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

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 4417-4417 ◽  
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.

Procoagulant Activity of Sulfated Polysaccharides

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 4419-4419
Author(s):  
Michael Dockal ◽  
Sabine Knappe ◽  
Erwin Panholzer ◽  
Michael Palige ◽  
Hartmut J. Ehrlich ◽  
...  
Keyword(s):  
Tissue Factor ◽  
Tissue Factor Pathway Inhibitor ◽  
Laminaria Japonica ◽  
Procoagulant Activity ◽  
Sulfated Polysaccharides ◽  
Molecular Characteristics ◽  
Procoagulant Effect ◽  
Tissue Factor Pathway ◽  
Dose Dependent ◽  
Selection Of

Abstract Abstract 4419 BAX513, a Laminaria japonica-derived fucoidan and other non-anticoagulant sulfated polysaccharides (NASPs) have been shown to improve clotting in FVIII- and FIX-deficient plasma (Liu et al. Thromb Haemost 2006; 95:68). In this study we assessed the procoagulant activities of fucoidans derived from a variety of brown sea algae species, and correlated the activity with molecular weight (MW) and degree of sulfation. Highly purified fucoidan preparations were studied in FVIII-inhibited whole blood by tissue factor-triggered thromboelastography (TEG). The procoagulant activity was characterized by calibrated automated thrombography (CAT) in FVIII- and FIX-deficient plasma and in combination with established hemophilia therapeutics. A dilute prothrombin time assay based on tissue factor pathway inhibitor supplementation (TFPI-dPT) was used to demonstrate the dose-dependent TFPI-inhibiting effect of BAX513 (EC50 = 0.18 ± 0.03 μ g/mL) in FVIII-deficient plasma. TEG in normal and FVIII-inhibited blood showed a dose-dependent procoagulant effect of most compounds where the optimal concentrations (1-100 nM) were dependent on the MW of the fucoidan. In FVIII-inhibited blood BAX513 at concentrations of ~10 nM (1.2 μ g/mL) completely normalized the TEG parameters. In contrast to sulfated fucoidans, undersulfated fucoidan hardly affected thrombin generation (TG). By CAT, the procoagulant window of NASPs in hemophilic plasma spanned more than two orders of magnitude with maximum effects being equivalent to (mU/mL) 730–940 FVIII, 32–80 FIX and 590–1230 FEIBA. NASPs combined with FVIII, FEIBA or FVIIa had an additive procoagulant effect. The optimal selection of molecular characteristics of NASPs will support the development of alternative hemophilia therapies. Disclosures: Dockal: Baxter Innovations GmbH: Employment. Knappe:Baxter Innovations GmbH: Employment. Panholzer:Baxter Innovations GmbH: Employment. Palige:Baxter Innovations GmbH: Employment. Ehrlich:Baxter Innovations GmbH: Employment. Scheiflinger:Baxter Innovations GmbH: Employment.

Pharmacokinetics of Full Length and Two-Domain Tissue Factor Pathway Inhibitor in Combination with Heparin in Rabbits

1993 ◽  
Vol 70 (03) ◽  
pp. 454-457 ◽  
Author(s):  
Claus Bregengaard ◽  
Ole Nordfang ◽  
Per Østergaard ◽  
Jens G L Petersen ◽  
Giorgio Meyn ◽  
...  
Keyword(s):  
Tissue Factor ◽  
Tissue Factor Pathway Inhibitor ◽  
Anticoagulant Activity ◽  
Fold Increase ◽  
Volume Of Distribution ◽  
Full Length ◽  
Heparin Binding ◽  
Extrinsic Pathway ◽  
C Terminus ◽  
Tissue Factor Pathway

SummaryTissue factor pathway inhibitor (TFPI) is a feed back inhibitor of the initial activation of the extrinsic pathway of coagulation. In humans, injection of heparin results in a 2-6 fold increase in plasma TFPI and recent studies suggest that TFPI may be important for the anticoagulant activity of heparin. Full length (FL) TFPI, but not recombinant two-domain (2D) TFPI, has a poly cationic C-terminus showing very strong heparin binding. Therefore, we have investigated if heparin affects the pharmacokinetics of TFPI with and without this C-terminus.FL-TFPI (608 U/kg) and 2D-TFPI (337 U/kg) were injected intravenously in rabbits with and without simultaneous intravenous injections of low molecular weight heparin (450 anti-XaU/kg).Heparin decreased the volume of distribution and the clearance of FL-TFPI by a factor 10-15, whereas the pharmacokinetics of 2D-TFPI were unaffected by heparin. When heparin was administered 2 h following TFPI the recovery of FL-TFPI was similar to that found in the group receiving the two compounds simultaneously, suggesting that the releasable pool of FL-TFPI is removed very slowly in the absence of circulating heparin.

scholarly journals Prothrombinase is protected from inactivation by tissue factor pathway inhibitor: competition between prothrombin and inhibitor*

1997 ◽  
Vol 323 (1) ◽  
pp. 33-37 ◽  
Author(s):  
Jo FRANSSEN ◽  
Irene SALEMINK ◽  
George M. WILLEMS ◽  
Tze-Chein WUN ◽  
H. Coenraad HEMKER ◽  
...  
Keyword(s):  
Tissue Factor ◽  
Rate Constant ◽  
Tissue Factor Pathway Inhibitor ◽  
Factor Xa ◽  
Full Length ◽  
Prothrombinase Complex ◽  
Factor Va ◽  
C Terminus ◽  
Length Variant ◽  
Tissue Factor Pathway

The inhibition of prothrombinase by tissue factor pathway inhibitor (TFPI) has been studied in the presence and absence of prothrombin. The rate constant of association of prothrombinase with full-length TFPI was 2.1×107 M-1ċs-1 and 0.05×107 M-1ċs-1 for the reaction with C-terminus truncated TFPI (TFPI1-161). The rate constant of dissociation was 0.65×10-4 s-1 in both cases. The rate constant of inhibition of prothrombinase by TFPI1-161 was similar to that of solution-phase factor Xa. In contrast, phospholipids and factor Va enhanced the association rate of the reaction between factor Xa and full-length TFPI by approx. 20-fold. Although TFPI, and in particular the full-length variant of the molecule, is a potent inhibitor of prothrombinase (overall inhibition constant of 3 pM), we also found that prothrombin competed very effectively with TFPI for the active site of factor Xa in the prothrombinase complex. A 50% reduction of the rate constant of inhibition was measured in the presence of 4 nM prothrombin, i.e. 0.2% of the plasma concentration of prothrombin. The physiological significance of TFPI as an inhibitor of prothrombinase activity is thus questionable.

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 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.

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.

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