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.

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.

scholarly journals TFPIβ is the GPI-anchored TFPI isoform on human endothelial cells and placental microsomes

Blood ◽  
2012 ◽  
Vol 119 (5) ◽  
pp. 1256-1262 ◽  
Author(s):  
Thomas J. Girard ◽  
Elodee Tuley ◽  
George J. Broze
Keyword(s):  
Endothelial Cells ◽  
Tissue Factor ◽  
Feedback Inhibition ◽  
Culture Media ◽  
Factor Viia ◽  
Factor Xa ◽  
Gpi Anchor ◽  
C Terminus ◽  
Before And After ◽  
Tissue Factor Pathway

Abstract Tissue factor pathway inhibitor (TFPI) produces factor Xa-dependent feedback inhibition of factor VIIa/tissue factor-induced coagulation. Messages for 2 isoforms of TFPI have been identified. TFPIα mRNA encodes a protein with an acidic N-terminus, 3 Kunitz-type protease inhibitor domains and a basic C-terminus that has been purified from plasma and culture media. TFPIβ mRNA encodes a form in which the Kunitz-3 and C-terminal domains of TFPIα are replaced with an alternative C-terminus that directs the attachment of a glycosylphosphatidylinositol (GPI) anchor, but whether TFPIβ protein is actually expressed is not clear. Moreover, previous studies have suggested that the predominant form of TFPI released from cells by phosphatidylinositol-specific phospholipase C (PIPLC) treatment is TFPIα, implying it is bound at cell surfaces to a separate GPI-anchored coreceptor. Our studies show that the form of TFPI released by PIPLC treatment of cultured endothelial cells and placental microsomes is actually TFPIβ based on (1) migration on SDS-PAGE before and after deglycosylation, (2) the lack of a Kunitz-3 domain, and (3) it contains a GPI anchor. Immunoassays demonstrate that, although endothelial cells secrete TFPIα, greater than 95% of the TFPI released by PIPLC treatment from the surface of endothelial cells and from placental microsomes is TFPIβ.

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.

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.

Tissue Factor Pathway Inhibitor in Tetracycline-induced Pleuritis in Rabbits

1998 ◽  
Vol 79 (03) ◽  
pp. 649-655 ◽  
Author(s):  
Usha Pendurthi ◽  
Siegfried Pueblitz ◽  
Kathleen Koenig ◽  
Todd Williams ◽  
Vijay Mohan Rao ◽  
...  
Keyword(s):  
Tissue Factor ◽  
Pleural Fluid ◽  
Tissue Factor Pathway Inhibitor ◽  
Factor Viia ◽  
Plasma Concentrations ◽  
Coagulation Factors ◽  
Lung Fibroblasts ◽  
Fibrin Deposition ◽  
Tissue Factor Pathway ◽  
Pleural Injury

SummaryPleural fibrin deposition that promotes loculation and fibrosis after pleural injury is initiated by tissue factor (TF). In this study, we sought to determine if tissue factor pathway inhibitor (TFPI), an inhibitor of the TF-factor VIIa complex, was likewise expressed in tetracycline (TCN)-induced pleural injury and, if so, whether TFPI was locally elaborated. Pleural fluid TFPI activity approximated that of plasma by 24 h and doubled by 3 days after intrapleural TCN. By contrast, pleural fluid coagulation factors VII and V remained below plasma concentrations at these intervals. Immunohistochemical studies demonstrated TF, TFPI and fibrin localized in pleural and subpleural tissues and within intrapleural adhesions. TFPI activity and mRNA were also elaborated by rabbit pleural mesothelial cells and lung fibroblasts. TFPI is locally expressed and pleural fluid TFPI exceeds plasma levels during TCN-induced pleural injury. Resident cells as well as extravasation likely contribute to intrapleural TFPI. TFPI expression temporally and anatomically approximates that of TF and may limit TF-induced fibrin deposition in evolving TCN-induced pleuritis.

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