Acute Release of Tissue Factor Pathway Inhibitor after In Vivo Thrombin Generation in Baboons

1999 ◽  
Vol 82 (12) ◽  
pp. 1652-1658 ◽  
Author(s):  
Egbert Kruithof ◽  
Vijay Kakkar ◽  
Florea Lupu ◽  
Cristina Lupu
Keyword(s):  
Tissue Factor ◽  
Thrombin Generation ◽  
Tissue Factor Pathway Inhibitor ◽  
Plasma Concentrations ◽  
Factor Xa ◽  
Treatment Groups ◽  
Tissue Factor Pathway ◽  
Positive Correlations

SummaryTissue factor pathway inhibitor (TFPI), the major downregulator of the procoagulant activity of tissue factor (TF), is synthesised by endothelial cells (EC) and acutely released in vitro after thrombin stimulation. Expression of TF on EC and subsequent thrombin generation occurs in vivo during sepsis or malignancy, inducing disseminated intravascular coagulation (DIC). The present study investigates the changes in plasma TFPI in relation to markers of in vivo thrombin generation induced by injection of factor Xa (FXa)/phospholipids in baboons at dosages leading to partial (48%) or complete fibrinogen depletion. The plasma concentrations of thrombin-antithrombin III (TAT) and fibrinopeptide A (FpA), as markers of in vivo generation of thrombin, were strongly enhanced after injection of FXa/phospholipids. TFPI levels, whether measured as antigen or activity, increased significantly in both treatment groups within few minutes, and were dependent on the dose of FXa/phospholipids. Significant positive correlations between plasma levels of TFPI and of TAT or FpA were observed. Altogether, our results indicate that experimentally induced in vivo generation of thrombin causes the acute release of TFPI, high-lighting a possible novel function of thrombin in downregulation of the coagulation process, potentially relevant for the outcome of DIC.

Effect of Depolymerized Holothurian Glycosaminoglycan (DHG) on Tissue Factor Pathway Inhibitor: In Vitro and In Vivo Studies

1997 ◽  
Vol 78 (02) ◽  
pp. 864-870 ◽  
Author(s):  
Hideki Nagase ◽  
Kei-ichi Enjyoji ◽  
Yu-ichi Kamikubo ◽  
Keiko T Kitazato ◽  
Kenji Kitazato ◽  
...  
Keyword(s):  
Tissue Factor ◽  
Tissue Factor Pathway Inhibitor ◽  
Factor Xa ◽  
Free Form ◽  
Initial Reaction ◽  
Extrinsic Pathway ◽  
Factor Xa Inhibition ◽  
Tissue Factor Pathway

SummaryDepolymerized holothurian glycosaminoglycan (DHG) is a glycosaminoglycan extracted from the sea cucumber Stichopus japonicusSelenka. In previous studies, we demonstrated that DHG has antithrombotic and anticoagulant activities that are distinguishable from those of heparin and dermatan sulfate. In the present study, we examined the effect of DHG on the tissue factor pathway inhibitor (TFPI), which inhibits the initial reaction of the tissue factor (TF)-mediated coagulation pathway. We first examined the effect of DHG on factor Xa inhibition by TFPI and the inhibition of TF-factor Vila by TFPI-factor Xa in in vitro experiments using human purified proteins. DHG increased the rate of factor Xa inhibition by TFPI, which was abolished either with a synthetic C-terminal peptide or with a synthetic K3 domain peptide of TFPI. In contrast, DHG reduced the rate of TF-factor Vila inhibition by TFPI-factor Xa. Therefore, the effect of DHG on in vitroactivity of TFPI appears to be contradictory. We then examined the effect of DHG on TFPI in cynomolgus monkeys and compared it with that of unfractionated heparin. DHG induced an increase in the circulating level of free-form TFPI in plasma about 20-fold when administered i.v. at 1 mg/kg. The prothrombin time (PT) in monkey plasma after DHG administration was longer than that estimated from the plasma concentrations of DHG. Therefore, free-form TFPI released by DHG seems to play an additive role in the anticoagulant mechanisms of DHG through the extrinsic pathway in vivo. From the results shown in the present work and in previous studies, we conclude that DHG shows anticoagulant activity at various stages of coagulation reactions, i.e., by inhibiting the initial reaction of the extrinsic pathway, by inhibiting the intrinsic Xase, and by inhibiting thrombin.

Antibodies to β2-Glycoprotein I Associated with Antiphospholipid Syndrome Suppress the Inhibitory Activity of Tissue Factor Pathway Inhibitor

2000 ◽  
Vol 84 (10) ◽  
pp. 653-656 ◽  
Author(s):  
Irene Salemink ◽  
Ron Blezer ◽  
George Willems ◽  
Monica Galli ◽  
Edouard Bevers ◽  
...  
Keyword(s):  
Tissue Factor ◽  
Blood Coagulation ◽  
Tissue Factor Pathway Inhibitor ◽  
Factor Xa ◽  
Anionic Phospholipid ◽  
Normal Plasma ◽  
Increased Risk ◽  
Tissue Factor Pathway

SummaryAnionic phospholipid membranes have a dual role in blood coagulation: they are essential for the initiation and propagation as well as for the limitation and termination of the blood coagulation process. Patients with the anti-phospholipid syndrome (APS) carrying antibodies against complexes of anionic phospholipids and plasma proteins, show in vitro inhibited phospholipid dependent coagulation reactions, whereas in vivo the presence of these antibodies is associated with an increased risk of thrombosis. In this study we focussed on the effects of these anti-phospholipid antibodies on the regulation of TF-mediated factor Xa (FXa) generation in plasma. We hypothesized that anti-phospholipid antibodies interfere with the phospholipiddependent inhibition by tissue factor pathway inhibitor (TFPI) of TFinduced coagulation. Indeed, total-IgG, anti-cardiolipin-IgG (aCL) and anti-β2GPI-IgG, isolated from patient plasmas, all stimulated TF-induced FXa generation in normal plasma. This enhanced FXa generation was not observed when the patient’s IgG was depleted of anti-β2GPI-IgG or when normal plasma was depleted of β2GPI or TFPI. Taken together, these data indicate that antibodies to β2GPI, circulating in patients with APS, suppress TFPI-dependent inhibition of TF-induced coagulation, which results in an increased FXa generation.

Peptides Inhibiting Tissue Factor Pathway Inhibitor Improve Hemostasis in Mice

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 24-24
Author(s):  
Michael Dockal ◽  
Rudolf Hartmann ◽  
Thomas Polakowski ◽  
Osterkamp Frank ◽  
Hartmut J. Ehrlich ◽  
...  
Keyword(s):  
Blood Loss ◽  
Tissue Factor ◽  
Thrombin Generation ◽  
Tissue Factor Pathway Inhibitor ◽  
Route Of Administration ◽  
Factor X ◽  
Treatment Groups ◽  
Tissue Factor Pathway ◽  
All Treatment

Abstract Abstract 24 Tissue factor pathway inhibitor (TFPI) is an activated (a) factor X (FXa)-dependent inhibitor of the extrinsic factor X (FX) activation complex and efficiently regulates the extrinsic pathway of coagulation. We are developing peptide inhibitors of TFPI with a view to improving hemostasis in hemophilia. The goal is to inhibit the interaction of TFPI with the factor Xa (FXa)-tissue factor (TF)-factor VIIa (FVIIa) complex, and thereby enhance thrombin formation to the extent that a stable clot is formed. Successful development of these peptides could allow treatment of hemophilia via a non-intravenous route of administration. By screening mRNA display libraries, we identified a de novo peptide which binds to and efficiently inhibit TFPI. The peptide was optimized by iterative amino acid substitution resulting in affinity-improved peptide with a well-characterized structure-activity relation. The affinity of the peptide was analyzed by Biacore and ELISA experiments. One of our optimized peptides bound to immobilized TFPI with an affinity below 1 nM. Kunitz domain 1 of TFPI was identified as the interaction site by NMR studies. We characterized the inhibitory activity of this TFPI binding peptide using in vitro model assay systems including inhibition of FXa and of the extrinsic tenase (TF-FVIIa-PL-Ca2+ complex). Inhibition of cell-associated TFPI was verified in a TF- and TFPI-dependent cell-based assay using TNFalpha-stimulated human umbilical vein endothelial cells (HUVECs) as a source of TF and TFPI. Inhibition of plasma TFPI was probed by thrombin generation experiments using FVIII-deficient patient plasma and ROTEM experiments using FVIII-inhibited whole blood. Furthermore, a cross-reactivity of the peptide to murine TFPI was demonstrated in thrombin generation experiments and in murine FXa inhibition model assays. Conjugation of a 40-kDa polyethyleneglycol (PEG) to the peptide base structure resulted in a peptide with significantly reduced renal clearance. The in vivo terminal half-life of this 40kD-PEG modified peptide was 21.5h and 19.8h after 1 mg/kg i.v and s.c. administration, respectively, with 73% bioavailability after s.c. administration. In a tail-tip bleeding model, administration of an anti-murine-TFPI antibody and 40kD-PEG-peptide in combination with sub-therapeutical doses of recombinant coagulation factors (10 IU/kg of rFVIII in FVIII ko mice and rFIX in FIX ko mice) led to a marked reduction of blood loss compared with buffer-treated or rFVIII/rFIX-treated mice. In addition, we established a murine nail-cut model using C57Bl6 wild-type mice for testing in vivo efficacy in a normal FVIII background as well. The TFPI sensitivity of the nail-cut model was verified by an anti-murine TFPI antibody. 40kD-PEG-peptide significantly reduced blood loss after i.v. and s.c. administration in all treatment groups compared with vehicle-treated animals. Furthermore, the i.v. administration of the peptide was well tolerated in all animals across all treatment groups without any signs of acute toxicity. In conclusion, we identified a low-molecular-weight peptide which efficiently inhibit all forms of naturally occurring TFPI proteins, including plasma TFPI and TFPI in vivo. Our results demonstrate that targeting TFPI efficiently improves hemostasis in hemophilia and provide an in vivo proof of concept for a non i.v. route of administration. Thus, the TFPI inhibitory peptides could be useful to prevent bleeding in hemophilia patients. Moreover, this new approach would probably allow treatment of inhibitor patients. Disclosures: Dockal: Baxter Innovations GmbH: Employment. Hartmann:Baxter Innovations GmbH: Employment. Polakowski:3B Pharmaceuticals GmbH: Employment. Frank:3B Pharmaceuticals GmbH: Employment. Ehrlich:Baxter Innovations GmbH: Employment. Schiviz:Baxter Innovations GmbH: Employment. Hoellriegl:Baxter Innovations GmbH: Employment. Muchitsch:Baxter Innovations GmbH: Employment. Reinecke:3B Pharmaceuticals GmbH: Employment. Scheiflinger:Baxter Innovations GmbH: Employment.

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.

Recombinant full-length tissue factor pathway inhibitor fails to bind to the cell surface: implications for catabolism in vitro and in vivo

Blood ◽  
2000 ◽  
Vol 95 (6) ◽  
pp. 1973-1978 ◽  
Author(s):  
Guyu Ho ◽  
Masaaki Narita ◽  
George J. Broze ◽  
Alan L. Schwartz
Keyword(s):  
Tissue Factor ◽  
Tissue Factor Pathway Inhibitor ◽  
Feedback Inhibition ◽  
Factor Xa ◽  
Full Length ◽  
Pharmacokinetic Studies ◽  
Dependent Manner ◽  
Carboxy Terminus ◽  
Tissue Factor Pathway

Abstract Tissue factor pathway inhibitor (TFPI) plays a key role in the regulation of tissue factor-initiated blood coagulation secondary to loss of the integrity of the blood vessel wall. TFPI is a naturally occurring Kunitz-type protease inhibitor that inhibits coagulation factor Xa and, in a factor Xa-dependent manner, mediates feedback inhibition of the factor VIIa/tissuefactor catalytic complex. In vivo full-length TFPI is thought to be primarily bound to the vascular endothelium and the high affinity binding requires an intact carboxy terminus. Here we describe a full-length TFPI molecule, expressed in mouse C127 cells (TFPIC127), which exhibits virtually no cellular binding yet contains the intact carboxy terminus. This TFPI (TFPIC127) is neither internalized nor degraded via the TFPI endocytic receptor, LDL-receptor–related protein. Pharmacokinetic studies of TFPIC127 in vivo demonstrate a 10-fold prolongation in the plasma half-life, compared with that of bacterial recombinant TFPI.

Inhibition of restenosis by tissue factor pathway inhibitor: in vivo and in vitro evidence for suppressed monocyte chemoattraction and reduced gelatinolytic activity

Blood ◽  
2004 ◽  
Vol 103 (5) ◽  
pp. 1653-1661 ◽  
Author(s):  
Christoph W. Kopp ◽  
Thomas Hölzenbein ◽  
Sabine Steiner ◽  
Rodrig Marculescu ◽  
Helga Bergmeister ◽  
...  
Keyword(s):  
Tissue Factor ◽  
Tissue Factor Pathway Inhibitor ◽  
Early Stage ◽  
Thrombus Formation ◽  
Matrix Metalloproteinase 2 ◽  
Monocyte Chemoattractant Protein 1 ◽  
Markers Of Inflammation ◽  
Tissue Factor Pathway

AbstractActivation of inflammatory and procoagulant mechanisms is thought to contribute significantly to the initiation of restenosis, a common complication after balloon angioplasty of obstructed arteries. During this process, expression of tissue factor (TF) represents one of the major physiologic triggers of coagulation that results in thrombus formation and the generation of additional signals leading to vascular smooth muscle cell (VSMC) proliferation and migration. In this study, we have investigated the mechanisms by which inhibition of coagulation at an early stage through overexpression of tissue factor pathway inhibitor (TFPI), an endogenous inhibitor of TF, might reduce restenosis. In a rabbit femoral artery model, percutaneous delivery of TFPI using a recombinant adenoviral vector resulted in a significant reduction of the intimamedia ratio 21 days after injury. Investigating several markers of inflammation and coagulation, we found reduced neointimal expression of monocyte chemoattractant protein-1 (MCP-1), lesional monocyte infiltration, and expression of vascular TF, matrix metalloproteinase-2 (MMP-2), and MMP-9. Moreover, overexpression of TFPI suppressed the autocrine release of platelet-derived growth factor BB (PDGF-BB), MCP-1, and MMP-2 in response to factors VIIa and Xa from VSMCs in vitro and inhibited monocyte TF activity. These results suggest that TFPI exerts its action in vivo through not only thrombotic, but also nonthrombotic mechanisms.

scholarly journals Factor XI contributes to thrombin generation in the absence of factor XII

Blood ◽  
2009 ◽  
Vol 114 (2) ◽  
pp. 452-458 ◽  
Author(s):  
Dmitri V. Kravtsov ◽  
Anton Matafonov ◽  
Erik I. Tucker ◽  
Mao-fu Sun ◽  
Peter N. Walsh ◽  
...  
Keyword(s):  
Tissue Factor ◽  
Thrombin Generation ◽  
Factor Xa ◽  
Factor Ix ◽  
Factor Xii ◽  
Factor Xi ◽  
Factor Xii Deficiency ◽  
Low Concentrations

Abstract During surface-initiated blood coagulation in vitro, activated factor XII (fXIIa) converts factor XI (fXI) to fXIa. Whereas fXI deficiency is associated with a hemorrhagic disorder, factor XII deficiency is not, suggesting that fXI can be activated by other mechanisms in vivo. Thrombin activates fXI, and several studies suggest that fXI promotes coagulation independent of fXII. However, a recent study failed to find evidence for fXII-independent activation of fXI in plasma. Using plasma in which fXII is either inhibited or absent, we show that fXI contributes to plasma thrombin generation when coagulation is initiated with low concentrations of tissue factor, factor Xa, or α-thrombin. The results could not be accounted for by fXIa contamination of the plasma systems. Replacing fXI with recombinant fXI that activates factor IX poorly, or fXI that is activated poorly by thrombin, reduced thrombin generation. An antibody that blocks fXIa activation of factor IX reduced thrombin generation; however, an antibody that specifically interferes with fXI activation by fXIIa did not. The results support a model in which fXI is activated by thrombin or another protease generated early in coagulation, with the resulting fXIa contributing to sustained thrombin generation through activation of factor IX.

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