scholarly journals Enhanced Antifibrinolytic Efficacy of a Plasmin-Specific Kunitz-Inhibitor (60-Residue Y11T/L17R with C-Terminal IEK) of Human Tissue Factor Pathway Inhibitor Type-2 Domain1

2020 ◽  
Vol 9 (11) ◽  
pp. 3684
Author(s):  
Kanagasabai Vadivel ◽  
Anne K. Zaiss ◽  
Yogesh Kumar ◽  
Frank M. Fabian ◽  
Ayman E. A. Ismail ◽  
...  
Keyword(s):  
Tissue Factor ◽  
Human Tissue ◽  
Active Sites ◽  
Tissue Factor Pathway Inhibitor ◽  
Clot Lysis ◽  
Dependent Manner ◽  
Kunitz Inhibitor ◽  
Tissue Factor Pathway ◽  
Inhibitor Type

Current antifibrinolytic agents reduce blood loss by inhibiting plasmin active sites (e.g., aprotinin) or by preventing plasminogen/tissue plasminogen activator (tPA) binding to fibrin clots (e.g., ε-aminocaproic acid and tranexamic acid); however, they have adverse side effects. Here, we expressed 60-residue (NH2NAE…IEKCOOH) Kunitz domain1 (KD1) mutants of human tissue factor pathway inhibitor type-2 that inhibit plasmin as well as plasminogen activation. A single (KD1-L17R-KCOOH) and a double mutant (KD1-Y11T/L17R- KCOOH) were expressed in Escherichia coli as His-tagged constructs, each with enterokinase cleavage sites. KD1-Y11T/L17R-KCOOH was also expressed in Pichia pastoris. KD1-Y11T/L17R-KCOOH inhibited plasmin comparably to aprotinin and bound to the kringle domains of plasminogen/plasmin and tPA with Kd of ~50 nM and ~35 nM, respectively. Importantly, compared to aprotinin, KD1-L17R-KCOOH and KD1-Y11T/L17R-KCOOH did not inhibit kallikrein. Moreover, the antifibrinolytic potential of KD1-Y11T/L17R-KCOOH was better than that of KD1-L17R-KCOOH and similar to that of aprotinin in plasma clot-lysis assays. In thromboelastography experiments, KD1-Y11T/L17R-KCOOH was shown to inhibit fibrinolysis in a dose dependent manner and was comparable to aprotinin at a higher concentration. Further, KD1-Y11T/L17R-KCOOH did not induce cytotoxicity in primary human endothelial cells or fibroblasts. We conclude that KD1-Y11T/L17R-KCOOH is comparable to aprotinin, the most potent known inhibitor of plasmin and can be produced in large amounts using Pichia.

The effect of human tissue factor pathway inhibitor-2 on the growth and metastasis of fibrosarcoma tumors in athymic mice

Blood ◽  
2004 ◽  
Vol 103 (3) ◽  
pp. 1069-1077 ◽  
Author(s):  
Hitendra Singh Chand ◽  
Xin Du ◽  
Duan Ma ◽  
Hector David Inzunza ◽  
Shintaro Kamei ◽  
...  
Keyword(s):  
Gene Expression ◽  
Tissue Factor ◽  
Human Tissue ◽  
Tissue Factor Pathway Inhibitor ◽  
Lung Metastases ◽  
Athymic Mice ◽  
Ecm Remodeling ◽  
Kunitz Inhibitor ◽  
Tissue Factor Pathway

AbstractHuman tissue factor pathway inhibitor-2 (TFPI-2) is a matrix-associated Kunitz inhibitor that inhibits the plasmin- and trypsin-mediated activation of zymogen matrix metalloproteinases involved in tumor progression, invasion, and metastasis. To directly assess its role in tumor growth and metastasis in vivo, we stably transfected HT-1080 fibrosarcoma cells expressing either fully active wild-type human TFPI-2 (WT) or inactive R24Q TFPI-2 (QT) and examined their ability to form tumors and metastasize in athymic mice in comparison to mock-transfected cells (MT). MT and QT fibrosarcoma tumors grew 2 to 3 times larger than WT tumors. Tumor metastasis was confined to the lung and was observed in 75% of mice treated with either MT or QT cells, whereas only 42% of mice treated with WT cells developed lung metastases. Real-time quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) analyses of each tumor group revealed 3- to 6-fold lower levels of murine vascular endothelial growth factor gene expression in WT tumors in relation to either MT or QT tumors. Comparative tumor gene expression analysis revealed that several human genes implicated in oncogenesis, invasion, metastasis, apoptosis, and angiogenesis had significantly altered levels of expression in WT tumors. Our collective data demonstrate that secretion of inhibitory TFPI-2 by a highly metastatic tumor cell markedly inhibits its growth and metastasis in vivo by regulating pericellular extracellular matrix (ECM) remodeling and angiogenesis. (Blood. 2004;103:1069-1077)

Experimental Haemorrhagic Effect of Two-Domain Non-Glycosylated Tissue Factor Pathway Inhibitor Compared to Low Molecular Weight Heparin

1996 ◽  
Vol 75 (04) ◽  
pp. 585-589 ◽  
Author(s):  
Jan Holst ◽  
Bengt Lindblad ◽  
Stefan E Matthíasson ◽  
Ulf Stjernquist ◽  
Mirella Ezban ◽  
...  
Keyword(s):  
Tissue Factor ◽  
Tissue Factor Pathway Inhibitor ◽  
Bleeding Time ◽  
Low Molecular Weight ◽  
Prophylactic Dose ◽  
Significant Prolongation ◽  
Kunitz Inhibitor ◽  
Rat Gastric Mucosa ◽  
Natural Inhibitor ◽  
Tissue Factor Pathway

SummaryThe glycosylated multivalent three-domain Kunitz inhibitor TFPI is a natural inhibitor of tissue factor-FVIIa complex in the presence of FXa. TFPI has an experimental antithrombotic capacity indistinguishable from LMWH in a prophylactic dose, regardless of glycosylation and of the third domain. An inherited equilibrium between antithrombosis and haemorrhage exists. The aim of the study was to evaluate whether a two-domain non-glycosylated TFPI (117QTFPI1−161) has a bleeding potential in a rat gastric mucosa model. Groups; placebo, LMWH (tinzaparin) 60 and 250 anti-Xa IU/kg and 117QTFPIM61−161 1.0 and 10.0 mg/kg, given i.v. (bolus injection), randomised double dummy design.All actively treated groups significantly prolonged both the bleeding volume (493-984 Μl) and the bleeding time (10-20 min) compared to placebo (41 Μl, 2 min). It was not possible to distinguish a difference between the lower dose of LMWH and 117QTFPI1−161 in either parameter (p = 0.23-0.71). The two doses of 117QTFPI1−161 caused elevation of plasma-TFPI, 18 and 150 times baseline value. Both LMWH doses (0.6-3.2 anti-Xa IU/ml) and both 117QTFPI1−161 doses (0.2-2.7 anti-Xa IU/ml), caused significant effect in the anti-Xa assay, however 117QTFPI1−161 significantly less. Only the largest dose of 117QTFPI1−161 caused significant prolongation in the APTT assay (34 s). Both doses of LMWH caused significant prolongation (60-300 s). LMWH was the only substance to prolong the dilute-PT assay.Non-glycosylated two-domain 1.0 mg/kg TFPI, yielding supra-physiological plasma concentration, has an experimental haemorrhagic potential indistinguishable from LMWH in a prophylactic dose. The effect mediated by this type of TFPI could primarily be due to an inhibition of FXa.

scholarly journals Procoagulant activity in patients with combined type 2 diabetes and coronary artery disease: No effects of long-term exercise training

2017 ◽  
Vol 14 (2) ◽  
pp. 144-151 ◽  
Author(s):  
Vibeke Bratseth ◽  
Rune Byrkjeland ◽  
Ida U Njerve ◽  
Svein Solheim ◽  
Harald Arnesen ◽  
...  
Keyword(s):  
Type 2 Diabetes ◽  
Exercise Training ◽  
Tissue Factor ◽  
Thrombin Generation ◽  
Tissue Factor Pathway Inhibitor ◽  
Ex Vivo ◽  
Tissue Factor Pathway ◽  
Artery Disease ◽  
Total Tissue

We investigated the effects of 12-month exercise training on hypercoagulability in patients with combined type 2 diabetes mellitus and coronary artery disease. Associations with severity of disease were further explored. Patients ( n = 131) were randomized to exercise training or a control group. Blood was collected at inclusion and after 12 months. Tissue factor, free and total tissue factor pathway inhibitor, prothrombin fragment 1 + 2 (F1 + 2) and D-dimer were determined by enzyme-linked immunosorbent assay and ex vivo thrombin generation by the calibrated automated thrombogram assay. Tissue factor and ex vivo thrombin generation increased from baseline to 12 months ( p < 0.01, all), with no significant differences in changes between groups. At baseline, free and total tissue factor pathway inhibitor significantly correlated to fasting glucose ( p < 0.01, both) and HbA1c ( p < 0.05, both). In patients with albuminuria ( n = 34), these correlations were strengthened, and elevated levels of D-dimer, free and total tissue factor pathway inhibitor ( p < 0.01, all) and decreased ex vivo thrombin generation ( p < 0.05, all) were observed. These results show no effects of exercise training on markers of hypercoagulability in our population with combined type 2 diabetes mellitus and coronary artery disease. The association between poor glycaemic control and tissue factor pathway inhibitor might indicate increased endothelial activation. More pronounced hypercoagulability and increased tissue factor pathway inhibitor were demonstrated in patients with albuminuria.

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.

scholarly journals Thromboresistance of Balloon-Injured Porcine Carotid Arteries After Local Gene Transfer of Human Tissue Factor Pathway Inhibitor

Circulation ◽  
2000 ◽  
Vol 101 (3) ◽  
pp. 289-295 ◽  
Author(s):  
Pierre Zoldhelyi ◽  
Janice McNatt ◽  
Harnath S. Shelat ◽  
Yasutaka Yamamoto ◽  
Zhi-Qiang Chen ◽  
...  
Keyword(s):  
Gene Transfer ◽  
Tissue Factor ◽  
Human Tissue ◽  
Tissue Factor Pathway Inhibitor ◽  
Carotid Arteries ◽  
Tissue Factor Pathway
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