Factor Vlla/Tissue Factor-Catalyzed Activation of Factors IX and X on a Cell Surface and in Suspension: A Kinetic Study

1992 ◽  
Vol 67 (06) ◽  
pp. 654-659 ◽  
Author(s):  
L Vijaya Mohan Rao ◽  
Thomas Robinson ◽  
An D Hoang
Keyword(s):  
Cell Surface ◽  
Tissue Factor ◽  
Intact Cell ◽  
Surface Membrane ◽  
Factor Ix ◽  
Factor X ◽  
Ovarian Carcinoma Cell Line ◽  
Cell Lysate ◽  
A Cell ◽  
The Difference

SummaryThe kinetics of activation of factor IX and factor X by factor Vila was studied in the presence of various sources of tissue factor: (1) a surface membrane of human ovarian carcinoma cell line, OC-2008 (2) the cell lysate (of 002008) and (3) reconstituted purified human tissue factor. The rates of activation of factors IX and X were monitored in activation peptide release assays using tritiated substrates. The results indicate that the apparent K m values for factor IX and factor X were similar for a given tissue factor, but varied with tissue factor source. The source of tissue factor greatly influenced the apparent differences in V max for factors IX and X. When a surface of monolayer provided tissue factor, the Vmax of factor IX was only 2-3 fold lower than factor X, but when either a cell lysate or purified tissue factor was the source of cofactor activity, the difference in V max rose to about 8-10 fold. Although, the tissue factor apoprotein in the cells was expressed entirely on the outer surface membrane, the activity of tissue factor on the intact cell surface was 50 to 100fold lower than in the lysed cell preparation.

Studies of the Mechanism for Enhanced Cell Surface Factor VIIa/Tissue Factor Activation of Factor X on Fibroblast Monolayers after Their Exposure to N-Ethylmaleimide

1994 ◽  
Vol 72 (06) ◽  
pp. 848-855 ◽  
Author(s):  
Dzung The Le ◽  
Samuel I Rapaport ◽  
L Vijaya Mohan Rao
Keyword(s):  
Cell Surface ◽  
Tissue Factor ◽  
Factor Viia ◽  
Cell Damage ◽  
Specific Binding ◽  
Surface Membrane ◽  
Annexin V ◽  
Factor X ◽  
Binding Studies ◽  
Anionic Phospholipid

SummaryFibroblast monolayers constitutively expressing surface membrane tissue factor (TF) were treated with 0.1 mM N-ethylmaleimide (NEM) for 1 min to inhibit aminophospholipid translocase activity without inducing general cell damage. This resulted in increased anionic phospholipid in the outer leaflet of the cell surface membrane as measured by the binding of 125I-annexin V and by the ability of the monolayers to support the generation of prothrombinase. Specific binding of 125I-rVIIa to TF on NEM-treated monolayers was increased 3- to 4-fold over control monolayers after only brief exposure to 125I-rVIIa, but this difference progressively diminished with longer exposure times. A brief exposure of NEM-treated monolayers to rVIIa led to a maximum 3- to 4-fold enhancement of VIIa/TF catalytic activity towards factor X over control monolayers, but, in contrast to the binding studies, this 3- to 4-fold difference persisted despite increasing time of exposure to rVIIa. Adding prothrombin fragment 1 failed to diminish the enhanced VIIa/TF activation of factor X of NEM-treated monolayers. Moreover, adding annexin V, which was shown to abolish the ability of NEM to enhance factor X binding to the fibroblast monolayers, also failed to diminish the enhanced VIIa/TF activation of factor X. These data provide new evidence for a possible mechanism by which availability of anionic phospholipid in the outer layer of the cell membrane limits formation of functional VIIa/TF complexes on cell surfaces.

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.

scholarly journals Binding of factor VIIa to tissue factor permits rapid antithrombin III/heparin inhibition of factor VIIa

Blood ◽  
1993 ◽  
Vol 81 (10) ◽  
pp. 2600-2607 ◽  
Author(s):  
LV Rao ◽  
SI Rapaport ◽  
AD Hoang
Keyword(s):  
Catalytic Activity ◽  
Tissue Factor ◽  
Antithrombin Iii ◽  
Carcinoma Cell ◽  
Factor Viia ◽  
Surface Membrane ◽  
Factor X ◽  
Ovarian Carcinoma Cell Line ◽  
Coagulant Activity ◽  
At Iii

Abstract Because free factor VIIa is inactivated only very slowly by a plasma concentration of antithrombin III (AT III) even in the presence of heparin, it has been assumed that AT III plays no significant role in regulating the initiation of tissue factor-dependent blood coagulation. However, in the present study, we present evidence that factor VIIa bound to tissue factor, unlike free factor VIIa, is readily inactivated by AT III in the presence of heparin. In a reaction mixture containing calcium ions and approximately equimolar concentrations of relipidated tissue factor (8.9 nmol/L) and factor VIIa (10 nmol/L), AT III (100 micrograms/mL) plus heparin (1 U/mL) inhibited 50% of the factor VIIa coagulant activity of the reaction mixture within 5 minutes. AT III/heparin was also shown to inhibit the catalytic activity towards factor X of factor VIIa/tissue factor complexes formed on monolayers of an ovarian carcinoma cell line (OC-2008) that constitutively expresses surface membrane tissue factor. AT III, even in the absence of exogenously added heparin, substantially inhibited the functional activity of factor VIIa/cell surface tissue factor complexes on intact monolayers. AT III alone and AT III/heparin, to a greater extent, also inhibited factor VIIa on “nonfunctional” factor VIIa/tissue factor complexes on intact monolayers, with resultant inhibition of their expression of factor VIIa/tissue factor catalytic activity toward factor X after cell lysis. The potential physiologic significance of these findings is discussed.

scholarly journals Binding of factor VIIa to tissue factor permits rapid antithrombin III/heparin inhibition of factor VIIa

Blood ◽  
1993 ◽  
Vol 81 (10) ◽  
pp. 2600-2607
Author(s):  
LV Rao ◽  
SI Rapaport ◽  
AD Hoang
Keyword(s):  
Catalytic Activity ◽  
Tissue Factor ◽  
Antithrombin Iii ◽  
Carcinoma Cell ◽  
Factor Viia ◽  
Surface Membrane ◽  
Factor X ◽  
Ovarian Carcinoma Cell Line ◽  
Coagulant Activity ◽  
At Iii

Because free factor VIIa is inactivated only very slowly by a plasma concentration of antithrombin III (AT III) even in the presence of heparin, it has been assumed that AT III plays no significant role in regulating the initiation of tissue factor-dependent blood coagulation. However, in the present study, we present evidence that factor VIIa bound to tissue factor, unlike free factor VIIa, is readily inactivated by AT III in the presence of heparin. In a reaction mixture containing calcium ions and approximately equimolar concentrations of relipidated tissue factor (8.9 nmol/L) and factor VIIa (10 nmol/L), AT III (100 micrograms/mL) plus heparin (1 U/mL) inhibited 50% of the factor VIIa coagulant activity of the reaction mixture within 5 minutes. AT III/heparin was also shown to inhibit the catalytic activity towards factor X of factor VIIa/tissue factor complexes formed on monolayers of an ovarian carcinoma cell line (OC-2008) that constitutively expresses surface membrane tissue factor. AT III, even in the absence of exogenously added heparin, substantially inhibited the functional activity of factor VIIa/cell surface tissue factor complexes on intact monolayers. AT III alone and AT III/heparin, to a greater extent, also inhibited factor VIIa on “nonfunctional” factor VIIa/tissue factor complexes on intact monolayers, with resultant inhibition of their expression of factor VIIa/tissue factor catalytic activity toward factor X after cell lysis. The potential physiologic significance of these findings is discussed.

scholarly journals Functional tissue factor is entirely cell surface expressed on lipopolysaccharide-stimulated human blood monocytes and a constitutively tissue factor-producing neoplastic cell line.

1989 ◽  
Vol 109 (1) ◽  
pp. 389-395 ◽  
Author(s):  
T A Drake ◽  
W Ruf ◽  
J H Morrissey ◽  
T S Edgington
Keyword(s):  
Cell Surface ◽  
Tissue Factor ◽  
Intact Cell ◽  
Surface Membrane ◽  
Coagulation Factors ◽  
Cell Types ◽  
Neoplastic Cell ◽  
Carcinoma Cells ◽  
Blood Monocytes ◽  
Intact Cells

Tissue factor (TF) is an integral membrane glycoprotein which, as the receptor and essential cofactor for coagulation factors VII and VIIa (FVII and FVIIa, respectively), is the primary cellular activator of the coagulation protease cascade. Previous studies on the procoagulant activity of a variety of cell types (either lysed or in the intact state) have variously been interpreted as showing that TF is either stored intracellularly or is present in a cryptic form in the surface membrane. Using mAbs to TF, we have directly investigated the subcellular localization and functional activity of TF in lipopolysaccharide-stimulated blood monocytes and J82 bladder carcinoma cells. Blocking of surface TF of viable cells with inhibitory anti-TF mAbs abolished greater than 90% of TF activity of the intact cells as well as of lysed cells. Furthermore, quantitative analysis of the binding of FVII and anti-TF mAb to J82 cells demonstrated that all surface-expressed TF molecules were capable of binding the ligand, FVII. By immunoelectron microscopy, TF was present only in the surface membrane of monocytes and J82 cells, although the latter also contained apparently inactive TF antigen in multivesicular bodies. On the intact cell surface the catalytic activity of the TF-FVIIa complex was investigated and found to be markedly less relative to cell lysates. Membrane alterations that affect the cofactor activity of TF may be a means of regulating the extent of initiation of the coagulation protease cascade in various cellular settings.

scholarly journals Inhibition of tissue factor/factor VIIa activity in plasma requires factor X and an additional plasma component

Blood ◽  
1985 ◽  
Vol 66 (1) ◽  
pp. 204-212
Author(s):  
NL Sanders ◽  
SP Bajaj ◽  
A Zivelin ◽  
SI Rapaport
Keyword(s):  
Tissue Factor ◽  
Factor Viia ◽  
Factor Ix ◽  
Factor Vii ◽  
Factor X ◽  
Additional Material ◽  
Plasma Reaction ◽  
Progress Curve ◽  
Peptide Release ◽  
Release Data

A study was carried out to explore requirements for the inhibition of tissue factor-factor VIIa enzymatic activity in plasma. Reaction mixtures contained plasma, 3H-factor IX or 3H-factor X, tissue factor (vol/vol 2.4% to 24%), and calcium. Tissue factor-factor VIIa activity was evaluated from progress curves of activation of factor IX or factor X, plotted from tritiated activation peptide release data. With normal plasma, progress curves exhibited initial limited activation followed by a plateau indicative of loss of tissue factor-factor VIIa activity. With hereditary factor X-deficient plasma treated with factor X antibodies, progress curves revealed full factor IX activation. Adding only 0.4 micrograms/mL factor X (final concentration) could restore inhibition. Inhibition was not observed in purified systems containing 6% to 24% tissue factor, factor VII, 0.5 micrograms/mL, factor IX, 13 micrograms/mL, and factor X up to 0.8 micrograms/mL, but could be induced by adding barium-absorbed plasma to the reaction mixture. Thus, both factor X and an additional material in plasma were required for inhibition. The amount of factor X needed appeared related to the concentration of tissue factor; adding more tissue factor at the plateau of a progress curve induced further activation. These results also indicate that inhibited reaction mixtures contained active free factor VII(a). Preliminary data suggest that inhibition may stem from loss of activity of the tissue factor component of the tissue factor- factor VII(a) complex.

scholarly journals In Hemophilia Α Plasma Treated with Emicizumab, Factor IX Activation By Factor VIIa Drives Thrombin Generation

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 17-17
Author(s):  
Dougald Monroe ◽  
Mirella Ezban ◽  
Maureane Hoffman
Keyword(s):  
Factor Viii ◽  
Tissue Factor ◽  
Plasma Levels ◽  
Thrombin Generation ◽  
Hemophilia A ◽  
Factor Viia ◽  
Factor Ix ◽  
Factor X ◽  
Dose Dependent

Background.Recently a novel bifunctional antibody (emicizumab) that binds both factor IXa (FIXa) and factor X (FX) has been used to treat hemophilia A. Emicizumab has proven remarkably effective as a prophylactic treatment for hemophilia A; however there are patients that still experience bleeding. An approach to safely and effectively treating this bleeding in hemophilia A patients with inhibitors is recombinant factor VIIa (rFVIIa). When given at therapeutic levels, rFVIIa can enhance tissue factor (TF) dependent activation of FX as well as activating FX independently of TF. At therapeutic levels rFVIIa can also activate FIX. The goal of this study was to assess the role of the FIXa activated by rFVIIa when emicizumab is added to hemophilia A plasma. Methods. Thrombin generation assays were done in plasma using 100 µM lipid and 420 µM Z-Gly-Gly-Arg-AMC with or without emicizumab at 55 µg/mL which is the clinical steady state level. The reactions were initiated with low (1 pM) tissue factor (TF). rFVIIa was added at concentrations of 25-100 nM with 25 nM corresponding to the plasma levels achieved by a single clinical dose of 90 µg/mL. To study to the role of factor IX in the absence of factor VIII, it was necessary to create a double deficient plasma (factors VIII and IX deficient). This was done by taking antigen negative hemophilia B plasma and adding a neutralizing antibody to factor VIII (Haematologic Technologies, Essex Junction, VT, USA). Now varying concentrations of factor IX could be reconstituted into the plasma to give hemophilia A plasma. Results. As expected, in the double deficient plasma with low TF there was essentially no thrombin generation. Also as expected from previous studies, addition of rFVIIa to double deficient plasma gave a dose dependent increase in thrombin generation through activation of FX. Interestingly addition of plasma levels of FIX to the rFVIIa did not increase thrombin generation. Starting from double deficient plasma, as expected emicizumab did not increase thrombin generation since no factor IX was present. Also, in double deficient plasma with rFVIIa, emicizumab did not increase thrombin generation. But in double deficient plasma with FIX and rFVIIa, emicizumab significantly increased thrombin generation. The levels of thrombin generation increased in a dose dependent fashion with higher concentrations of rFVIIa giving higher levels of thrombin generation. Conclusion. Since addition of FIX to the double deficient plasma with rFVIIa did not increase thrombin generation, it suggests that rFVIIa activation of FX is the only source of the FXa needed for thrombin generation. So in the absence of factor VIII (or emicizumab) FIX activation does not contribute to thrombin generation. However, in the presence of emicizumab, while rFVIIa can still activate FX, FIXa formed by rFVIIa can complex with emicizumab to provide an additional source of FX activation. Thus rFVIIa activation of FIX explains the synergistic effect in thrombin generation observed when combining rFVIIa with emicizumab. The generation of FIXa at a site of injury is consistent with the safety profile observed in clinical use. Disclosures Monroe: Novo Nordisk:Research Funding.Ezban:Novo Nordisk:Current Employment.Hoffman:Novo Nordisk:Research Funding.

scholarly journals Inhibition of tissue factor/factor VIIa activity in plasma requires factor X and an additional plasma component

Blood ◽  
1985 ◽  
Vol 66 (1) ◽  
pp. 204-212 ◽  
Author(s):  
NL Sanders ◽  
SP Bajaj ◽  
A Zivelin ◽  
SI Rapaport
Keyword(s):  
Tissue Factor ◽  
Factor Viia ◽  
Factor Ix ◽  
Factor Vii ◽  
Factor X ◽  
Additional Material ◽  
Plasma Reaction ◽  
Progress Curve ◽  
Peptide Release ◽  
Release Data

Abstract A study was carried out to explore requirements for the inhibition of tissue factor-factor VIIa enzymatic activity in plasma. Reaction mixtures contained plasma, 3H-factor IX or 3H-factor X, tissue factor (vol/vol 2.4% to 24%), and calcium. Tissue factor-factor VIIa activity was evaluated from progress curves of activation of factor IX or factor X, plotted from tritiated activation peptide release data. With normal plasma, progress curves exhibited initial limited activation followed by a plateau indicative of loss of tissue factor-factor VIIa activity. With hereditary factor X-deficient plasma treated with factor X antibodies, progress curves revealed full factor IX activation. Adding only 0.4 micrograms/mL factor X (final concentration) could restore inhibition. Inhibition was not observed in purified systems containing 6% to 24% tissue factor, factor VII, 0.5 micrograms/mL, factor IX, 13 micrograms/mL, and factor X up to 0.8 micrograms/mL, but could be induced by adding barium-absorbed plasma to the reaction mixture. Thus, both factor X and an additional material in plasma were required for inhibition. The amount of factor X needed appeared related to the concentration of tissue factor; adding more tissue factor at the plateau of a progress curve induced further activation. These results also indicate that inhibited reaction mixtures contained active free factor VII(a). Preliminary data suggest that inhibition may stem from loss of activity of the tissue factor component of the tissue factor- factor VII(a) complex.

scholarly journals Studies of a mechanism inhibiting the initiation of the extrinsic pathway of coagulation

Blood ◽  
1987 ◽  
Vol 69 (2) ◽  
pp. 645-651 ◽  
Author(s):  
LV Rao ◽  
SI Rapaport
Keyword(s):  
Tissue Factor ◽  
Factor Viia ◽  
Factor Xa ◽  
Factor Ix ◽  
Factor Vii ◽  
Factor X ◽  
Extrinsic Pathway ◽  
Mechanism Of Inhibition ◽  
Peptide Release ◽  
Release Assay

Abstract We have extended earlier studies (Blood 66:204, 1985) of a mechanism of inhibition of factor VIIa/tissue factor activity that requires a plasma component (called herein extrinsic pathway inhibitor or EPI) and factor Xa. An activated peptide release assay using 3H-factor IX as a substrate was used to evaluate inhibition. Increasing the tissue factor concentration from 20% to 40% (vol/vol) overcame the inhibitory mechanism in normal plasma but not in factor VII-deficient plasma supplemented with a low concentration of factor VII. A second wave of factor IX activation obtained by a second addition of tissue factor to plasma with a normal factor VII concentration was almost abolished by supplementing the reaction mixture with additional EPI and factor X. Factor Xa's active site was necessary for factor Xa's contribution to inhibition, but preliminary incubation of factor Xa with EPI in the absence of factor VIIa/tissue factor complex or of factor VIIa/tissue factor complex in the absence of EPI did not replace the need for the simultaneous presence of factor Xa, factor VIIa/tissue factor, calcium, and EPI in an inhibitory reaction mixture. Inhibition of factor VIIa/tissue factor was reversible; both tissue factor and factor VIIa activity could be recovered from a dissociated, inhibited factor VIIa/tissue factor complex. EPI appeared to bind to a factor VIIa/tissue factor complex formed in the presence of factor Xa but not to a factor VIIa/tissue factor complex formed in the absence of factor Xa.

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