Biological Explanation of Clinically Observed Elevation of TFPI Plasma Levels After Treatment with TFPI-Antagonistic Aptamer BAX 499

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1104-1104 ◽  
Author(s):  
Michael Dockal ◽  
Robert Pachlinger ◽  
Rudolf Hartmann ◽  
Sabine Knappe ◽  
Benny Sorensen ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Plasma Levels ◽  
Thrombin Generation ◽  
Proteolytic Processing ◽  
Full Length ◽  
Model Systems ◽  
Cell Culture Supernatant ◽  
Clinical Samples ◽  
Dependent Manner ◽  
Molar Excess

Abstract Abstract 1104 BAX 499, an aptamer that binds with a high affinity to TFPI (KD ∼0.1 nM), blocks the anticoagulant action of TFPI in plasma and model systems. BAX 499 was recently tested in a phase 1 safety, tolerability and ex vivo efficacy study in hemophilia patients. The study was prematurely stopped due to an increased number of bleeding events. Baxter subsequently performed several analyses to investigate why clinical safety was inconsistent with preclinical observations. Analysis of plasma levels of full length TFPI (fl-TFPI) and BAX 499, of global hemostatic parameters via thrombin generation (CAT) and thromboelastography (ROTEM) demonstrated that BAX 499 induced a considerable increase in fl-TFPI plasma levels: over 25-fold at the highest dose (72 mg, SubQ). Pharmacodynamic assessment showed that high fl-TFPI concentrations substantially reduce thrombin generation even at excess BAX 499 plasma concentrations (∼1 μM). This strongly suggests that the BAX 499-induced rise in plasma fl-TFPI levels caused the increased bleeding. Assessment of biological mechanisms underlying the increase in fl-TFPI plasma levels showed that BAX 499 can potentially raise plasma levels by 1) increasing the synthesis of TFPI by endothelial cells, 2) affecting the distribution of TFPI which is bound to the endothelial surface, stored in platelets and circulated in plasma, 3) interfering with the metabolism of plasma TFPI, i.e. preventing the conversion of full length to truncated TFPI by proteases and 4) affecting TFPI clearance. BAX 499 treatment of Human Umbilical Vein Endothelial Cells (HUVECs) hardly affected TFPI mRNA production, indicating that BAX 499 has virtually no effect on TFPI synthesis at the transcriptional level. Treatment of HUVECs with BAX 499 stimulated release of TFPI to the cell culture supernatant in a dose dependent manner. FACS analysis showed that BAX 499 has a minimal influence on cell surface TFPI of endothelial cells but, like Heparin, is able to mobilize and release intracellularly-stored TFPI. TFPI released from HUVECs, however, is too low to explain the increased plasma fl-TFPI levels detected in clinical samples. Most TFPI circulating in blood is truncated (80–90%) and bound to lipoproteins. The Lys86-Gln90 region in TFPI is a hot spot for proteolytic cleavage by a wide variety of proteases such as elastase. Cleavage results in the simultaneous removal of TFPI Kunitz 1 domain and loss in activity. BAX 499 (1μM) caused a ∼4-fold decrease in elastase-catalyzed cleavage of TFPI, showing that BAX 499 can delay the proteolytic processing of full length TFPI. BAX 499 was tested in Biacore studies for the interference of fl- TFPI binding to low density lipoprotein receptor-related protein 1 (LRP1), involved in receptor-mediated endocytosis.BAX 499 (1 μM) strongly reduced TFPI binding to LRP1. Pharmacokinetic studies in mice showed that injected human fl-TFPI is rapidly cleared from their circulation and that administration of TFPI with a molar excess of a non-PEGylated variant of BAX 499 resulted in prolonged TFPI clearance. This effect was even more pronounced when TFPI was dosed with PEGylated BAX 499. In conclusion, the elevated TFPI plasma levels in hemophilia patients treated with aptamer BAX 499 can be explained as follows: BAX 499 releases intracellularly-stored TFPI, impacts its metabolism and prolongs the circulatory half life of fl-TFPI, most likely due to binding of BAX 499 to the Kunitz 3-C terminus domain of TFPI, a region required for fl-TFPI clearance. The net result of these effects is elevated plasma fl-TFPI, which even at a molar excess of BAX 499 retains anti-coagulant activity. Disclosures: No relevant conflicts of interest to declare.

scholarly journals The C-Terminus of Tfpiα Inhibits Factor V Activation By Protecting the Arg1545 Cleavage Site

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 257-257
Author(s):  
Peter van Doorn ◽  
Jan Rosing ◽  
Simone Wielders ◽  
Tilman M. Hackeng ◽  
Elisabetta Castoldi
Keyword(s):  
Thrombin Generation ◽  
Full Length ◽  
Model Systems ◽  
Factor V ◽  
Dependent Manner ◽  
High Affinity ◽  
C Terminus ◽  
Dose Dependent ◽  
Acidic Region ◽  
Basic Region

Abstract Coagulation factor V (FV) is the inactive precursor of FVa, which acts as an essential cofactor of factor Xa (FXa) in the prothrombinase complex. FV is maintained in the inactive state by the interaction between a basic and acidic region in the B-domain. The C-terminus of tissue factor pathway inhibitor-α (TFPIα) is highly homologous to the FV basic region and also binds to the acidic region of FV. In fact, a large fraction of plasma TFPIα circulates in complex with FV. Thanks to this interaction, FV acts as a cofactor of TFPIα in the inhibition of FXa and TFPIα inhibits prothrombinase complexes containing forms of FVa that retain the acidic region. However, when FV is activated through cleavage at Arg709, Arg1018 and Arg1545 by FXa or thrombin, it loses its anticoagulant properties and becomes a strong procoagulant. Recently, a FV splicing variant (FV-short) that lacks the basic region and binds TFPIα with high affinity has been described. FV-short is present in all individuals and represents ~5% of all plasma FV. To gain more insight in the functional implications of the FV-TFPIα interaction, we studied the effects of a peptide identical to the TFPIα C-terminus (TFPIα C-term) on thrombin generation in plasma and on FV activation in model systems. All major findings were confirmed with full-length TFPIα. TFPIα C-term (0-5 µM) prolonged the lag time and decreased the peak height of tissue factor- and FXa-triggered thrombin generation in a dose-dependent manner. These effects were more pronounced at low procoagulant stimuli and in the presence of plasma TFPIα. TFPIα C-term also inhibited thrombin generation in FV-depleted plasma reconstituted with FV, but not in FV-depleted plasma reconstituted with FVa, suggesting an effect on FV activation and/or prothrombinase. In model systems, TFPIα C-term inhibited the activation of purified FV by FXa and thrombin in a dose-dependent manner. This could be due to inhibition of FV proteolysis and/or to inhibition of prothrombinase in the assay used to quantify FVa activity. Therefore, FV activation was also followed by SDS-PAGE and Western blotting. This showed that TFPIα C-term (1 µM) interferes with FV proteolysis by both FXa and thrombin by selectively impairing cleavage of FV at Arg1545, which is located close to the FV acidic region (residues 1493-1537). The effect of TFPIα C-term on FV activation by thrombin was 3-fold stronger for FV-short than for full-length FV, in line with their respective affinities for the TFPIα C-terminus. Full-length TFPIα (10 nM) also inhibited FV cleavage at Arg1545 and delayed FV activation by thrombin. Its effect was also more pronounced on FV-short than on FV. In summary, binding of the TFPIα C-terminus to the acidic region of FV inhibits FV activation by FXa or thrombin by blocking access to the Arg1545 cleavage site. Since cleavage at this site marks the transition of FV from an anticoagulant form (TFPIα-cofactor) to a procoagulant form (FXa-cofactor), this may represent an important new anticoagulant function of TFPIα. The main target of this anticoagulant mechanism is presently unclear, but it is unlikely to be intact FV, whose plasma concentration is 100-fold higher than the TFPIα concentration. More likely candidates are low-abundance FV species that lack the basic region but retain the acidic region, and therefore bind TFPIα with high affinity, such as FV-short, early FV activation intermediates and/or platelet FV. Supported by grant nr. 2014-1 from the Dutch Thrombosis Foundation. Disclosures No relevant conflicts of interest to declare.

Design Of a Most Efficient Inhibitor Of TFPI By Molecular Fusion Of Two Inhibitory Peptides

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3564-3564
Author(s):  
Michael Dockal ◽  
Rudolf Hartmann ◽  
Thomas Polakowski ◽  
Johannes Brandstetter ◽  
Willibald Kammlander ◽  
...  
Keyword(s):  
Amino Acids ◽  
Endothelial Cells ◽  
Cell Surface ◽  
Platelet Activation ◽  
Research Funding ◽  
Thrombin Generation ◽  
Fusion Peptide ◽  
Peptide Inhibitors ◽  
Model Systems ◽  
Inhibitory Peptide

Abstract TFPI is an important inhibitor of the extrinsic coagulation pathway. It efficiently inhibits TF-FVIIa and FXa by quaternary complex formation. Plasma contains various truncated forms of TFPI which are poor inhibitors, and full length (fl)TFPI (0.3 – 0.5 nM) which is the most active TFPI in plasma. flTFPI is released from platelets upon activation, and increases flTFPI concentrations locally up to 30-fold. Most intravascular TFPI (∼80%) is associated with endothelial cells. Both endothelial forms, TFPIa and TFPIb, are similarily effective inhibitors of FX activation on the endothelial cell surface. Inhibition of TFPI in hemophilia models with blocking antibodies, aptamers or peptide inhibitors improves hemostasis and may become an option to treat hemophilia. Recently, we presented peptide inhibitors of TFPI that enhance coagulation in hemophilia models. Two optimized peptides, JBT-A7 and JBT-B5, efficiently blocked inhibitory activity of TFPI and bound to distinct binding sites. We demonstrated the crystal structure of JBT-A7, a linear TFPI inhibitory peptide composed of 20 amino acids, bound to NtermK1 (TFPI 1-83). JBT-B5, a cyclic TFPI inhibitory peptide of 23 amino acids, co-crystallized with TFPI KD1-KD2 (TFPI 22-150). Overlaying the KD1 structure in the KD1-KD2/JBT-B5 and the NTermK1/JBT-A7 complex provided atomic details for linking the two peptide entities. Binding of peptides to TFPI and TFPI fragments was studied by BioCore. The TFPI inhibitory potential of the resulting fusion peptide was tested in model systems (FXa inhibition and TF-FVIIa catalyzed FX activation) and global hemostatic assays (TF-triggered thrombin generation) using hemophilia plasma. To model situations of increased TFPI concentration, both model and plasma assays were carried out at TFPI concentrations up to 10 nM, which is 40-50-fold higher than the physiological flTFPI plasma concentration. To characterize the inhibition of platelet TFPI, we used platelets isolated from blood samples and platelet rich plasma from different donors. Binding of a biotinylated fusion peptide on living HUVE cells was assessed by fluorescence activated cell sorting (FACS) and fluorescence microscopy. Inhibition of cell surface TFPI was analyzed on cultivated HUVECs stimulated with TNFa for TF expression. We monitored FXa generation by the TFPI-dependent cell surface FX activation complex by conversion of an FXa-specific fluorogenic substrate. The overlay of the crystal structures of KD1-KD2/JBT-B5 and the NTermKD1/JBT-A7 complexes revealed non-overlapping epitopes and close proximity of the termini of both peptides. The distance could be bridged by an approximately ten amino acid linker. A fusion peptide with a 10-serine-linker was synthesized and showed highly improved dissociation in Biacore experiments and most efficiently inhibited TFPI activity in the model assays. In contrast, single peptides only partially inhibit TFPI especially at high TFPI concentrations. In thrombin generation assays using hemophilia plasma, the fusion peptide showed a substantially higher ability than the single peptides to increase the thrombin peak even at elevated TFPI. The fusion peptide efficiently inhibited TFPI released from platelets and improved thrombin generation in TFPI deficient plasma reconstituted with platelets as the only source of TFPI released upon platelet activation. The fusion peptide was also shown to bind TFPI on the surface of living HUVECs. This is consistent with its binding epitopes on KD1 and KD2 which result in inhibition of cell surface TFPI in a cell based FX activation assay. Thus, we demonstrate that a molecular fusion peptide most efficiently inhibits all physiologic forms of TFPI. X-ray structures of binary and ternary peptide TFPI complexes provided atomic details for linking two single peptides to generate a fusion peptide that most efficiently blocks TFPI in plasma, released from platelets and associated with endothelial cells. It most efficiently neutralizes TFPI even at substantially elevated concentrations occurring at sites of platelet activation. Our observations support the notion that targeting TFPI with TFPI inhibitors is a promising novel strategy to mitigate the bleeding risk in hemophilia patients. Disclosures: Dockal: Baxter Innovations GmbH, Vienna, Austria: Employment. Hartmann:Baxter Innovations GmbH, Vienna, Austria: Employment. Polakowski:3B Pharmaceuticals, Berlin, Germany: Employment. Brandstetter:Baxter Innovations GmbH, Vienna, Austria: Research Funding. Kammlander:Baxter Innovations GmbH, Vienna, Austria: Employment. Panholzer:Baxter Innovations GmbH, Vienna, Austria: Employment. Redl:Baxter Innovations GmbH, Vienna, Austria: Employment. Osterkamp:3B Pharmaceuticals, Berlin, Germany: Employment. Rosing:Baxter Innovations GmbH, Vienna, Austria: Consultancy, Research Funding. Scheiflinger:Baxter Innovations GmbH, Vienna, Austria: Employment.

scholarly journals Effect of blood flow on thrombin generation is dependent on the nature of the thrombogenic surface

Blood ◽  
1994 ◽  
Vol 84 (7) ◽  
pp. 2206-2213 ◽  
Author(s):  
A Diquelou ◽  
S Lemozy ◽  
D Dupouy ◽  
B Boneu ◽  
K Sakariassen ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Blood Flow ◽  
Shear Rate ◽  
Plasma Levels ◽  
Thrombin Generation ◽  
Type Iii Collagen ◽  
Fibrin Deposition ◽  
Positive Correlation ◽  
Shear Rates ◽  
Fibrin Formation

We have investigated the influence of blood flow on thrombin generation, fibrin formation, and fibrin deposition on procoagulant and nonprocoagulant surfaces. Nonanticoagulated human blood was drawn for 5 minutes directly from an antecubital vein over stimulated endothelial cells expressing tissue factor and over human type III collagen fibrils, positioned in parallel-plate perfusion chambers. The shear rates at these surfaces were 50, 650, and 2,600 s-1. Deposition of platelets and fibrin was measured by morphometry. Thrombin and fibrin formation was determined by measuring prothrombin fragments 1 + 2 (F 1 + 2), thrombin-antithrombin III complexes, (T-AT) and fibrinopeptide A (FPA) in blood effluent from the perfusion chamber at the end of the 5- minute perfusion period. On procoagulant endothelial cells, the thrombi were primarily composed of fibrin. The fibrin deposition (81%, 21%, and 2% at 50, 650, and 2,600 s-1, respectively) and plasma levels of F 1 + 2, T-AT and FPA were shear rate dependent and highest at 50 s-1. There was a positive correlation between F 1 + 2 and T-AT and the fibrin deposition (P < .01). In contrast, the collagen surface triggered primarily thrombi that were composed of platelets. The platelet thrombi and plasma levels of F 1 + 2 and T-AT were also dependent on the shear rate, but highest at 650 and 2,600 s-1. F 1 + 2 and T-AT reached the same level as observed with procoagulant endothelial cells at the higher shear rates. There was a positive correlation between F 1 + 2 and T-AT and the platelet thrombus formation (P < .05), confirming the predominant role of platelets in thrombin generation. Thus, thrombin formation is strongly influenced by the blood flow, and this effect depends on the composition of the thrombogenic surface.

Effect of Increased Tissue Factor Pathway Inhibitor (TFPI) Levels On Factor Xa Inhibition and Global Hemostasis in the Presence of TFPI-Antagonistic Aptamer BAX 499.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2207-2207 ◽  
Author(s):  
Michael Dockal ◽  
Rudolf Hartmann ◽  
Sabine Knappe ◽  
Michael Palige ◽  
Willibald Kammlander ◽  
...  
Keyword(s):  
Inhibitory Activity ◽  
Whole Blood ◽  
Plasma Levels ◽  
Thrombin Generation ◽  
Anticoagulant Activity ◽  
Reaction System ◽  
Factor Xa ◽  
Model Systems ◽  
Bleeding Tendency ◽  
Fold Excess

Abstract Abstract 2207 In a recent hemophilia clinical trial, patients' TFPI plasma levels substantially increased after intravenous or subcutaneous administration of TFPI-inhibitory aptamer BAX 499. To better understand the consequences of elevated plasma full-length (fl)-TFPI and the potency of BAX 499, the TFPI-inhibitory activity of a wide concentration range of BAX 499 was tested at levels up to 10 nM fl-TFPI in two model systems and in two global hemostatic coagulation assays. Factor Xa (FXa) inhibition assay measured the influence of BAX 499 on the interaction between TFPI and FXa. BAX 499 at 1000 nM efficiently inhibited fl-TFPI within the physiological concentration (< 0.5 nM). However, with increasing TFPI, inhibition of TFPI by BAX499 became considerably less efficient, reflected by increasing EC50 and decreasing maximum inhibition. At substantially elevated TFPI concentrations (10 nM) EC50 increased ∼20-fold, with TFPI retaining ∼70% of its FXa inhibitory activity. Similar results were observed in a more complex reaction system (TF-FVIIa-catalyzed FX activation). These results suggest that BAX 499 is a partial inhibitor of TFPI, efficiently neutralizing TFPI at low concentrations and less efficiently inhibiting TFPI at high TFPI levels. In thrombin generation assays with FVIII-inhibited plasma, a substantial excess of BAX 499 was required to compensate the anticoagulant activity of increasing fl-TFPI. Based on peak thrombin values, fl-TFPI levels that could be neutralized by a given BAX 499 concentration were determined. For example, a ∼50-fold excess of BAX 499 neutralized 0.2 nM TFPI added to FVIII-inhibited plasma, whereas a 140-fold excess was required for neutralization of 7.3 nM fl-TFPI added. An equivalent ROTEM experiment performed in FVIII-inhibited whole blood confirmed these findings. In summary, the data showed that even a high concentration of BAX 499 (1 μM) was not able to counteract the strong inhibitory effect of 10 nM fl-TFPI on FXa, on TF-FVIIa-catalyzed FX activation, on thrombin generation in plasma and clot formation in whole blood, due to the partially inhibitory properties of BAX 499. The current study offers an explanation for increased bleeding tendency in hemophilia patients associated with the ≥25-fold elevated fl-TFPI plasma levels after BAX 499 administration. BAX 499 does not appear to be able to compensate the anticoagulant activity of increased TFPI levels caused by partial inhibition in patients treated with BAX 499. Disclosures: No relevant conflicts of interest to declare.

Serine Proteases Of Coagulation Modulate Tissue Factor Expression and Monocyte-Induced Thrombin Generation

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3470-3470
Author(s):  
Flora Lemaire ◽  
Philippe Nguyen ◽  
Sonia BEN Hadj Kalifa ◽  
Marie Christine Mulpas ◽  
Nathalie Hezard
Keyword(s):  
Endothelial Cells ◽  
Tissue Factor ◽  
Serine Proteases ◽  
Protein C ◽  
Thrombin Generation ◽  
Complete Agreement ◽  
Dependent Manner ◽  
Human Monocytes ◽  
Rt Pcr ◽  
Thromb Thrombolysis

Abstract Background As part of their role in immune response and inflammation, monocytes exhibit a potent procoagulant phenotype which is mediated by Tissue Factor (TF), the main trigger of coagulation. TF expression by monocytes is induced by various agonists such as lipopolysaccarides (LPS) and proinflammatory cytokines. We recently reported that Factor Xa could induce TF expression (Ben-Hadj–Khalifa J Thromb Thrombolysis 2011). The aim of the present study is to evaluate, in a model of highly purified human monocytes, the effect of 2 major serine proteases, namely factor VIIa, which activates Xa, and activated Protein C (APC), which inhibits thrombin generation but also exerts potent cytoprotection. Methods Human monocytes were purified by elutriation of cytapheresis material obtained from healthy donors. Informed consent was obtained. Cell viability (trypan blue-negative cells) was > 98%, the quality of elutriation was estimated by the percentage of CD14+ cells (> 95 %). Monocytes (5.106 cells/mL) were activated for 5 h, by FVIIa (0.1, 0.4, 2 µM, Novo Nordisk) or APC (30. 50, 100 nM, Diagnostica Stago) at 37°C in a 5% CO2 humidified atmosphere. FXa (0.025 U/mL, Diagnostica Stago) was used as a positive control. TF expression was studied using real-time RT-PCR, Western Blotting (WB), and thrombin generation assay (TGA). Our experimental conditions have previously been reported (Ben-Hadj–Khalifa J Thromb Thrombolysis 2011). Results 1) FVIIa : TF mRNA and protein were not detected in response to FVIIa. FVIIa-activated monocytes supported thrombin generation. However, the kinetics of thrombin generation was slow compared with FXa (table 1). 2) APC : Monocytes dose-dependently expressed TF mRNA and protein in response to APC, with a complete agreement between RT-PCR and WB. APC-stimulated monocytes supported a strong thrombin generation, in a dose-dependent manner. The expression of TF in response to APC was consistently higher than in response to FXa, whatever the assay (figure 1) Discussion / Conclusion The effect of FVIIa and APC on monocyte TF expression has never been previously reported. We could not demonstrate the ability of FVIIa to induce TF expression. However, these experiments were performed in the absence of exogenous TF. As it is reported that FVIIa activates PARs inside the complex FVIIa-TF, or FVIIa-TF-FXa (Rollin Hematologie 2012, Camerer J Cell Biology 2000), we will repeat the experiments in the presence of TF. Unexpectedly, whereas we failed to detect TF expression in RT-PCR and WB, we observed an effect of VIIa on monocyte-induced thrombin generation. It raises the question of the dependency towards TF expression of our model. We report for the first time that APC is a strong inducer of TF expression by monocytes. This observation is unexpected since APC is a major coagulation inhibitor and cytoprotective. The expression of TF is currently associated with cell activation or apoptosis induction. The cytoprotective effect of APC has been described for endothelial cells and macrophages, but with different pathways for the activation of PAR-1, which is Endothelial Protein C Receptor (ECPR)-dependent in endothelial cells and CD11b/CD18 dependent for macrophages (Van de Poll, Current Opin Infections Des 2011). Interestingly, monocytes not only express PAR-1 and b2 integrins, but also EPCR and thrombomodulin (TM), the endothelial cofactor of thrombin, for the activation of PC. Further investigations including signaling pathway studies are required to elucidate this paradigm. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Effect of blood flow on thrombin generation is dependent on the nature of the thrombogenic surface

Blood ◽  
1994 ◽  
Vol 84 (7) ◽  
pp. 2206-2213 ◽  
Author(s):  
A Diquelou ◽  
S Lemozy ◽  
D Dupouy ◽  
B Boneu ◽  
K Sakariassen ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Blood Flow ◽  
Shear Rate ◽  
Plasma Levels ◽  
Thrombin Generation ◽  
Type Iii Collagen ◽  
Fibrin Deposition ◽  
Positive Correlation ◽  
Shear Rates ◽  
Fibrin Formation

Abstract We have investigated the influence of blood flow on thrombin generation, fibrin formation, and fibrin deposition on procoagulant and nonprocoagulant surfaces. Nonanticoagulated human blood was drawn for 5 minutes directly from an antecubital vein over stimulated endothelial cells expressing tissue factor and over human type III collagen fibrils, positioned in parallel-plate perfusion chambers. The shear rates at these surfaces were 50, 650, and 2,600 s-1. Deposition of platelets and fibrin was measured by morphometry. Thrombin and fibrin formation was determined by measuring prothrombin fragments 1 + 2 (F 1 + 2), thrombin-antithrombin III complexes, (T-AT) and fibrinopeptide A (FPA) in blood effluent from the perfusion chamber at the end of the 5- minute perfusion period. On procoagulant endothelial cells, the thrombi were primarily composed of fibrin. The fibrin deposition (81%, 21%, and 2% at 50, 650, and 2,600 s-1, respectively) and plasma levels of F 1 + 2, T-AT and FPA were shear rate dependent and highest at 50 s-1. There was a positive correlation between F 1 + 2 and T-AT and the fibrin deposition (P < .01). In contrast, the collagen surface triggered primarily thrombi that were composed of platelets. The platelet thrombi and plasma levels of F 1 + 2 and T-AT were also dependent on the shear rate, but highest at 650 and 2,600 s-1. F 1 + 2 and T-AT reached the same level as observed with procoagulant endothelial cells at the higher shear rates. There was a positive correlation between F 1 + 2 and T-AT and the platelet thrombus formation (P < .05), confirming the predominant role of platelets in thrombin generation. Thus, thrombin formation is strongly influenced by the blood flow, and this effect depends on the composition of the thrombogenic surface.

AV513 Enhances Tissue Factor Activity by Inhibiting TFPI in Hemophilia A Plasma: Potential TFPI Target Sites and Potentiation of Factor VIII Activity.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 497-497 ◽  
Author(s):  
Sabine Knappe ◽  
Nicholas Kripalani ◽  
Susannah Patarroyo-White ◽  
Kirk W. Johnson ◽  
Srinivasa Prasad
Keyword(s):  
Human Plasma ◽  
Tissue Factor ◽  
Thrombin Generation ◽  
Hemophilia A ◽  
Brown Seaweed ◽  
Full Length ◽  
Dependent Manner ◽  
Severe Hemophilia ◽  
Low Concentrations ◽  
Dose Dependent

Abstract AV513, a sulfated polysaccharide prepared from brown seaweed and enriched for fucoidan accelerates clotting in hemophilia A and B plasma. Prior studies have demonstrated that AV513 functions mainly by inhibiting TFPI activity. In this report, we have further studied AV513 mechanism of action using human plasma thromboelastography (TEG) model and a thrombin generation assay in FVIII deficient plasma. In the TEG assay, AV513 enhanced tissue factor (TF) induced clotting in human hemophilia A pooled plasma in a dose-dependent manner. A dose-dependent potentiation effect by AV513 was observed with reduction of TEG R time to normal levels at 0.5 μg/mL of AV513. An AV513 dose dependent increase in thrombin generation was observed in hemophilia A pooled plasma with a 3-fold increase in peak thrombin generation at 2.5 μg/mL AV513 (basal: 40 nM vs. treated: 140 nM). When TFPI was immunodepleted from hemophilia A plasma (FVIII/TFPI deficient), sub-optimal concentrations of TF enhanced thrombin generation which was not further enhanced by addition of AV513. The increase in thrombin generation in FVIII /TFPI deficient plasma was inhibited by 93% after the addition of exogenous full-length TFPI or by 47% after the addition of truncated rTFPI (amino acids 1–161) that lacks the Kunitz 3 domain and the C-terminal tail. AV513 at low concentrations reversed exogenous full length TFPI activity almost completely in FVIII/TFPI-deficient plasma but had only a small effect on truncated TFPI. In a human plasma TEG combination study of AV513 and low concentrations of rFVIII (ReFacto), AV513 at a concentration of 0.25 μg/mL reduced the TEG R time by 35% in severe hemophilia A plasma compared to 10 mU/mL of rFVIII alone. These results indicate that AV513 functions as a pro-coagulant by inhibiting TFPI at least in part via targeting the C-terminal region. Moreover, AV513, in the presence of low rFVIII (ReFacto) levels, improves severe hemophilia A plasma clotting - as measured by TEG - beyond that observed with either agent alone.

scholarly journals Filamin-A is required for the incorporation of tissue factor into cell-derived microvesicles

2014 ◽  
Vol 111 (04) ◽  
pp. 647-655 ◽  
Author(s):  
Anthony Maraveyas ◽  
Camille Ettelaie ◽  
Mary Collier
Keyword(s):  
Endothelial Cells ◽  
Plasma Membrane ◽  
Tissue Factor ◽  
Thrombin Generation ◽  
Cytoplasmic Domain ◽  
Cytoskeletal Protein ◽  
Dependent Manner ◽  
Filamin A ◽  
Partial Suppression ◽  
Protease Activated Receptor 2

SummaryWe previously reported that the incorporation of tissue factor (TF) into cell-derived microvesicles (MVs) is regulated by the phosphorylation of the cytoplasmic domain of TF. Since the cytoskeletal protein filamin-A is known to bind to the cytoplasmic domain of TF in a phosphorylation-dependent manner, the involvement of filamin-A in the incorporation of TF into MVs was examined. Endothelial cells were transfected to express TF, whereas MDA-MB-231 cells were used to examine endogenously expressed TF. MV release was induced by activating protease-activated receptor-2 (PAR2). Partial suppression of filamin-A expression using two different filamin-A siRNA sequences resulted in significant reductions in the incorporation of TF antigen into MVs as determined by TF-ELISA and western blot analysis, and was reflected in reduced thrombin-generation and FXa-generation capacities of these MVs. Deletion of the cytoplasmic domain of TF also resulted in reduced incorporation of TF into MVs, whereas the suppression of filamin-A expression had no additional effect on the incorporation of truncated TF into MVs. Partial suppression of filamin-A expression had no effect on the number and size distribution of the released MVs. However, >90% suppression of filamin-A expression resulted in increased MV release, possibly as a result of increased instability of the plasma membrane and underlying cytoskeleton. In conclusion, the presence of filamin-A appears to be essential for the incorporation of TF into MVs following PAR2 activation, but is not required for the process of MV formation and release following PAR2 activation.

Elevated prothrombin results in clots with an altered fiber structure: a possible mechanism of the increased thrombotic risk

Blood ◽  
2003 ◽  
Vol 101 (8) ◽  
pp. 3008-3013 ◽  
Author(s):  
Alisa S. Wolberg ◽  
Dougald M. Monroe ◽  
Harold R. Roberts ◽  
Maureane Hoffman
Keyword(s):  
Plasma Levels ◽  
Thrombin Generation ◽  
Fibrin Clot ◽  
Length Ratio ◽  
Dependent Manner ◽  
Thrombotic Risk ◽  
Increased Risk ◽  
Clot Structure ◽  
Dose Dependent ◽  
Fibrin Clots

AbstractIndividuals with elevated prothrombin levels are at increased risk of venous thrombosis. To understand the mechanism behind this observation, we studied the effect of prothrombin concentration on thrombin generation and fibrin clot structure. The pattern of thrombin generation was directly related to the prothrombin level at all concentrations tested. From 0% to 300% of normal plasma levels of prothrombin, increasing the prothrombin concentration increased the initial rate, peak, and total amount of thrombin generated. Importantly, fibrin clot structure was also affected by the prothrombin concentration. Fibrin clots made from prothrombin concentrations less than 10% of plasma levels were weak and poorly formed. Fibrin clots made at 10% to 100% of plasma levels of prothrombin had similar fiber structures (mass-to-length ratio; μ). However, the fiber mass-to-length ratio decreased with increasing prothrombin levels more than 100% of plasma levels, in a dose-dependent manner. These results suggest that increased levels of prothrombin alter thrombin generation and clot structure. Specifically, elevated prothrombin levels produce clots with reduced fibrin mass-to-length ratios compared with normal clots. We hypothesize that this alteration in fibrin clot structure is an important determinant of the risk of thrombosis.

scholarly journals Potential Role of Circulating Endoglin in Hypertension via the Upregulated Expression of BMP4

Cells ◽  
2020 ◽  
Vol 9 (4) ◽  
pp. 988 ◽  
Author(s):  
Eunate Gallardo-Vara ◽  
Luis Gamella-Pozuelo ◽  
Lucía Perez-Roque ◽  
José L. Bartha ◽  
Irene Garcia-Palmero ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Cardiovascular Diseases ◽  
Pregnant Women ◽  
Plasma Levels ◽  
Molecular Mechanisms ◽  
Proteolytic Processing ◽  
Gene Transcript ◽  
Multisystem Disorder ◽  
Transcript Levels

Endoglin is a membrane glycoprotein primarily expressed by the vascular endothelium and involved in cardiovascular diseases. Upon the proteolytic processing of the membrane-bound protein, a circulating form of endoglin (soluble endoglin, sEng) can be released, and high levels of sEng have been observed in several endothelial-related pathological conditions, where it appears to contribute to endothelial dysfunction. Preeclampsia is a multisystem disorder of high prevalence in pregnant women characterized by the onset of high blood pressure and associated with increased levels of sEng. Although a pathogenic role for sEng involving hypertension has been reported in several animal models of preeclampsia, the exact molecular mechanisms implicated remain to be identified. To search for sEng-induced mediators of hypertension, we analyzed the protein secretome of human endothelial cells in the presence of sEng. We found that sEng induces the expression of BMP4 in endothelial cells, as evidenced by their proteomic signature, gene transcript levels, and BMP4 promoter activity. A mouse model of preeclampsia with high sEng plasma levels (sEng+) showed increased transcript levels of BMP4 in lungs, stomach, and duodenum, and increased circulating levels of BMP4, compared to those of control animals. In addition, after crossing female wild type with male sEng+ mice, hypertension appeared 18 days after mating, coinciding with the appearance of high plasma levels of BMP4. Also, serum levels of sEng and BMP4 were positively correlated in pregnant women with and without preeclampsia. Interestingly, sEng-induced arterial pressure elevation in sEng+ mice was abolished in the presence of the BMP4 inhibitor noggin, suggesting that BMP4 is a downstream mediator of sEng. These results provide a better understanding on the role of sEng in the physiopathology of preeclampsia and other cardiovascular diseases, where sEng levels are increased.

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