Tissue Factor Mediated Activation of Prothrombin Complex Concentrates (PCCs) Is Differently Inhibited by Dabigatran, Rivaroxaban and Apixaban. Potential Clinical Implications

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 3410-3410
Author(s):  
Jawed Fareed ◽  
Nasir Sadeghi ◽  
Daniel Kahn ◽  
Josephine Cunanan ◽  
Kimberly Bartosiak ◽  
...  
Keyword(s):  
Tissue Factor ◽  
Thrombin Generation ◽  
Conflicts Of Interest ◽  
Ex Vivo ◽  
Oral Anticoagulants ◽  
Protein G ◽  
Prothrombin Complex Concentrates ◽  
Sds Page ◽  
Activation Products ◽  
Tissue Factors

Abstract Abstract 3410 The newly developed oral anticoagulants represent specific antithrombin (dabigatran, Boehinger, Ingelheim) and antifactor Xa agents (rivaroxaban, Bayer Health Care/Jhonsen) and apixaban, Bristol Myers Squibb/Pfizer). Prothrombin Complex Concentrates (PCCs) such as profilnine® and beriplex® are reported to partially neutralize the anticoagulant effects of these agents. Since these PCCs are capable of generating factor Xa and thrombin, the newer anticoagulants may be neutralized differentially by the proteases generated by PCCs. Coagulation and thrombosis are activated substantially by tissue factor in vivo. The purpose of this study is to compare the inhibitory effects of dabigatran, rivaroxaban and apixaban in tissue factor mediated thrombin generation using profilnine, by utilizing various approaches to characterize activation products including thrombin. Materials and Methods Dabigatran, rivaroxaban, and apixaban were synthesized and/or commercially obtained. Profilnine (Grifols Biologicals Inc.) was also commercially obtained. One commercial lot of a recombinant thrombin preparation Recothrom® was obtained from ZymoGenetics Inc for the development of polyclonal antibodies. To generate specific antisera, individual groups of rabbits (n = 3–6) were challenged repeatedly with human recombinant thrombin, over a 9-month period. At the end of this time the antisera from each rabbit was collected and pooled. Immunglobulin (IgGs) were isolated using a protein G column (HiTrap Protein G HP – GE Helathcare Bio-Science Crop). Buffered profilnine (2.5 u/ml) was activated with routinely used tissue factor reagents by adding commercially available PT reagents such as thromboplastin C, neoplastinPlus, and simplastin at a 1:4 ratio and incubated for 30 minutes. The activation of profilnine was measured by using thrombin generation utilizing a fluorogenic substrate method (Technoclone) and the protease generation profile was evaluated using mass spectrometry method (SELDI), SDS-PAGE analysis and immunoblotting using a specific antithrombin (Recothrombin) antibody to profile the activation products. Similar studies were carried out in profilnine supplemented with graded amount of various oral anticoagulants in the concentration range of 0–2.5ug/ml. Results All tissue factors produce varying degrees of time dependent activation of profilnine as measured by consumption of prothrombin peak at 71 KDa and generation of thrombin peaks at 3l–37 KDa as observed in the SELDI. Varying amounts of prothrombin generation at 52 KDa was also evident. Distinct immunoblot for thrombin in western blotting analysis was consistent with SDS-PAGE and SELDI analysis showing the generation of thrombin. The anti-Xa agents blocked the generation of thrombin whereas dabigatran failed to produce this effect. This phenomenon was also observed in all three methods used to study generation of the thrombin when using other PCCs such as octaplex and thromboplex activated by various tissue factors. In the fluorometric thrombin generation assays both apixaban and rivaroxaban produced a relatively stronger inhibition of thrombin generation (IC50= 20–200ng/ml) wheras > 500ng/ml for dabigatran in various PCCs. Conclusion These results suggest that in contrast to dabigatran both rivaroxaban and apixaban produce a much stronger inhibition of tissue factor mediated generation of the thrombin in PCCs. Inhibition of the functional generation of thrombin was weaker with dabigatran in contrast to apixiban and rivaroxiban. The observed ex-vivo neutralization profile of these agents by PCCs may be due to the differential interactions with the protease generated during their activation. These differences along with the compositional variations in the PCCs should be taken into account while considering prothrombin complexes for the neutralization of new oral anticoagulants. Disclosures: No relevant conflicts of interest to declare.

Oral Anti-Factor Xa and Factor IIa Agent Mediated Inhibition Of Tissue-Factor Mediated Generation Of Thrombin In Prothrombin Complex Concentrates

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 4810-4810
Author(s):  
Daneyal Syed ◽  
Debra Hoppensteadt ◽  
Daniel Kahn ◽  
Job Harenberg ◽  
Jawed Fareed
Keyword(s):  
Tissue Factor ◽  
Oral Anticoagulant ◽  
Thrombin Generation ◽  
Conflicts Of Interest ◽  
Factor Xa ◽  
Onset Time ◽  
Thrombin Inhibitors ◽  
Bleeding Complications ◽  
Prothrombin Complex Concentrates ◽  
Inhibition Of Thrombin

Introduction Several oral anti-factor IIa and factor Xa agents have recently been developed. These include the thrombin inhibitors Ximelagatran/Melagatran (M) and Dabigatran Etexilate/Dabigatran (D), which require endogenous conversion to the active agents D and M. The factor Xa inhibitors, Rivaroxaban (R) and Apixaban (A), are anti-Xa agents that do not require any endogenous activation. Ximelagatran was withdrawn from the market due to adverse reactions. Dabigatran, Rivaroxaban, and Apixaban are approved for various clinical indications. Antagonism of the anticoagulant effect may be required in bleeding complications. Contradictory results were reported for the efficacy of various prothrombin complex concentrates (PCCs) with these new oral anticoagulants (NOACs). The purpose of this study was to determine the differences in the thrombin generation inhibitory profiles of the newer oral anticoagulant agents. Methods Commercially available PCCs namely Octaplex and Beriplex, were used as a source of Factors II, VII, IX and X. To investigate the effect of each of these agents, a working solution of 1U/ml of both PCCs were supplemented in a graded concentration of 0-1250ng/ml with M, D, R and A. Thrombin generation studies were carried out using a thromboplastin activator (RC High, Technoclone Vienna, Austria). Total thrombin generated was measured in terms of nM’s. The IC-50 for each agent was calculated individually. The time course of thrombin generation was also measured following the kinetic profiles and AUC. Results Dabigatran and Melagatran produced relatively weaker inhibition of thrombin generation with the IC-50 values ranging from 410-110ng/ml in Beriplex and 350-1120ng/ml in Octaplex. Both Rivaroxaban and Apixaban produced strong inhibition of thrombin generation, with the IC-50 ranging from 58-62ng/ml in Octaplex; whereas, in Beriplex these values ranged from 48-50ng/ml. The onset time for thrombin generation and total thrombin formation was concentration dependent. The kinetics of thrombin generation with A and R were distinct from D and M. At concentrations below 310ng/ml the total amount of thrombin generated was comparable to the control; however, its formation was delayed. In both systems, D exhibited the weakest thrombin generation inhibitory potential. While the onset time of thrombin generation was delayed at concentrations below 310ng/ml the levels were comparable to or higher than the control. Discussion This data suggests that PCC’s such as Octaplex and Beriplex can be used to generate thrombin and it’s inhibition by new oral anticoagulant drugs. Octaplex generates much higher amount of thrombin than Beriplex at equivalent units. These results also show that in comparison to the oral anti-Xa agents, the oral anti-IIa agents are relatively weaker inhibitors of thrombin generation. These studies also suggest that the differential inhibition of the generation of thrombin through tissue factor by the anti-Xa and IIa agents may contribute to the potential neutralization profile of PCC’s for these drugs. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Procoagulant activity in patients with combined type 2 diabetes and coronary artery disease: No effects of long-term exercise training

2017 ◽  
Vol 14 (2) ◽  
pp. 144-151 ◽  
Author(s):  
Vibeke Bratseth ◽  
Rune Byrkjeland ◽  
Ida U Njerve ◽  
Svein Solheim ◽  
Harald Arnesen ◽  
...  
Keyword(s):  
Type 2 Diabetes ◽  
Exercise Training ◽  
Tissue Factor ◽  
Thrombin Generation ◽  
Tissue Factor Pathway Inhibitor ◽  
Ex Vivo ◽  
Tissue Factor Pathway ◽  
Artery Disease ◽  
Total Tissue

We investigated the effects of 12-month exercise training on hypercoagulability in patients with combined type 2 diabetes mellitus and coronary artery disease. Associations with severity of disease were further explored. Patients ( n = 131) were randomized to exercise training or a control group. Blood was collected at inclusion and after 12 months. Tissue factor, free and total tissue factor pathway inhibitor, prothrombin fragment 1 + 2 (F1 + 2) and D-dimer were determined by enzyme-linked immunosorbent assay and ex vivo thrombin generation by the calibrated automated thrombogram assay. Tissue factor and ex vivo thrombin generation increased from baseline to 12 months ( p < 0.01, all), with no significant differences in changes between groups. At baseline, free and total tissue factor pathway inhibitor significantly correlated to fasting glucose ( p < 0.01, both) and HbA1c ( p < 0.05, both). In patients with albuminuria ( n = 34), these correlations were strengthened, and elevated levels of D-dimer, free and total tissue factor pathway inhibitor ( p < 0.01, all) and decreased ex vivo thrombin generation ( p < 0.05, all) were observed. These results show no effects of exercise training on markers of hypercoagulability in our population with combined type 2 diabetes mellitus and coronary artery disease. The association between poor glycaemic control and tissue factor pathway inhibitor might indicate increased endothelial activation. More pronounced hypercoagulability and increased tissue factor pathway inhibitor were demonstrated in patients with albuminuria.

Impact of long-term testosterone treatment on plasma levels of free TFPI and TF-induced thrombin generation ex vivo in elderly men with low testosterone levels

2009 ◽  
Vol 102 (11) ◽  
pp. 945-950 ◽  
Author(s):  
Ingvild Agledahl ◽  
Johan Svartberg ◽  
John-Bjarne Hansen ◽  
Ellen Brodin
Keyword(s):  
Tissue Factor ◽  
Plasma Levels ◽  
Thrombin Generation ◽  
Ex Vivo ◽  
Favorable Effect ◽  
Elderly Men ◽  
Testosterone Treatment ◽  
Testosterone Levels ◽  
Low Testosterone ◽  
One Year

SummaryMen have a higher incidence of cardiovascular disease (CVD) than women of similar age, and it has been suggested that testosterone may influence the development of CVD. Recently, we demonstrated that elderly men with low testosterone levels had lower plasma levels of free tissue factor pathway inhibitor (TFPI) Ag associated with shortened tissue factor (TF)-induced coagulation initiation in a population based case-control study. Our hypothesis was that one year of testosterone treatment to physiological levels in elderly men would increase the levels of free TFPI Ag in plasma and have a favorable effect on TF-induced coagulation. Twenty-six men with low testosterone levels (≤11.0 nM) were randomly assigned to treatment with intramuscular testosterone depot injections (testosterone undecanoate 1,000 mg) or placebo in a double-blinded study. Each participant received a total of five injections, at baseline, 6, 16, 28 and 40 weeks, and TF-induced thrombin generation ex vivo and plasma free TFPI Ag were measured after one year. At the end of the study total and free testosterone levels were significantly higher in the testosterone treated group (14.9 ± 4.5 nM vs. 8.1 ± 2.4 nM; p<0.001, and 363.3 ± 106.6 pM vs. 187.3 ± 63.2 pM; p<0.001, respectively). Testosterone treatment for one year did neither cause significant changes in TF-induced thrombin generation ex vivo nor changes in plasma levels of free TFPI Ag. In conclusion, normalising testosterone levels by testosterone treatment for 12 months in elderly men did not affect TF-induced coagulation or plasma TFPI levels. The potential antithrombotic role of testosterone therapy remains to be elucidated.

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

2009 ◽  
Vol 101 (03) ◽  
pp. 471-477 ◽  
Author(s):  
Ingvild Agledahl ◽  
Johan Svartberg ◽  
Bjarne Hansen ◽  
Ellen Brodin
Keyword(s):  
Tissue Factor ◽  
Thrombin Generation ◽  
Tissue Factor Pathway Inhibitor ◽  
Ex Vivo ◽  
Elderly Men ◽  
Lower Plasma ◽  
Testosterone Levels ◽  
Initiation Phase ◽  
Tissue Factor Pathway ◽  
Low Testosterone

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

Trace FVIII Augments Rfviia Induced Thrombin Generation and Improves Hemostasis in Hemophilia A Patients with Inhibitors: A clinical Cohort Study

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 40-40
Author(s):  
Tami Livnat ◽  
Uri Martinowitz ◽  
Shirley Azar-Avivi ◽  
Ariela Zivelin ◽  
Gili Kenet
Keyword(s):  
Thrombin Generation ◽  
Hemophilia A ◽  
Conflicts Of Interest ◽  
Ex Vivo ◽  
Therapy Response ◽  
Response To Therapy ◽  
Individual Therapy ◽  
Severe Bleeding ◽  
Inhibitor Level

Abstract Abstract 40 Treatment of Hemophilia A patients with inhibitors is challenging, as correlation between inhibitor level and hemostatic response to therapy may be limited. Thrombin generation (TG) assays may be used to monitor hemostasis and/or predict patients' response to various bypass agents. Since combination of excess FVIII and bypassing agents may potentiate improved TG in inhibitor plasma tested in-vitro, we aimed to define the therapeutic feasibility of co-administration of rFVIIa and FVIII in hemophilia A patients with inhibitors. Patients and Methods: Following consent, blood was sampled from 15 hemophilia patients (age: 0.5–46y) with inhibitor (0.5–668 BU). Platelet poor plasma (PPP) was prepared, spiked and incubated with excess FVIII. Ex-vivo kinetics of FVIII neutralization over time was evaluated by sequential measurements of residual FVIII activity. We then used recalcification induced-TG (performed in PPP supplemented with 4μM phospholipids), to measure the ex-vivo response to increasing concentrations of FVIII (0–200%) and rFVIIa (0–6.8μg/ml), alone or in combination. Based upon these ex-vivo studies, an individually tailored therapeutic regimen of concomitant bolus doses of rFVIIa and FVIII was applied to nine hemophilia patients with inhibitors. Results: FVIII ex- vivo measurements post incubation detected either rapid or slow neutralization- not correlating with inhibitor level. Flat baseline TG curves were recorded for all inhibitor patients, with variable responses to FVIII and/or rFVIIa. Combined spiking with FVIII and rFVIIa dramatically increased rFVIIa induced ETP (762.7 ±305.7 as compared to 339.3±179.9 nM/min with rFVIIa only) and peak height (48.7±23.6 vs 23.7±16.6) in all patients' plasma samples. Based upon individual ex vivo assays, concomitant bolus doses of rFVIIa (120–200 mcg/kg) and FVIII (50–100 U/Kg), were applied to 9 patients, for a total of 333 episodes during study period (February 2010-Septemeber 2012). Patients during immune tolerance received rFVIIa prophylaxis with combined rFVIIa/FVIII dosing applied 3 times weekly. For most mild- moderate joint bleeds hemostasis was defined as satisfactory following a single combined dose. Severe bleeding episodes or target joint bleeds responded to 2–8 (median:3) combined doses, applied every 12 hours. During study period the median number of spontaneous joint bleeds decreased from 4 to 1 per month. Neither thrombosis nor any other complications evolved. Conclusions: Prediction of individual therapy response may be achieved by pre-analytical studies, assessing FVIII neutralization kinetics as well as ex-vivo TG responses to combined bypass/FVIII therapy. Such studies enabled treatment of inhibitor patients according to individually tailored regimens. We confirmed for the first time that the in- vitro advantage of combining FVIII and rFVIIa, indeed accounts for improved hemostasis and may safely be applied to inhibitor patients. Disclosures: No relevant conflicts of interest to declare.

The Transfer Of Dabigatran and Rivaroxaban Across a Dually Perfused Isolated Human Placental Cotyledon – Implications For Therapy In Pregnancy

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1142-1142 ◽  
Author(s):  
Priya Bapat ◽  
Reuven Kedar ◽  
Nir Melamed ◽  
Jeremy Matlow ◽  
Angelika Lubetsky ◽  
...  
Keyword(s):  
Placental Transfer ◽  
Conflicts Of Interest ◽  
Ex Vivo ◽  
Oral Anticoagulants ◽  
Elective Caesarean Section ◽  
Reproductive Age ◽  
Clotting Factors ◽  
Fetal Circulation ◽  
Normal Ranges ◽  
Risk Pregnancy

Abstract Background Anticoagulant therapy is often required in cases of high-risk pregnancy for the prophylaxis of venous thromboembolism following surgery, atrial fibrillation, and congestive heart failure, and for the prevention of pregnancy loss in thrombophilic women. During pregnancy, the concentrations of many blood-clotting factors rise, thereby increasing the need for anticoagulants that are safe to use throughout gestation. Dabigatran (Pradaxa®) and rivaroxaban (Xarelto®) are newer generation oral anticoagulants that are increasingly being prescribed to women of reproductive age for the treatment of thromboembolic disorders. Dabigatran acts by directly inhibiting thrombin, and rivaroxaban acts as a direct factor Xa inhibitor. However, the information regarding fetal safety and placental transfer of these drugs is currently lacking. If there is limited transfer of either drug across the placenta, then it may not increase the risk of bleeding in the fetus. The objective of this study was to determine the transplacental kinetics of dabigatran and rivaroxaban. Methods Placentae were obtained with informed consent after elective caesarean section of healthy term pregnancies in Toronto, Ontario. The transplacental transfer of dabigatran and rivaroxaban were separately assessed using ex vivo dual perfusion of an isolated human placental cotyledon. Dabigatran, at a concentration of 35 ng/ml, was added to the maternal circulation at the start of the experimental phase. Maternal and fetal samples were taken throughout the pre-experimental (1 h) and experimental (3 h) phases for measurement of dabigatran and markers of placental viability. Separate placenta perfusions with rivaroxaban were conducted at an initial maternal concentration of 250 ng/ml. The perfused drug was measured in maternal and fetal samples using liquid chromatography-mass spectrometry (LC/MS). Results There was slow transfer of dabigatran from the maternal to fetal circulation. The fetal-to-maternal (F:M) concentration ratio was 0.33 ± 0.13 after 3 hours (n=3). In contrast, the transfer of rivaroxaban from maternal to fetal circulation was much more rapid, as characterized by a F:M ratio of 0.72 ± 0.12 at 3 hour (n=4), suggesting rapid equilibrium between maternal and fetal circulations. Placental viability markers for all perfusions were within normal ranges. Conclusions This is the first direct evidence of the transfer of dabigatran and rivaroxaban across the human placenta from the mother to fetus. It suggests less fetal exposure to dabigatran. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Thrombin Generation Assay and Its Application in the Clinical Laboratory

2016 ◽  
Vol 62 (5) ◽  
pp. 699-707 ◽  
Author(s):  
Armando Tripodi
Keyword(s):  
Liver Disease ◽  
Thrombin Generation ◽  
Clinical Laboratory ◽  
Ex Vivo ◽  
Myeloproliferative Neoplasms ◽  
Oral Anticoagulants ◽  
Relative Strength ◽  
Nonalcoholic Fatty Liver ◽  
Thrombin Generation Assay

Abstract BACKGROUND A gap exists between in vivo and ex vivo coagulation when investigated by use of the coagulation tests prothrombin time (PT) and activated partial thromboplastin time (APTT). The thrombin generation assay (TGA) has been developed to fill this gap. CONTENT TGA evaluates thrombin generation (resulting from the action of the procoagulant driver) and decay (resulting from the action of the anticoagulant driver), thus assessing the balance between the two. Coagulation of the test plasma (platelet poor or platelet rich) is activated by small amounts of tissue factor and phospholipids, and the reaction of thrombin generation is continuously monitored by means of a thrombin-specific fluorogenic substrate. Among the parameters derived from the thrombin-generation curve, the most important is the endogenous thrombin potential, defined as the net amount of thrombin that test plasmas can generate on the basis of the relative strength of the pro- and anticoagulant drivers. TGA is therefore the candidate assay to investigate hypo- or hypercoagulability. SUMMARY From my analysis of the literature, I draw the following conclusions. There is strong evidence that TGA is helpful to elucidate coagulation mechanisms in various clinical conditions that until recently were poorly understood (chronic liver disease; diabetes; inflammatory bowel disease, myeloproliferative neoplasms, nonalcoholic fatty liver disease). TGA is a promising laboratory tool for investigating hemorrhagic coagulopathies and monitoring replacement therapy in hemophiliacs, predicting the risk of recurrent venous thromboembolism after a first event, and monitoring patients on parenteral or oral anticoagulants. These applications require clinical trials in which TGA results are combined with specific clinical end points.

Tissue Factor-Bearing Microparticles Derived from Tumor Cells: Impact on Coagulation Activation.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1757-1757 ◽  
Author(s):  
Monica Davila ◽  
Ali Amirkhosravi ◽  
Enriqueta Coll ◽  
Robles D. Liza ◽  
John Francis
Keyword(s):  
Flow Cytometry ◽  
Tissue Factor ◽  
Tumor Cells ◽  
Whole Blood ◽  
Thrombin Generation ◽  
Ex Vivo ◽  
Annexin V ◽  
Cancer Tissue ◽  
Cancer Associated Thrombosis ◽  
Mcf 7

Abstract Thromboembolic disease is a frequent complication in cancer. Tissue factor (TF), shown to be involved in tumor growth and metastasis, is also considered to play a central role in the pathogenesis of cancer-associated thrombosis. Circulating TF-bearing microparticles (TF+ MPs) have been found in the plasma of patients with different malignancies and are thought to contribute to their hypercoagulable state. Although numerous studies have focused on TF+ MPs derived from blood cells, there is no information available on the pathological relevance of MPs originating from tumor cells. We conducted a study to detect, enumerate and characterize the procoagulant activity (PCA) of MPs released specifically from tumor cells. MPs from high (MDA-231) and low (MCF-7) TF-expressing human breast carcinoma cells were generated ex vivo in whole blood or in buffer under stirring conditions for 45 minutes. The numbers (MPs/ml) of total and TF-expressing tumor-derived particles (TMPs) in cell-free plasmas were measured by flow cytometry using FITC-labeled annexin V and a PE-labeled monoclonal anti-TF antibody respectively. The PCA of TMPs was measured by a one stage clotting assay and a highly sensitive fluorogenic thrombin generation assay. In order to evaluate the PCA of circulating TMPs, we injected 2x106 TF+ MPs derived from MDA-231 cells into mice via the tail vein. Human TF antigen and activity were measured in cell-free mouse plasmas at various intervals (5–420 min) after injections by ELISA and clotting assay, respectively. MPs less than 1μm in diameter were released from tumor cells in both buffer and whole blood by stirring. TMPs positive for TF consisted of approximately 40% of the annexin V+ MPs, and such particles derived from as low as 1x105 MDA-231 cells could be enumerated reliably (2.5x104 MPs/105 cells). By ultracentrifugation of cell-free plasmas, we confirmed that TF antigen was associated entirely with the MP fraction. TMPs derived from as few as 450 MDA-231 cells shortened plasma recalcification times from 525 ± 114 to 265 ± 148 (P<0.01), and significantly accelerated thrombin generation as evidenced by a 3 fold shortening in lag time, and a 2 fold increase in the rate of thrombin formation and thrombin concentration. No PCA was detected with MCF-7-derived TMPs. The PCA of TMPs was inhibited completely by a blocking anti-TF monoclonal antibody, but not by saturating concentrations of annexin V (an inhibitor of phospholipid PCA) or corn trypsin inhibitor (an inhibitor of the intrinsic pathway). Five minutes following the injection of TMPs into mice, appreciable levels of human TF antigen and activity were detected in cell-free plasmas. Both TF activity and antigen decreased over time and were detectable no longer than 30 minutes after injection, indicating a rapid clearance of circulating TMPs in vivo. In contrast, when TMPs were incubated in human whole blood ex vivo for various intervals, TF activity remained unchanged in cell-free plasmas for at least 5 hrs and TF antigen was not detected by flow cytometry on any blood corpuscles, including platelets, at all intervals. However, when whole blood containing TMPs was clotted by recalcification, no TF activity could be detected in the serum, indicating the incorporation of TMPs in formed clots. In summary, MPs bearing active TF are released readily from tumor cells and can significantly activate coagulation even at very low concentrations. These results provide new insights towards the potential contribution of TMPs to the pathogenesis of cancer-associated thrombosis.

Impact Of Thrombin Generation, Tissue Factor Activity and Thrombomodulin Activity On The Positivity Of Assisted Reproductive Technique In Infertile Women

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3630-3630
Author(s):  
Emmanuelle Mathieu d'Argent ◽  
Patrick Van Dreden ◽  
Marjorie Comtet ◽  
Vassiliki Galea ◽  
Hela Ketatni ◽  
...  
Keyword(s):  
Tissue Factor ◽  
Thrombin Generation ◽  
Conflicts Of Interest ◽  
Factor Xa ◽  
Hormone Treatment ◽  
Control Group ◽  
Significant Finding ◽  
Assisted Reproductive Technique ◽  
Infertile Women ◽  
Reproductive Techniques

Abstract Introduction Women undergoing assisted reproductive techniques (ART) are given gonadotrophins to promote the development of multiple follicles within their ovaries. This treatment may be associated with a risk of ovarian hyperstimulation syndrome and venous or arterial thrombosis. The association of clinical and biological criteria of hypercoagulability might contribute to the identification of patients at risk and probably could predict success of ART. The aim of this study was to evaluate thrombin generation, thrombomodulin activity, tissue factor (TF) activity, and procoagulant phospholipids in selected women undergoing ART. We also assessed the potential correlation between the levels of the studied biomarkers and the outcome of the ART. Material and Method A cohort of 27 infertile women eligible for ART and 30 healthy age matched women was studied. All patients were Caucasian aged 33.75 ± 3.52 years and weight 61.08 ± 8.10 kg. Women included in the study did not present any personal or family history of VTE or known thrombophilia. They did not present any signs of hemorrhagic syndrome and did not suffer from any known autoimmune disease. Blood samples were taken under fasting conditions at the following time-points: at the inclusion (T0), between the 5th and 8th day of ovarian stimulation with gonadotrophines (T1) and at the day of HCG administration (T2). Thrombin generation (TG) in plasma was assessed using the Calibrated Automated Thrombogram assay (using, PPP-Reagent-5pM TF from Diagnostica Stago, France), Plasma levels of thrombomodulin activity (TMa), and TF activity (TFa) were measured by home-made tests, Procoagulant phospholipids (PPL) dependent clotting time was measured using a factor Xa-based assay (STA-R Procoag-PPL, Diagnostica Stago, France). Results The endogenous thrombin potential (ETP), PPL, TMa and TFa were significantly higher in the ART-T0 group as compared to the control group. At ART-T2 a significant increase of TG was observed as compared to ART-T0. At ART-T0 44.5%, 44.4 % and 33.3 % of women had ETP, TFa and TMa higher than the Upper Normal Limit respectively (UNL = mean+2 S.D.). Among with negative ART 89% and 91.7% showed TMa and TFa levels > UNL at ART-T0. At T1 50% of women had a least one parameter of TG higher than the UNL. At ART T2 65.2 % of women had TG > UNL. At the same time, 87.5% and 83.4% of women with negative ART had levels of TMa and TFa > UNL. Conclusion This study analyzed the profile of thrombin generation in infertile women eligible for ART and investigated the influence of hormone treatment with gonadotropins and HCG on TG and levels of TMa and TFa. Hypercoagulability, in terms of increased ETP is present in 46% of infertile women eligible for ART. These women remain in a hypercoagulable state throughout the entire period of hormone treatment. The most significant finding of this study was that 33% of patients show a value superior to the UNL for thrombomodulin and 45% for tissue factor. Interestingly 89%of women with negative ART had TMa higher than the UNL. Respectively 91% of women with negative ART had TFa levels higher than the UNL Disclosures: No relevant conflicts of interest to declare.

Protein S As a Potential Treatment for FIX-Derived Deep Vein Thrombosis

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 2277-2277
Author(s):  
Vijaya Satish Sekhar Pilli ◽  
Willium Plautz ◽  
Rinku Majumder ◽  
Paolo Simioni
Keyword(s):  
Deep Vein Thrombosis ◽  
Thrombin Generation ◽  
Conflicts Of Interest ◽  
Ex Vivo ◽  
Protein S ◽  
Factor Ix ◽  
Wild Type ◽  
Vein Thrombosis ◽  
Deep Vein

Abstract Background: Every year, 0.1-0.2% of the USA population experiences deep vein thrombosis (DVT). Two causes of DVT are increased Factor IX (FIX) levels and hyperactivating mutations in FIX (FIX Padua variant- R338L and Malmo variant T148A). In principle, inhibition of activated FIX (FIXa) should alleviate DVT. Previous in vitro studies demonstrated that the anticoagulant Protein S (PS) inhibits the intrinsic pathway mediated by wild type FIXa, making PS an attractive candidate to treat DVT. Aims: To establish Protein S as a remedy for FIX-mediated DVT/Padua/Malmo Methods: Anisotropy, clotting assays, thrombin generation assays, co-localization, co-immunoprecipitation, and bleeding assays. Results: We further explored the physiological relevance of the PS-FIXa interaction and PS-mediated inhibition of FIXa by ex vivo (co-immunoprecipitation) and in vivo (co-localization) studies. Because PS can inhibit FIXa in vivo, we used competitive, direct anisotropy assays and co-immunoprecipitation assays to measure the efficiency PS and hyperactive FIXa (R338L) interaction. Interestingly, the results demonstrated that FIXa R338L has lost its affinity towards PS compared with wild type FIXa. The same finding was obtained by ex vivo thrombin generation assays and FXa generation assays supplemented with various concentrations of PS. Thus, to be inhibited, hyperactive FIX requires a greater amount of PS compared with wild type FIXa. We are further confirming this finding with mouse models. Conclusion: Addition of PS to plasma inhibits both wild type and R338L FIXa and extends clotting time. Previous studies showed that the addition of PS has no significant negative effects. Thus, we conclude that PS supplementation potentially constitutes a novel and effective treatment for FIX-mediated DVT. Disclosures No relevant conflicts of interest to declare.

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