Activation Of Human Factor VII By Activated Factors IX And X In Purified Systems

1981 ◽  
Author(s):  
D R Masys ◽  
S P Bajaj ◽  
S I Rapaport
Keyword(s):  
Gel Electrophoresis ◽  
Relative Efficiency ◽  
Human Factor ◽  
Polyacrylamide Gel Electrophoresis ◽  
Factor Ix ◽  
Factor Vii ◽  
Activate Factor ◽  
Factor X ◽  
One Stage ◽  
Amplification Loop

Factor VII activity, as measured in a one-stage clotting assay, increases when whole blood is clotted in glass. Prior studies in this laboratory using factor-deficient plasmas suggested that this factor VII activation was due to activated factor IX (IXa). We therefore studied activation of VII by IXa and by activated factor X (Xa) in purified systems. Human factors II, VII, IX, and X were each purified to homogeneity as judged by SDS-polyacrylamide gel electrophoresis. Reaction mixtures of VII, IXa or Xa, and other cofactors and enzymes were made, and subsampled for VII activity. The activation state of VII was judged by comparison of one-stage clotting assay to a coupled amidolytic assay using a synthetic substrate. In the presence of phospholipid (PL) and calcium (Ca), both IXa and Xa activated VII 25 fold; however, Xa was roughly 800 times more efficient than IXa. In the absence of PL, Xa was roughly 20 times more efficient than IXa, in Ca-containing solutions. Only slight activation of VII by either enzyme occurred in the absence of Ca. The addition of prothrombin (II) markedly slowed activation of VII by both Xa and IXa; however, this effect did not occur if fully-decarboxylated II was used. The addition of anti thrombin III and thrombin-modified factor VIII at physiologic concentrations did not change rates of VII activation by IXa or Xa.These results confirm the ability of IXa and Xa to activate factor VII at physiologic concentrations in purified systems. The higher relative efficiency of Xa over IXa under all conditions studied contrasts strikingly with observations in whole plasma systems where the VII activation measurable after clotting is greater in X-deficient than in IX-deficient plasma. The activation of VII by Xa and IXa may serve as an amplification loop in the generation of clotting by either “intrinsic” or “extrinsic” cascades.

scholarly journals Activation of human factor VII by activated factors IX and X

Blood ◽  
1982 ◽  
Vol 60 (5) ◽  
pp. 1143-1150 ◽  
Author(s):  
DR Masys ◽  
SP Bajaj ◽  
SI Rapaport
Keyword(s):  
Human Factors ◽  
Factor Viii ◽  
Active Site ◽  
Human Factor ◽  
Antithrombin Iii ◽  
Factor Ix ◽  
Factor Vii ◽  
Activate Factor ◽  
Factor X ◽  
Activated Factor Vii

Factor VII clotting activity increases about five-fold when blood is clotted in glass. Prior studies suggested that this results from activation induced by activated factor IX (IXa). However, in purified systems containing phospholipid and calcium, activated factor X (Xa) is known to activate factor VII rapidly. Therefore, we studied activation of factor VII by IXa and X, in systems using purified human factors. Concentrations of IXa and Xa were calculated from total activated protein concentrations rather than from active site concentrations. In the presence of phospolipid and calcium, both IXa and Xa activated factor VII 25-fold; however, Xa was roughly 800 times more efficient than IXa. Without added phospholipid, activation of factor VII by both Xa and IXa was markedly slowed, and Xa was roughly 20 times more efficient than IXa. When both phospholipid and calcium were omitted, activation of factor VII by either enzyme was negligible. Adding normal prothrombin, but not decarboxylated prothrombin, substantially slowed activation of factor VII by both Xa and IXa. Adding thrombin-activated factor VIII and antithrombin-III did not change rates of factor VII activation by either enzyme. These results from purified systems do not provide an explanation for the prior data from plasma systems.

scholarly journals Activation of human factor VII by activated factors IX and X

Blood ◽  
1982 ◽  
Vol 60 (5) ◽  
pp. 1143-1150 ◽  
Author(s):  
DR Masys ◽  
SP Bajaj ◽  
SI Rapaport
Keyword(s):  
Human Factors ◽  
Factor Viii ◽  
Active Site ◽  
Human Factor ◽  
Antithrombin Iii ◽  
Factor Ix ◽  
Factor Vii ◽  
Activate Factor ◽  
Factor X ◽  
Activated Factor Vii

Abstract Factor VII clotting activity increases about five-fold when blood is clotted in glass. Prior studies suggested that this results from activation induced by activated factor IX (IXa). However, in purified systems containing phospholipid and calcium, activated factor X (Xa) is known to activate factor VII rapidly. Therefore, we studied activation of factor VII by IXa and X, in systems using purified human factors. Concentrations of IXa and Xa were calculated from total activated protein concentrations rather than from active site concentrations. In the presence of phospolipid and calcium, both IXa and Xa activated factor VII 25-fold; however, Xa was roughly 800 times more efficient than IXa. Without added phospholipid, activation of factor VII by both Xa and IXa was markedly slowed, and Xa was roughly 20 times more efficient than IXa. When both phospholipid and calcium were omitted, activation of factor VII by either enzyme was negligible. Adding normal prothrombin, but not decarboxylated prothrombin, substantially slowed activation of factor VII by both Xa and IXa. Adding thrombin-activated factor VIII and antithrombin-III did not change rates of factor VII activation by either enzyme. These results from purified systems do not provide an explanation for the prior data from plasma systems.

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.

Genetic Polymorphism of Factor IX Gene in Greek Patients with Thrombophilia.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 4121-4121
Author(s):  
Pantelis P.E. Makris ◽  
Michel M. Iskas ◽  
Rigini R. Papi ◽  
Dimitrios D.K. Kiriakidis
Keyword(s):  
Vitamin K ◽  
Polyacrylamide Gel Electrophoresis ◽  
Control Sample ◽  
Coagulation Factor ◽  
Factor Ix ◽  
University Hospital ◽  
Activate Factor ◽  
Factor X ◽  
Coagulation Factor Ix ◽  
Pcr Products

Abstract Introduction. Coagulation factor IX plays an important intermediate role in the activation of blood coagulation. It is located within the blood plasma as a zymogen, in its inactivated state. Factor IX is dependent on the presence of Vitamin K. The structure of factor IX closely resembles the structures of many other Vitamin K dependent plasma proteins, such as prothrombin, factor X and protein C. After being activated, Factor IX forms a complex with calcium ions, membrane phospholipids and coagulation factor VIIIa to activate factor X. The exact locus of the coagulation factor IX gene was found to exist in the Xq26-q27 region of the X chromosome. The FIX gene spans 34 kb and contains eight exons. Over 300 different mutations have been identified in the FIX gene, all of which result in the production of inactive FIX, causing hemophilia B. Aim. In this study we searched for mutations in the FIX gene which result in an increased activity of FIX thus being the cause of thrombophilia syndromes. Material: A total of 108 individuals from unrelated families were involved in this study, presenting thrombophilic syndromes. A control sample from a healthy non-thrombophilic individual was also used. Total DNA from the above individuals was supplied to us by the Haemostasis and Thrombosis Unit of AHEPA University Hospital, Thessaloniki, Greece. According to HAT (Heparin Antithrombin Test, Makris, Van Dreden 1998) method a mixture of human antithrombin and heparin is added in the plasma and partial thromboplastin time is estimated. 97% of normal individuals exhibit prolonged time values in this test, whereas in our patients the time was significantly reduced. However, after the addition of recombined human FIX (rhFIX) in the mixture, prolongation of PTT is noted. Methods: The promoter region and the eight exons of the FIX gene were amplified by PCR using seven labelled primer pairs specific for these regions, that were described previously in literature. The amplification reactions were performed in a MJ Research P200 thermal cycler while the Tm of each primer pair was optimised as shown in the table. PCR products were analyzed using LI-COR DNA analyzer which is based on fragment separation by polyacrylamide gel electrophoresis. With this method PCR products presenting up to a 1 bp difference in their molecular weight create distinct bands on the gel and thus an insertion, or deletion of a base can be detected. However, no such differentiation was present among the samples examined. Assuming that the potential mutations could involve point mutations and thus be undetectable by the above method, the samples were sequenced and compared with the control. Sequencing the promoter and the 8 exons sites of the FIX gene of the most high risk cases. A point mutation was detected in four of the samples. The mutation was a single base change (ACT →GCT) located at the 21975 bp of the FIX gene, in exon 6. This mutation causes a significant change, replacing the Thr194 residue with an Ala residue (T194A). The sequencing pattern of one of these patients and the control is shown in the figure. Figure Figure

scholarly journals Studies of the activation of factor VII bound to tissue factor [see comments]

Blood ◽  
1996 ◽  
Vol 87 (9) ◽  
pp. 3738-3748 ◽  
Author(s):  
LV Rao ◽  
T Williams ◽  
SI Rapaport
Keyword(s):  
Tissue Factor ◽  
Factor Viia ◽  
Tissue Injury ◽  
Factor Xa ◽  
Inhibitory Effect ◽  
Factor Ix ◽  
Factor Vii ◽  
Activate Factor ◽  
Factor X ◽  
Ovarian Carcinoma Cell Line

Experiments were performed to evaluate activation of factor VII bound to relipidated tissue factor (TF) in suspension and to TF constitutively expressed on the surface of an ovarian carcinoma cell line (OC-2008). Activation was assessed by measuring cleavage of 125I- factor VII and by the ability of unlabeled factor VII to catalyze activation of a variant factor IX molecule that, after activation, cannot back-activate factor VII. Factor Xa was found to effectively activate factor VII bound to TF relipidated in either acidic or neutral phospholipid vesicles. Autoactivation of factor VII bound to TF in suspension was dependent on the preparation of TF apoprotein used and the technique of its relipidation. This highlights the need for caution in extrapolating data from TF in suspension to the activation of factor VII bound to cell surfaces during hemostasis. A relatively slow activation of factor VII bound to OC-2008 monolayers in the absence of added protease was observed consistently. Antithrombin in the presence or absence of heparin prevented this basal activation, whereas TF pathway inhibitor (TFPI/factor Xa complexes had only a limited inhibitory effect. Adding a substrate concentration of factor X markedly enhanced basal activation of factor VII, but both TFPI/factor Xa and antithrombin/heparin abolished this enhancement. Overall, our data are compatible with the hypothesis that not all factor VII/TF complexes formed at a site of tissue injury are readily activated to factor VIIa (VIIa)/TF complexes during hemostasis. The clinical significance of this is discussed.

Structure of the Human Factor VII Gene

1987 ◽  
Author(s):  
Patrick J O'hara ◽  
Frank A Grant ◽  
A Betty ◽  
J Haldmen ◽  
Mark J Murray
Keyword(s):  
Serine Protease ◽  
Vitamin K ◽  
Protein C ◽  
Tandem Repeats ◽  
Human Factor ◽  
Factor Ix ◽  
Factor Vii ◽  
Factor X ◽  
X Protein ◽  
Additional Exon

Factor VII is a member of a family of vitamin K-dependent, gamma-carboxylated plasma protein which includes factor IX, factor X, protein C, protein S and prothrombin. Activated factor VII (factor Vila) is a plasma serine protease which participates in a cascade of reactions leading to the coagulation of blood. Two overlapping genomic clones containing sequences encoding human factor VII were isolated and characterized. The complete sequence of the gene was determined and found to span 12.8 kilobases. The mRNA for factor VII as demonstrated by cDNA cloning is polyadenylated at multiple sites but contains only one AAUAAA poly-A signal sequence. The mRNA can undergo alternative splicing forming one transcript containing eight segments as exons and another with an additional exon which encodes a larger pre-pro leader sequence. The portion of the pre-pro leader coded for by the additional exon has no known counterpart in the other vitamin K-dependent proteins. The positions of the introns with respect to the amino acid sequence encoded by the eight essential exons of factor VII are the same as those present in factor IX, factor X, protein C and the first three exons of prothrombin. These exons code for domains generally conserved among members of this gene family, including a pre-pro leader (the essential exon la and alternative exon lb), a gamma-carboxylated domain (exons 2 and 3) a growth factor domain (exons 4 and 5) an activation region (exon 6) and a serine protease (exon 8). The corresponding introns in these genes are dissimilar with respect to size and sequence, with the exception of the third intron in factor VII and protein C. Four introns and a portion of exon 8 in factor VII contain regions made up of tandem repeats of oligonucleotide monomer elements. More than a quarter of the intron sequences and more than a third of the 3' untranslated portion of the mRNA transcript consist of these minisatellite tandem repeats. This type of structure is responsible for polymorphisms due to allelic variation in repeat copy number in other areas of the human genome. Tandem repeats can evolve as a result of random crossover in DNA whose sequence is not maintained by selection. This suggests that much of the sequence information present in the introns and untranslated portion of the message is dispensable.

scholarly journals Activation of Human Factor X

1977 ◽  
Author(s):  
Carolyn L. Orthner ◽  
Sam Morris ◽  
David P. Kosow
Keyword(s):  
Molecular Weight ◽  
Heavy Chain ◽  
Gel Electrophoresis ◽  
Human Factor ◽  
Polyacrylamide Gel Electrophoresis ◽  
Factor Xa ◽  
Polyacrylamide Gel ◽  
Coagulation Cascade ◽  
Molecular Forms ◽  
Factor X

Factor X is the zymogen of the proteolytic coagulation enzyme Factor Xa. Since the activation of Factor X to Factor Xa may be a rate limiting step of the coagulation cascade we have begun investigations of the mechanism of this reaction. Human Factor X has been purified 6000-fold from human plasma and the final product is over 95% pure as judged by Polyacrylamide gel electrophoresis. Human Factor X has a monomeric molecular weight of 75,000 and consists of two chains held together by a disulphide bridge. The molecular weight of the heavy chain is 56,000 and that of the light chain is 17,500. The venom coagulant protein of V. russelli (RVV-X) activates human Factor X by cleaving the heavy chain. When fully activated, human Factor Xa shows two bands on Polyacrylamide gel electrophoresis indicating that human Factor Xa like the bovine enzyme has two molecular forms.The kinetic mechanism of the activation reaction has been investigated utilizing the chromogenic Factor Xa substrate Bz-Ile-Glu-Gly-Arg-p-Nitroanilide (S-2222). The reaction has an absolute requirement for Ca; Mg cannot substitute for Ca, however Mg can increase the Vmax of Xa formation in the presence of suboptimal concentrations of Ca. Both Ca and Mg effects exhibit positive cooperativity. Our data indicate that human Factor X has at least three cooperative metal binding sites some of which are specific for Ca.

scholarly journals Factor IXa-factor VIIIa-cell surface complex does not contribute to the basal activation of the coagulation mechanism in vivo

Blood ◽  
1992 ◽  
Vol 79 (8) ◽  
pp. 2039-2047 ◽  
Author(s):  
KA Bauer ◽  
PM Mannucci ◽  
A Gringeri ◽  
F Tradati ◽  
S Barzegar ◽  
...  
Keyword(s):  
Tissue Factor ◽  
Plasma Concentrations ◽  
Factor Ix ◽  
Factor Vii ◽  
Coagulation System ◽  
Activate Factor ◽  
Factor X ◽  
Factor Ixa ◽  
Tissue Factor Pathway ◽  
Activation Peptide

Abstract We have infused recombinant factor VIIa into patients with hereditary factor VII deficiency with marked reductions in plasma concentrations of factor IX activation peptide (FIXP), factor X activation peptide (FXP), and prothrombin activation fragment F1+2. These investigations show substantial elevations in these markers of coagulation activation and thereby demonstrate that the factor VII-tissue factor pathway is largely responsible for the activation of factor IX as well as factor X in the basal state (ie, the absence of thrombosis or provocative stimuli). We have administered a monoclonal antibody purified factor IX concentrate to individuals with hemophilia B. These studies show an increase in the plasma levels of FIXP that were initially greatly decreased, but no change in FXP or F1+2. We have also infused highly purified factor VIII concentrate into patients with hemophilia A. The data demonstrate no significant changes in the plasma concentrations of FXP and F1+2. The above observations indicate that factor IXa generated by the factor VII-tissue factor pathway is unable to activate factor X under basal conditions. Based upon the above findings, we outline a model of blood coagulation system function under basal conditions, and suggest a process by which the generation of factor Xa and thrombin might be accelerated during normal hemostasis and in the setting of thrombotic disorders.

scholarly journals Activation of human factor VII in the initiation of tissue factor- dependent coagulation

Blood ◽  
1986 ◽  
Vol 68 (3) ◽  
pp. 685-691 ◽  
Author(s):  
LV Rao ◽  
SI Rapaport ◽  
SP Bajaj
Keyword(s):  
Tissue Factor ◽  
Factor Ix ◽  
Factor Vii ◽  
Activate Factor ◽  
Factor Xii ◽  
Factor X ◽  
Clotting Time ◽  
Activated Factor Vii ◽  
Peptide Release ◽  
Minimal Generation

Abstract We have used activation peptide release assays to compare factor VII and activated factor VII (VIIa) activation of factor X, normal factor IX (IXN), and a variant factor IX (IXBmLE), which, after activation, is unable to back-activate factor VII. In purified systems, factor VII and VIIa each rapidly activated factor X, but after a one minute lag for factor VII. VIIa also readily activated both IXN and IXBmLE. Factor VII initially failed to activate substantial amounts of either IXN or IXBmLE; on further incubation factor VII activated IXN but not IXBmLE. Activation of IXN began when approximately 10% of factor VII had been converted to VIIa, as measured by 125I-factor VII radioactivity profiles. Adding factor VII to VIIa slowed its activation of IXBmLE. However, in the presence of factor X, factor VII alone rapidly activated IXBmLE. Unlike purified systems, 1 nmol/L VIIa added to factor VII-deficient plasma failed to activate factor IX. Increasing factor VII to 10 nmol/L (plasma concentration) either as native VII or VIIa yielded similar activation curves for factor IX and similar activation curves for factor X. Adding 5% VIIa to factor X-deficient plasma and to factor XII-deficient plasma substantially shortened the dilute tissue factor clotting time of only the former. These data support the hypothesis that factor VII/tissue factor complex initiates tissue factor-dependent clotting through a minimal generation of Xa. This Xa then rapidly back-activates a small amount of factor VII, following which the rates of activation of both factors IX and X increase dramatically.

In Vitro Characterization of High Purity Factor IX Concentrates for the Treatment of Hemophilia B

1995 ◽  
Vol 73 (04) ◽  
pp. 584-591 ◽  
Author(s):  
Steven A Limentani ◽  
Kerry P Gowell ◽  
Steven R Deitcher
Keyword(s):  
High Purity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Factor Ix ◽  
Factor Vii ◽  
Dominant Factor ◽  
Factor X ◽  
Reducing Conditions ◽  
Sds Page ◽  
Activation Products

SummaryThis study employed sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis and immunoblotting to assess the purity of seven high purity factor IX concentrates: Aimafix (Aima), AlphaNine-SD (Alpha Therapeutic), Factor IX VHP (Biotransfusion), Immunine (Immuno), Mononine (Armour Pharmaceutical), Nanotiv (Kabi Pharmacia), and 9MC (Blood Products Laboratory). The mean specific activity of these products ranged from 68 U factor IX/mg (Aimafix) to 246 U factor IX/mg (Mononine). SDS-PAGE analysis showed that the highest purity product, Mononine, had a single contaminating band under non-reducing conditions. Two additional bands were detected when this product was analyzed under reducing conditions. All other products had multiple contaminating bands that were more apparent under reducing than non-reducing conditions. The immunoblot for factor IX showed a dominant factor IX band for all products. In addition, visible light chain of factor IX was detected for AlphaNine-SD, Factor IX VHP, Immunine, Mononine, Nanotiv, and 9MC, suggesting that the factor IX in these products had undergone partial activation to factor IXa. Another contaminating band was visible at 49,500 for all of the products except 9MC. In addition to this band, high molecular weight contaminants were apparent for some products, most notably AlphaNine-SD. The identity of these bands is unknown. Immunoblotting failed to demonstrate factor VII as a contaminant of any of the high purity products, although factor Vila could be detected in some lots of Immunine, Nanotiv, and 9MC by a clot-based assay. Factor X contaminated Aimafix, AlphaNine-SD, Factor IX VHP, Immunine, Nanotiv, and 9MC, but activation products of factor X were not detected. Prothrombin contaminated all of the products except Mononine. Activation products of prothrombin were identified for three of four lots of Immunine and for one lot of Factor IX VHP. These results thus demonstrate that high purity factor IX concentrates differ substantially in the degree to which they are contaminated by potentially thrombogenic materials.

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