scholarly journals Pathways to Blood Coagulation Product I Formation

Blood ◽  
1963 ◽  
Vol 21 (6) ◽  
pp. 745-754 ◽  
Author(s):  
THEODORE H. SPAET ◽  
JOSÉ CINTRON
Keyword(s):  
Sodium Citrate ◽  
Deae Cellulose ◽  
Factor Ix ◽  
Rabbit Serum ◽  
Stable Complex ◽  
Normal Rabbit Serum ◽  
Factor V ◽  
Factor X ◽  
Cent Sodium ◽  
Autoprothrombin C

Abstract The basic reagent used was an eluate obtained from barium sulfate used to adsorb various sera. When this eluate was prepared from normal rabbit serum, it responded to treatment with coagulants from adsorbed plasma, with Stypven, or with 25 per cent sodium citrate to give products with similar if not identical properties. With each preparation a stable complex formed with cephalin which withstood washing, was relatively heat-stable, was inactivated by adsorbed serum, and which required factor V for optimal prothrombin conversion. In eluates prepared from human serum, normal activation occurred in the absence of factor IX, but was defective in the absence of factor X. A preparation of factor X purified by DEAE cellulose chromatography was activated by 25 per cent sodium citrate. It is suggested that product I, the product of Stypven activation, and autoprothrombin C represent similar or identical reagents; it is further suggested that factor X is their common precursor.

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

1976 ◽  
Vol 36 (01) ◽  
pp. 104-114 ◽  
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.

Characterization Of The In Vitro “Factor VIII Bypassing Activity”

1981 ◽  
Author(s):  
D L Aronson ◽  
J Bagley
Keyword(s):  
Factor Viii ◽  
Early Stage ◽  
Deae Cellulose ◽  
Factor Ix ◽  
Factor Xii ◽  
Factor V ◽  
Factor X ◽  
Hemostatic Effect ◽  
Prolonged Aptt

The in vitro correction of the prolonged APTT of hemophilic plasma has been ascribed to an uncharacterized entity “Factor VIII Bypassing Activity.” Such products also correct the prolonged APTT plasma deficient in Factor IX, Factor X and Factor XII, but not of Factor V deficient plasma. Correction of the APTT in Factor VIII deficient plasma by early stage coagulants such as Factor XIIa, Kallikrein and Factor IXa is minimal. These results indicate that this in vitro activity acts at the level of either the activation of Factor X or the activation of prothrombin.A coagulant has been prepared from serum by barium precipitation, heparin-agarose, DEAE cellulose and high pressure liquid chromatography (HPLC). The in vitro coagulant properties are similar to “activated” prothrombin complex (Autoplex) and the biologic and chemical properties are identical to activated Factor X.Infusion of the partially purified serum coagulant into normal dogs was well tolerated and, in contrast to Factor IX concentrates, gave no signs of DIC. Infusion into bleeding hemophilic dogs had no hemostatic effect. It is concluded that a major portion of the in vitro potency of activated prothrombin concentrates is due to activated Factor X, a material which when infused has no in vivo hemostatic effect.Acknowledgments - The authors gratefully acknowledge the studies of Dr. Henry Kingdon in hemophilic dogs.

Spectrophotometric Determination of Factor Xa Generation in Factor IX Concentrates

1977 ◽  
Vol 37 (03) ◽  
pp. 535-540 ◽  
Author(s):  
D. S Pepper ◽  
D Banhegyi ◽  
Ann Howie
Keyword(s):  
Intrinsic Factor ◽  
Factor Xa ◽  
Factor Ix ◽  
Factor V ◽  
Factor X ◽  
Generation Times ◽  
Incubation Periods ◽  
Multiple Sample ◽  
Recording Spectrophotometer

SummaryPrevious work from this department, concerned with testing the potential thrombogenicity of therapeutic factor IX concentrates, demonstrated that following recalcification of factor IX concentrates thrombin was generated within 3-30 minutes of incubation (Sas et al. 1975). The test developed (known as the TGt 50 test) is a two-stage assay and was thus found to be time consuming, tedious and tended to become inaccurate with long incubation periods and a large number of samples. A semiautomatic version of the test is reported in which the synthetic peptide Bz-ILE-GLU-GLY-ARG-pNA (S-2222) is added to recalcified, diluted factor IX concentrate in the micro-cuvette of a multiple sample recording spectrophotometer. Information can be obtained on (a) the amount of Xa (if any) present prior to recalcification (b) the initial amount of Xa formed and (c) the time taken to activate all factor X to Xa. Direct graphical interpretation shows a number of qualitative differences between commercial preparations, but by either of the criteria (b) or (c) above, it is possible to place the different products into “activated” and “non activated” groups such that both the Xa generation times and TGt 50 tests identify the same two groups of products. This agreement also indicates that the TGt 50 test is independent of the intrinsic factor V levels in the various concentrates.

Prothrombin Activation Is Increased among Asymptomatic Carriers of the Prothrombin G20210A and Factor V Arg506Gln Mutations

2000 ◽  
Vol 84 (09) ◽  
pp. 396-400 ◽  
Author(s):  
Steve Humphries ◽  
Belinda Smillie ◽  
Lily Li ◽  
Jacqueline Cooper ◽  
Samad Barzegar ◽  
...  
Keyword(s):  
Factor Viia ◽  
Conclusive Evidence ◽  
Factor Ix ◽  
Factor Vii ◽  
Prothrombin G20210a ◽  
Factor V ◽  
Factor X ◽  
Coagulation Proteins ◽  
Prothrombin Activation

SummaryThe risk of venous thrombosis is increased in individuals who carry specific genetic abnormalities in blood coagulation proteins. Among Caucasians, the prothrombin G20210A and factor V Arg506Gln (FV R506Q) mutations are the most prevalent defects identified to date. We evaluated their influence on markers of coagulation activation among participants in the Second Northwick Park Heart Study, which recruited healthy men (aged 50–61 years) from nine general medical practices in England and Wales. They were free of clinical vascular disease and malignancy at the time of recruitment. Genotypes for the two mutations were analyzed using microplate array diagonal gel electrophoresis, and coagulation markers (factor XIIa; activation peptides of factor IX, factor X, and prothrombin; fibrinopeptide A) were measured by immunoassay. Factor VII coagulant activity and factor VIIa levels were determined by a functional clotting assay. Among 1548 men genotyped for both mutations, 28 (1.8%) and 52 (3.4%) were heterozygous for prothrombin G20210A and FV R506Q, respectively. The only coagulation marker that was significantly associated with the two mutations was prothrombin activation fragment F1+2 [mean ± SD, 0.88 ± 0.32 nmol/L in men with prothrombin G20210A (p = 0.002) and 0.89 ± 0.30 in men with FV R506Q (p = 0.0001) versus 0.72 ± 0.24 among non-carriers for either mutation]. This data provides conclusive evidence that heterozygosity for the prothrombin G20210A as well as the FV R506Q mutations in the general population leads to an increased rate of prothrombin activation in vivo.

Removal from Bovine Prothrombin of the Substrate for Russell’s Viper Venom

1969 ◽  
Vol 21 (02) ◽  
pp. 203-216 ◽  
Author(s):  
J. H Milstone ◽  
N Oulianoff
Keyword(s):  
Calcium Chloride ◽  
Barium Sulfate ◽  
Deae Cellulose ◽  
Factor V ◽  
Factor X ◽  
Bovine Plasma ◽  
Russell’S Viper ◽  
Repeated Passage ◽  
Almost All ◽  
Filter Cakes

SummaryBovine prothrombin was prepared by adsorption on barium sulfate. After elution, it was passed through thick filter-cakes of Standard Super-Cel, which removed some venom substrate (factor X). Almost all the remaining venom substrate was removed by repeated passage through columns of DEAE-cellulose. Finally, the ratio of venom substrate to prothrombin was considerably less than 1/1,000 that of plasma. The prothrombin was also poor in factor V. It yielded very little thrombin upon incubation with Russell’s viper venom, factor V, phospholipid and calcium chloride. However, inclusion of bovine plasma at a final dilution of 1/10,000 caused the mixture to produce thrombin rapidly. This system offers promise for the assay of venom substrate in plasma.Thrombokinase derived from bovine plasma was able, at 0.000071 mg/ml, to substitute for both the venom and its substrate in thrombin-producing systems. However, with this small amount of thrombokinase, phospholipid was indispensable. The system was sensitive to 0.00001 mg phospholipid/ml.With 1,000 times as much thrombokinase, prothrombin was activated without addition of accessory factors, in the presence of oxalate. Removal of venom substrate did not affect this response of prothrombin. Nor did removal of venom substrate from the prothrombin prevent its activation by crystallized trypsin in the presence of oxalate.

Substrate Specificity Of The Coagulant Enzymes From The Venom Of Vipera Russellii Russellii And Vipera Russellii Siamensis

1981 ◽  
Author(s):  
Kathrine Kovach Lavine ◽  
Craig M Jackson
Keyword(s):  
Gel Electrophoresis ◽  
Protein C ◽  
Sodium Dodecyl ◽  
Activated Protein C ◽  
Factor Ix ◽  
Factor V ◽  
Chromogenic Substrate ◽  
Factor X ◽  
Substrate Specificities ◽  
Carboxyglutamic Acid

Two enzymes that increase the rate of coagulation, are present in the venom of Vipera russellii and have been widely used as tools in investigating coagulation. Venoms from two subspecies of Vipera russellii are available, but the differences between these subspecies has not previously been investigated. The substrate specificities of these two enzymes and possible differences between the same enzyme from the two subspecies of Russell’s viper have not been extensively characterized. The “Factor X activator” activates not only Factor X, but Factor IX and Protein C. The relative rates of activation of these zymogens have been investigated by both their chromogenic substrate hydrolase activity and sodium dodecyl sulfate gel electrophoresis and have been observed to be markedly different. Factor X is activated far more rapidly than either Protein C or Factor IX. The “Factor X activator” from V. r. russellii and V. r. siamensis differ in their activities on these zymogens as well. Elimination of the region of the light chain of Factor X that contains the gamma carboxyglutamic acid residues also markedly reduces the rate at which it can be activated.The “Factor V activator”, which mimics thrombin in activating Factor V, appears to be much more specific. Little or no difference in the rate of Factor V activation by the enzymes from the two subspecies has been observed. Most importantly, the “Factor V activator” does not activate Protein C, making it a particularly useful tool for investigation of Factor V when the effects of Factor V degradation by activated Protein C are to be avoided.

Molecular evolution of the vertebrate blood coagulation network

2003 ◽  
Vol 89 (03) ◽  
pp. 420-428 ◽  
Author(s):  
Colin Davidson ◽  
Robert Hirt ◽  
Kalpana Lal ◽  
Philip Snell ◽  
Greg Elgar ◽  
...  
Keyword(s):  
Blood Coagulation ◽  
Serine Proteases ◽  
Large Scale ◽  
Factor Ix ◽  
Supplementary Information ◽  
Factor Vii ◽  
Factor V ◽  
Factor X ◽  
Sequence Alignments ◽  
Comparative Sequence

SummaryIn mammalian blood coagulation 5 proteases, factor VII (FVII), factor IX (FIX), factor X (FX), protein C (PC) and prothrombin act with two cofactors factor V and factor VIII to control the generation of fibrin. Biochemical evidence and molecular cloning data have previously indicated that blood coagulation involving tissue factor, prothrombin and fibrinogen is present in all vertebrates. Using degenerate RT-PCR we have isolated and characterized novel cDNAs with sequence identity to the blood coagulation serine proteases and cofactors from chicken and the puffer fish (Fugu rubripes). Sequence alignments, phylogenetic and comparative sequence analysis all support the existence of the Gla-EGF1-EGF2-SP domain serine proteases FVII, FIX, FX, PC and the A1-A2-B-A3-C1-C2 domain protein cofactors FV and FVIII in these species. These results strongly suggest that the blood coagulation network is present in all jawed vertebrates and evolved before the divergence of tetrapods and teleosts over 430 million years ago; and that vertebrate blood coagulation may have benefited from two rounds of gene or whole genome duplication. Sequences identified in Fugu coding for additional FVII-like, FIX-like and PC-like sequences support the possibility of further tandem and large-scale duplications in teleosts. Comparative sequence analyses of amino acid residues in the active site region suggest these additional sequences have evolved new and as yet unknown functions.Supplementary information to this article available at both http://europium.csc.mrc.ac.uk and www.thrombosis-online.com

Treatment of Hereditary Protein C Deficiency with Stanozolol

1987 ◽  
Vol 57 (01) ◽  
pp. 020-024 ◽  
Author(s):  
A W Broekmans ◽  
J Conard ◽  
R G van Weyenberg ◽  
M H Horellou ◽  
C Kluft ◽  
...  
Keyword(s):  
Plasma Protein ◽  
Protein C ◽  
Protein S ◽  
Fold Increase ◽  
Factor Ix ◽  
Factor Vii ◽  
Type I ◽  
Factor V ◽  
Factor X ◽  
Protein C Deficiency

SummaryFive type I protein C deficient male patients received 5 mg stanozolol b.i.d. during 4 weeks. After four weeks of treatment plasma protein C activity increased from 0.42 to 0.74 U/ml and protein C antigen from 0.49 to 0.75 U/ml. This approximately 1.6 fold increase in plasma protein C was accompanied by an increase in factor II antigen (1.5 fold), factor V activity (1.6 fold), factor X antigen (1.1 fold), antithrombin III antigen (1.3 fold) and heparin cofactor II antigen (1.5 fold), while the concentration of factor VII, factor VIII, and factor IX activity, and of protein S antigen remained unchanged. Prothrombin fragment F1+2, measured in two patients, increased 1.3 fold. In addition to its effect on procoagulant and anticoagulant factors stanozolol had profibrinolytic effects, reflected in an increase in tPA activity and in the concentration of plasminogen. These data indicate that in type I protein C deficient patients stanozolol increases the concentrations of both procoagulant and anticoagulant factors and favours fibrinolysis. The efficacy of stanozolol in preventing thrombotic disease in type I protein C deficient patients, however, remains to be established. During the four weeks of stanozolol treatment no thrombotic manifestations were observed in the protein C deficient patients.

Autoprothrombin C in Irregular Blood Clotting

1962 ◽  
Vol 08 (01) ◽  
pp. 001-020
Author(s):  
Walter H. Seegers ◽  
Eva Marciniak
Keyword(s):  
Blood Clotting ◽  
Hemophilia A ◽  
Deae Cellulose ◽  
Hemophilia B ◽  
Factor Vii ◽  
Factor X ◽  
The Common ◽  
Autoprothrombin C ◽  
Derivatives Of ◽  
Autocatalytic Activation

SummaryIn this study autoprothrombin C was considered in relationship to the hemorrhagic diseases. Concentrates of autoprothrombin C were made from purified prothrombin known to be homogeneous by several criteria. These autoprothrombin C preparations were free of thrombin, and were almost a single component when analyzed by centrifugation. Autoprothrombin C corrected the partial thromboplastin time and produced prothrombin consumption in all the plasmas obtained from patients with hemorrhagic diseases except parahemophilia. In the case of hemophilia B prothrombin consumption was obtained with the addition of purified prothrombin or purified autoprothrombin II. In the case of Stuart plasma prothrombin consumption was rapid after the addition of purified prothrombin or purified prothrombin chromatographed on Amberlite ICR-50, but not after the addition of purified prothrombin that was chromatographed on DEAE cellulose. The latter prothrombin is an abnormal prothrombin molecule and does not readily yield autoprothrombin C. It is concluded that Stuart prothrombin is abnormal and that this is a molecular disease. There is no need to postulate the existence of factor X to account for the irregular prothrombin activation in Stuart plasma. The most important question considered was how can autoprothrombin C be generated from prothrombin to promote the autocatalytic activation of prothrombin. Certain prothrombin molecules do not yield this enzyme, but normal prothrombin does. However, it does so only under certain conditions of activation. By applying the new knowledge of prothrombin chemistry to blood clotting irregularities in hemorrhagic diseases the following main considerations highlight the common deviations: 1. Abnormal prothrombin molecule, 2. Changes related to the derivatives of prothrombin, 3. Accessories needed for the generation of autoprothrombin C so it can function in auto-catalysis are irregular, and 4. Accessories needed for the function of autoprothrombin C after it is out of the prothrombin molecule are irregular. In the first group is Stuart Plasma. In the second group falls hemophilia B (autoprothrombin II) and the so-called factor VII deficiency (autoprothrombin I). In the third group is hemophilia A. In the fourth group is parahemophilia, and the platelet abnormalities.

Generation Of Coagulation Factors By The Isolated Rat Liver Perfused With A Synthetic Blood Substitute

1981 ◽  
Author(s):  
C A Owen ◽  
E J W Bowie
Keyword(s):  
Factor Viii ◽  
Rat Liver ◽  
Blood Substitute ◽  
Factor Ix ◽  
Factor Vii ◽  
Clotting Factor ◽  
Isolated Perfused Rat Liver ◽  
Factor Xii ◽  
Factor V ◽  
Factor X

Measuring the release of small amounts of a clotting factor from an isolated perfused rat liver is difficult if the perfusate already contains some of the factor. Further, platelet-containing perfusates generate a coagulant activity that may invalidate clotting assays.We have successfully employed a completely synthetic blood substitute for rat liver perfusions. The perfusate is “Fluosol-43” generously furnished by Alpha Therapeutic Corporation. The oxygen-carrying perfluorochemical is FC-43 (perfluorotributylamine) and the substitute for albumin is hydroxyethyl starch. Using the Brauer perfusion technique, we found that rat livers in 5 hours released an average of 2.3% of the normal plasma concentration of prothrombin, 8.4% factor V, 16.2% factor VII, 7.0% factor IX, 3.7% factor X, 28.3% factor XI and 12.3% factor XII. Antithrombin III and plasminogen were also generated.Only minute amounts of factor VIII were released unless serum, cryoprecipitate or cryoprecipitate-free plasma was added; then the yield was 8.8% on average. The more “venom factor” (platelet aggregability with Bothrops alternata venom) added to the synthetic perfusate, the more factor VIII was released.

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