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1.
Enzyme-linked coagulation assay. II. A sensitive assay for tissue factor and factors II, VII, and X 总被引:2,自引:0,他引:2
We have developed a solid-phase clotting assay which uses peroxidase-fibrinogen in solution and fibrinogen bound to microtiter plates as a substrate for the thrombin generated from the clotting cascade. We have developed this assay for measurement of the extrinsic pathway factors thromboplastin (tissue factor, factor III), VII and VIIa, X, and II. Using long incubation times (40-90 min), thromboplastin could be measured in extracts of human brain at very low concentrations. Specificity for thromboplastin was demonstrated by showing a requirement for factors II, V, X, and VII but not for VIII, IX, XI, or XII; both substrate plasmas monodeficient in single factors and mixtures of the pure factors were used in demonstrating this specificity. The assay was modified to measure factors II, VII, VIIa, and X using appropriate deficient plasmas. The limit of detection was 2-3 orders of magnitude lower than a one-stage clotting test for all factors assayed. This assay has the advantages of convenience, specificity comparable to standard clotting tests, and high sensitivity. 相似文献
2.
Josic D Hoffer L Buchacher A 《Journal of chromatography. B, Analytical technologies in the biomedical and life sciences》2003,790(1-2):183-197
A review is given of preparative methods for the isolation of the vitamin K-dependent clotting factors II, VII, IX, X and clotting inhibitor protein C, all derived from human plasma. Factor II, activated factor VII and activated protein C are also obtained from recombinant animal cells. The methods for their purification are described. The problem of difference in posttranslational modifications between plasma derived and recombinant protein is discussed with regard to therapeutic proteins. 相似文献
3.
The 24-hr variations in clotting times and vitamin K-dependent blood coagulation factors were studied in rats kept on a 12-hr light-dark cycle (light on: 0600-1800 hours). Clotting times were determined under a binocular microscope by measuring the time required for the formation of the first fibrin thread. Factors II, VII and X were analyzed by the prothrombin test while the factor IX was quantified using the activated partial thromboplastin time assay. Results indicated that the clotting times were significantly longer during the dark (activity) period with a peak at 1:00 and a trough at 17:00. Similarly, a variation was found in factor activity levels: prothrombin (II), factor VII and factor X had higher activities during the light span (rest period). The highest activities found at 13:00 and 09:00 were statistically different from the minimum activity levels obtained at 21:00. Factor IX did not show a significant circadian variation. 相似文献
4.
Enzyme-linked coagulation assay. III. Sensitive immunoassays for clotting factors II, VII, and X 总被引:3,自引:0,他引:3
G J Doellgast 《Analytical biochemistry》1987,162(1):102-114
The solid-phase clotting assay utilizing fibrinogen coated on the wells of a microtiter plate and peroxidase-fibrinogen in solution as a substrate for thrombin (enzyme-linked coagulation assay, ELCA) has been modified for use as an immunoassay. Direct inhibition of factors II, VII, and X by polyclonal (rabbit) antibodies and of factor X by monoclonal antibodies has been demonstrated at high dilution of these antibodies and detection of the specific factors using ELCA. Using plates coated with a second antibody (goat anti-mouse IgG) as well as fibrinogen, monoclonal antibodies to factors X and VII were measured by binding the active factor to the plate and detection of the bound factor using ELCA. The assay was very sensitive, permitting the detection of as little as 0.2 ng/ml (30 pg/assay) of monoclonal antibody, or less than 0.4 ng/ml (60 pg/assay) of factor Xa. When plates were coated with monoclonal antibody to factor X and fibrinogen, the assay permitted the identification of distinct epitope specificities for two monoclonal antibodies to factor X by distinct competition of the monoclonal antibodies added in the solution phase for binding of factor Xa to the plate. This assay could be applied generally for immunoassay of clotting factors, and could have application in general as an immunoassay amplification system. 相似文献
5.
Charles D. Lox 《Life sciences》1984,34(14):1357-1363
Male Sprague-Dawley rats were injected with either a single subcutaneous dose of 75 mg of secobarbital, or once daily injections of 20 mg of secobarbital for 7 days. Plasma was collected prior to treatment and 18 hours later (75 mg) or 8 and 15 days later (20 mg). Plasma was analyzed for the platelet count (PLT), prothrombin time (PT), fibrinogen (FIB), and coagulation factor activities for factors II, V, VII, IX, and X. Treatment with a single subcutaneous injection of 75 mg of secobarbital caused statistically significant alterations in every clotting activity measured whereas 7 days of treatment with 20 mg once daily resulted in only 2 clotting factors being abnormal. These two factors returned to pretreatment levels following 7 days of withdrawal of secobarbital. The data indicates that a single larger dose of secobarbital is more influential on hepatic synthesized clotting factor activity than is longer treatment with a lesser dose. 相似文献
6.
Dr. V. Ivaskevicius 《Medizinische Genetik》2008,20(2):204-210
A complex network of hemostasis proteins maintains the blood flow and integrity of the vascular system. Molecular biology techniques have led to identification and cloning of the corresponding genes, providing the basis for development of various recombinant clotting factor concentrates. Analysis of these genes allowed for phenotype and genotype correlations in patients with hemorrhagic or thromboembolic disorders and analysis of structure and function relationships of the involved proteins. Excepting coagulation factors VIII and X, deficiencies in factors fibrinogen, II, V, VII, X, XI, and XII (except in dysfibrinogenemia) accompanying a tendency to bleed are inherited, autosomally recessive traits and represent 3–5% of all inherited coagulation factor deficiencies. The prevalences for homozygous forms in the general population vary between 1:500,000 for factor VII deficiency and 1:1 million for factor V deficiency. 相似文献
7.
J. de Grouchy Marie-Dominique Dautzenberg Catherine Turleau Suzette Beguin Françoise Chavin-Colin 《Human genetics》1984,66(2-3):230-233
Summary Blood clotting factors were investigated in a patient with trisomy 8 mosaicism, a patient with an r(13), and a patient with distal trisomy 13q. Results are compatible with the assignment of the structural genes of factors VII and X to 13q34 and the existence of a regulatory mechanism associated with chromosome 8 controlling the expression of factor VII alone. 相似文献
8.
J C Mestries S Martin D Janodet F Herodin P Gourmelon M Fatome 《Comptes rendus de l'Académie des sciences. Série III, Sciences de la vie》1991,313(6):227-232
Neutron-gamma irradiation of the baboon at lethal dose altered the plasma clotting factors and induced a fibrinoformation alteration which occurred shortly before death. These disturbances, which were not found after gamma irradiation, could explain the importance of the haemorrhagic syndrome. Treatment by P.P.S.B. (factors II, VII, X and IX) counteracted the alterations of the plasma clotting factors, but had no influence on the lethality nor on the fibrinoformation alteration which seems to be an important cause of death. 相似文献
9.
Carol K. Kasper 《The Western journal of medicine》1970,113(1):4-8
A concentrate containing plasma clotting factors II, VII, IX and X was used to secure hemostasis for a herniorrhaphy, an osteotomy of a femur, a cup arthroplasty of a hip, and a tonsillectomy in patients with factor IX deficiency. After single infusions of concentrate, the net increase in plasma factor IX activity was 0.7 to 1.0 percent for each in-vitro unit of factor IX infused per kilogram of body weight. After large infusions of concentrate in two patients, the disappearance pattern of factor IX had two phases: a first component with half-disappearance times of 4.4 and 6 hours, and a second component with half-disappearance times of 26 and 32.6 hours. 相似文献
10.
The salivary glands of Dermacentor andersoni ticks possess anticoagulant activities that can alter the clotting time of rabbit whole blood. Salivary gland extracts from female ticks inhibit both the intrinsic and extrinsic coagulation systems, and maximal activities against both pathways occur when the ticks attain about 250 mg feeding weight. These anticoagulants are directed against both coagulation factors V and VII, but they do not affect factors II or X. Despite this salivary anticoagulant activity, heavily tick-infested rabbits suffer no visible alteration of their peripheral blood coagulability and have no detectable circulating fibrin degradation products, suggesting that the ticks do not secrete a factor with fibrinolytic activity. 相似文献
11.
The gene coding for coagulation factor X was studied in a family segregating chromosomal abnormalities involving chromosomes 13 and 6. An individual monosomic for 13q34 was deficient in levels of clotting factors VII and X, while her brother, who is trisomic for 13q34, had elevated levels. DNA dosage studies with a cloned human factor X gene demonstrated that the low levels of factor X expression in the individual with the chromosome 13q34 deletion were due to the absence of one copy of the factor X structural gene. This confirms the assignment of the human gene coding for factor X to 13q34. 相似文献
12.
Masaaki Kurata Takeshi Iidaka Naoko Yamasaki Yukiko Sasayama Yoshimasa Hamada 《Experimental Animals》2005,54(2):189-192
A battery of simple tests for profiling abnormalities of vitamin K-dependent coagulation factors encountered in drug-toxicity studies was verified in rats treated with warfarin (3 and 10 mg/kg, p.o). The thrombotest, or hepaplastin-test, is useful as a follow-up test after routine screening tests for coagulation abnormalities based on PT and APTT, to rule out other coagulation-factor abnormalities. Measurement of coagulation factor activities (factors II, VII, IX and X) using factor-deficient human plasmas provides direct evidence of decreased activities of vitamin K-dependent factors. Furthermore, Echis carinatus venom coagulation time, together with factor II activity, allows us to confirm the generation of PIVKA-II. 相似文献
13.
A Girolami 《Folia haematologica (Leipzig, Germany : 1928)》1980,107(1):131-136
Congenital factor VII deficiency is a well known clotting disorder first identified in 1951. Several congenital abnormalities of factor VII have been described during the past decade. These abnormalities were all characterized by the presence of a discrepancy between factor VII clotting activity and factor VII cross reacting material or antigen. Recently other factor VII abnormalities have been described which showed peculiar sensitivities to ox-brain thromboplastins as compared to thromboplastins of other origin. These discoveries have complicated the differential diagnosis of prothrombin complex factors deficiences and abnormalities. A correct diagnosis may be reached only by means of a battery of tests which employ tissue thromboplastins of different origin. 相似文献
14.
Synthesis, purification, and characterization of an Arg152----Glu site-directed mutant of recombinant human blood clotting factor VII 总被引:2,自引:0,他引:2
Coagulation factor VII circulates in blood as a single-chain zymogen of a serine protease and is converted to its activated two-chain form, factor VIIa, by cleavage of an internal peptide bond located at Arg152-Ile153. Previous studies using serine protease active-site inhibitors suggest that zymogen factor VII may possess sufficient proteolytic activity to initiate the extrinsic pathway of blood coagulation. In order to assess the putative intrinsic proteolytic activity of single-chain factor VII, we have constructed a site-specific mutant of recombinant human factor VII in which arginine-152 has been replaced with a glutamic acid residue. Mutant factor VII was purified in a single step from culture supernatants of baby hamster kidney cells transfected with a plasmid containing the sequence for Arg152----Glu factor VII using a calcium-dependent, murine anti-factor VII monoclonal antibody column. Purified mutant factor VII was indistinguishable from plasma-derived or recombinant wild-type factor VII by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and migrated as a single band with an apparent molecular weight of 50,000. The average specific activity of several mutant factor VII preparations was 0.00025 unit/micrograms, or 0.01% of that observed for recombinant wild-type factor VII preparations. The clotting activity of mutant factor VII was, however, completely inhibited following incubation with dansyl-Glu-Gly-Arg chloromethyl ketone, suggesting that the apparent clotting activity of mutant factor VII was due to a contaminating serine protease.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
15.
Ninety studies on 58 patients undergoing chronic warfarin therapy included Quick prothrombin times, partial thromboplastin times, thromboplastin generation tests and assays for clotting factors II, V, VII, VIII, IX, X, XI and XII. The results indicate no benefit from supplementation of the Quick tests by any of these other procedures. It is suggested that the Quick test uniformly performed, using a standard uniform thromboplastin, would be the procedure of choice. 相似文献
16.
The propeptide region of clotting factor IX is a signal for a vitamin K dependent carboxylase: evidence from protein engineering of amino acid -4. 总被引:1,自引:0,他引:1
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Homologous "propeptide" regions are present in a family of vitamin K-dependent mammalian proteins, including clotting factors II, VII, IX, X, protein C, protein S and bone "gla" proteins. To test the hypothesis that the propeptide is a signal for the correct gamma-carboxylation of the adjacent gamma-carboxy region, we have mutated amino acid -4 of human factor IX from an arginine to a glutamine residue, by M13-directed site-specific mutagenesis of a cDNA clone. After expression of mutant factor IX in dog kidney cells, we find that it is secreted into the medium in a precursor form containing the propeptide, and is inefficiently gamma-carboxylated compared to the control, wild-type, recombinant factor IX. This result supports the hypothesis that the propeptide region is required for efficient gamma-carboxylation of factor IX in dog kidney cells. Furthermore, it confirms previous results that arginine at amino acid -4 is required for correct propeptide processing. 相似文献
17.
Tissue factor, the physiologic trigger of blood clotting, is the membrane-anchored protein cofactor for the plasma serine protease, factor VIIa. Tissue factor is hypothesized to position and align the active site of factor VIIa relative to the membrane surface for optimum proteolytic attack on the scissile bonds of membrane-bound protein substrates such as factor X. We tested this hypothesis by raising the factor VIIa binding site above the membrane surface by creating chimeras containing the tissue factor ectodomain linked to varying portions of the membrane-anchored protein, P-selectin. The tissue factor/P-selectin chimeras bound factor VIIa with high affinity and supported full allosteric activation of factor VIIa toward tripeptidyl-amide substrates. That the active site of factor VIIa was raised above the membrane surface when bound to tissue factor/P-selectin chimeras was confirmed using resonance energy transfer techniques in which appropriate fluorescent dyes were placed in the active site of factor VIIa and at the membrane surface. The chimeras were deficient in supporting factor X activation by factor VIIa due to decreased k(cat). The chimeras were also markedly deficient in clotting plasma, although incubating factor VII or VIIa with the chimeras prior to the addition of plasma restored much of their procoagulant activity. Interestingly, all chimeras fully supported tissue factor-dependent factor VII autoactivation. These studies indicate that proper positioning of the factor VII/VIIa binding site on tissue factor above the membrane surface is important for efficient rates of activation of factor X by this membrane-bound enzyme/cofactor complex. 相似文献
18.
Zymogen and procofactor concentrations in physiological biochemical systems (PBS) have not yet been explained. The problem in question is to determine optimal plasma clotting factor (factors II, VII, IX, and X and cofactors V and VIII) concentrations for coagulation system (CS) as a whole. Constrained optimization technique is used to solve this problem. The constraint is determined by the ability of the CS to perform its physiological function--thrombin generation (and hence clot formation)--under vessel injury conditions. The constraint statement is based on the CS dynamics equations. In solving the problem the Lagrange multiplier is used. A hypothesis is advanced that this problem can be solved based on principle of minimum protein consumption subject to an above constraint. The results obtained indicate that the optimal clotting factor concentrations are in good agreement with those measured by biochemical techniques. A comparison between the theoretical results and experimental data lends support for our hypothesis that zymogen and procofactor concentrations in the CS (and, probably, for other biochemical systems) are determined by the principle of minimum protein consumption. 相似文献
19.
Geoffrey K. Isbister Kalana Maduwage Fiona E. Scorgie Seyed Shahmy Fahim Mohamed Chandana Abeysinghe Harendra Karunathilake Margaret A. O’Leary Christeine A. Gnanathasan Lisa F. Lincz 《PLoS neglected tropical diseases》2015,9(8)
Background
Russell’s viper envenoming is a major problem in South Asia and causes venom induced consumption coagulopathy. This study aimed to investigate the kinetics and dynamics of venom and clotting function in Russell’s viper envenoming.Methodology/Principal Findings
In a prospective cohort of 146 patients with Russell’s viper envenoming, we measured venom concentrations, international normalised ratio [INR], prothrombin time (PT), activated partial thromboplastin time (aPTT), coagulation factors I, II, V, VII, VIII, IX and X, and von Willebrand factor antigen. The median age was 39y (16–82y) and 111 were male. The median peak INR was 6.8 (interquartile range[IQR]:3.7 to >13), associated with low fibrinogen [median,<0.01g/L;IQR:<0.01–0.9g/L), low factor V levels [median,<5%;IQR:<5–4%], low factor VIII levels [median,40%;IQR:12–79%] and low factor X levels [median,48%;IQR:29–67%]. There were smaller reductions in factors II, IX and VII over time. All factors recovered over 48h post-antivenom. The median INR remained >3 at 6h post-antivenom but had reduced to <2, by 24h. The aPTT had also returned to close to normal (<50sec) at 24h. Factor VII, VIII and IX levels were unusually high pre-antivenom, median peak concentrations of 393%, 307% and 468% respectively. Pre-antivenom venom concentrations and the INR (r = 0.20, p = 0.02) and aPTT (r = 0.19, p = 0.03) were correlated (non-parametric Spearman analysis).Conclusions
Russell’s viper coagulopathy results in prolonged aPTT, INR, low fibrinogen, factors V, VIII and X which recover over 48h. Severity of clotting abnormalities was associated with venom concentrations. 相似文献20.
A study has been performed on the effect of Norinyl-1 and Ortho-Novin, two low-dose oral contraceptives, on blood-clotting factors. Ortho-Novin contains twice the amount of hormone as Norinyl-1. It was therefore possible to observe whether any changes detected were related to the dose of oestrogen-progestin combination. The women were tested in parallel with matched normal female controls and a group in the third trimester of pregnancy. Significant rises in factor VII and X levels were found with both low-dose preparations from the third month onwards. There was no difference between patients on Norinyl-1 and Ortho-Novin, and hence the clotting changes do not appear to be dose-dependent. The long-term effects on clotting factors of these low-dose oral contraceptive preparations remain to be investigated. 相似文献