Impact of V617F JAK2 Mutation on Monocyte Tissue Factor and Procoagulant Activity in Patients with Essential Thrombocythemia(ET) or Polycythemia VERA (PV)

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 3736-3736
Author(s):  
Anna Falanga ◽  
Alfonso Vignoli ◽  
Marina Marchetti ◽  
Laura Russo ◽  
Marina Panova-Noeva ◽  
...  
Keyword(s):  
Tissue Factor ◽  
Blood Coagulation ◽  
Thrombin Generation ◽  
Procoagulant Activity ◽  
Jak2v617f Mutation ◽  
Wild Type ◽  
Coagulation Activation ◽  
Allele Burden ◽  
Generation Capacity

Abstract Clinical data suggest an increased thrombotic risk in patients with ET or PV carrying the JAK2V617F mutation. Laboratory data from our group show that ET patients carrying the JAK2V617F mutation are characterized by an enhanced platelet and neutrophil activation status (Falanga et al, Exp Hem 2007) and blood coagulation activation (Marchetti et al, Blood 2008), as compared to JAK2 wild-type ET. Since monocytes significantly contribute to blood coagulation activation as an important source of circulating tissue factor (TF), in this study we aimed to characterize the prothrombotic phenotype of monocytes from ET and PV patients and to evaluate whether and to what extent it is influenced by the JAK2V617F mutation. Twenty-four ET patients (10 JAK2 wild-type; 14 JAK2V617F carriers with 2%–35% mutant allele burden), 27 PV patients (all JAK2V617F carriers, 16 with 9%– 44% and 11 with 60%–100% allele burden, respectively), and 20 age-matched healthy subjects (controls, C) were enrolled into the study. Monocyte-associated TF antigen was measured on the cell surface by whole blood flow-cytometry, in both basal condition and after in vitro stimulation by bacterial endotoxin (lypopolysaccharide, LPS), as well as in cell lysates by ELISA. Monocyte procoagulant activity was evaluated by the Calibrated Automated Thrombogram (CAT) as the capacity of isolated monocyte lysates to induce thrombin generation in normal pool plasma. In basal conditions, significantly (p<0.05) higher surface levels of TF were measured on monocytes from ET (17.1±3.2% positive cells) and PV (24.4±3.7% positive cells) patients compared to C (8.2±1.9% positive cells). Similarly, the total TF antigen content of cell lysates was significantly increased in patients compared to C. The analysis of the data according to JAK2V617F mutational status, showed a gradient of increased TF expression starting from JAK2V617F negative patients (11.7±2.5%), versus JAK2V617F ET and PV subjects with <50% allele burden (20.3±3.6% and 23.2±2.8%, respectively), versus JAK2V617F PV patients with >50% allele burden (26.1±4.2%). The in vitro LPS stimulation significantly increased TF expression on monocytes from all study subjects and C compared to non-stimulated monocytes (p<0.05 for all groups), with a more elevated expression by monocytes from PV and ET patients compared to C. However, the relative increase in TF expression was greater in C (=3.7 fold) compared to both ET (=2.2 fold) and PV (=2 fold) patients. As observed in basal conditions, LPS-induced TF was higher in JAK2V617F positive patients as compared to negative, with the highest expression in JAK2V617F PV carriers with >50% allele load. Thrombin generation induced by monocytes from ET and PV patients was significantly increased compared to controls, as determined by significantly higher thrombin peaks (ET=145±12, PV=142±17, C=72.2±5 nM), and quantity of thrombin generated in time, i.e. the endogenous thrombin potential (ETP) (ET=1143±34, PV=1074±64, C=787±58 nM*min). The JAK2V617F PV subjects with >50% allele burden presented with the highest thrombin generation capacity (peak= 184±34 nM; ETP= 1268±32 nM). Our data indicate that the expression of the JAK2V617F mutation in ET and PV patients may confer to monocytes a different hemostatic phenotype in terms of increased expression of surface TF and thrombin generation capacity. These findings are in agreement with the previous observation of a hypercoagulable state associated with this mutation and suggest a new mechanism linking hemostatic cellular phenotypic alteration to genetic dysfunction in patients with myeloproliferative disease.

The Impact of Factor IX Level on Thrombin Generation and Bleeding During Warfarin Anticoagulation

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 541-541
Author(s):  
Yesim Dargaud ◽  
Maureane Hoffman ◽  
Claude Negrier ◽  
Leana Lefrapper ◽  
Dougald M. Monroe
Keyword(s):  
Tissue Factor ◽  
Vitamin K ◽  
Intracranial Haemorrhage ◽  
Thrombin Generation ◽  
Factor Ix ◽  
Target Range ◽  
Significant Difference ◽  
Generation Capacity ◽  
The Impact

Abstract Abstract 541 Bleeding occurs in from 10 – 16% of warfarin-treated patients. Having a PT-INR in the target range is associated with better outcomes. However, even patients with an INR in the target range of 2–3 can suffer bleeding, suggesting that INR does not perfectly reflect the therapeutic effect of warfarin. The goal of our studies was to determine whether the level of specific coagulation factors could predict the risk of bleeding while the INR was in the target range. We modeled warfarin anticoagulation in our previously published in vitro cell based-model by adjusting the levels of vitamin K-dependent factors to those of patients with an INR of 2–3. We then examined the effect of variations in the level of FIX. The cogulation reactions were initiated by monocyte-expressed tissue factor (assayed at 1pM). Variation in FIX had a marked effect on thrombin generation. However, in plasma with the same levels of factors, as expected, variations in FIX had no effect on the PT-INR. Thus, we hypothesized that a subject with a lower FIX level than average may have a lower level of thrombin generation than is indicated by the INR. The INR might, therefore, underestimate the level of anticoagulation in such a subject. If s/he is maintained in the “therapeutic range” as measured by the INR, s/he will actually be over-anticoagulated and prone to hemorrhage. A prospective, single centre clinical study has been carried out to test this hypothesis in warfarinized patients. Between October 2010 and June 2011, 312 consecutive patients admitted to the emergency department of Edouard Herriot Hospital in Lyon, with an INR between 1.8 and 3.2, were included in the study after obtaining informed consent. Twenty six patients were admitted for a bleeding episode, 18 for recurrent thrombosis and 268 for other medical reasons. Patients presenting with bleeding, 17 males and 9 females, were aged 74±14 years old compared to the rest of the patients aged 76±14. Among the 26 bleeders, 7 had a spontaneous intracranial haemorrhage, 2 had a trauma-induced intracranial haemorrhage, 12 presented a gastrointestinal bleeding and 5 exhibited muscle hematomas, severe epistaxis or urinary tract bleeding. PT-INR and vitamin K-dependent factor levels were determined in all patients. Thrombin generation capacity in platelet poor plasma was measured using Calibrated Automated Thrombin generation assay (Thrombinoscope bv, Maastricht, The Netherlands), with tissue factor 1pM and phospholipids PC:PS:PE 4μM. No statistically significant difference was observed in the PT-INR of bleeding patients (INR=2.4±0.4) and those having a thrombosis (INR=2.5±0.5) or patients admitted for other reasons (INR=2.6±0.2). Plasma prothrombin and factor × levels were also similar in all three groups. However, a statistically lower plasma factor IX activity was observed in bleeders (p=0.01, Mann Whitney test) compared to other groups, 47.6±20 IU/dL vs. 63±33 IU/dL. In all the warfarinized subjects with an INR between 1.8 and 3.2, no correlation was found between thrombin generation capacity and PT-INR results (p=0.85, Spearman correlation test). However, a statistically significant correlation was observed between thrombin generation capacity and factor IX levels (p=0.0002). In patients, presenting with warfarin-related haemorrhage, the endogenous thrombin potential (ETP) was significantly lower at 340±335 nM.min (p=0.05) then that of warfarinized subjects who did not suffer bleeding (ETP 406±215 nM.min). These data support our hypothesis based on our in vitro results and show that patients who bleed when their PT-INR is in the target range 2 – 3 might have defective thrombin generation related to a lower level of factor IX than expected. Thus, our results suggest that the appropriate target INR level might not be the same for all patients. Those with factor IX levels that differ significantly from the mean of the population might be managed best by selecting a target INR that is based on the level of thrombin generation. Of course, a “target range” for parameters of thrombin generation during warfarin therapy would need to be developed if the assay were to be used for this purpose. Disclosures: No relevant conflicts of interest to declare.

Effects of Recombinant Factor VIIa (NovoSeven®) on Restoring Thrombin Generation in Patients with Hemophilia A and Antibodies to Factor VIII.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 1161-1161
Author(s):  
Sabine Eichinger ◽  
Barbara Lubszcyk ◽  
Karl Zwiauer ◽  
Andreas Gleiss ◽  
Peter Quehenberger ◽  
...  
Keyword(s):  
Factor Viii ◽  
Thrombin Generation ◽  
Hemophilia A ◽  
Factor Viia ◽  
Controlled Study ◽  
Coagulation Activation ◽  
Before And After ◽  
Generation Capacity ◽  
D Dimer

Abstract Introduction: Development of antibodies (ab) against factor VIII (FVIII) is a serious complication of replacement therapy in patients with hemophilia A. In case of bleeding patients with FVIII ab are treated with agents that induce hemostasis independently of FVIII. Recombinant activated factor VIIa (rVIIa) shows clinical efficacy, but its effects on hemostatic system need still to be fully elucidated. Methods: In an open controlled study, we investigated thrombin generation (peak thrombin) and parameters of coagulation activation [D-Dimer, prothrombin fragment F1+2 (F1+2)] in 5 patients with hemophilia A and FVIII ab, and in 5 healthy age-matched controls before and after intravenous bolus infusion of rVIIa (90 μg/kg bodyweight NovoSeven®, NovoNordisk, Denmark) (in hemophiliacs only). All parameters were measured in plasma before and 0.5, 1, 2, 3, and 4 hours after rVIIa infusion by use of commercially available assays (Technothrombin®TGA, Technoclone, Austria; Asserachrom®D-Di, Diagnostica Stago, France; Enzygnost F1+2, Dade Behring, Germany). Results: At baseline, hemophilia A patients had markedly lower mean (min-max) peak thrombin levels than controls [0.12 (0.0–0.6) nM vs. 186.9 (116.0–254.4) nM]. Mean (min-max) F1+2 levels did not significantly differ between patients and controls [160.7 (89.8–331.3) pmol/l vs. 160.8 (104.4–242.3) pmol/l]. Notably, D-Dimer levels were significantly higher in hemophiliacs than controls [1087.5 (174.8–3882.4) ng/ml vs. 146.3 (87.2–289.8) ng/mL]. FVIIa levels reached a mean (min-max) maximum of 28 (24–32) U/ml after 0.5 hours in all patients. After infusion, a considerable increase in mean (min-max) peak thrombin levels to 40.7 (28.3–51.6) nM was seen. Time to maximum levels was 30 minutes in three patients and 60 minutes in two. For each of the five patients the peak thrombin level was substracted from the level of its matched control at the same time point. The mean of these differences was 168.7 nM (95% CI 82.6–254.8), which translates into 80.2% (95% CI 65.4% – 88.6%) lower peak thrombin levels in haemophiliacs with FVIII ab. F1+2 significantly increased in all patients [mean (min-max) maximum levels 292.5 (175.1–464.3) pmol/l]; time to maximum levels varied from 2 to 4 hours. D-Dimer levels remained almost unchanged in all patients. Conclusion: Patients with hemophilia A and FVIII ab have low in vitro thrombin generation and F1+2 levels. After rVIIa infusion, coagulation activation as measured by F1+2 levels is slightly increased, and thrombin generation capacity is restored by 20% compared to healthy controls. Measurement of peak thrombin could be useful to monitor procoagulant treatment of patients with hemophilia A and FVIII ab.

Tissue factor procoagulant activity of plasma microparticles is increased in patients with early-stage prostate cancer

2009 ◽  
Vol 101 (06) ◽  
pp. 1147-1155 ◽  
Author(s):  
Brigitte Spath ◽  
Martin Friedrich ◽  
Felix Kyoung-Hwan Chun ◽  
Guy Marx ◽  
Ali Amirkhosravi ◽  
...  
Keyword(s):  
Prostate Cancer ◽  
Tissue Factor ◽  
Thrombin Generation ◽  
Early Stage ◽  
Critical Role ◽  
Procoagulant Activity ◽  
Dependent Manner ◽  
Phase Reaction ◽  
Coagulation Activation ◽  
D Dimer

SummaryTissue factor (TF) plays a critical role in tumour growth and metastasis, and its enhanced release into plasma in association with cellular microparticles (MPs) has recently been associated with pathological cancer progression. We have previously demonstrated significantly elevated levels of plasma TF antigen as well as systemic coagulation and platelet activation in patients with localised prostate cancer. In this prospective study, we used a highly sensitive one-stage clotting assay to measure preoperative TF-specific procoagulant activity (PCA) of plasma MPs in 68 consecutive patients with early-stage prostate cancer to further explore the relevance of circulating TF in this tumour entity. Automated calibrated thrombography was used to monitor thrombin generation in cell-free plasma samples in the absence of exogenous TF or phospholipids. Compared to healthy male controls (n=20), patients had significantly increased levels of both D-dimer and TF-specific PCA of plasma MPs (p<0.001). Furthermore, MP-associated TF PCA was higher in patients with (n=29) than in those without (n=39) laboratory evidence of an acute-phase reaction (p=0.004) and decreased to normal levels within one week after radical prostatectomy. Overall, we found a significant correlation between TF-specific PCA of plasma MPs and plasma D-dimer (p=0.002), suggesting that plasma MPs contributed to in-vivo coagulation activation in a TF-dependent manner. Thrombin generation in plasma was also significantly increased in patients compared to controls (p<0.01). Collectively, our findings suggest that TF-specific PCA of plasma MPs contributes to intravascular coagulation activation in patients with early-stage prostate cancer and may represent a potential link between hypercoagulability, inflammation, and disease progression.

Characterization of the Procoagulant Activity of Two Different Microparticle Species.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 1143-1143
Author(s):  
Ludwig Traby ◽  
Marietta Kollars ◽  
Khalid Shoumariyeh ◽  
Ansgar Weltermann ◽  
Paul Alexander Kyrle ◽  
...  
Keyword(s):  
Cell Culture ◽  
Endothelial Cells ◽  
Thrombin Generation ◽  
Procoagulant Activity ◽  
Clot Formation ◽  
Coagulation Activation ◽  
Thrombin Generation Assay ◽  
Normal Plasma ◽  
Activation Assay

Abstract Abstract 1143 Background: Microparticles (MP) exhibit procoagulant activity by exposure of tissue factor (TF) and consecutive factor (F) × activation via binding to activated FVII (FVIIa). We have shown that TF-negative MP derived from endothelial cells (EMP) after exposure to cisplatin induce thrombin generation in vitro in a TF-independent manner (Lechner et al., J. Thromb. Haemost. 2007). The procoagulant properties of TF-negative MP are not well characterized. We therefore aimed to investigate the mechanisms of coagulation activation by TF-negative EMP (obtained from human pulmonary microvascular endothelial cells - HMVEC-L) in comparison to TF-abundant MP derived from a cancer cell line (A431-MP). Methods: MP were obtained by ultracentrifugation (100,000 × g for 1 h) of cell culture supernatant. The procoagulant activity was measured in normal plasma and in plasmas deficient in coagulation factors VII, VIII, IX, X, XI, and XII, respectively, by an in vitro thrombin generation assay (Technothrombin TGA, Technoclone, Austria), by activated partial thromboplastin (aPTT), prothrombin time (PT) and a chromogenic FIX activation assay. Antibodies to TF and FVII (both American Diagnostica, USA) were used. Plasmas used in the thrombin generation assay were MP-depleted by ultracentrifugation before use. Results: EMP and A431-MP induced in vitro thrombin generation in normal plasma. In vitro thrombin generation induced by EMP was abolished in plasmas deficient in FVIII or FIX, and was markedly reduced in FXI-deficient plasma. In FVII-deficient plasma, normal thrombin generation after addition of EMP was seen. In contrast, A431-MP-triggered thrombin generation was abolished in FVII-deficient plasma, but was not influenced by depletion of FVIII, FIX or FXI. In FX-deficient plasma, thrombin generation could not be triggered by addition of either EMP or A431-MP. Thrombin generation was normal after addition of EMP or A431-MP to FXII-deficient plasma (Figure). In a modified aPTT system using kaolin as surface activator, EMP and A431-MP induced clot formation in normal plasma. In a modified PT system only A431-MP but not EMP induced clot formation. We then investigated whether MP can directly activate FIX. In a plasma-free environment FIX activation by A431-MP was much more pronounced than by EMP (14-fold vs. 2.4 fold increase compared to a control experiment with cell culture medium only). FIX activation by A431-MP was blocked by the addition of antibodies to TF and FVII, while no such effect was seen after addition to EMP. Conclusions: Our findings show that both TF-positive and TF-negative MP exhibit procoagulant activity. If TF is expressed (A431-MP), coagulation activation is triggered via FVII/FVIIa, which is abolished in FVII-deficient plasma or by the addition of antibodies to TF or FVII. TF-negative EMP induce thrombin generation via the intrinsic coagulation pathway by activating FXI and FIX, as shown by the generation of activated FIX in a FIX activation assay. EMP are also able to induce clot formation in an aPTT based system. We surmise that the procoagulatory effect of EMP may be due to their high phospholipid content or to a specific phospholipid surface composition. The results of our FIX activation assay also confirm a direct FIX activation by TF/FVIIa, which can be abolished by addition of antibodies to TF or FVII. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Differential roles of factors IX and XI in murine placenta and hemostasis under conditions of low tissue factor

2020 ◽  
Vol 4 (1) ◽  
pp. 207-216 ◽  
Author(s):  
Steven P. Grover ◽  
Clare M. Schmedes ◽  
Alyson C. Auriemma ◽  
Emily Butler ◽  
Molly L. Parrish ◽  
...  
Keyword(s):  
Tissue Factor ◽  
Thrombin Generation ◽  
Critical Role ◽  
Wild Type ◽  
Genetic Studies ◽  
Acute Hemorrhage ◽  
Expected Frequency ◽  
Low Levels ◽  
Coagulation Pathway

Abstract The intrinsic tenase complex (FIXa-FVIIIa) of the intrinsic coagulation pathway and, to a lesser extent, thrombin-mediated activation of FXI, are necessary to amplify tissue factor (TF)-FVIIa–initiated thrombin generation. In this study, we determined the contribution of murine FIX and FXI to TF-dependent thrombin generation in vitro. We further investigated TF-dependent FIX activation in mice and the contribution of this pathway to hemostasis. Thrombin generation was decreased in FIX- but not in FXI-deficient mouse plasma. Furthermore, injection of TF increased levels of FIXa-antithrombin complexes in both wild-type and FXI−/− mice. Genetic studies were used to determine the effect of complete deficiencies of either FIX or FXI on the survival of mice expressing low levels of TF. Low-TF;FIX−/y male mice were born at the expected frequency, but none survived to wean. In contrast, low-TF;FXI−/− mice were generated at the expected frequency at wean and had a 6-month survival equivalent to that of low-TF mice. Surprisingly, a deficiency of FXI, but not FIX, exacerbated the size of blood pools in low-TF placentas and led to acute hemorrhage and death of some pregnant dams. Our data indicate that FIX, but not FXI, is essential for survival of low-TF mice after birth. This finding suggests that TF-FVIIa–mediated activation of FIX plays a critical role in murine hemostasis. In contrast, FXI deficiency, but not FIX deficiency, exacerbated blood pooling in low-TF placentas, indicating a tissue-specific requirement for FXI in the murine placenta under conditions of low TF.

scholarly journals Regulation of macrophage procoagulant responses by the tissue factor cytoplasmic domain in endotoxemia

Blood ◽  
2007 ◽  
Vol 109 (12) ◽  
pp. 5251-5259 ◽  
Author(s):  
Jasimuddin Ahamed ◽  
Frank Niessen ◽  
Toru Kurokawa ◽  
Young Kyung Lee ◽  
Gourab Bhattacharjee ◽  
...  
Keyword(s):  
Tissue Factor ◽  
Cytoplasmic Domain ◽  
Interferon Γ ◽  
Wild Type ◽  
Coagulation Activation ◽  
Proinflammatory Response ◽  
Protease Activated Receptors ◽  
Lps Challenge

Abstract Tissue factor (TF) is the primary initiator of coagulation, and the TF pathway mediates signaling through protease-activated receptors (PARs). In sepsis, TF is up-regulated as part of the proinflammatory response in lipopolysaccharide (LPS)–stimulated monocytes leading to systemic coagulation activation. Here we demonstrate that TF cytoplasmic domain–deleted (TFΔCT) mice show enhanced and prolonged systemic coagulation activation relative to wild-type upon LPS challenge. However, TFΔCT mice resolve inflammation earlier and are protected from lethality independent of changes in coagulation. Macrophages from LPS-challenged TFΔCT mice or LPS-stimulated, in vitro–differentiated bone marrow–derived macrophages show increased TF mRNA and functional activity relative to wild-type, identifying up-regulation of macrophage TF expression as a possible cause for the increase in coagulation of TFΔCT mice. Increased TF expression of TFΔCT macrophages does not require PAR2 and is specific for toll-like receptor, but not interferon γ receptor, signaling. The presence of the TF cytoplasmic domain suppresses ERK1/2 phosphorylation that is reversed by p38 inhibition leading to enhanced TF expression specifically in wild-type but not TFΔCT mice. The present study demonstrates a new role of the TF cytoplasmic domain in an autoregulatory pathway that controls LPS-induced TF expression in macrophages and procoagulant responses in endotoxemia.

Characterization of the Procoagulant Potential of Endothelial Colonies Forming Cells and Comparison with Two Cell Therapy Products: Relevance for Vascular Cell Therapy

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 5317-5317
Author(s):  
Wendy Cuccuini ◽  
Claire Tournois ◽  
Gael Poitevin ◽  
Bernard Pignon ◽  
Marie Antoinette Sevestre ◽  
...  
Keyword(s):  
Cell Therapy ◽  
Cord Blood ◽  
Tissue Factor ◽  
Thrombin Generation ◽  
Mononuclear Cells ◽  
Procoagulant Activity ◽  
Inflammatory Conditions ◽  
Cell Based Therapy ◽  
Cd34 Cells

Abstract Abstract 5317 Background: Endothelial colonies forming cells (ECFC) are good candidates for cell-based therapy in cardiovascular diseases. Concerns have been raised about the potential risks of ECFC-based cell therapy, in terms of thrombogenicity, particularly in inflammatory conditions. We published that cord blood ECFC (cb-ECFC) express Tissue Factor (TF) in response to TNFα. TF/FVIIa interaction did not modify in vitro the non-procoagulant properties of TNFα stimulated cb-ECFC [Cuccuini et al, JTH 2010]. We also reported, in a monocyte model, a co-expression of TF and metalloproteinase MMP-9, with possible consequences in terms of haemostasis and angiogenesis [Poitevin et al, JTH 2008]. Aim of the study: The objective of this study was: 1- to further evaluate the procoagulant potential of ECFC and the capacity to exhibit MMP activity; 2- to evaluate whether TF and MMP could be detected in two different cell therapy products (CTP). Methods: ECFC were obtained in culture after isolation of CD34+ cells from cord blood (as previously described). CTP were obtained from an ongoing clinical trial evaluating cell therapy to improve vascularization in chronic critical limb ischemia (NIH NCT00533104). Two different CTP were analyzed: 1- Peripheral blood mononuclear cells (PB-MNC) obtained by cytapheresis without any mobilization (n= 11); 2- Bone marrow mononuclear cells (BM-MNC) (n= 11). CTP are characterized in flow cytometry, in terms of cell composition, including CD34+ cells; CD34+/CD133+/VEGF-R2+ cells were considered as endothelial progenitor cells. Results: cb-ECFC can express procoagulant TF in response to TNFa (10 ng/mL). The expression of TF was compared with purified monocytes, CD34+ cells, mature endothelial cells (HUVEC), PB-MNC and BM-MNC. TF mRNA were detected in CD34+ cells. As microparticles (MPs) are strongly procoagulant, we evidenced that cb-ECFC produced CD146+/Annexin V+/TF+ MPs in response to TNFa. These MPs can trigger thrombin generation. The analysis of conditioned supernatants of cb-ECFC in response to TNFa indicated that the majority of TF is borne by MP, but a small amount of soluble TF is present, in the absence of MP. This soluble TF does not induce thrombin generation. Using Q-PCR, we showed that cb-ECFC expressed Tissue Factor Pathway Inhibitor (TFPI). This expression of TFPI mRNA was not increased by TNFa. The analysis of TNFa-conditioned cb-ECFC supernatants showed that the amount of TFPI protein is a 20 fold lower than plasmatic levels of free-TFPI. We next examined the presence of TF mRNA in both CTP. TF mRNA were estimated to a 2 fold higher in BM-MNC than in cb-ECFC (baseline). On the contrary, TF mRNA were a 5 fold lower in PB-MNC than in cb-ECFC. In both CTP, the alternatively spliced form (as-TF) represented a very minor part of TF. The percentage of CD14+ cells was respectively 17 % and 26 % in BM-MNC and in PB-MNC. The amount of TF mRNA in PB-MNC was not statistically different from unstimulated purified monocytes. On the contrary, TF mRNA were a 7 fold higher in BM-MNC in comparison with PB-MNC or monocytes. The percentage of CD34+ cells was low: 2.6 % in BM-MNC and 0.1 % in PB-MNC. TF mRNA were higher in CD34+ cells than in BM-MNC (3 fold) and in PB-MNC (27 fold). As we previously reported a co-regulation of TF with MMP, we first analyzed the expression of MMP-2 and MMP-9 in cb-ECFC. Using Q-PCR, we found that cb-ECFC strongly expressed MMP-2 whereas the expression of MMP-9 was very weak. This profile was comparable in cb-ECFC and HUVEC. Using zymography, we confirmed the expression of MMP-2 but not MMP-9, in response to TNFa. We next analyzed CTP. In BM-MNC, MMP-2 and MMP-9 were expressed at a very high level. In PB-MNC, MMP-2 and MMP-9 could be detected but at a very low level. Conclusion: Taken together, our results show that ECFC, BM-MNC and PB-MNC can express TF. The procoagulant activity of ECFC is also the consequence of MP formation in inflammatory conditions. The profile of MMP in CTP remains to be clarified in terms of cell origin and possible influence on thrombotic and angiogenic properties. In our hands, the inhibition of TF can limit procoagulant activity with no effect on proangiogenic properties in vitro. In terms of clinical relevance, one single patient treated with PB-MNC, developed a deep vein thrombosis at the site of injection. It is therefore mandatory to evaluate the procoagulant potential of CTP in the context of vascular repair. Disclosures: No relevant conflicts of interest to declare.

Blockade of GPIIb/IIIa by eptifibatide and tirofiban does not alter tissue factor induced thrombin generation in human endotoxemia

2003 ◽  
Vol 90 (12) ◽  
pp. 1054-1060 ◽  
Author(s):  
Ulla Derhaschnig ◽  
Christine Pachinger ◽  
Ingrid Schweeger-Exeli ◽  
Claudia Marsik ◽  
Bernd Jilma
Keyword(s):  
Tissue Factor ◽  
Thrombin Generation ◽  
Animal Experiments ◽  
Low Grade ◽  
Coagulation Activation ◽  
Double Blind ◽  
Human Endotoxemia ◽  
Parallel Group ◽  
Factor Α

SummaryActivated platelets facilitate thrombin generation by providing a catalytic surface on which coagulation activation occurs. The glycoprotein (GP) IIb/IIIa receptor might play a major role in this process as shown by in vitro and animal experiments. However, it is controversial whether the GPIIb/IIIa receptor facilitates tissue factor-induced thrombin generation in humans as well. We therefore investigated whether two clinically used GPIIb/IIIa antagonists (tirofiban and eptifibatide) may blunt TF-induced coagulation in humans.Thirty male volunteers received 2 ng/kg endotoxin and standard doses of eptifibatide, tirofiban or placebo over 5 hours in a randomized, double-blind, placebo-controlled, double-dummy parallel-group trial. Markers of thrombin generation (prothrom-bin fragment 1+2, thrombin-antithrombin complexes), fibrinoly-sis (D-dimer, plasmin-antiplasmin complexes) as well as inflammatory markers (interleukin-6, tumor necrosis factor-α) were measured by enzyme linked immunoasssays, TF-mRNA expression was quantified by RT-PCR. Neither eptifibatide nor tirofiban influenced LPS-induced coagulation activation or fibrinolytic activity. Additionally, the increase of TNF-α and IL-6 was similar in all groups.In conclusion, GPIIb/IIIa blockade with eptifibatide or tirofiban did not influence TF-induced coagulation activation in human low grade endotoxemia.

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