Monocyte IL-10 produced in response to lipopolysaccharide modulates thrombin generation by inhibiting tissue factor expression and release of active tissue factor-bound microparticles

2007 ◽  
Vol 97 (04) ◽  
pp. 598-607 ◽  
Author(s):  
Stéphane Poitevin ◽  
Eva Cochery-Nouvellon ◽  
Annick Dupont ◽  
Philippe Nguyen
Keyword(s):  
Tissue Factor ◽  
Thrombin Generation ◽  
Interleukin 10 ◽  
Inhibitory Effect ◽  
Original Method ◽  
Integrated System ◽  
Dependent Manner ◽  
Highly Sensitive ◽  
Procoagulant Effect ◽  
Kinetics Of

SummaryLipopolysaccharide (LPS)-stimulated monocytes are known to have a procoagulant effect. This property is currently explained by the fact that monocytes, in response to LPS, can express tissue factor (TF) and undergo a process of membrane microvesiculation. Interleukin-10 (IL-10) has been shown to downregulate TF expression and inhibit procoagulant activity (PCA). In order to further characterize the inhibitory effect of IL-10 on LPS-induced PCA, we used the integrated system of analysis of kinetics of thrombin generation in normal plasma (thrombinography). For this, we developed an original method of elutriation allowing to obtain a highly purified monocyte preparation, under endotoxin-free conditions. Thrombin generation was measured using a highly sensitive and specific fluorogenic method which we adapted to inhibit the contact factor pathway. Results show that recombinant human IL-10 decreased the kinetics of thrombin generation in a dose-dependent manner. Furthermore, the inhibition of endogenous IL-10 released by monocytes in response to LPS is associated with an increase in the kinetics of thrombin generation. We demonstrated that this effect was a consequence of the up-regulation of TF expression and TF-bound microparticle release. In conclusion, we report that IL-10 can regulate thrombin generation in conditions close to physiology as allowed by thrombinography, and that endogenous IL-10 regulates TF expression and release of active TF-bound microparticles by a negative feed back loop through IL-10 receptor α

scholarly journals Physiological concentrations of tissue factor pathway inhibitor do not inhibit prothrombinase

Blood ◽  
1996 ◽  
Vol 87 (5) ◽  
pp. 1845-1850 ◽  
Author(s):  
AE Mast ◽  
GJ Jr Broze
Keyword(s):  
Tissue Factor ◽  
Calcium Ions ◽  
Thrombin Generation ◽  
Tissue Factor Pathway Inhibitor ◽  
Factor Viia ◽  
Factor Xa ◽  
Inhibitory Effect ◽  
Factor V ◽  
Dependent Manner ◽  
Tissue Factor Pathway

Tissue factor pathway inhibitor (TFPI) is a Kunitz-type serine proteinase inhibitor that directly inhibits factor Xa and, in a factor Xa dependent manner, inhibits the factor VIIa/tissue factor catalytic complex. The inhibitory effect of TFPI in prothrombin activation assays using purified components of the prothrombinase complex was examined. When factor Xa is added to mixtures containing TFPI, prothrombin, calcium ions, and nonactivated platelets or factor V and phospholipids, TFPI significantly reduces subsequent thrombin generation, and the inhibitory effect is enhanced by heparin. If factor Xa is preincubated with calcium ions and thrombin-activated platelets or factor Va and phospholipids to permit formation of prothrombinase before the addition of prothrombin and physiologic concentrations of TFPI (< 8 nmol/L), minimal inhibition of thrombin generation occurs, even in the presence of heparin. Thus, contrary to results in amidolytic assays with chromogenic substrates, prothrombinase is resistant to inhibition by TFPI in the presence of its physiological substrate, prothrombin. Higher concentrations of TFPI (approximately 100 nmol/L), similar to those used in animal studies testing for therapeutic actions of TFPI, do effectively block prothrombinase activity.

Physiological concentrations of tissue factor pathway inhibitor do not inhibit prothrombinase

Blood ◽  
1996 ◽  
Vol 87 (5) ◽  
pp. 1845-1850 ◽  
Author(s):  
AE Mast ◽  
GJ Jr Broze
Keyword(s):  
Tissue Factor ◽  
Calcium Ions ◽  
Thrombin Generation ◽  
Tissue Factor Pathway Inhibitor ◽  
Factor Viia ◽  
Factor Xa ◽  
Inhibitory Effect ◽  
Factor V ◽  
Dependent Manner ◽  
Tissue Factor Pathway

Abstract Tissue factor pathway inhibitor (TFPI) is a Kunitz-type serine proteinase inhibitor that directly inhibits factor Xa and, in a factor Xa dependent manner, inhibits the factor VIIa/tissue factor catalytic complex. The inhibitory effect of TFPI in prothrombin activation assays using purified components of the prothrombinase complex was examined. When factor Xa is added to mixtures containing TFPI, prothrombin, calcium ions, and nonactivated platelets or factor V and phospholipids, TFPI significantly reduces subsequent thrombin generation, and the inhibitory effect is enhanced by heparin. If factor Xa is preincubated with calcium ions and thrombin-activated platelets or factor Va and phospholipids to permit formation of prothrombinase before the addition of prothrombin and physiologic concentrations of TFPI (< 8 nmol/L), minimal inhibition of thrombin generation occurs, even in the presence of heparin. Thus, contrary to results in amidolytic assays with chromogenic substrates, prothrombinase is resistant to inhibition by TFPI in the presence of its physiological substrate, prothrombin. Higher concentrations of TFPI (approximately 100 nmol/L), similar to those used in animal studies testing for therapeutic actions of TFPI, do effectively block prothrombinase activity.

The Effect of Active Site-inhibited Factor VIIa on Tissue Factor-initiated Coagulation Using Platelets before and after Aspirin Administration

1997 ◽  
Vol 78 (04) ◽  
pp. 1202-1208 ◽  
Author(s):  
Marianne Kjalke ◽  
Julie A Oliver ◽  
Dougald M Monroe ◽  
Maureane Hoffman ◽  
Mirella Ezban ◽  
...  
Keyword(s):  
Tissue Factor ◽  
Active Site ◽  
Large Scale ◽  
Thrombin Generation ◽  
Factor Viia ◽  
Dependent Manner ◽  
Antithrombotic Agent ◽  
Before And After ◽  
Ic50 Values

SummaryActive site-inactivated factor VIIa has potential as an antithrombotic agent. The effects of D-Phe-L-Phe-L-Arg-chloromethyl ketone-treated factor VIla (FFR-FVIIa) were evaluated in a cell-based system mimicking in vivo initiation of coagulation. FFR-FVIIa inhibited platelet activation (as measured by expression of P-selectin) and subsequent large-scale thrombin generation in a dose-dependent manner with IC50 values of 1.4 ± 0.8 nM (n = 8) and 0.9 ± 0.7 nM (n = 7), respectively. Kd for factor VIIa binding to monocytes ki for FFR-FVIIa competing with factor VIIa were similar (11.4 ± 0.8 pM and 10.6 ± 1.1 pM, respectively), showing that FFR-FVIIa binds to tissue factor in the tenase complex with the same affinity as factor VIIa. Using platelets from volunteers before and after ingestion of aspirin (1.3 g), there were no significant differences in the IC50 values of FFR-FVIIa [after aspirin ingestion, the IC50 values were 1.7 ± 0.9 nM (n = 8) for P-selectin expression, p = 0.37, and 1.4 ± 1.3 nM (n = 7) for thrombin generation, p = 0.38]. This shows that aspirin treatment of platelets does not influence the inhibition of tissue factor-initiated coagulation by FFR-FVIIa, probably because thrombin activation of platelets is not entirely dependent upon expression of thromboxane A2.

In Vitro Effect of Danaparoid Sodium (Orgaran®) on Thrombin Generation after Minimal Tissue Factor Pathway Activation.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 4151-4151
Author(s):  
Ismail Elalamy ◽  
Anna D. Petropoulou ◽  
Mohamed Hatmi ◽  
Meyer M. Samama ◽  
Grigoris T. Gerotziafas
Keyword(s):  
Molecular Weight ◽  
Tissue Factor ◽  
Thrombin Generation ◽  
Lag Time ◽  
Dependent Manner ◽  
Pathway Activation ◽  
Coagulation Tests ◽  
Vitro Effect ◽  
Routine Coagulation

Abstract Introduction: Orgaran® (Org 10172) is a low molecular weight heparinoid which consists of natural sulphated glycosaminoglycans (heparan, dermatan, chondroitin sulphate). It has a mean molecular weight of approximately 6 kDa (4–10 kDa), an excellent bioavailability following subcutaneous administration and an anti-Xa/anti-IIa activity ratio superior to 22. It is the anticoagulant of choice in patients developping Heparin-Induced Thrombocytopenia (HIT), whereas its’ use is also proposed for surgical thromboprophylaxis. Orgaran® has no effect on routine coagulation tests (aPTT, PT, TT). Thrombin generation test(TG) is a global clotting assay proven to be sensitive to the anticoagulant effect of LMWHs and specific FXa inhibitors (i.e. fondaparinux and BAY-597939). In this in vitro study, we determined the tissue factor (TF)-induced TG inhibition potency of Orgaran® using the Thrombogram-Thrombinoscope® assay. Materials and Methods: TG was assessed after TF pathway activation in Platelet Rich Plasma (PRP) (1.5x105 platelets/μl) using diluted thromboplastin (Dade Innovin®, 1:1000 final dilution). The clotting process is provoked by a physiologically relevant TF concentration. Orgaran® was added to control plasma from 8 healthy volunteers at five different final concentrations (0.2, 0.4, 0.6, 0.8 and 1IU anti-Xa/ml). TG was initiated by adding the triggering solution containing CaCl2 and the fluorogenic substrate. The analyzed TG parameters are the lag time, the maximal concentration of thrombin (Cmax), the time to reach Cmax (Tmax), the TG velocity and the endogenous thrombin potential (ETP). Results: Orgaran® prolonged significantly the lag time and the Tmax at a concentration over 0.40 IU anti-Xa/ml (p<0.05). At the lowest studied concentration (0.20 IU anti-Xa/ml), lag time and Tmax were only prolonged by 12%, whereas their maximal prolongation (around 50%) was observed at 1IU anti-Xa/ml. Furthermore, Orgaran® inhibited ETP, Cmax and TG velocity in an almost linear dose dependent manner. A significant inhibition of ETP, Cmax and TG velocity was obtained at concentrations superior to 0.20 IU anti-Xa/ml. (p<0.05). At the highest studied concentration (1IU anti-Xa/ml) Orgaran® suppressed all TG parameters by about 80% (Table 1). Conclusion: Orgaran® exhibited a significant inhibitory activity of in vitro TG. At concentrations achieved in clinical practice (prophylactic or therapeutic dose), Orgaran® modified in vitro TG profile while it has no effect on routine coagulation tests. Thus, TG assay is a sensitive method for monitoring Orgaran® and this test requires a clinical prospective evaluation. Table 1. Determination of IC20 and IC50 anti-Xa inhibitory concentrations of Orgaran® on TG parameters Lag Time Tmax ETP Cmax Velocity IC: Inhibitory Concentration * or Concentration increasing 20% and 50% the lag time and the Tmax respectively IC 20 (IU/ml) 0.30 0.30 0.18 0.18 0.15 IC 50 (IU/ml) 0.83 >1 0.30 0.50 0.35 1IU anti-Xa/ml 53% 47% 68% 76% 84%

scholarly journals The Antithrombotic Potential of Tinzaparin and Enoxaparin Upon Thrombin Generation Triggered In Vitro by Human Ovarian Cancer Cells IGROV1

2016 ◽  
Vol 23 (2) ◽  
pp. 155-163 ◽  
Author(s):  
Mouna Sassi ◽  
Taher Chakroun ◽  
Elisabeth Mbemba ◽  
Patrick Van Dreden ◽  
Ismail Elalamy ◽  
...  
Keyword(s):  
Ovarian Cancer ◽  
Cancer Cells ◽  
Tissue Factor ◽  
Thrombin Generation ◽  
Fold Increase ◽  
Inhibitory Effect ◽  
Ovarian Cancer Cells ◽  
Ovarian Adenocarcinoma ◽  
Venous Thromboembolic Events

Background: A documented relationship between ovarian cancer and thrombosis does exist. Low-molecular-weight heparins (LMWHs) are cornerstone drugs in the primary prevention and treatment of venous thromboembolic events in patients with cancer. However, cancer cells may alter the efficiency of these antithrombotic agents. Objective: We aimed to characterize the procoagulant phenotype of human epithelial ovarian adenocarcinoma cells, IGROV1, and to compare the capacity of tinzaparin and enoxaparin to inhibit thrombin generation triggered by these cells. Methods: Thrombin generation induced by different concentrations of IGROV1 cells on platelet poor plasma (PPP) was assessed by the calibrated automated thrombogram assay. Tissue factor (TF) expression was studied using Western blot analysis. Then, the experimental model of thrombin generation was used to compare the inhibitory effect of clinically relevant concentrations of both tinzaparin and enoxaparin. The inhibitory concentration 50 (IC50) of the mean rate index and the endogenous thrombin potential and the 2-fold increase in lag time were analyzed on the basis of the anti-Xa and anti-IIa activities of the LMWHs. Results: IGROV1 cells suspended into PPP resulted in a significant increase in thrombin generation in the absence of any exogenous source of TF and phospholipids. Tissue factor was expressed by IGROV1 cells. Tinzaparin was a more potent inhibitor of thrombin generation than enoxaparin. The inhibition of thrombin generation induced by IGROV1 cancer cells depended mainly on the anti-Xa activity of the LMWHs. Conclusion: This experimental study in ovarian cancer cells demonstrates that the antithrombotic activity of LMWHs is not completely predicted by the anti-Xa or anti-IIa activities measured in PPP.

scholarly journals Monitoring Compound-Related Effects on Coagulability in Rats and Cynomolgus and Rhesus Monkeys by Thrombin Generation Kinetic Measurement

2020 ◽  
Vol 39 (3) ◽  
pp. 207-217
Author(s):  
F. Poitout-Belissent ◽  
D. Culang ◽  
D. Poulin ◽  
R. Samadfan ◽  
S. Cotton ◽  
...  
Keyword(s):  
Tissue Factor ◽  
Thrombin Generation ◽  
In Vivo Studies ◽  
Coagulation Cascade ◽  
Dependent Manner ◽  
Kinetic Assay ◽  
Thrombin Generation Assay ◽  
Coefficients Of Variation

Thrombin generation assay (TGA) is a sensitive method for the assessment of the global clotting potential of plasma. This kinetic assay can detect both hypocoagulable and hypercoagulable conditions: delayed or reduced thrombin generation leading to a prolonged clotting time, or induced thrombin activity, shifting the coagulation cascade toward thrombosis. The purpose of this study is to qualify the TGA in nonhuman primates (NHP) and rats for its use during nonclinical in vivo and in vitro studies. Blood was drawn from nonanesthetized animals, and platelet-poor plasma was obtained after double centrifugation; coefficients of variation were <10% for all derived parameters of thrombin generation assessed with 5 pM of tissue factor. Thrombin generation was evaluated in vitro in rat and NHP plasmas with ascending doses of unfractionated heparin (UFH), recombinant tissue factor, and anticoagulant compounds. Thrombin generation was decreased with UFH and anticoagulant compounds, but was increased in the presence of tissue factor, in a dose-dependent manner. In a rat model of inflammation, animals were administered a low dose of lipopolysaccharides. Thrombin generation measurements were decreased 3 hours post-LPS administration with a nadir at 24 hours, while thrombin–antithrombin complexes reached a peak at 8 hours, supporting an earlier production of thrombin. In conclusion, these data demonstrated that TGA can be performed in vitro for screening of compounds expected to have effects on coagulation cascade, and thrombin generation can be measured at interim time points during nonclinical in vivo studies in rats and NHP.

Differential Procoagulant Phenotype of Pancreatic and Breast Cancer Cells Related to Different Tissue Factor Activity.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 3992-3992
Author(s):  
Grigoris T. Gerotziafas ◽  
Ismail Elalamy ◽  
Marie-Paule Roman ◽  
Claudine Prengel ◽  
Elisabeth Verdy ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Cancer Cells ◽  
Tissue Factor ◽  
Breast Cancer Cells ◽  
Thrombin Generation ◽  
Human Platelet ◽  
Procoagulant Activity ◽  
Dependent Manner ◽  
Mcf7 Cells ◽  
Platelet Poor Plasma

Abstract Tissue factor (TF) expressed by some cancer cells is implicated in metastasis and angiogenesis. The influence of cancer cells on blood coagulation has not been adequately studied. We evaluated the procoagulant potential of pancreatic and breast cancer cells (BXPC3 and MCF7 cell lines respectively) when they are in contact with human platelet-poor plasma (PPP). At 40% and 90% confluence, adhesive cultures of BXPC3 and MCF7 cells were treated with trypsine according to standardized procedure and cancer cells were suspended in normal human platelet poor plasma (PPP) at increasing concentrations. Coagulation was triggered by CaCl2 addition and thrombin generation (TG) was monitored using the Calibrated Automated Thrombogram-Thrombinoscope® (Biodis-France). In some experiments, cancer cells were incubated for 30 min with a polyclonal specific anti-TF antibody (American Diagnostics). Cancer cells accelerated TG by decreasing significantly lag-time, and time to Peak of thrombin (ttPeak) but they did not significantly influence the endogenous thrombin potential. BXPC3 had significantly more potent procoagulant activity compared to MCF7 cells. Incubation of cancer cells with anti-TF antibody resulted in a concentration dependent inhibition of their procoagulant effect. The IC50 of the anti-TF antibody for TG induced by BXPC3 was about 10-fold higher to that for MCF7. Pancreatic cancer cells (BXPC3) and breast cancer cells (MCF7) accelerate thrombin generation of human plasma in a TF-dependent manner. BXPC3 have more potent procoagulant activity than MCF7 probably due to increased TF expression.

Modulating the Factor V Procofactor to Cofactor Transition Using Recombinant B-Domain Fragments

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 376-376
Author(s):  
Matthew W Bunce ◽  
Mettine H.A. Bos ◽  
Rodney M. Camire
Keyword(s):  
Thrombin Generation ◽  
Inhibitory Effect ◽  
Dependent Manner ◽  
Clotting Factors ◽  
Basic Peptide ◽  
Membrane Bound ◽  
Cofactor Activity ◽  
Acidic Region ◽  
Basic Region ◽  
Prothrombin Activation

Abstract Abstract 376 Activation of clotting factors from their inactive precursor states by limited and discrete proteolysis is a hallmark of coagulation. Prothrombinase, the physiological activator of prothrombin, is comprised of the serine protease factor Xa (FXa) and its cofactor factor Va (FVa). The precursor of FVa, FV, is an inactive procofactor and cannot participate to any significant degree in prothrombinase. It is well established that the large central B domain of FV (domain organization of A1-A2-B-A3-C1-C2) stabilizes the inactive procofactor state, however the mechanism by which this occurs is poorly defined. Since FVa has such a profound impact on thrombin generation and clot formation, defining the mechanism by which FV is kept inactive may reveal a unique way to target the cofactor for therapeutic gain. We previously demonstrated that deletion of a conserved region of the B domain enriched in basic residues converts FV to an active cofactor (JBC 2007, 282: 15033). Here we provide new insights into the mechanism by which the basic region of the B-domain contributes to maintaining FV as an inactive procofactor. Using a bacterial expression system, we generated FV B-domain fragments and assessed their ability to disrupt the procofactor to cofactor transition. The FV variant FV-810 (residues 811–1491 deleted), which lacks over 80% of the B-domain including the basic region, possesses activity comparable to FVa in clotting assays, thrombin generation assays, and in vitro prothrombin activation reactions. Addition of a FV B-domain fragment containing the basic region (residues 951–1008; ‘basic peptide') inhibited FV-810 activity in all three assays. As a control, no inhibitory effect was observed with a non-homologous peptide from the FVIII B-domain. Surprisingly, the basic peptide did not inhibit FVa in these assays, nor did it inhibit FV-810 or other FV variants that had been pre-incubated with thrombin. These results indicate that additional B-domain sequences present in FV-810 must somehow contribute to the inhibitory effect of the basic region. Analysis of the B-domain sequences within FV-810 and other cofactor-like FV variants revealed a concentration of acidic residues between Thr1492 and Asn1538 that may help to stabilize FV in an inactive state most likely through an electrostatic interaction with the basic region. In support of this model, FV BR152, a variant that possesses the basic region but lacks the acidic region, has FVa-like activity in clotting assays and prothrombin activation assays. Unlike the basic peptide, a peptide derived from the acidic region (residues 1492–1538) did not inhibit the activity of FV BR152, indicating that the acidic region must be located proximal to the A3 domain in order to inhibit FV. To further probe the mechanism by which the basic peptide functions, we directly monitored FXa binding to FV-810 on phospholipid vesicles using fluorescence anisotropy. In the absence of the basic peptide, labeled FXa bound to membrane-bound FV-810 with a Kd of ∼1 nM in a 1:1 stoichiometry. Addition of the FV basic peptide significantly reduced FXa binding to FV-810 in a dose-dependent manner, indicating competition between FXa and the basic peptide for FV-810. Consistent with the results of the functional assays, the basic peptide had no effect on FXa binding to membrane-bound FVa. Taken together our data support a model in which the basic B-domain region of FV inhibits the cofactor activity of FV through a functional interaction with an acidic region proximal to the A3 domain, thereby masking a FXa binding site that is exposed following thrombin cleavage of FV at Arg1545. These findings offer important new insights into how FV is kept as an inactive procofactor and provide key mechanistic clues into how the B-domain participates in this process. Moreover, these results demonstrate a novel strategy to regulate FV cofactor activity by using functional elements of the B-domain to shift an active cofactor back to an inactive procofactor. 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.

Fibrinogen Attentuates Thrombin Decay In a Concentration-Dependent Manner

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 2346-2346 ◽  
Author(s):  
Romy Kremers ◽  
Rob Wagenvoord ◽  
Coenraad Hemker
Keyword(s):  
Decay Rate ◽  
Tissue Factor ◽  
Thrombin Generation ◽  
Decay Rates ◽  
Plasma Fibrinogen ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Human Fibrinogen ◽  
First Order ◽  
Pseudo First Order

Abstract Introduction Clotting of blood(-plasma) is caused by the first traces of thrombin that appear. The bulk of thrombin emerges in the clot and is subsequently inactivated. Fibrin(-ogen) has a stimulatory effect on the amount of thrombin activity observed. This can be due to stimulation of prothrombin conversion, to attenuation of thrombin inhibition or both. Thrombin is known to bind reversibly to specific sites on the E domain of fibrin and to remain active when bound. We tested the hypothesis that this binding makes thrombin less accessible to inhibitors and thus fosters the presence of active thrombin. Thrombin decay is caused by serpins, mainly antithrombin (AT), and by α2Macroglobulin (α2M). Aim In this study we aim to determine the effect of fibrin(-ogen) on the rate of thrombin decay and on the contribution of α2M to total decay. Methods Thrombin generation (TG) in normal pool plasma in the presence or absence of fibrinogen was triggered with 5 pM tissue factor (TF) and measured by calibrated automated thrombinography (CAT). Thrombin decay was measured by triggering thrombin generation with high (50 pM) tissue factor (TF) causing all prothrombin to be converted within 3 minutes, so that the part of the curve after 3 min is governed by thrombin decay alone. Total thrombin decay rate was calculated as the pseudo first order decay constant of that part of the curve. The α2M-dependent thrombin decay rate was measured as the pseudo first order constant with which the amidolytically active α2M-thrombin complex forms. AT and α2M levels were determined by functional assays that were developed in house. Fibrinogen was measured by the von Clauss method. Defibrination was done by adding reptilase and winding out the formed fibrin. Anonymized plasma samples remaining from routine analyses were obtained from the clinical routine laboratory (n=77), including samples from patients in which a shift in the ratio of AT and α2M could be expected (liver cirrhosis, nephrotic syndrome). Pearson’s correlation analysis was used to establish the relationships between thrombin decay rates and inhibitor concentrations, and the influence of plasma fibrinogen concentration on these reactions. Results Thrombin generation and thrombin decay were measured in normal pooled plasma, defibrinated normal pooled plasma and defibrinated normal pooled plasma spiked with 1, 2 or 3 g/L purified human fibrinogen. The removal of fibrinogen from plasma decreases thrombin generation by 40% (p<0.001) and the addition of purified human fibrinogen to defibrinated plasma concentration-dependently restores TG. In addition, plasma defibrination causes the rate of total thrombin decay to increase significantly from 0.36 min-1 to 0.47 min-1 (p<0.001). This effect can be reversed as well by the addition of purified human fibrinogen. Thrombin decay by α2M is inhibited by fibrinogen in a concentration-dependent manner (36% at 3 g/L fibrinogen) (p<0.001). No significant concentration dependence was found for AT-dependent decay. In the patient samples fibrinogen levels varied between 1.20 and 4.79 g/L with a mean value of 2.79 g/L (± 0.62 g/L). AT concentrations ranged from 0.58 µM to 3.08 µM and α2M levels from 1.85 µM to 7.73 µM. Both total and AT-dependent thrombin decay rates were significantly correlated with the plasma AT levels (p<0.001) and α2M-dependent thrombin decay was significantly correlated with the plasma α2M concentration (p<0.001). Plasma fibrinogen levels were significantly and inversely correlated with the rate of thrombin decay by α2M, but not AT (p=0.001). Conclusion The presence of fibrin(-ogen) decreases the decay rate of thrombin in a concentration-dependent manner, primarily by its influence on α2M-dependent thrombin inactivation. This causes thrombin generation to increase. Together, these results suggest that elevated fibrinogen levels may predispose individuals to thrombosis by protecting thrombin. Disclosures: No relevant conflicts of interest to declare.

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