The Interaction of Contact Product and Factor IX

1964 ◽  
Vol 11 (01) ◽  
pp. 155-166 ◽  
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.

The Formation of Intermediate Product I in a Purified System The Role of Factor IX or of its Precursor and of a Hageman Factor-PTA Fraction

1961 ◽  
Vol 6 (02) ◽  
pp. 224-234 ◽  
Author(s):  
E. T Yin ◽  
F Duckert
Keyword(s):  
Intermediate Product ◽  
Factor Ix ◽  
Assay Method ◽  
Lag Phase ◽  
Factor X ◽  
Haemophilia B ◽  
Hageman Factor ◽  
One Stage ◽  
The One

Summary1. The role of two clot promoting fractions isolated from either plasma or serum is studied in a purified system for the generation of intermediate product I in which the serum is replaced by factor X and the investigated fractions.2. Optimal generation of intermediate product I is possible in the purified system utilizing fractions devoid of factor IX one-stage activity. Prothrombin and thrombin are not necessary in this system.3. The fraction containing factor IX or its precursor, no measurable activity by the one-stage assay method, controls the yield of intermediate product I. No similar fraction can be isolated from haemophilia B plasma or serum.4. The Hageman factor — PTA fraction shortens the lag phase of intermediate product I formation and has no influence on the yield. This fraction can also be prepared from haemophilia B plasma or serum.

scholarly journals Plasma factor IX levels in patients given hexoestrol or stilboestrol to suppress lactation

BMJ ◽  
1969 ◽  
Vol 4 (5675) ◽  
pp. 82-84 ◽  
Author(s):  
C. A. Hakim ◽  
M. G. Elder ◽  
D. F. Hawkins
Keyword(s):  
Factor Ix ◽  
Plasma Factor

Measurement of Human Igf-II Using Sephacryl Extraction: A Rapid and Reliable Assay Method

1996 ◽  
Vol 33 (3) ◽  
pp. 201-208 ◽  
Author(s):  
Barbara H Mason ◽  
Michele A Tatnell ◽  
Ian M Holdaway
Keyword(s):  
Growth Factor ◽  
Binding Proteins ◽  
Complete Removal ◽  
Assay Method ◽  
Insulin Like Growth Factor ◽  
Serum Concentrations ◽  
Igf Binding Proteins ◽  
Factor Ii ◽  
Igf I ◽  
Igfbp 3

Measurement of insulin-like growth factor II (IGF-II) in human serum is complicated by the presence of IGF binding proteins and usually involves cumbersome extraction procedures followed by radioimmunoassay. We have utilized an extraction process developed for measuring insulin-like growth factor II in ovine serum using Sephacryl HR100, and have applied this to the extraction of human samples followed by radioimmunoassay for human IGF-II. The assay yielded 98% recovery of unlabelled IGF-II, parallelism between dilutions of eluate and the standard curve, complete removal of binding proteins and near-complete removal of IGF-I, and intra- and interassay coefficients of variation of 5% and 9%, respectively. The normal range for serum IGF-II in women was 490–1056 μg/L, and IGF-II levels were positively correlated with serum concentrations of insulin-like growth factor binding protein-3 (IGFBP-3) but not with IGF-I levels. Mean serum concentrations of IGF-II were reduced below normal in a number of hypopituitary patients and children with short stature and IGF-II concentrations in these subjects correlated positively with IGF-I and IGFBP-3. In acromegalic patients IGF-II levels were usually normal and were negatively correlated with IGF-I concentrations. From our experience with the above results the present assay appears particularly suitable for clinical measurements and research projects where high sample throughput is required.

Immunochemical Characterization of a Factor IX Inhibitor Following Anamnestic Response

1975 ◽  
Author(s):  
H. R. Roberts ◽  
C. R. Fuller ◽  
H. Worden ◽  
J. Stuart ◽  
H. Reisner ◽  
...  
Keyword(s):  
Light Chain ◽  
Gel Filtration ◽  
Complete Removal ◽  
Igg Subclasses ◽  
Factor Ix ◽  
Biologically Active ◽  
Anamnestic Response ◽  
Polyclonal Igg ◽  
Monospecific Antisera ◽  
A Minor

We previously characterized a human inhibitor for Factor IX in patient P.W.B, with Hemophilia B as an IgG4, λ immunoglobulin of restricted electrophoretic mobility. This restriction to a minor IgG subclass led us to characterize a second Factor IX inhibitor occurring in patient R. J. after an anamnestic response to Factor IX. On preparative zone electrophoresis the inhibitor migrated with a broad zone of mobility in the anodal portion of the γ peak and was restricted to the anodal portion of the IgG containing fractions. Gel filtration on calibrated 1.5 M sepharose columns revealed inhibitor activity in fractions corresponding to a molecular weight of 150,000. The inhibitor was further characterized by the technique of antibody neutralization using monospecific antisera to immunoglobulin classes, subclasses and light chain types in the zone of antibody excess. The inhibitor was completely neutralized by antibody to IgG whereas antisera to IgA, IgM, IgD and IgE had no effect. Neutralization was abolished by absorption of the IgG antiserum with purified IgG. Neutralization with antisera specific for light chains indicated a mixture of light chain types with an estimated ϰ/λ ratio of 6/1. Neutralization with antisera specific for IgG subclasses revealed a mixture of IgG subclasses. The Factor IX inhibitor was thus characterized as a polyclonal IgG immunoglobulin. Sepharose conjugates of R. J. globulin effect complete removal of Factor IX from normal plasma on an immunoabsorbent column and biologically active Factor IX may be eluted with 1600-fold purification.

EXPRESSION IN MAMMALIAN CELLS OF FUSION PROTEINS BETWEEN HUMAN FACTORS IX AND VII

1987 ◽  
Author(s):  
K L Berkner ◽  
S J Busby ◽  
J Gambee ◽  
A Kumar
Keyword(s):  
Fusion Protein ◽  
Fusion Proteins ◽  
Factor Ix ◽  
Biologically Active ◽  
Factor Vii ◽  
Clotting Factor ◽  
Clotting Factors ◽  
Mammalian Expression ◽  
Plasma Factor ◽  
Gla Domain

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.

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

1966 ◽  
Vol 251 (1) ◽  
pp. 43-50 ◽  
Author(s):  
Harold R. Roberts ◽  
Gwendolyn P. Gross ◽  
William P. Webster ◽  
Ivan I. Dejanov ◽  
George D. Penick
Keyword(s):  
Factor Ix ◽  
Plasma Factor

Fluorogenic Assays for Functional Factor VIIa and Tissue Factor.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1740-1740
Author(s):  
Sara J. Ethier ◽  
Saulius Butenas ◽  
Richard J. Jenny
Keyword(s):  
Tissue Factor ◽  
Specific Activity ◽  
Factor Viia ◽  
Factor Vii ◽  
Assay Method ◽  
Sensitivity Index ◽  
Functional Assay ◽  
Plasma Samples ◽  
Functional Basis

Abstract Blood coagulation is initiated when cryptic tissue factor (TF) becomes exposed on the surface of vascular cells where it can bind circulating factor VIIa (fVIIa). The resulting fVIIa/TF enzyme complex catalyzes the activation of certain blood zymogens that propagate the coagulation event. As a key enzyme for the initiation of coagulation, circulating fVIIa levels have been viewed as an indicator of hemostatic potential and may also be an indicator for the risk of developing cardiovascular disease. The formation of the fVIIa/TF complex is also the basis of specific coagulation assays. Natural or synthetic TF is employed in the prothrombin time (PT assay) where it is used to initiate coagulation in vitro. Therefore, fVIIa and TF play important roles both in vivo and in vitro. Despite such important roles, there have been no simple methods described for measuring the active forms of either protein. This report describes the initial development of an assay for fVIIa and TF that employs the use of an Aminonaphthalenesulfonamide-based (ANSN) fluorogenic substrate. The ANSN substrate is hydrolyzed by fVIIa in a TF-dependent manner, and by titrating fVIIa into a system containing excess TF, the assay becomes sensitive to the fVIIa concentration. Inversely, by titrating TF into a system containing excess fVIIa, the assay becomes sensitive to the TF concentration. This approach has led to the development of both a rapid kinetic and an end-point assay method for each analyte providing detection ranges down to 156 and 1.56 pM respectively. The utility of these assays is displayed by the following applications. Thromboplastin reagents are generally qualified against a W.H.O. standard and are assigned a numerical value known as the International Sensitivity Index (ISI). Theoretically the ISI value should be affected by the quantity and quality of TF in each preparation. An inverse relationship between ISI value and the amount of functional TF would support this theory. To prove this relationship the TF assay was employed to examine two thromboplastin reagents with ISI values of 1.84 and 1.01. The concentration of functional TF measured by this assay was 10.8 nM and 14.6 nM respectively. Because most commercial thromboplastin reagents are prepared by adding TF on a mass basis (as opposed to a functional basis), target ISI values are often missed, leading to an excessive number of failed lots. The TF functional assay provides a method for assigning a specific activity to each TF preparation. By adding TF on a functional basis, lot-to-lot variability would be minimized and target ISI values would be easily achieved. To substantiate this claim, we have determined the functional activity of seven different lots of purified recombinant human TF. With each lot analyzed at a fixed concentration, we observed a broad range of specific activity (range: 8,250 U/sec/mg -14,500 U/sec/mg). These data support the need for a functional TF assay to be incorporated in the manufacturing process of thromboplastin reagents. Lastly, the utility of the fVIIa assay was demonstrated in an experiment using factor VII-deficient plasma samples that were spiked with known quantities of fVIIa. Plasma samples spiked with fVIIa in the range of 0.5-2nM returned mean assay values within 71–100% of the expected values. These data demonstrate the feasibility of using the fVIIa assay to monitor fVIIa levels in hemophilia patients receiving recombinant fVIIa therapy. Further studies will address the circulating concentrations of fVIIa and TF in “normal” and disease state plasma samples.

scholarly journals 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

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.

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

Blood ◽  
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.

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

1996 ◽  
Vol 76 (06) ◽  
pp. 0965-0969 ◽  
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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