The Assay of Factor VII in its Native and Activated Forms

1979 ◽  
Author(s):  
T.J. Snape ◽  
D. Griffiths

The preparation and use for factor VII assay of an artificial human factor VII-deficient substrate plasma is reported. Results of factor VII assays carried out using this plasma as substrate were closely comparable with values obtained using congenitally deficient plasma.Neither of these assay systems distinguishes between factor VII in its native and activated forms. The activation state of factor VII in plasma and in a therapeutic factor VII concentrate has been investigated using an amidolytic assay (Seligsohn et al, 1978, Blood, 52, 978) and also by a method involving conventional assay before and after incubation with heparin (Godal et al, 1974, Thromb. Res., 5, 773). The results suggest that factor VII in the concentrate is unactivated and is stable with respect to attempts to activate it by cold activation in the presence of contact activators.

1998 ◽  
Vol 79 (01) ◽  
pp. 104-109 ◽  
Author(s):  
Osamu Takamiya

SummaryMurine monoclonal antibodies (designated hVII-B101/B1, hVIIDC2/D4 and hVII-DC6/3D8) directed against human factor VII (FVII) were prepared and characterized, with more extensive characterization of hVII-B101/B1 that did not bind reduced FVIIa. The immunoglobulin of the three monoclonal antibodies consisted of IgG1. These antibodies did not inhibit procoagulant activities of other vitamin K-dependent coagulation factors except FVII and did not cross-react with proteins in the immunoblotting test. hVII-DC2/D4 recognized the light chain after reduction of FVIIa with 2-mercaptoethanol, and hVIIDC6/3D8 the heavy chain. hVII-B101/B1 bound FVII without Ca2+, and possessed stronger affinity for FVII in the presence of Ca2+. The Kd for hVII-B101/B1 to FVII was 1.75 x 10–10 M in the presence of 5 mM CaCl2. The antibody inhibited the binding of FVII to tissue factor in the presence of Ca2+. hVII-B101/B1 also inhibited the activation of FX by the complex of FVIIa and tissue factor in the presence of Ca2+. Furthermore, immunoblotting revealed that hVII-B101/B1 reacted with non-reduced γ-carboxyglutaminic acid (Gla)-domainless-FVII and/or FVIIa. hVII-B101/B1 showed a similar pattern to that of non-reduced proteolytic fragments of FVII by trypsin with hVII-DC2/D4 on immunoblotting test. hVII-B101/B1 reacted differently with the FVII from the dysfunctional FVII variant, FVII Shinjo, which has a substitution of Gln for Arg at residue 79 in the first epidermal growth factor (1st EGF)-like domain (Takamiya O, et al. Haemosta 25, 89-97,1995) compared with normal FVII, when used as a solid phase-antibody for ELISA by the sandwich method. hVII-B101/B1 did not react with a series of short peptide sequences near position 79 in the first EGF-like domain on the solid-phase support for epitope scanning. These results suggested that the specific epitope of the antibody, hVII-B101/B1, was located in the three-dimensional structure near position 79 in the first EGF-like domain of human FVII.


1976 ◽  
Vol 36 (01) ◽  
pp. 104-114 ◽  
Author(s):  
D. L Aronson ◽  
A. J Mustafa

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.


1960 ◽  
Vol 04 (02) ◽  
pp. 253-260 ◽  
Author(s):  
Franco Gobbi

SummaryThe fractionation properties of human Factor VIII (antihaemophilic factor, AHF, antihaemophilic globulin) have been studied using a plasma of congenital afibrinogenaemia as a starting material.From a fibrinogen-free plasma, Factor VIII does not precipitate with ethanol at a final concentration of 8%; on the contrary the maximum yield is reached at an ethanol concentration of 25%.With a precipitation method carried out by a one to ten dilution of plasma with distilled water and acidification by N/10 hydrochloric acid to a pFI 5.2, Factor VIII does not precipitate with the euglobulin fraction; when normal plasma is used, such a precipitation is almost complete.With the salting-out fractionation method by ammonium sulphate, Factor VIII precipitates at a concentration between 25 and 33% of saturation either from fibrinogen-free and from normal human plasma.A non-specific thromboplastic activity appears in the fractions prepared by every method. This activity, which is probably due to the activation of seric accelerators, is easily removed by Al(OH)s adsorption. Thus, in order to insure the specificity of Factor VIII assays, the preliminary adsorption of the fractions is indispensable before testing their antihaemophilic activity.Fibrinogen and Factor VIII have different and definite precipitation patterns. When these two factors are associated the fractionation properties of AHF appear quite modified, showing a close similarity to those of fibrinogen. This fact can explain the technical difficulties encountered in the attempt to purify the antihaemophilic factor, and the lack of reproducible procedures for removing fibrinogen without affecting Factor VII.


1995 ◽  
Vol 73 (02) ◽  
pp. 239-242 ◽  
Author(s):  
E M Bladbjerg ◽  
T Tholstrup ◽  
P Marckmann ◽  
B Sandström ◽  
J Jespersen

SummaryThe mechanisms behind dietary effects on fasting coagulant activity of factor VII (FVII: C) are not clarified. In the present study of 15 young volunteers, two experimental diets differing in composition of saturated fatty acids (C18:0 [diet S] or C12:0 + C14:0 [diet ML]) were served for 3 weeks each. Fasting blood samples were collected before and after the dietary regimen and analysed for triglycerides, FVII:C, and protein concentrations of FVII, FII, FX, protein C, CRP, albumin, fibrinogen, and F1+2. FVII:C was significantly reduced on diet S compared with diet ML. This was accompanied by a decrease in FVII protein, F1+2 and the vitamin K-dependent proteins FII, FX, and protein C. In contrast, no changes were observed in triglycerides, FVII:C/FVII: Ag, albumin and CRP. Fibrinogen was increased on diet S compared with diet ML. Our findings suggest that the change in fasting FVII:C was part of a general change in concentrations of vitamin K-dependent proteins.


1986 ◽  
Vol 56 (03) ◽  
pp. 250-255 ◽  
Author(s):  
C Boyer ◽  
M Wolf ◽  
C Rothschild ◽  
M Migaud ◽  
J Amiral ◽  
...  

SummaryA new solid phase enzyme-linked immunosorbent assay (ELISA) was developed for the quantitation of human Factor VII antigen (F VII Ag), using a monospecific rabbit anti-F VII antiserum. Anti-F VII F(ab′)2 fragments were adsorbed to polystyrene plates. The binding of serial dilutions of control or test plasma, containing F VII, was detected by incubation with peroxidase-labeled anti- FV II IgG followed by the addition of hydrogen peroxyde and O-phenylenediamine. This ELISA is specific, sensitive (detection limit: 0.05%) and accurate (coefficient of variation: 1.5-4% for within- and 1.6-9% for between-assays). F VII coagulant activity (F VII C) and F VII Ag were determined in large populations of controls and patients. In normal plasma (n = 38), F VII Ag ranged from 83 to 117% and the correlation coefficient between F VII Ag and F VII C was 0.94. In patients with severe (F VII C inf. 1%) congenital F VII deficiency (n = 5), F VII Ag was undetectable in two cases (inf. 0.05%) and markedly reduced (0.35 to 5.6%) in the three other cases. In patients with liver cirrhosis (n = 15), F VII Ag ranged from 21 to 59% and was in good correlation with F VII C (r = 0.84). In dicoumarol treated patients (n = 15), the levels of F VII Ag ranged from 51% to 79% and a poor correlation (r = 0.52) with F VIIC was observed. In “compensated” DIC (n = 5), levels of F VII Ag varied from 60 to 186%, with significantly higher F VII C levels (from 143 to 189%). In contrast, in “decompensated” DIC (n = 7), low F VII Ag and F VII C levels were observed (from 7 to 27%). In patients with deep-vein thrombosis (n = 25), high levels of F VII Ag (from 102 to 136%) and F VII C (from 110 to 150%) were demonstrated. In surgical patients, no significant difference was observed before and one day after intervention.


1981 ◽  
Author(s):  
J Katz ◽  
E Rodriguez ◽  
C Madani ◽  
D Hicks ◽  
H E Branson

Thirty-two newborns with elevated capillary hematocrits >65% were studied. Twenty-two newborns required plasmaexchange transfusion. All had central (venous) hematocrits >65% and had symptoms referrable to complications associated with this syndrome. Of the 22, 15 were appropriate-for-gestational age, 5 were small-for-gestational age, and 2 were large-for-gestational age. Viscosity measurements in the 10 newborns who did not require plasma-exchanges showed increased viscosity in 2 in the slow shear rates associated with bloodflow in the smaller vessels. Coagulation data before and after plasma exchange did not show a hypercoagulable state: PT-14.2±0.7 and 12.9±1.2 secs, PTT 49.9±3.6 and 42.2±3.2 secs, factor VII 73±5 and 78±5%, factor VIII 103±10 and 94±10%, AT III levels were low 14±1.2 and 17±1.3 mg/dl, fibrin degradation products were <10μg/ml, fibrin monomer was not detected, plasminogen levels were 5±0.8 and 7±0.9mg/dl, fibrinogen levels were 203±9.8 and 200±11.8 mg%. Vitamin K dependent factors were reduced factor V 44±6 and 49±11%, factor VII 77±5 and 86±5%, factor IX 28±2 and 42±3%, factor X 35±4 and 62±6%, factor XI 55±5 and 84±9%, factor XII 47±5 and 63±5%. Statistical significant differences were found only with factors IX, X, XI and XII. Thrombocytopenia was present in 6 patients (20% incidence) and post plasma exchange the platelet counts rose significantly and in 2 patients within 3 days reached normal levels. No statistical difference in the platelet counts were noted before and after the plasma-exchange and were similar to the levels determined in 10 newborn controls. Neonatal polycythemia with thrombocytopenia may indicate a more severe disorder, with hematocrits in the 6 patients >70%. It is suggested that the mechanism of the thrombocytopenia may be aggregates of platelets that deaggregate following plasmaexchange. The complications associated with neonatal polycythemia appear related to hyperviscosity, erythrocyte and platelet “sludging” in the smaller vessels.


1997 ◽  
Vol 20 (3) ◽  
pp. 163-165 ◽  
Author(s):  
M. Camici ◽  
L. Evangelisti ◽  
P. Balestri ◽  
L. Cioni ◽  
P. Fundi ◽  
...  

The Authors evaluated the behavior of protein C activity, factor X and factor VII coagulant activity and serum lipoprotein(a) before and after dialytic treatment in patients on maintenance hemodialysis. They observed depressed protein C activity that significantly (p<0.005) increased and became normal immediately after hemodialysis while factor X and factor VII increased (p<0.01; p<0.05) despite heparinization together with amount of serum lipoprotein(a). In vitro incubation (30 'at 37°C) of uremic and healthy blood showed a decrease in serum lipoprotein(a) concentration. After heparin addition (final concentration 0.5 U/ml) lipoprotein(a) increased in the uremic blood only. The clinical and physiopathological implications of these results are discussed.


Author(s):  
George J. Broze ◽  
Philip W. Majerus
Keyword(s):  

1986 ◽  
Vol 51 (0) ◽  
pp. 531-541 ◽  
Author(s):  
K. Berkner ◽  
S. Busby ◽  
E. Davie ◽  
C. Hart ◽  
M. Insley ◽  
...  
Keyword(s):  

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