scholarly journals Caveolae optimize tissue factor–Factor VIIa inhibitory activity of cell-surface-associated tissue factor pathway inhibitor

2012 ◽  
Vol 443 (1) ◽  
pp. 259-266 ◽  
Author(s):  
Susan A. Maroney ◽  
Paul E. Ellery ◽  
Jeremy P. Wood ◽  
Josephine P. Ferrel ◽  
Catherine E. Bonesho ◽  
...  
Keyword(s):  
Tissue Factor ◽  
Inhibitory Activity ◽  
Tissue Factor Pathway Inhibitor ◽  
Factor Viia ◽  
Umbilical Vein ◽  
Anticoagulant Activity ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Factor Xa ◽  
Tissue Factor Pathway

TFPI (tissue factor pathway inhibitor) is an anticoagulant protein that prevents intravascular coagulation through inhibition of fXa (Factor Xa) and the TF (tissue factor)–fVIIa (Factor VIIa) complex. Localization of TFPI within caveolae enhances its anticoagulant activity. To define further how caveolae contribute to TFPI anticoagulant activity, CHO (Chinese-hamster ovary) cells were co-transfected with TF and membrane-associated TFPI targeted to either caveolae [TFPI–GPI (TFPI–glycosylphosphatidylinositol anchor chimaera)] or to bulk plasma membrane [TFPI–TM (TFPI–transmembrane anchor chimaera)]. Stable clones had equal expression of surface TF and TFPI. TX-114 cellular lysis confirmed localization of TFPI–GPI to detergent-insoluble membrane fractions, whereas TFPI–TM localized to the aqueous phase. TFPI–GPI and TFPI–TM were equally effective direct inhibitors of fXa in amidolytic assays. However, TFPI–GPI was a significantly better inhibitor of TF–fVIIa than TFPI–TM, as measured in both amidolytic and plasma-clotting assays. Disrupting caveolae by removing membrane cholesterol from EA.hy926 cells, which make TFPIα, CHO cells transfected with TFPIβ and HUVECs (human umbilical vein endothelial cells) did not affect their fXa inhibition, but significantly decreased their inhibition of TF–fVIIa. These studies confirm and quantify the enhanced anticoagulant activity of TFPI localized within caveolae, demonstrate that caveolae enhance the inhibitory activity of both TFPI isoforms and define the effect of caveolae as specifically enhancing the anti-TF activity of TFPI.

scholarly journals Factor Xa Cleavage of Tissue Factor Pathway Inhibitor Is Associated with Loss of Anticoagulant Activity

1998 ◽  
Vol 80 (08) ◽  
pp. 273-280 ◽  
Author(s):  
Irene Salemink ◽  
Jo Franssen ◽  
George Willems ◽  
Coenraad Hemker ◽  
Anguo Li ◽  
...  
Keyword(s):  
Tissue Factor ◽  
Inhibitory Activity ◽  
Tissue Factor Pathway Inhibitor ◽  
Factor Viia ◽  
Anticoagulant Activity ◽  
Factor Xa ◽  
Peptide Bond ◽  
Factor X ◽  
Tissue Factor Pathway ◽  
Factor Assay

SummaryTissue factor : factor VIIa induced activation of blood coagulation is inhibited by the complex between factor Xa and tissue factor pathway inhibitor (factor Xa : TFPI). We recently reported that phospholipid-bound factor Xa reduces the high binding affinity of factor Xa : TFPI for negatively charged phospholipids by a partial degradation of TFPI (17). The present study was undertaken to elucidate the factor Xa cleavage sites in TFPI and to delineate the consequences of this proteolysis with respect to the inhibitory activity of factor Xa : TFPI. We found that phospholipid-bound factor Xa cleaves in TFPI the peptide bonds between Lys86-Thr87 and Arg199-Ala200. Interestingly, Arg199 is the P1 residue of the third Kunitz-type protease inhibitor domain. The fast cleavage of the Arg199-Ala200 bond results in a 50-70% reduction of the anticoagulant activity of factor Xa : TFPI, as determined with a dilute tissue factor assay, but is not associated with a diminished inhibitory activity of factor Xa : TFPI towards TF : factor VIIa catalyzed activation of factor X. On the other hand, the slower cleavage of the Lys86-Thr87 peptide bond was associated with both a diminished anticoagulant and anti-TF : factor VIIa activity. Dissociation of factor Xa from the cleaved TFPI was not observed. These data provide evidence for a dual role of factor Xa since it is the essential cofactor in the TFPI-controlled regulation of TF-dependent coagulation as well as a catalyst of the inactivation of TFPI.

scholarly journals Small Peptides Blocking Inhibition of Factor Xa and Tissue Factor-Factor VIIa by Tissue Factor Pathway Inhibitor (TFPI)

2013 ◽  
Vol 289 (3) ◽  
pp. 1732-1741 ◽  
Author(s):  
Michael Dockal ◽  
Rudolf Hartmann ◽  
Markus Fries ◽  
M. Christella L. G. D. Thomassen ◽  
Alexandra Heinzmann ◽  
...  
Keyword(s):  
Tissue Factor ◽  
Tissue Factor Pathway Inhibitor ◽  
Factor Viia ◽  
Anticoagulant Activity ◽  
Tight Binding ◽  
Factor Xa ◽  
Factor X ◽  
Intermediate Segment ◽  
Tissue Factor Pathway ◽  
Α Helix

Tissue factor pathway inhibitor (TFPI) is a Kunitz-type protease inhibitor that inhibits activated factor X (FXa) via a slow-tight binding mechanism and tissue factor-activated FVII (TF-FVIIa) via formation of a quaternary FXa-TFPI-TF-FVIIa complex. Inhibition of TFPI enhances coagulation in hemophilia models. Using a library approach, we selected and subsequently optimized peptides that bind TFPI and block its anticoagulant activity. One peptide (termed compound 3), bound with high affinity to the Kunitz-1 (K1) domain of TFPI (Kd ∼1 nm). We solved the crystal structure of this peptide in complex with the K1 of TFPI at 2.55-Å resolution. The structure of compound 3 can be segmented into a N-terminal anchor; an Ω-shaped loop; an intermediate segment; a tight glycine-loop; and a C-terminal α-helix that is anchored to K1 at its reactive center loop and two-stranded β-sheet. The contact surface has an overall hydrophobic character with some charged hot spots. In a model system, compound 3 blocked FXa inhibition by TFPI (EC50 = 11 nm) and inhibition of TF-FVIIa-catalyzed FX activation by TFPI (EC50 = 2 nm). The peptide prevented transition from the loose to the tight FXa-TFPI complex, but did not affect formation of the loose FXa-TFPI complex. The K1 domain of TFPI binds and inhibits FVIIa and the K2 domain similarly inhibits FXa. Because compound 3 binds to K1, our data show that K1 is not only important for FVIIa inhibition but also for FXa inhibition, i.e. for the transition of the loose to the tight FXa-TFPI complex. This mode of action translates into normalization of coagulation of hemophilia plasmas. Compound 3 thus bears potential to prevent bleeding in hemophilia patients.

Proteolysis of Tissue Factor Pathway Inhibitor (TFPI) by Plasmin: Effect on TFPI Activity

1998 ◽  
Vol 80 (09) ◽  
pp. 423-427 ◽  
Author(s):  
Anguo Li ◽  
Tze-Chein Wun
Keyword(s):  
Tissue Factor ◽  
Tissue Factor Pathway Inhibitor ◽  
Factor Viia ◽  
Anticoagulant Activity ◽  
Limited Proteolysis ◽  
Factor Xa ◽  
Clot Lysis ◽  
Plasma Clot ◽  
Amino Terminal ◽  
Tissue Factor Pathway

SummaryAn important regulator of the initiation of blood coagulation is the plasma glycoprotein, tissue factor pathway inhibitor (TFPI). TFPI inhibits factor Xa and factor VIIa/tissue factor complex, thereby dampens the proteolytic cascade of the tissue factor pathway. Plasma clot lysis is primarily mediated by the fibrinolytic enzyme, plasmin, which is generated through limited proteolysis of plasminogen by endogenous or exogenously administered plaminogen activators. In this study, the interaction of plasmin with recombinant E. coli-derived TFPI (rTFPI) was examined. Plasmin was found to cause a time and concentration dependent proteolysis of rTFPI, resulting in the decrease of anti-factor Xa (measured by chromogenic substrate assay) and anticoagulant (measured by tissue factor-induced clotting assay) activities. Amino-terminal sequencing of the proteolytic fragments revealed that plasmin cleaved rTFPI at K86-T87, R107-G108, R199-A200, K249-G250, and K256-R257. Western blot analysis showed that proteolysis of exogenously added rTFPI also occurred in plasma supplemented with urokinase, and this is accompanied by decrease of anticoagulant activity. These changes were abolished by addition of aprotinin, an inhibitor of plasmin. These data indicate that TFPI is susceptible to proteolysis when plasma fibrinolytic system is activated. The results taken together suggest that plasmin degradation of TFPI may contribute to rethrombosis after thrombolysis, and may contribute to the variability of the efficacy of TFPI in various thrombolysis/reocclusion studies reported previously.

Abstract 43: Prothrombinase Assembled with Factor V Leiden is Resistant to Inhibition by Tissue Factor Pathway Inhibitor α Lowering the Procoagulant Threshold for Initiation of Coagulation

2016 ◽  
Vol 36 (suppl_1) ◽  
Author(s):  
Jeremy P Wood ◽  
Lisa M Baumann Kreuziger ◽  
Susan A Maroney ◽  
Rodney M Camire ◽  
Alan E Mast
Keyword(s):  
Tissue Factor ◽  
Thrombin Generation ◽  
Tissue Factor Pathway Inhibitor ◽  
Platelet Rich Plasma ◽  
Anticoagulant Activity ◽  
Factor V Leiden ◽  
Factor Xa ◽  
Factor V ◽  
Activation Threshold ◽  
Tissue Factor Pathway

Factor V (FV) assembles with factor Xa (FXa) into prothrombinase, the enzymatic complex that converts prothrombin to thrombin. Tissue factor pathway inhibitor α (TFPIα) inhibits prothrombinase by high affinity interactions with FXa-activated FV and the FXa active site, thereby blocking the initiation of coagulation. FV Leiden (FVL) is strongly linked to venous thrombosis through its resistance to degradation by activated protein C (aPC), which enhances the propagation of coagulation. FVL combined with a 50% reduction in TFPI causes severe thrombosis and perinatal lethality in mice, suggesting that FVL also promotes the initiation of coagulation. To examine this possibility, thrombin generation assays initiated with limiting FXa were performed with control or FVL plasma and platelet-rich plasma (PRP). The activation threshold for thrombin generation was 10 to 20 pM FXa in 10 control plasmas, but was 5 pM in 4 of 10 homozygous FVL plasmas. FVL PRP had a similar decrease in the activation threshold. The differences in activation threshold were totally normalized by an anti-TFPI antibody, while exogenous TFPIα and a FV-binding peptide that mimics TFPIα had reduced anticoagulant activity in FVL plasma, revealing that the procoagulant effects of FVL in these assays rely on TFPIα. Next, FVL plasmas were studied in fibrin clot formation assays, as they are sensitive to small amounts of thrombin. In reactions activated with 0.5 pM FXa, 1 of 8 control plasmas, compared to 7 of 8 homozygous FVL plasmas, clotted within 60 minutes, with differences again normalized by the anti-TFPI antibody. In prothrombinase activity assays using purified proteins, TFPIα was a 1.7-fold weaker inhibitor of prothrombinase assembled with FVL compared to FV. Thus, in addition to its aPC-mediated effect on the propagation of coagulation, FVL is resistant to TFPIα inhibition, exerting a procoagulant effect on coagulation initiation. This is evident in responses to small stimuli, where TFPIα blocks clotting in plasmas with FV but not FVL. The TFPIα-mediated modulation of the procoagulant threshold may explain the severe perinatal thrombosis in FVL mice with decreased TFPI and be clinically relevant in the clotting associated with oral contraceptives, which cause acquired TFPI deficiency.

Comparison of Recombinant Tissue Factor Pathway Inhibitors Expressed in Human SK Hepatoma, Mouse C127, Baby Hamster Kidney, and Chinese Hamster Ovary Cells

1992 ◽  
Vol 68 (01) ◽  
pp. 054-059 ◽  
Author(s):  
Tze-Chein Wun ◽  
Kuniko K Kretzmer ◽  
Mark O Palmier ◽  
Kathleen C Day ◽  
Margaret D Huang ◽  
...  
Keyword(s):  
Tissue Factor ◽  
Chinese Hamster Ovary ◽  
Chinese Hamster Ovary Cells ◽  
Sodium Dodecyl ◽  
Chinese Hamster ◽  
Factor Xa ◽  
Baby Hamster Kidney ◽  
Carboxy Terminus ◽  
Mammalian Expression ◽  
Tissue Factor Pathway

SummaryRecombinant tissue factor pathway inhibitor (rTFPI) has been expressed in four mammalian expression systems using human SK hepatoma, mouse C127, baby hamster kidney (BHK), and Chinese hamster ovary (CHO) cells as hosts. On sodium dodecyl sulfate polyacrylamide gel electrophoresis, the immunoaffinity purified rTFPIs all show broad bands and the mean molecular weight of SK hepatoma and C127 rTFPIs (M r ~ 38,000) appear larger than those of BHK and CHO rTFPIs (M r ~ 35,000). All these proteins inhibit factor Xa and appear to bind factor Xa with 1:1 stoichiometry. The ability of these proteins to inhibit tissue factor-induced coagulation in plasma was examined using a prothrombin time assay. The relative activities of SK rTFPI:C127 rTFPI:BHK rTFPI:CHO rTFPI were found to be 28:15:2.1:1. By Western blot using specific antisera against the amino- and carboxy-termini of TFPI as probes, it is found that all the immunoaffinity purified rTFPIs possess approximately equal amounts of the amino terminus, but the C127 and BHK rTFPIs are deficient in carboxy terminus and the CHO rTFPI is essentially devoid of this region of the protein. Mono S chromatography allowed separation of the full-length and the truncated molecules with high and low anticoagulant activities, respectively. The above results suggest that proteolysis of the carboxy terminus of TFPI occurs to different extent when TFPI is expressed in different cells and that the carboxy terminal region of the TFPI molecule is important for the inhibition of tissue factor-induced coagulation. Recombinant and non-recombinant SK hepatoma cells appear to produce TFPI with the least amount of proteolysis compared with other cell systems tested and SK TFPI functionally resembles TFPI circulating in the blood.

Localization of tissue factor pathway inhibitor to lipid rafts is not required for inhibition of factor VIIa/tissue factor activity

2003 ◽  
Vol 89 (01) ◽  
pp. 65-73 ◽  
Author(s):  
Garnet Jack ◽  
Keith Page ◽  
Tina Tetzloff ◽  
Connie Hall ◽  
Alan Mast ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Tissue Factor ◽  
Lipid Rafts ◽  
Tissue Factor Pathway Inhibitor ◽  
Factor Viia ◽  
Factor Xa ◽  
Hek293 Cells ◽  
Membrane Domains ◽  
Gpi Anchor ◽  
Tissue Factor Pathway

SummaryTissue factor pathway inhibitor (TFPI) abrogates coagulation initiated by the factor VIIa/tissue factor catalytic complex. While the gene structure of TFPI suggests that it is a secreted protein, a large pool of TFPI is associated with the vascular endothelium through its affinity for a glycosylphosphatidylinositol (GPI)-linked membrane protein. Inhibition of tissue factor by TFPI coincides with the translocation of quaternary complexes containing tissue factor, factor VIIa, factor Xa, and TFPI to detergent-insoluble plasma membrane domains rich in cholesterol, sphingomyelin, and GPI-linked proteins known as lipid rafts and caveolae. It is not known if localization of TFPI to these membrane domains is required for its inhibition of tissue factor procoagulant activity. We generated chimeric TFPI molecules linked directly to the plasma membrane via a GPI anchor or hydrophobic transmembrane domain and expressed these in HEK293 cells that produce tissue factor but not endogenous TFPI. The GPI-anchored chimera was exclusively enriched in detergent-insoluble membrane fractions while the transmembrane molecule was not. Transfectants expressing equal levels of the GPI-linked or transmembrane TFPI displayed equal anticoagulant potency as assessed by tissue factor-mediated conversion of factor X to factor Xa. Disruption of lipid rafts with cyclodextrin likewise had no effect on the inhibitory activity of the transmembrane or GPI-linked TFPI chimeras in HEK293 cells, nor on endogenous TFPI expressed by ECV304 cells. Thus, we conclude that the GPI anchor and membrane localization to lipid rafts does not enhance inhibition of factor VIIa/ tissue factor by cell-surface associated TFPI.

Aptamer ARC19499 mediates a procoagulant hemostatic effect by inhibiting tissue factor pathway inhibitor

Blood ◽  
2011 ◽  
Vol 117 (20) ◽  
pp. 5514-5522 ◽  
Author(s):  
Emily K. Waters ◽  
Ryan M. Genga ◽  
Michael C. Schwartz ◽  
Jennifer A. Nelson ◽  
Robert G. Schaub ◽  
...  
Keyword(s):  
Tissue Factor ◽  
Tissue Factor Pathway Inhibitor ◽  
Hemophilia A ◽  
Factor Viia ◽  
Factor Xa ◽  
Coagulation Factor ◽  
Factor Ix ◽  
Intrinsic Pathway ◽  
Extrinsic Pathway ◽  
Tissue Factor Pathway

Abstract Hemophilia A and B are caused by deficiencies in coagulation factor VIII (FVIII) and factor IX, respectively, resulting in deficient blood coagulation via the intrinsic pathway. The extrinsic coagulation pathway, mediated by factor VIIa and tissue factor (TF), remains intact but is negatively regulated by tissue factor pathway inhibitor (TFPI), which inhibits both factor VIIa and its product, factor Xa. This inhibition limits clot initiation via the extrinsic pathway, whereas factor deficiency in hemophilia limits clot propagation via the intrinsic pathway. ARC19499 is an aptamer that inhibits TFPI, thereby enabling clot initiation and propagation via the extrinsic pathway. The core aptamer binds tightly and specifically to TFPI. ARC19499 blocks TFPI inhibition of both factor Xa and the TF/factor VIIa complex. ARC19499 corrects thrombin generation in hemophilia A and B plasma and restores clotting in FVIII-neutralized whole blood. In the present study, using a monkey model of hemophilia, FVIII neutralization resulted in prolonged clotting times as measured by thromboelastography and prolonged saphenous-vein bleeding times, which are consistent with FVIII deficiency. ARC19499 restored thromboelastography clotting times to baseline levels and corrected bleeding times. These results demonstrate that ARC19499 inhibition of TFPI may be an effective alternative to current treatments of bleeding associated with hemophilia.

scholarly journals Tissue factor pathway inhibitor: the carboxy-terminus is required for optimal inhibition of factor Xa

Blood ◽  
1992 ◽  
Vol 79 (8) ◽  
pp. 2004-2010 ◽  
Author(s):  
R Wesselschmidt ◽  
K Likert ◽  
T Girard ◽  
TC Wun ◽  
GJ Jr Broze
Keyword(s):  
Tissue Factor ◽  
Tissue Factor Pathway Inhibitor ◽  
Factor Viia ◽  
Factor Xa ◽  
Reversible Inhibitor ◽  
Normal Plasma ◽  
Encounter Complex ◽  
Factor Xa Inhibition ◽  
Tissue Factor Pathway ◽  
Inhibit Factor

Abstract Tissue factor pathway inhibitor (TFPI) is a multivalent Kunitz-type protease inhibitor that binds to and inactivates factor Xa directly, and in a factor Xa-dependent fashion inhibits the factor VIIa/tissue factor catalytic complex. TFPI is a slow, tight-binding, competitive, and reversible inhibitor of factor Xa, in which the formation of an initial encounter complex between TFPI and factor Xa is followed by slow isomerization to a final, tightened complex. Wild-type recombinant TFPI (rTFPI), expressed in mouse C127 cells, separates into two forms on heparin-agarose chromatography that elute at 0.3 mol/L and 0.6 mol/L NaCl. Western blot analysis shows that both forms contain the N- terminus of full-length TFPI, but only rTFPI(0.6) is recognized by an antibody directed against the C-terminus. rTFPI(0.3) and rTFPI(0.6) inhibit factor Xa with 1:1 stoichiometry and inhibit factor VIIa/tissue factor equally in an endpoint-type assay. However, rTFPI(0.6) is a more potent inhibitor than rTFPI(0.3) of coagulation in normal plasma induced by either factor Xa or tissue factor. The initial inhibition of factor Xa (less than 5 seconds) produced by rTFPI(0.6) is several-fold greater than that produced by rTFPI(0.3), presumably reflecting a lower Ki of the immediate encounter complex between factor Xa and TFPI. The differential effect of these forms of TFPI on tissue factor-induced coagulation in normal plasma appears to be directly related to their ability to inhibit factor Xa. To confirm the role of the C-terminal region of TFPI in optimal factor Xa inhibition, a carboxy-terminal mutant of rTFPI, which is truncated after leucine 252 and thus lacks the basic sequence K T K R K R K K Q R V K (residues 254–265), was expressed in C127 cells. This form of rTFPI elutes from heparin-agarose at 0.28 mol/L NaCl and inhibits factor Xa at a rate that is slower than rTFPI(0.3). The Ki(final)s for factor Xa inhibition by rTFPI(0.6), rTFPI(0.3), and rTFPI1–252 are 3.1 +/- 0.6, 19.6 +/- 0.8, and 19.6 +/- 3.0 pmol/L, respectively.

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