Factor V from Human Plasma

1961 ◽  
Vol 05 (01) ◽  
pp. 001-020
Author(s):  
Douglas M. Surgenor ◽  
Nancy A. Wilson ◽  
Anne S. Henry
Keyword(s):  
Human Serum ◽  
Human Plasma ◽  
Kinetic Data ◽  
Human Factor ◽  
Kinetic Studies ◽  
Partial Purification ◽  
Factor V ◽  
Two Stage ◽  
Plasma Factor ◽  
Tissue Thromboplastin

SummaryA method is described for the partial purification of a human plasma factor which accelerates the conversion of prothrombin to thrombin in the presence of tissue thromboplastin. This factor may be dried from the frozen state, and may be kept in stable dry form for long periods of time. The quantitative assay of this activity is done in a classical two-stage prothrombin system using tissue thromboplastin and calcium. From its properties, it is concluded that this activity corresponds to factor V, labile factor and plasma Ac-globulin.Chemical and kinetic studies reveal that human factor V is active in plasma and is destroyed by thrombin. Human serum has little or no factor V activity.These results thus fail to support the postulated activation of factor V during clotting. All of the kinetic data are consistent with an enzymatic role for factor V in the formation of tissue prothrombin activator (thromboplastin).

Plasma Factor V Activation Is Prevented by Activated Protein C in the Presence of Phospholipid Vesicles, Not Platelets

1993 ◽  
Vol 69 (02) ◽  
pp. 124-129 ◽  
Author(s):  
Susan Solymoss ◽  
Kim Thi Phu Nguyen
Keyword(s):  
Western Blotting ◽  
Protein C ◽  
Thrombin Generation ◽  
Blood Platelets ◽  
Activated Protein C ◽  
Phospholipid Vesicles ◽  
Factor V ◽  
Two Stage ◽  
One Stage ◽  
Plasma Factor

SummaryActivated protein C (APC) is a vitamin K dependent anticoagulant which catalyzes the inactivation of factor Va and VIIIa, in a reaction modulated by phospholipid membrane surface, or blood platelets. APC prevents thrombin generation at a much lower concentration when added to recalcified plasma and phospholipid vesicles, than recalcified plasma and platelets. This observation was attributed to a platelet associated APC inhibitor. We have performed serial thrombin, factor V one stage and two stage assays and Western blotting of dilute recalcified plasma containing either phospholipid vesicles or platelets and APC. More thrombin was formed at a given APC concentration with platelets than phospholipid. One stage factor V values increased to higher levels with platelets and APC than phospholipid and APC. Two stage factor V values decreased substantially with platelets and 5 nM APC but remained unchanged with phospholipid and 5 nM APC. Western blotting of plasma factor V confirmed factor V activation in the presence of platelets and APC, but lack of factor V activation with phospholipid and APC. Inclusion of platelets or platelet membrane with phospholipid enhanced rather than inhibited APC catalyzed plasma factor V inactivation. Platelet activation further enhanced factor V activation and inactivation at any given APC concentration.Plasma thrombin generation in the presence of platelets and APC is related to ongoing factor V activation. No inhibition of APC inactivation of FVa occurs in the presence of platelets.

scholarly journals The Effects of Injection of Human Factor VIII Antibody Into Rabbits

Blood ◽  
1973 ◽  
Vol 42 (4) ◽  
pp. 509-521 ◽  
Author(s):  
S. M-C. Shen ◽  
D. I. Feinstein ◽  
S. I. Rapaport
Keyword(s):  
Factor Viii ◽  
Human Factor ◽  
Rabbit Model ◽  
Kinetic Studies ◽  
Factor Vii ◽  
Factor V ◽  
Initial Values ◽  
Human Factor Viii ◽  
Hemolytic Complement Activity ◽  
Antigen Antibody

Abstract Rabbits were injected with an immunoglobulin fraction of human serum containing a factor VIII antibody. Factor VIII levels fell abruptly, persisted below 10% of a rabbit plasma standard for 12 hr, and returned to normal by 120-168 hr. The factor VIII antigen-antibody reaction did not result in Intravascular clotting as evaluated by kinetic studies with 125I-fibrinogen. However, small falls in factor V and factor VII levels were observed over a 6-hr period after the injection. Platelets fell to about one-half of initial values within 15 min, rose to 80% of initial levels over 2 hr, and subsequently declined to 65%-70% of initial levels. WBC levels fell to below 20% of initial values 2 hr after the injection but returned to about 75% of initial values by 6 hr. Total hemolytic complement activity was unaffected. Animals made granulocytopenic with nitrogen mustard and animals with hereditary C'6 deficiency behaved similarly to normal animals. One may conclude that the injection of human factor VIII antibody into rabbits produces a rabbit model with impaired intrinsic coagulation suitable for studies of the mechanism of endotoxin-induced intravascular clotting.

Preparation and use of a Combined Reagent for the Two-Stage Assay of Factor VIII Procoagulant Activity

1979 ◽  
Author(s):  
N. Pettet ◽  
V.K. Hirst
Keyword(s):  
Factor Viii ◽  
Close Agreement ◽  
Comparative Assessment ◽  
Procoagulant Activity ◽  
Factor V ◽  
Two Stage ◽  
Freeze Dried ◽  
Significant Difference ◽  
Plasma Factor ◽  
Assay Methods

Activated serum, bovine factor V and phospholipid, diluted In barbitone sodium saline pH 7.3 were prepared as a pooled reagent and freeze-dried in aliquots. Normal plasma with CPD as anticoagulant was used as the substrate. The reagent was easily prepared, is convenient to use and has been assessed in three independent trials. Repeated assays performed using the International standard for factor VIII and the British Standards for plasma factor VIII and concentrate factor VIII provided combined potency estimates which showed no significant difference from the values stated for these standards. In a group study, where seven participants performed two-stage assays using their normal reagents and techniques, the potency estimates obtained by RPL using the combined reaqent were in close agreement with those obtained by workers with other assay methods. Quality control measurements of procoagulant activity in factor VIII concentrate performed in duplicate at RPL and plasma Fractionation Laboratory PFL , Oxford over a period of several months provided a satisfactory comparative assessment of the combined reagent assay and the two-stage factor VIII assay used at PFL. Use of the combined reagent in a simplified two-stage assay has been studied and its suitability in automatic techniques has beer shown to be acceptable.

Isolation of a New Enzyme from Human Plasma Fractions and its Effects on Coagulation

1979 ◽  
Author(s):  
Michael H. Coan ◽  
Duane D. Schroeder ◽  
Milton M. Mozen
Keyword(s):  
Human Plasma ◽  
Human Factor ◽  
Active Form ◽  
Factor V ◽  
Diisopropyl Fluorophosphate ◽  
Human Factor Viii ◽  
Plasma Fractions ◽  
Related Proteins

A new hydrolase enzyme has been isolated from human plasma fractions. This enzyme has been purified to homogeneity by adsorption to and elution from DEAE-5ephadex, Benzamidine- EACA-Sepharose, and Heparin-5epharose. The molecular weight, as judged by SDSpolyacrylamide gel electrophoresis, is 70,000 daltons; and there are 2 peptide chains (45,000 and 25,000) after reduction. This enzyme, isolated in an active form, hydrolyzes 5-2238 (H-D-Phe-Pip-Arg-PNA), a chromogenic substrate (AB Kabi) , at a rapid rate, S-225l to a slishtly lesser extent, and S-2222, S-2302, and 5-2160 about equally and much more slowly. CaCl2 greatly enhances its activity. The enzyme is inhibited by antithrombin III especially in the presence of heparin, but poorly by soybean trypsin inhibitor or by diisopropyl fluorophosphate. The enzyme binds to and can be eluted from insoluble barium salts. In vitro, the protein will activate and degrade human Factor VIII, will activate human Factor V, and will inhibit epinephrine-induced platelet aggregation; however, the in vivo function is unknown . Comparison with known properties of protein C and other coagulation-related proteins indicates. that this enzyme has not been previously described.

Human Plasma And Platelet Factor V Levels As Measured By Radioimmunoassay

1981 ◽  
Author(s):  
P B Tracy ◽  
J M Peterson ◽  
M E Nesheim ◽  
J A Katzmann ◽  
K G Mann
Keyword(s):  
Human Factor ◽  
Specific Activity ◽  
Human Platelets ◽  
Prolonged Storage ◽  
Factor V ◽  
Platelet Factor ◽  
Average Value ◽  
Standard Curve ◽  
Plasma Factor ◽  
Bioassay Data

Highly purified human Factor V was used for the development of a competitive double antibody radioimmunoassay (RIA) using 125I-human Factor V, burro anti-human Factor V antisera as the primary antibody and goat anti-burro antisera as the precipitating antibody. The standard curve allows the detection of as little as 20 ng of Factor V per ml of plasma. With this specific RIA for human Factor V, we have measured the level of Factor V in the plasma and platelets of normal individuals. The normal level of Factor V in plasma ranges from 4 to 14 μg per ml, with the average value equal to 7.0 ± 2.0 μg/ml (n = 64; 33 females; 31 males; 22 to 61 years of age). There appeared to be no correlation between antigen levels and age or sex. Factor V clotting assays were consistent with the RIA data for any given plasma preparation providing freshly drawn plasma was used in the bioassay. The bioassay data were quantitated based upon the specific activity of purified plasma Factor V; 1.7 units of Factor V equals 1 μg of protein. Plasma Factor V antigen levels were not affected by lyophilization of the plasma, prolonged storage of the plasma at -20°C or intentional conversion of the plasma to serum. The levels of Factor V present in washed human platelets were also determined using the RIA. Assay of washed platelets lysed in 0.2% Triton X-100 indicated that 0.6 to 0.85 μg of Factor V was present per 2.5 × 108 platelets (4400 to 6200 molecules of Factor V per platelet). This result is in marked contrast to our observations for the bovine system, where we found that bovine platelets possess approximately 400 to 800 molecules of Factor V per platelet, and plasma Factor V levels range from 30 to 50 μg per ml. In the bovine system, the platelets possess approximately 1% of the total Factor V present, while in human blood, the platelets possess as much as 10 to 15% of the total Factor V present.

Improved Purification Method Of Human Plasma Factor V And Applications Of A Rabbit Anti-Human Factor V Antibody

1981 ◽  
Author(s):  
C Breederveld ◽  
T Bruin ◽  
A Sturk ◽  
Th Hakvoort ◽  
J W ten Cate
Keyword(s):  
Human Factor ◽  
Specific Binding ◽  
Blood Platelets ◽  
Rabbit Antiserum ◽  
Factor V ◽  
Binding Studies ◽  
Neutralizing Activity ◽  
Fab Fragments ◽  
Α2 Macroglobulin ◽  
Plasma Factor

Human factor V, purified from citrated plasma by a method recently described by us (Bolnuis et al 1979) was found to be slightly contaminated with α2-macroglobulin. A rabbit antiserum to this preparation, obtained by frequent subcutaneous immunization, showed factor V procoagulant neutralizing activity. Immune electrophoresis demonstrated precipitation lines with α2-macroglobulin. This contamination was succesfully removed from the factor V preparation by affinity chromatography with specific raboit anti human α2-macroglobulin bound to Sepharose 2B. The antiserum to the original, impure factor V preparation showed no more precipitation arcs after absorption with 3% normal plasma and ammonium sulphate precipitation of the complexes. Factor V procoagulant neutralizing activity titre was estimated 1: 8 (50% neutralization after 1 hour incubation at 37°C.) This antiserum was used for the following studies: 1. An inhibitor neutralization assay was developed for measuring antigenic activities of the factor V molecule. Antigen concentration in a range from 12.5-100% could be reliably measured. 2. Fab-fragments, obtained by papain digestion of the purified IgG (by protein A SePharose chromatography) were labeled with 125I by an immobilized lactoperoxidase metnod. These Fab-fragments fully retained their factor V procoagulant neutralizing activity. 125I-Fab-frag ments were used in specific binding studies to both non-activated and ADP and thrombin-activated human blood platelets.

A High Titre Circulating Inhibitor of Human Factor V: Clinical, Biochemical and Immunological Features and its Treatment by Plasmapheresis

1975 ◽  
Author(s):  
G. S. Grace ◽  
P. Wolf
Keyword(s):  
Human Factor ◽  
Kinetic Studies ◽  
High Titre ◽  
Factor V ◽  
Anamnestic Response ◽  
Normal Plasma ◽  
History Of ◽  
Viii And Ix ◽  
Assay Methods ◽  
Very High

A 44 year old male with a two month history of epistaxis and haematuria was admitted because of melaena. He had not received antibiotics or blood transfusions. Haemostatic studies showed a complete absence of Factor V activity and this was not corrected by the addition of an equal part of normal plasma. Incubation studies showed that the inhibitor was specific for human Factor V. Factor VIII and IX activities were low by one stage assay methods but were normal after the addition of bovine Factor V.The titre of the antibody was very high; a 900 fold dilution causing a 50% reduction in the Factor V level of an equal volume of normal plasma on incubation at 37° C for 30 mins. The inhibitor appeared in the second protein peak on Sephadex G200 fractionation and was neutralized by antihuman IgG antiserum. Kinetic studies showed the inhibition to be first order with respect to Factor V and inhibitor.The patient was treated with plasmapheresis on a IBM Celltrifuge. Four litres of plasma were removed on each of four occasions over a period of 14 days. The antibody titre dropped from 900 to about 100. Despite the addition of immunosuppresive therapy the antibody has persisted at a titre of about 200 and has now been present over 5 months. No anamnestic response occurred following multiple transfusions.

Kinetics of Human Blood Coagulation Induced by Trypsin

1964 ◽  
Vol 12 (01) ◽  
pp. 307-330 ◽  
Author(s):  
E. T Yin
Keyword(s):  
Human Serum ◽  
Trypsin Inhibitor ◽  
Soybean Trypsin Inhibitor ◽  
Factor Vii ◽  
Factor V ◽  
Factor X ◽  
Serum Factor ◽  
Clotting System ◽  
Plasma Factor ◽  
Kinetics Of

Summary1. Trypsin is a potent activator of purified human serum factor X. In the presence of calcium the activation was greatly enhanced.2. Trypsin did not activate purified human serum factor VII or crude human plasma factor V, with or without calcium.3. A new trypsin inhibitor, Benzamidine-HCl (shown to be superior to soybean trypsin inhibitor) was used. The Benzamidine-HCl - trypsin complex did not interfere in the clotting system, whereas the soybean trypsin inhibitor-trypsin complex did.4. Benzamidine-HCl was used to inactivate the trypsin at any desired stage during the activation of factor X.5. The trypsin activated factor X was found to be very stable.6. In the presence of activated factor X, factor V, cephalin and calcium, a very potent prothrombin activator was formed.7. An apparent time-consuming reaction was observed when activated factor X was incubated with factor V in the presence of calcium, which required the subsequent addition of cephalin to convert prothrombin to thrombin.8. The trypsin clotting mechanism is analogous to the clotting of blood by Russell’s viper venom.

scholarly journals Radioimmunoassay of factor V in human plasma and platelets

Blood ◽  
1982 ◽  
Vol 60 (1) ◽  
pp. 59-63 ◽  
Author(s):  
PB Tracy ◽  
LL Eide ◽  
EJ Bowie ◽  
KG Mann
Keyword(s):  
Platelet Count ◽  
Human Plasma ◽  
Whole Blood ◽  
Human Factor ◽  
Factor V ◽  
Single Chain ◽  
Average Value ◽  
Double Antibody ◽  
Antibody Competition

Abstract Homogeneous, single-chain human factor V was used to develop a double antibody competition radioimmunoassay to measure factor V concentrations in plasma and platelets. Standard curves were constructed that allow for the detection of as little as 20 ng factor V/ml of plasma. Normal factor V concentrations range from 4 to 14 micrograms/ml of plasma with an average value of 7.0 +/- 2.0 micrograms/ml (n = 64). No correlation was observed between antigen levels and age or sex. The radioimmunoassay data are consistent with factor V clotting assays, providing freshly drawn plasma is used in the bioassay. Radioimmunoassay of washed platelets indicate that 0.63–1.93 microgram of factor V is present per 2.5 X 10(8) platelets (4612–14128 molecules of the factor V platelet). When normalized to individual hematocrits and platelet count, the data indicated that platelets contribute approximately 18%-25% of the factor V found in whole blood. In addition, two individuals with functionally deficient factor V were examined and found to be deficient in both antigen and activity.

Sign in / Sign up

Export Citation Format

Share Document