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 A soluble tissue factor-annexin V chimeric protein has both procoagulant and anticoagulant properties

Blood ◽  
2006 ◽  
Vol 107 (3) ◽  
pp. 980-986 ◽  
Author(s):  
Xin Huang ◽  
Wei-Qun Ding ◽  
Joshua L. Vaught ◽  
Roman F. Wolf ◽  
James H. Morrissey ◽  
...  
Keyword(s):  
Tissue Factor ◽  
Blood Coagulation ◽  
Vascular Injury ◽  
Factor Viia ◽  
Chimeric Protein ◽  
Annexin V ◽  
Factor X ◽  
Low Concentrations ◽  
Finite Period ◽  
Factor X Activation

AbstractTissue factor (TF) initiates blood coagulation, but its expression in the vascular space requires a finite period of time. We hypothesized that targeting exogenous tissue factor to sites of vascular injury could lead to accelerated hemostasis. Since phosphatidylserine (PS) is exposed on activated cells at sites of vascular injury, we cloned the cDNA for a chimeric protein consisting of the extracellular domain of TF (called soluble TF or sTF) and annexin V, a human PS-binding protein. Both the sTF and annexin V domains had ligand-binding activities consistent with their native counterparts, and the chimera accelerated factor X activation by factor VIIa. The chimera exhibited biphasic effects upon blood coagulation. At low concentrations it accelerated blood coagulation, while at higher concentrations it acted as an anticoagulant. The chimera accelerated coagulation in the presence of either unfractionated or low-molecular-weight heparins more potently than factor VIIa and shortened the bleeding time of mice treated with enoxaparin. The sTF-annexin V chimera is a targeted procoagulant protein that may be useful in accelerating thrombin generation where PS is exposed to the vasculature, such as may occur at sites of vascular injury or within the vasculature of tumors.

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.

Glycosylphosphatidylinositol Anchored TFPI As Main Regulator of Factor X Activation On the Surface of Endothelial Cells.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2210-2210
Author(s):  
Michael Dockal ◽  
Robert Pachlinger ◽  
Angelina Baldin-Stoyanova ◽  
Fabian Knofl ◽  
Nadja Ullrich ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Cell Surface ◽  
Tissue Factor ◽  
Conflicts Of Interest ◽  
Factor Viia ◽  
Umbilical Vein ◽  
Surface Proteins ◽  
Enzyme Linked Immunosorbent Assay ◽  
Factor X ◽  
Extrinsic Pathway

Abstract Abstract 2210 Tissue factor pathway inhibitor (TFPI) is a key regulator of factor X (FX) activation in the extrinsic pathway of blood coagulation. TFPI inhibits FXa generation by formation of a quaternary complex consisting of factor VIIa (FVIIa), tissue factor (TF), FXa and TFPI. The main portion (∼80%) of TFPI in humans is reportedly associated with endothelial cells. We used human umbilical vein endothelial cells (HUVECs) as a model to obtain further insight into the function of TFPIα and the glycosylphosphatidylinositol (GPI) anchored TFPI form, which represents TFPIα bound to GPI-anchored surface proteins and/or TFPIβ. In contrast to TFPIα, which consists of 3 Kunitz domains (KD) and a basic C-terminal part, GPI-anchored TFPIβ lacks the third Kunitz domain (KD3) and the basic C–terminal region due to alternative splicing. In TFPIβ these two domains are replaced by a sequence that adds a GPI anchor to the protein linking it to the cell membrane. Treatment of HUVECs with phosphatidylinositol phospholipase C (PI-PLC) that cleaves GPI-anchors and subsequent fluorescence activated cell sorting (FACS) on living cells showed that GPI-anchored TFPI represents about 70–80% of cell surface TFPI. Staining of TFPI on and in fixed and permeabilized cells (total TFPI) demonstrated that GPI-anchored cell surface TFPI contributes to ∼20% of total cellular TFPI. Enzyme-linked immunosorbent assay (ELISA) showed that PI-PLC treatment released a TFPI protein lacking the KD3 and basic C-terminus. These findings strongly suggest that TFPIβ is the predominant GPI-anchored form of TFPI on HUVECs. FX activation assays performed on the cell surface of PI-PLC treated living HUVECs showed the importance of GPI-anchored TFPI on extrinsic Xase complex activity. PI-PLC treatment resulted in increased FX activation. Although GPI-anchored TFPI displays ∼70–80% of cell surface TFPI, overall FXa generation was increased only by ∼50%. In conclusion, HUVEC surface TFPI is predominantly TFPIβ, and GPI-anchored TFPI is the main but not sole regulator of FX activation on the surface of HUVECs. Disclosures: No relevant conflicts of interest to declare.

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 Ligand-induced protease receptor translocation into caveolae: a mechanism for regulating cell surface proteolysis of the tissue factor-dependent coagulation pathway.

1996 ◽  
Vol 133 (2) ◽  
pp. 293-304 ◽  
Author(s):  
J R Sevinsky ◽  
L V Rao ◽  
W Ruf
Keyword(s):  
Cell Surface ◽  
Tissue Factor ◽  
Cell Biology ◽  
Feedback Inhibition ◽  
Factor Viia ◽  
Coagulation Cascade ◽  
Low Density ◽  
Factor X ◽  
Quaternary Complex ◽  
Enzymatic Complex

The ability to regulate proteolytic functions is critical to cell biology. We describe events that regulate the initiation of the coagulation cascade on endothelial cell surfaces. The transmembrane protease receptor tissue factor (TF) triggers coagulation by forming an enzymatic complex with the serine protease factor VIIa (VIIa) that activates substrate factor X to the protease factor Xa (Xa). Feedback inhibition of the TF-VIIa enzymatic complex is achieved by the formation of a quaternary complex of TF-VIIa, Xa, and the Kunitz-type inhibitor tissue factor pathway inhibitor (TFPI). Concomitant with the downregulation of TF-VIIa function on endothelial cells, we demonstrate by immunogold EM that TF redistributes to caveolae. Consistently, TF translocates from the Triton X-100-soluble membrane fractions to low-density, detergent-insoluble microdomains that inefficiently support TF-VIIa proteolytic function. Downregulation of TF-VIIa function is dependent on quaternary complex formation with TFPI that is detected predominantly in detergent-insoluble microdomains. Partitioning of TFPI into low-density fractions results from the association of the inhibitor with glycosyl phosphatidylinositol anchored binding sites on external membranes. Free Xa is not efficiently bound by cell-associated TFPI; hence, we propose that the transient ternary complex of TF-VIIa with Xa supports translocation and assembly with TFPI in glycosphingolipid-rich microdomains. The redistribution of TF provides evidence for an assembly-dependent translocation of the inhibited TF initiation complex into caveolae, thus implicating caveolae in the regulation of cell surface proteolytic activity.

scholarly journals Influence of mutations in tissue factor on the fine specificity of macromolecular substrate activation

1997 ◽  
Vol 321 (3) ◽  
pp. 787-794 ◽  
Author(s):  
Sabine DITTMAR ◽  
Wolfram RUF ◽  
Thomas S. EDGINGTON
Keyword(s):  
Tissue Factor ◽  
Factor Viia ◽  
Factor Xa ◽  
Catalytic Efficiency ◽  
Factor Ix ◽  
Factor X ◽  
Wild Type ◽  
Binding Studies ◽  
Fine Specificity ◽  
Fibronectin Type Iii

The C-terminal fibronectin-type-III-like module of the tissue factor (TF) extracellular domain plays a requisite role in the activation of macromolecular substrates by factor VIIa (VIIa) in complex with TF. Unlike the mutations Lys165→Ala, Lys166→Ala in TF, which prevent efficient proteolysis of factor X, we found that the coagulant defect of a site-specific Trp158→Arg, Ser160→Gly replacement mutant of TF is largely attributable to the inability of TF to efficiently support the activation of the bound zymogen VII to the active protease VIIa. Binding studies demonstrated comparable affinity of binding of VIIa or VII by wild-type TF and TFR158G160. In comparison with wild-type TF, the catalytic efficiency of factor X activation was reduced 56-fold with TFA165A166 as the cofactor, but only 3.5-fold with TFR158G160. The activation of VII bound to TF by factor Xa or VIIa was reduced 2-fold in the presence of TFR158G160 and 7Ő8-fold with TFA165A166. This suggests that the molecular recognition of VII in complex with TF by the enzymes TFŐVIIa and factor Xa are similar. Generation of factor IXa by TFR158G160ŐVIIa was unaltered, but reduced 2-fold with TFA165A166. In addition, the mutations affected the cleavage of the two scissile bonds of factor IX differently, providing further support for the idea that the cofactor, TF, influences the fine specificity of activation of macromolecular substrates by the TFŐVIIa complex.

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 Fusion proteins comprising annexin V and Kunitz protease inhibitors are highly potent thrombogenic site-directed anticoagulants

Blood ◽  
2005 ◽  
Vol 105 (10) ◽  
pp. 3902-3909 ◽  
Author(s):  
Hsiu-Hui Chen ◽  
Cristina P. Vicente ◽  
Li He ◽  
Douglas M. Tollefsen ◽  
Tze-Chein Wun
Keyword(s):  
Tissue Factor ◽  
Fusion Proteins ◽  
Tissue Factor Pathway Inhibitor ◽  
Factor Viia ◽  
Amyloid Β ◽  
Annexin V ◽  
Amyloid Β Protein ◽  
Anionic Phospholipid ◽  
Tissue Factor Pathway

AbstractThe anionic phospholipid, phosphatidyl-l-serine (PS), is sequestered in the inner layer of the plasma membrane in normal cells. Upon injury, activation, and apoptosis, PS becomes exposed on the surfaces of cells and sheds microparticles, which are procoagulant. Coagulation is initiated by formation of a tissue factor/factor VIIa complex on PS-exposed membranes and propagated through the assembly of intrinsic tenase (factor VIIIa/factor IXa), prothrombinase (factor Va/factor Xa), and factor XIa complexes on PS-exposed activated platelets. We constructed a novel series of recombinant anticoagulant fusion proteins by linking annexin V (ANV), a PS-binding protein, to the Kunitz-type protease inhibitor (KPI) domain of tick anticoagulant protein, an aprotinin mutant (6L15), amyloid β-protein precursor, or tissue factor pathway inhibitor. The resulting ANV-KPI fusion proteins were 6- to 86-fold more active than recombinant tissue factor pathway inhibitor and tick anticoagulant protein in an in vitro tissue factor–initiated clotting assay. The in vivo antithrombotic activities of the most active constructs were 3- to 10-fold higher than that of ANV in a mouse arterial thrombosis model. ANV-KPI fusion proteins represent a new class of anticoagulants that specifically target the anionic membrane-associated coagulation enzyme complexes present at sites of thrombogenesis and are potentially useful as antithrombotic agents.

Factors Affecting the lnteraction of Tissue Factor/Factor Vll with Factor X in a Heterogeneous Tubular Reactor

1991 ◽  
Vol 65 (02) ◽  
pp. 139-143 ◽  
Author(s):  
Cynthia H Gemmell ◽  
Vincet T Turitto ◽  
Yale Nemerson
Keyword(s):  
Steady State ◽  
Tissue Factor ◽  
Factor Viia ◽  
Transmembrane Protein ◽  
Strong Association ◽  
Tubular Reactor ◽  
Factor Xa ◽  
Phospholipid Bilayer ◽  
Factor Vii ◽  
Factor X

SummaryA novel reactor recently described for studying phospholipiddependent blood coagulation reactions under flow conditions similar to those occurring in the vasculature has been further charactenzed. The reactor is a capitlary whose inner wall is coated with a stable phospholipid bilayer (or two bilayers) containing tissue factor, a transmembrane protein that is required for the enzymatic activation of factor X by factor VIIa. Perfusion of the capillary at wall shear rates ranging from 25 s−1 to 1,200 s−1 with purified bovine factors X and VIIa led to steady state factor Xa levels at the outlet. Assay were performed using a chromogenic substrate, SpectrozymeTMFXa, or by using a radiometric technique. In the absence of Ca2+ or factor VIIa there was no product formation. No difference was noted in the levels of factor Xa achieved when non-activated factor VII was perfused. Once steady state was achieved further factor Xa production continued in the absence of factor VIIa implying a very strong association of factor VIIa with the tissue factor in the phospholipid membrane. In agreement with static vesicle-type studies the reactor was sensitive to wall tissue factor concentration, temperature and the presence of phosphatidylserine in the bilayer.

Activation of Factor X by Factor VIIa Complexed with Human-mouse Tissue Factor Chimeras Requires Human Exon 3

1996 ◽  
Vol 76 (03) ◽  
pp. 361-368 ◽  
Author(s):  
Carrie H Fang ◽  
T-C Lin ◽  
Arabinda Guha ◽  
Yale Nemerson ◽  
William H Konigsberg
Keyword(s):  
Tissue Factor ◽  
Human Factor ◽  
Factor Viia ◽  
Mouse Tissue ◽  
Factor X ◽  
Lower Affinity ◽  
E Coli ◽  
Sequence Elements ◽  
Specific Activities ◽  
Human And Mouse

SummaryIn an attempt to define sequence elements in human and mouse tissue factor (TF) that are responsible for the species specificity observed in their interaction with human factor VIIa (HVIIa), we constructed human-mouse chimeric TF cDNAs, inserted them into plasmid vectors, and induced their expression in E.coli. Assays for procoagulant activity were carried out with the resulting E. coli lysates using (HVIIa) human and mouse (MVIIa). The ratio of the procoagulant activities, HVIIa/MVIIa, revealed that human TF exon 3 was essential for activity when the TF:VIIa complex was formed with HVIIa. By ligating the maltose binding protein (MBP) gene to TF cDNAs it was possible to construct, express and purify MBP-TF chimeras as well as to estimate their specific activities. With selected MBP-TF chimeras and HVIIa we determined kinetic parameters for the activation of human factor X. Replacement of exon 3 in human TF cDNA with the corresponding exon from mouse TF cDNA resulted in both lower affinity for HVIIa and failure to convert bound HVIIa into a potent protease

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