43-50 ◽

Cited By ~ 14

Author(s):

Harold R. Roberts ◽

Gwendolyn P. Gross ◽

William P. Webster ◽

Ivan I. Dejanov ◽

George D. Penick

Keyword(s):

Factor Ix ◽

Plasma Factor

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Christmas disease in a Hovawart family resembling human hemophilia B Leyden is caused by a single nucleotide deletion in a highly conserved transcription factor binding site of the F9 gene promoter

10.1101/486373 ◽

2018 ◽

Author(s):

Bertram Brenig ◽

Lilith Steingräber ◽

Shuwen Shan ◽

Fangzheng Xu ◽

Marc Hirschfeld ◽

...

Keyword(s):

Coagulation Factor ◽

Bleeding Disorder ◽

Factor Ix ◽

Hemophilia B ◽

Start Codon ◽

Luciferase Reporter ◽

Single Nucleotide ◽

Single Nucleotide Deletion ◽

Nucleotide Deletion ◽

Plasma Factor

Hemophilia B is a classical monogenic X-chromosomal recessively transmitted bleeding disorder caused by genetic variants within the coagulation factor IX gene (F9). Although hemophilia B has been described in 32 dog breeds hitherto, it has not yet been reported in the Hovawart. Here we describe the identification of a Hovawart family transmitting typical signs of an X-linked bleeding disorder. Five males had been reported to suffer from recurrent hemorrhagic episodes, four of them had to be euthanized finally and one died due to severe blood loss. A blood sample of one of these males with only 2% of the normal concentration of plasma factor IX (FIX) together with samples of seven relatives including the mother and grandmother were provided for further analysis. Next generation sequencing of DNA of the mother and grandmother revealed a single nucleotide deletion in the F9 promoter (NC_006621.3:g.109,501,492delC; CanFam3.1). Genotyping of the deletion in 1,298 dog specimens (83 different breeds) including 720 Hovawarts revealed that the mutation was only present in the aforementioned Hovawart family. The deletion is located 73 bp upstream of the F9 start codon in the highly conserved overlapping DNA binding sites of hepatocyte nuclear factor 4α (HNF4α) and androgen receptor (AR). The deletion only abolishes binding of HNF4α as demonstrated by electrophoretic mobility shift assay (EMSA) using purified recombinant human HNF4α and a transient overexpression lysate of human AR with double-stranded DNA probes encompassing the mutated promoter region. Luciferase reporter assays using wild type and mutated promoter fragment constructs transfected into Hep G2 cells showed a 65.3% reduction in expression of the mutated promoter. The data presented here provide evidence that the deletion identified in the Hovawart family caused a rare type of hemophilia B resembling human hemophilia B Leyden.

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Separation of human plasma factor IX from HTLV-I or HIV by immunoaffinity chromatography using conformation-specific antibodies

Blood ◽

10.1182/blood.v70.5.1312.bloodjournal7051312 ◽

1987 ◽

Vol 70 (5) ◽

pp. 1312-1315

Author(s):

SA Limentani ◽

BC Furie ◽

BJ Poiesz ◽

R Montagna ◽

K Wells ◽

...

Keyword(s):

Human Plasma ◽

Leukemia Virus ◽

Immunoaffinity Chromatography ◽

Factor Ix ◽

Specific Antibodies ◽

Viral Contamination ◽

Human T Cell ◽

Plasma Factor ◽

Immunodeficiency Virus ◽

T Cell Leukemia Virus

Immunoaffinity chromatography using conformation-specific antibodies yields pure factor IX from human plasma in a single rapid, facile purification step. We evaluated this technique to determine whether factor IX can be separated from human T cell leukemia virus-I (HTLV-I) and human immunodeficiency virus (HIV) in plasma supplemented with these viruses. Viral content was determined with an enzyme-linked immunosorbent (ELISA) assay sensitive to 50 ng viral protein. Both HTLV- I and HIV coeluted with unbound protein. Neither HTLV-I nor HIV was detected in purified factor IX. We conclude that, to the limits of detection, factor IX purified by this method is free of viral contamination.

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Age and Sex Dependent Regulation of the Factor IX Gene in Mice

Thrombosis and Haemostasis ◽

10.1055/s-0038-1650693 ◽

1996 ◽

Vol 76 (06) ◽

pp. 0965-0969 ◽

Cited By ~ 12

Author(s):

Sumiko Kurachi ◽

Eri Hitomi ◽

Kotoku Kurachi

Keyword(s):

Reference Group ◽

Mrna Level ◽

Factor Ix ◽

Mouse Strains ◽

Female Group ◽

Human Populations ◽

Activity Levels ◽

Mrna Levels ◽

Animal Groups ◽

Plasma Factor

SummaryPlasma factor IX and liver factor IX mRNA levels in two normal mouse strains (B6D2F1 and BALB/CJNIA) were determined in relation to aging and sex of the animals. With male B6D2F1 mice, mean plasma factor IX activity levels for the 14 and 21-22 month-old animals were found to be 124% and 226%, respectively, of the 5 month-old group. Similarly, liver factor IX mRNA levels for the same age animal groups were 145% and 227%, respectively, of the reference group. Mean plasma factor IX levels for the same age female animals were 132% and 175%, respectively, and were accompanied by similarly elevated liver factor IX mRNA levels, 119 and 175%, respectively, of the 5 month-old female group. Factor IX activity and mRNA levels for the 5,14 and 21-22 month-old female animal groups were lower than those of the corresponding male age groups by 25, 20 and 37%, and 20,36 and 38%, respectively. With BALB/CJNIA mice, similar correlation was observed between the advancing age and substantial elevations in the factor IX mRNA level as well as on the unequal factor IX mRNA levels in females and males.These results indicate that the plasma factor IX level in both male and female mice is greatly elevated with aging, in general agreement with a similar phenomenon observed for human populations, and that this increase is due to a similar elevation in the factor IX mRNA level in the liver. In mice, both factor IX activity and mRNA levels are significantly higher in males than in females, which has not been described for humans.

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The Interaction of Contact Product and Factor IX

Thrombosis and Haemostasis ◽

10.1055/s-0038-1654816 ◽

1964 ◽

Vol 11 (01) ◽

pp. 155-166 ◽

Cited By ~ 3

Author(s):

A. D Cattan ◽

K. W. E Denson

Keyword(s):

Calcium Ions ◽

Complete Removal ◽

Factor Ix ◽

Assay Method ◽

Plasma Samples ◽

Active Product ◽

Complex Process ◽

Plasma Factor

Summary1. The activation of factor IX in recalcified plasma develops slowly and is accelerated by dilution of the resulting serum.2. The complex process of activation and decay of the active product, introduces error into the assay method.3. An excess or the complete removal of calcium ions eliminates the activation but does not prevent decay of the active product.4. The assay of factor IX in plasma gives results similar to those for serum obtained from celite exhausted plasma.5. Activation of factor IX is minimal in the serum obtained when plasma samples deficient in contact product, or the contact factors are clotted.6. Plasma factor IX is probably activated enzymatically by calcium ions and contact product. The presence in serum of an inhibitor of the process was not clearly demonstrated.

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Presence of Activated Factor VII in Factor IX Concentrates and its Persistence in the Circulation After Infusion

10.1055/s-0039-1684824 ◽

1979 ◽

Cited By ~ 2

Author(s):

U. Seligsohn ◽

C.K. Kasper ◽

B. Østerud ◽

S.I. Rapaport

Keyword(s):

Hemophilia A ◽

Factor Ix ◽

Factor Vii ◽

Amidolytic Activity ◽

Activated Factor Vii ◽

Plasma Factor ◽

Disappearance Time

Six brands of Factor IX concentrates were evaluated for their Factor VII content and for the presence of activated Factor VII through use of a coupled amidolytic assay, insensitive to activated Factor VII, and a clotting assay, sensitive to activated Factor VII. The Factor VII content of the concentrates studied (except for one concentrate purposely produced to exclude Factor VII) varied between 33 to 621 U per vial. All concentrates contained activated Factor VII, as indicated by ratios of Factor VII clotting activity to Factor VII amidolytic activity of from 1.6 to 21.5. Higher ratios were found in two brands of activated concentrates than in non-activated concentrates. In 10 patients infused with Factor IX concentrates, plasma Factor VII activity rose strikingly in the clotting assay but not in the amidolytic assay. Thus, the elevated Factor VII levels by the clotting assay after infusion of Factor IX concentrates stem from circulating activated Factor VII. A mean intravascular half-disappearance time of 144 min was found for activated Factor VII. Its persistence in the circulation makes it important to evaluate the possible role of activated Factor VII in the thrombogenicity of Factor IX concentrates and in their reported effectiveness in treating bleeding in Hemophilia A patients with inhibitors.

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Expression and Linkage of Genes for X-linked Hemophilias A and B in the Dog

Blood ◽

10.1182/blood.v41.4.577.577 ◽

1973 ◽

Vol 41 (4) ◽

pp. 577-585 ◽

Cited By ~ 25

Author(s):

K. M. Brinkhous ◽

P. D. Davis ◽

John B. Graham ◽

W. Jean Dodds

Keyword(s):

X Chromosome ◽

Hemophilia A ◽

Factor Ix ◽

Neonatal Lethality ◽

Plasma Factor ◽

Normal Males ◽

Linkage Of Genes ◽

Linkage Distance ◽

Antihemophilic Factor ◽

Preferential Inactivation

Abstract The linkage distance on the X chromosome between the genes for hemophilia A (classic hemophilia) and B (PTC deficiency, Christmas disease) was estimated directly by breeding two strains of dogs, each segregating for a different type of hemophilia. Gene expression was determined by bioassays of plasma factor VIII (antihemophilic factor) and factor IX (PTC, Christmas factor). Double heterozygotes in repulsion for both hemophilia A and B could be readily identified by intermediate plasma levels of both procoagulants. There was no evidence of a tendency toward preferential inactivation of the paternally derived X chromosome, and the procoagulant levels showed that random inactivation had occurred at both loci. When double heterozygotes were bred against normal males or males with hemophilia A and B, the progeny that resulted indicated that the genes recombined freely. Thus, the genes are at least 50 map units apart. The phenotypes of five new hemophilic genotypes are described as a result of the various crossbreedings, including males with double hemophilia AB. When both hemophilia genes are in the coupling phase, there is evidence of increased intrauterine or neonatal lethality in males. The data from this study, along with that on gene linkage of human hemophilia A and B, provide support for the thesis of homology of the X chromosome during speciation.

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Two Distinct Mutations Cause Severe Hemophilia B in Two Unrelated Canine Pedigrees

Thrombosis and Haemostasis ◽

10.1055/s-0037-1614374 ◽

1999 ◽

Vol 82 (10) ◽

pp. 1270-1275 ◽

Cited By ~ 11

Author(s):

Weikuan Gu ◽

James Catalfamo ◽

Jharna Ray ◽

Kunal Ray ◽

Marjory Brooks

Keyword(s):

Catalytic Domain ◽

Stop Codon ◽

Spontaneous Mutation ◽

Mammalian Species ◽

Donor Site ◽

Splice Junction ◽

Factor Ix ◽

Hemophilia B ◽

Acceptor Site ◽

Plasma Factor

SummaryThe molecular defects causing severe factor IX deficiency were identified in two distinct canine breed-variants. Both defects were associated with an absence of plasma factor IX coagulant activity and antigen. A large deletion mutation was found in 1 breed variant, spanning the entire 5’ region of the factor IX gene extending to exon 6. An approximately 5 kb insertion disrupted exon 8 of the second breed-variant. This insertion was associated with alternative splicing between a donor site 5’ and acceptor site 3’ to the normal exon 8 splice junction, with introduction of a new stop codon. The resultant transcript lacked most of the factor IX catalytic domain and 3’ untranslated region. Molecular analyses of canine hemophilia B define an experimental model for study of inhibitor formation and gene therapy strategies, and provide insight into spontaneous mutation mechanisms in the factor IX gene and on the X chromosome of mammalian species.

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