Effect of tissue factor pathway inhibitor on thrombin generation assay

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.

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Low Protein C, Tissue Factor Pathway Inhibitor, and Antithrombin allow Sufficient Thrombin Generation in Neonatal Plasma

33rd Hemophilia Symposium ◽

10.1007/978-3-642-18260-0_49 ◽

2004 ◽

pp. 297-301

Author(s):

G. Cvirn ◽

S. Gallistl ◽

T. Rehak ◽

G. Jürgens ◽

W. Muntean

Keyword(s):

Tissue Factor ◽

Protein C ◽

Thrombin Generation ◽

Tissue Factor Pathway Inhibitor ◽

Low Protein ◽

Tissue Factor Pathway

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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

Arteriosclerosis Thrombosis and Vascular Biology ◽

10.1161/atvb.36.suppl_1.43 ◽

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.

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Plasma free tissue factor pathway inhibitor (TFPI) levels and TF-induced thrombin generation ex vivo in men with low testosterone levels

Thrombosis and Haemostasis ◽

10.1160/th08-10-0667 ◽

2009 ◽

Vol 101 (03) ◽

pp. 471-477 ◽

Cited By ~ 7

Author(s):

Ingvild Agledahl ◽

Johan Svartberg ◽

Bjarne Hansen ◽

Ellen Brodin

Keyword(s):

Tissue Factor ◽

Thrombin Generation ◽

Tissue Factor Pathway Inhibitor ◽

Ex Vivo ◽

Elderly Men ◽

Lower Plasma ◽

Testosterone Levels ◽

Initiation Phase ◽

Tissue Factor Pathway ◽

Low Testosterone

SummaryLow testosterone levels in men have been associated with cardiovascular risk factors, some prothrombotic factors, and lately also an increased risk of both cardiovascular disease and all-cause mortality. Experimental studies have shown increased synthesis and release of tissue factor pathway inhibitor (TFPI) by physiological levels of testosterone in endothelial cells. Our hypothesis was that elderly men with low testosterone levels would have lower plasma levels of plasma free TFPI with subsequent increased thrombin generation. Elderly men with low (n=37) and normal (n=41) testosterone levels were recruited from a general population, and tissue factor (TF)-induced thrombin generation ex vivo and plasma free TFPI Ag were measured. Elderly men with low testosterone levels had lower plasma free TFPI Ag (10.9 ± 2.3 ng/ml vs. 12.3 ± 3.0 ng/ml, p=0.027) and shorter initiation phase of TF-induced coagulation assessed by lag-time (5.1 ± 1.0 min vs. 5.7 ± 1.3, p=0.039). The differences between groups remained significant and were strengthened after adjustment for waist circumference and other cardiovascular risk factors. Lag-time increased linearly across quartiles of plasma free TFPI Ag (p<0.001). Multiple regression analysis revealed that total and free testosterone were independent predictors of plasma free TFPI Ag. Our findings suggest that low testosterone levels in elderly men is associated with low plasma free TFPI Ag and subsequent shortened initiation phase of TF-induced coagulation.

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Tissue factor pathway inhibitor is the main determinant of thrombin generation in haemophilic patients

Haemophilia ◽

10.1111/hae.13679 ◽

2019 ◽

Vol 25 (2) ◽

pp. 343-348 ◽

Cited By ~ 8

Author(s):

Pierre Chelle ◽

Aurélie Montmartin ◽

Pauline Damien ◽

Michèle Piot ◽

Michel Cournil ◽

...

Keyword(s):

Tissue Factor ◽

Thrombin Generation ◽

Tissue Factor Pathway Inhibitor ◽

Main Determinant ◽

Tissue Factor Pathway

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Procoagulant Activity of Laminaria Japonica Derived Fucoidan (BAX513) Depends on the Interaction with the C-Terminus of Tissue Factor Pathway Inhibitor

Blood ◽

10.1182/blood.v116.21.4417.4417 ◽

2010 ◽

Vol 116 (21) ◽

pp. 4417-4417 ◽

Cited By ~ 1

Author(s):

Michael Palige ◽

Christoph Redl ◽

Sabine Knappe ◽

Hartmut J. Ehrlich ◽

Michael Dockal ◽

...

Keyword(s):

Tissue Factor ◽

Mechanism Of Action ◽

Thrombin Generation ◽

Tissue Factor Pathway Inhibitor ◽

Laminaria Japonica ◽

Full Length ◽

Procoagulant Activity ◽

C Terminus ◽

Tissue Factor Pathway ◽

Dose Dependent

Abstract Abstract 4417 BAX513, a fucoidan derived from the brown seaweed Laminaria japonica, and other non-anticoagulant sulfated polysaccharides (NASPs) improve coagulation in hemophilic blood and plasma. Fucoidans are heterogeneous, polysulfated molecules with procoagulant activities in a wide concentration range. Tissue factor pathway inhibitor (TFPI) has been described as a potential target for the procoagulant activity of NASPs (Liu et al. Thromb Haemost 2006; 95:68). In the current study, we investigated the interaction of BAX513 with TFPI proteins to gain a detailed understanding of the mechanism of action of BAX513. We used calibrated automated thrombography to monitor the activity of BAX513 in normal, FX and TFPI-deficient plasma. TFPI plasma levels were varied by the addition of truncated TFPI (TFPI1-160) and TFPI-domain specific antibodies. Initiating thrombin generation by addition of FXa to plasma deficient in both, FX and FVIII-showed a BAX513-dose dependent increase of thrombin generation, which was completely abolished when TFPI-specific polyclonal antibodies were present. Furthermore, when full-length TFPI was inhibited in plasma and instead supplemented with increasing amounts of TFPI 1–160, BAX513 did not show any activity. The data are further supported by surface plasmon resonance experiments (BiaCore) exploring the BAX513-TFPI interaction. A high affinity interaction was only observed for BAX513 with full-length TFPI but not for BAX513 with TFPI1-160. Our findings support a mechanism of action in which BAX513 acts as a potent dose-dependent TFPI antagonist that requires the highly charged C-terminus of TFPI to unfold its full potential. Understanding the mechanism of action of BAX513 supports the development of BAX513 as a promising new therapeutic for hemophiliacs and FVIII-inhibitor patients. Disclosures: Palige: Baxter Innovations GmbH: Employment. Redl:Baxter Innovations GmbH: Employment. Knappe:Baxter Innovations GmbH: Employment. Ehrlich:Baxter Innovations GmbH: Employment. Dockal:Baxter Innovations GmbH: Employment. Scheiflinger:Baxter Innovations GmbH: Employment.

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