scholarly journals Tissue Factor Pathway Inhibitor Gene Disruption Produces Intrauterine Lethality in Mice

Blood ◽  
1997 ◽  
Vol 90 (3) ◽  
pp. 944-951 ◽  
Author(s):  
Zhong-Fu Huang ◽  
Darryl Higuchi ◽  
Nina Lasky ◽  
George J. Broze
Keyword(s):  
Tissue Factor ◽  
Gene Disruption ◽  
Tissue Factor Pathway Inhibitor ◽  
Factor Viia ◽  
Factor Xa ◽  
Yolk Sac ◽  
Functional Assay ◽  
Wild Type ◽  
Kunitz Domain ◽  
Tissue Factor Pathway

Abstract Tissue factor pathway inhibitor (TFPI) is a multivalent Kunitz-type proteinase inhibitor that directly inhibits factor Xa and, in a factor Xa–dependent fashion, produces feedback inhibition of the factor VIIa/TF catalytic complex responsible for the initiation of coagulation. To further define the physiologic role of TFPI, gene-targeting techniques were used to disrupt exon 4 of the TFPI gene in mice. This exon encodes Kunitz domain-1 of TFPI, which is required for factor VIIa/TF inhibition. In mice heterozygous for TFPI gene-disruption, TFPIK1(+/−), an altered form of TFPI lacking Kunitz domain-1, circulates in plasma at a concentration ∼40% that of wild-type TFPI. TFPIK1(+/−) animals have plasma TFPI activity ∼50% that of wild-type mice, based on a functional assay that measures factor VIIa/TF inhibition, and have a normal phenotype. Sixty percent of TFPIK1(−/−) mice die between embryonic days E9.5 and E11.5 with signs of yolk sac hemorrhage. The extent of structural abnormalities within the yolk sac vascular system appears to mirror the condition of the embryo, suggesting that the embryonic and extra-embryonic tissues are both responding to same insult, presumably circulatory insufficiency. Organogenesis is normal in TFPIK1 null animals that progress beyond E11.5, but hemorrhage, particularly in the central nervous system and tail, is evident during later gestation and none of the TFPIK1(−/−) mice survive to the neonatal period. The presence of immunoreactive fibrin(ogen) in the liver and intravascular thrombi is consistent with the notion that unregulated factor VIIa/TF action and a consequent consumptive coagulopathy underlies the bleeding diathesis in these older embryos. Human TFPI-deficient embryos may suffer a similar fate because an individual with TFPI deficiency has not been identified.

scholarly journals Tissue Factor Pathway Inhibitor Gene Disruption Produces Intrauterine Lethality in Mice

Blood ◽  
1997 ◽  
Vol 90 (3) ◽  
pp. 944-951 ◽  
Author(s):  
Zhong-Fu Huang ◽  
Darryl Higuchi ◽  
Nina Lasky ◽  
George J. Broze
Keyword(s):  
Tissue Factor ◽  
Gene Disruption ◽  
Tissue Factor Pathway Inhibitor ◽  
Factor Viia ◽  
Factor Xa ◽  
Yolk Sac ◽  
Functional Assay ◽  
Wild Type ◽  
Kunitz Domain ◽  
Tissue Factor Pathway

Tissue factor pathway inhibitor (TFPI) is a multivalent Kunitz-type proteinase inhibitor that directly inhibits factor Xa and, in a factor Xa–dependent fashion, produces feedback inhibition of the factor VIIa/TF catalytic complex responsible for the initiation of coagulation. To further define the physiologic role of TFPI, gene-targeting techniques were used to disrupt exon 4 of the TFPI gene in mice. This exon encodes Kunitz domain-1 of TFPI, which is required for factor VIIa/TF inhibition. In mice heterozygous for TFPI gene-disruption, TFPIK1(+/−), an altered form of TFPI lacking Kunitz domain-1, circulates in plasma at a concentration ∼40% that of wild-type TFPI. TFPIK1(+/−) animals have plasma TFPI activity ∼50% that of wild-type mice, based on a functional assay that measures factor VIIa/TF inhibition, and have a normal phenotype. Sixty percent of TFPIK1(−/−) mice die between embryonic days E9.5 and E11.5 with signs of yolk sac hemorrhage. The extent of structural abnormalities within the yolk sac vascular system appears to mirror the condition of the embryo, suggesting that the embryonic and extra-embryonic tissues are both responding to same insult, presumably circulatory insufficiency. Organogenesis is normal in TFPIK1 null animals that progress beyond E11.5, but hemorrhage, particularly in the central nervous system and tail, is evident during later gestation and none of the TFPIK1(−/−) mice survive to the neonatal period. The presence of immunoreactive fibrin(ogen) in the liver and intravascular thrombi is consistent with the notion that unregulated factor VIIa/TF action and a consequent consumptive coagulopathy underlies the bleeding diathesis in these older embryos. Human TFPI-deficient embryos may suffer a similar fate because an individual with TFPI deficiency has not been identified.

Structure and Biology of Tissue Factor Pathway Inhibitor

2001 ◽  
Vol 86 (10) ◽  
pp. 959-972 ◽  
Author(s):  
Jens Birktoft ◽  
Sarah Steer ◽  
S. Paul Bajaj ◽  
Madhu Bajaj
Keyword(s):  
Tissue Factor ◽  
Tissue Factor Pathway Inhibitor ◽  
Factor Viia ◽  
Factor Xa ◽  
Knock Out ◽  
Linker Region ◽  
Disease States ◽  
Kunitz Domain ◽  
Tissue Factor Pathway ◽  
Basic Region

SummaryHuman tissue factor pathway inhibitor (TFPI) is a modular protein comprised of three Kunitz type domains flanked by peptide segments that are less structured. The sequential order of the elements are: an N-terminal acidic region followed by the first Kunitz domain (K1), a linker region, a second Kunitz domain (K2), a second linker region, the third Kunitz domain (K3), and the C-terminal basic region. The K1 domain inhibits factor VIIa complexed to tissue factor (TF) while the K2 domain inhibits factor Xa. No direct protease inhibiting functions have been demonstrated for the K3 domain. Importantly, the Xa-TFPI complex is a much more potent inhibitor of the VIIa-TF than TFPI by itself. Furthermore, the C-terminal basic region of TFPI is required for rapid physiologic inhibition of coagulation and is needed for the inhibition of smooth muscle cell proliferation. Although a number of additional targets for attachment have been reported, the C-terminal basic region appears to play an important role in binding of TFPI to cell surfaces. A primary site of TFPI synthesis is endothelium and the endothelium-bound TFPI contributes to the antithrombotic potential of the vascular endothelium. Further, increased levels of plasma TFPI under septic conditions may represent endothelial dysfunction. We have proposed that the extravascular cells that synthesize TF also synthesize TFPI providing dual components necessary for the regulation of clotting in their microenvironment. Like the TF synthesis in these cells is augmented by serum, so is the case with the TFPI gene expression. TFPI gene knock out mice reveal embryonic lethality suggesting a possible role of this protein in early development. Since TF-induced coagulation is thought to play a significant role in many disease states, including disseminated intravascular clotting, sepsis, acute lung injury and cancer, recombinant TFPI may be a beneficial therapeutic agent in these disease states to attenuate pathologic clotting. The purpose of this review is to outline recent developments in the field related to the structural specificity and biology of TFPI.

Factor Xa Enhances the Binding of Tissue Factor Pathway Inhibitor to Acidic Phospholipids

1996 ◽  
Vol 75 (05) ◽  
pp. 796-800 ◽  
Author(s):  
Sanne Valentin ◽  
Inger Schousboe
Keyword(s):  
Tissue Factor ◽  
Tissue Factor Pathway Inhibitor ◽  
Factor Xa ◽  
Microtitre Plate ◽  
C Terminus ◽  
Microtitre Plate Assay ◽  
Kunitz Domain ◽  
Tissue Factor Pathway ◽  
Acidic Phospholipids

SummaryIn the present study, the interaction between tissue factor pathway inhibitor (TFPI) and phospholipids has been characterized using a microtitre plate assay. TFPI was shown to bind calcium-independently to an acidic phospholipid surface composed of phosphatidylserine, but not a surface composed of the neutral phosphatidylcholine. The interaction was demonstrated to be dependent on the presence of the TFPI C-terminus. The presence of heparin (1 U/ml, unfractionated) was able to significantly reduce the binding of TFPI to phospholipid. The interaction of TFPI with phosphatidylserine was significantly decreased in the presence of calcium, but this was counteracted, and even enhanced, following complex formation of TFPI with factor Xa prior to incubation with the phospholipid surface. Moreover, a TFPI variant, not containing the third Kunitz domain and the C-terminus, was unable to bind to phospholipid. However, following the formation of a TFPI/factor Xa-complex this TFPI variant was capable of interacting with the phospholipid surface. This indicates that the role of factor Xa as a TFPI cofactor, at least in part, is to mediate the binding of TFPI to the phospholipid surface.

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.

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.

Factor XI Deficient Mice Have Reduced Platelet Accumulation and Fibrin Deposition after Laser Injury.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 218-218
Author(s):  
T. Regan Baird ◽  
David Gailani ◽  
Bruce Furie ◽  
Barbara C. Furie
Keyword(s):  
Tissue Factor ◽  
Factor Viia ◽  
Thrombus Formation ◽  
Factor Xa ◽  
Fibrin Deposition ◽  
Wild Type ◽  
Factor Xi ◽  
Factor Xia ◽  
Tissue Factor Pathway ◽  
Platelet Accumulation

Abstract Tissue factor exposure at sites of vascular injury results in the generation of factor Xa and thrombin. A current model of blood coagulation suggests that the amount of thrombin generated through this pathway is limited by the inhibition of the factor VIIa-tissue factor complex by tissue factor pathway inhibitor in the presence of factor Xa. The initial thrombin activates a number of hemostatic proteins including factor XI. Factor XIa then activates factor IX leading to generation of the tenase complex to maintain the thrombin flux. While in vitro studies support this hypothesis the importance of factor XI for thrombus formation in vivo remains unclear. We have examined thrombus formation upon laser injury to the arterioles (30–50 μm diameter) of the cremaster muscle in living mice lacking factor XI using digital multi-channel fluorescence intravital microscopy. Platelets were labeled with Alexa 488 conjugated murine CD41 Fab fragments by systemic infusion of the antibody. Maximum platelet accumulation in factor XI null mice (median of 35 thrombi in 4 mice) is only 25% of that of wild type mice (median of 40 thrombi in 4 mice) after injury (p<0.03). The time course of platelet accumulation is similar between both genotypes. Maximum platelet accumulation occurs in approximately 90 seconds (p<0.2). Fibrin deposition was observed simultaneously using an Alexa 660 conjugated anti-fibrin antibody that does not recognize fibrinogen. Maximum fibrin deposition in factor XI null mice is 50% that of wild type mice (p<0.001) and the rate of fibrin generation is slower in factor XI null mice. However, the time to achieve half maximal fibrin deposition is approximately the same in factor XI null mice (77 sec) compared to wild type mice (63.5 sec, p<0.09). These data suggest that the primary difference in response to laser induced injury between the factor XI null mice and wild type mice is the level of thrombin generated and supports the hypothesis that factor XI participates in maintaining thrombin flux after inhibition of the factor VII-tissue factor. The model above postulates a single source of tissue factor, the vessel wall, and further, that the tissue factor-factor VIIa complex formed from the exposed tissue factor is rapidly inactivated by tissue factor pathway inhibitor after the appearance of the initial factor Xa formed. In addition it has been suggested that a rapidly growing thrombus blocks access to vascular wall tissue factor. However we have recently observed that there is a P-selectin and P-selectin glycoprotein ligand 1 dependent pathway of blood coagulation that recruits blood borne tissue factor into a growing thrombus at sites of laser-induced vessel injury. Both vessel wall and blood borne tissue factor are required for normal thrombus formation. Our data suggest that although tissue factor is continuously recruited to the growing thrombus, factor XIa plays a significant role in thrombin generation.

Engineering Kunitz Domain of Human Tissue Factor Pathway Inhibitor-2 to Selectively Inhibit Fibrinolysis: An Antifibrinolytic Agent with Potential to Replace Aprotinin

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 24-24
Author(s):  
Madhu S Bajaj ◽  
Godwin I Ogueli ◽  
Amy E Schmidt ◽  
Sreejesh Shanker ◽  
S. Paul Bajaj
Keyword(s):  
Blood Loss ◽  
Tissue Factor ◽  
Tissue Factor Pathway Inhibitor ◽  
Factor Viia ◽  
Total Blood Loss ◽  
Total Blood ◽  
Antifibrinolytic Agent ◽  
Kunitz Domain ◽  
Factor Xia ◽  
Tissue Factor Pathway

Abstract Tissue factor pathway inhibitor-2 (TFPI-2) inhibits factor XIa, plasma kallikrein and factor VIIa/tissue factor; accordingly, it has been proposed for use as an anticoagulant. Full-length TFPI-2 or its isolated first Kunitz domain (KD1) also inhibits plasmin and therefore it has been proposed for use as an antifibrinolytic agent. However, the anticoagulant properties of TFPI-2 or KD1 would diminish its antifibrinolytic function. In this report, structure based investigations and analysis of the serine proteases profiles revealed that coagulation enzymes prefer a hydrophobic residue at the P2′ (nomenclature of Schechter and Berger, BBRC, 27:157–162, 1967) position in their substrates/inhibitors, whereas plasmin prefers a positively charged arginine residue at the corresponding position in its substrates/inhibitors. Based upon this observation, we changed the P2′ residue Leu17 (bovine pancreatic trypsin inhibitor/aprotinin numbering) in KD1 to Arg (KD1-L17R) and compared its inhibitory properties with the wild-type KD1 (KD1-WT). Both WT and KD1-L17R were expressed in E. Coli, folded and purified to homogeneity. Amino-terminal sequences and mass spectra revealed proper folding of the KD1-WT and KD1-L17R. As compared to KD1-WT, the KD1-L17R neither prolonged the activated partial thromboplastin time of normal plasma nor it inhibited factor XIa, plasma kallikrein or factor VIIa/tissue factor. Further, KD1-L17R inhibited plasmin with ~4-fold increased affinity. In a mouse liver laceration model of bleeding from small vessels, KD1-L17R reduced total blood loss by 84% compared with KD1-WT, which reduced total blood loss by 10%. Moreover, in this bleeding model, KD1-L17R was more effective than aprotinin (70% reduction), which has been used as an antifibrinolytic agent to decrease blood loss during major surgery. In this model, KD1-L17R was also more effective than the lysine analogue tranexamic acid (52% reduction). In additional studies, in a tail transection model of bleeding from a large vessel, KD1-L17R reduced total blood loss by 70% and was more effective than KD1-WT (46% reduction), aprotinin (43% reduction) and tranexamic acid (67% reduction). Notably, as compared to aprotinin, renal toxicity manifesting as multifocal tubular necrosis by histopathology was not observed with KD1-L17R or KD1-WT. In conclusion, KD1-L17R is a specific inhibitor of plasmin without anticoagulant properties and is more effective in reducing blood loss compared with known antifibrinolytic agents in clinical use.

scholarly journals Structural and functional characterization of tissue factor pathway inhibitor following degradation by matrix metalloproteinase-8

2002 ◽  
Vol 367 (2) ◽  
pp. 451-458 ◽  
Author(s):  
Anna C. CUNNINGHAM ◽  
Karen A. HASTY ◽  
Jan J. ENGHILD ◽  
Alan E. MAST
Keyword(s):  
Matrix Metalloproteinase ◽  
Tissue Factor ◽  
Tissue Factor Pathway Inhibitor ◽  
Proteolytic Enzymes ◽  
Functional Characterization ◽  
Factor Xa ◽  
Terminal Region ◽  
The Third ◽  
Kunitz Domain ◽  
Tissue Factor Pathway

Vascular injury results in the activation of coagulation and the release of proteolytic enzymes from neutrophils and connective- tissue cells. High concentrations of these inflammatory proteinases may destroy blood coagulation proteins, contributing to coagulation and bleeding disorders associated with severe inflammation. Matrix metalloproteinase-8 (MMP-8) is released from neutrophils at sites of inflammation and vascular disease. We have investigated the effect of MMP-8 degradation on the anticoagulant function of tissue factor pathway inhibitor (TFPI) as a potential pathological mechanism contributing to coagulation disorders. MMP-8 cleaves TFPI following Ser174 within the connecting region between the second and third Kunitz domains (kcat/Km75M-1·s-1) as well as following Lys20 within the NH2-terminal region. MMP-8 cleavage of TFPI decreases the anticoagulant activity of TFPI in factor Xa initiated clotting assays as well as the ability of TFPI to inhibit factor Xa in amidolytic assays. Yet, MMP-8 cleavage does not alter the ability of TFPI to inhibit trypsin. Since the inhibition of both factor Xa and trypsin is mediated by binding to the second Kunitz domain, these results suggest that regions of TFPI other than the second Kunitz domain may directly interact with factor Xa. 125I-factor Xa ligand blots of TFPI fragments generated following MMP-8 degradation were used for probing binding interactions between factor Xa and regions of TFPI, other than the second Kunitz domain. In experiments performed under reducing conditions that disrupt the Kunitz domain structure, 125I-factor Xa binds to the C-terminal fragment of MMP-8-degraded TFPI. This fragment contains portions of TFPI distal to Ser174, which include the third Kunitz domain and the basic C-terminal region. An altered form of TFPI lacking the third Kunitz domain, but containing the C-terminal region, was used to demonstrate that the C-terminal region directly interacts with factor Xa.

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