Normal Human Platelet-poor Plasma Research Articles

Comparison of Procoagulant and Proinflammatory Effects of Nuclear, Mitochondrial, and Bacterial DNA

Background: Sepsis is a syndrome in which infection triggers a systemic inflammatory and pro-coagulant response, with a prevalence of up to 1 case per 1000 and a mortality rate of up to 40%. Cell-free DNA (cfDNA) is elevated in sepsis, and correlates with mortality. This DNA may come from nuclear, mitochondrial, or bacterial sources. Nuclear and mitochondrial DNA may come from activated neutrophils which release neutrophil extracellular traps (NETs). CpG motifs on bacterial and mitochondrial DNA can stimulate inflammatory responses via TLR9, which is present on neutrophils, monocytes, and recently shown to be expressed on platelets. cfDNA can activate coagulation via the intrinsic pathway. cfDNA may thus play an important pathogenic role in sepsis. This study elucidates the relative effects of nuclear, mitochondrial, and bacterial DNA on inflammatory and pro-coagulant pathways.Methods: Mitochondrial DNA concentrations were measured by PCR using plasma samples from septic patients. Nuclear and mitochondrial DNA were purified from human embryonic kidney 293 cells, and bacterial DNA was from E. coli. Neutrophils from healthy donors were cultured with purified bacterial, mitochondrial, or nuclear DNA at 15µg/mL for 20h. IL-6 levels in the supernatants were measured by ELISA, and neutrophil viability was measured by flow cytometry for annexin-V binding and propidium iodide exclusion. The three types of DNA were added to either citrated normal human platelet-poor plasma or platelet-rich plasma, and continuous thrombin generation was measured (Technothrombin, Vienna, Austria). Light transmission aggregometry was performed in citrated platelet-rich plasma with co-treatment of a sub-threshold concentration of ADP and varying concentrations of DNA. Markers of platelet activation were measured by flow cytometry for P-selectin and activated integrin αIIbβ3. All reagents contained less than 0.06EU/mL of LPS by limulus amoebocyte lysate assay.Results: Cell-free mitochondrial DNA was elevated in plasma from septic patients compared to healthy controls. Bacterial, but not mitochondrial or nuclear, DNA increased neutrophil IL-6 secretion. Both mitochondrial and bacterial DNA increased neutrophil viability at 20h. At concentrations found in the plasma of critically-ill patients, mitochondrial, nuclear, and bacterial DNA increased thrombin generation in both platelet-poor plasma and platelet-rich plasma to a similar degree, and this effect was abolished by corn-trypsin inhibitor and reduced in FXII-depleted plasma, indicating dependence on the intrinsic pathway of coagulation. Independently of coagulation, nuclear DNA at high concentrations, such as may be seen in the NET micro-environment, was capable of causing aggregation of ADP pre-stimulated platelets in citrated plasma, which was accompanied by activation of integrin αIIbβ3and surface expression of P-selectin. This effect also occurred with synthetic phosphodiester oligonucleotides, and was abolished by DNase pre-digestion.Conclusions: cfDNA of bacterial origin can stimulate neutrophil IL-6 release, while both mitochondrial and bacterial DNA prolonged neutrophil viability. All types of DNA can activate coagulation via the contact pathway. DNA at high concentrations may be able to directly stimulate platelets. Total plasma cfDNA and cell-free mitochondrial DNA specifically are elevated in sepsis. Thus, nuclear, mitochondrial, and bacterial DNA may play distinct roles in the pathogenesis of sepsis. DisclosuresBhagirath:Pfizer: Research Funding.

Is Thrombin Time Useful To Guide Peri-Procedural Management For Patients On Dabigatran Etexilate?: An In Vitro Validation Study

IntroductionPossibilities to monitor the intensity of dabigatran etexilate (DE) treatment may be valuable before urgent intervention. The Working Group on Perioperative Haemostasis proposed that the drug plasma concentration ([]) should be less or equal to 30ng/mL. However, plasma levels where it is safe to carry out an invasive procedure or surgery have not been confirmed prospectively. In addition, no biological test has been correlated with bleeding risk. Literature showed heterogeneous data regarding the arrest of DE depending on the renal function and the haemorrhagic risk. In addition, physicians request guidance for patients excluded from the clinical trials. Consequently, there is still a need for a rapid and widely available biological test.Some authors make proposals based on activated Partial Thromboplastin Time (aPTT), Hemoclot Thrombin Inhibitor® (HTI) or Thrombin Time (TT). TT displayed several advantages over aPTT. However, TT is affected by numerous analytic variables.Therefore, the objectives of the present study were:1. To determine the optimal [thrombin] with a variety of instruments and reagents2. To assess the repeatability of TT at optimized conditions3. To compare the sensitivity and linearity of TT at residual [dabigatran] (DA) with those of aPTT and HTI4. To validate the fibrinogen reagent (with heparin inhibitor) for TT experiments MethodsDA was spiked at increasing [] in pooled citrated normal human platelet-poor plasma (NPP). The following [DA] were prepared: 0, 5, 10, 20, 30, 40 and 50ng/ml.1. Optimal [thrombin] determination.The optimal [thrombin] was defined with 2 reagents: bovine thrombin (HemosIL® TT) and human thrombin (STA®-Thrombin) on 4 instruments: STA-R Evolution®, ACLTOP® , CS2000i® and KC10®.TT higher than Tmax is not informative for the physician. Thus the optimized [thrombin] was defined as the maximum [] giving, in a reproducible way, a TT at 50ng/ml lower than Tmax and a TT at 0ng/ml higher than the minimum TT. The Tmax was set arbitrarily at 120 sec.2. RepeatabilityThe repeatability of optimized TT was assessed by running aliquots of NPP spiked with all [DA] on 10 consecutive days3. TT (1.5 NIH/ml, STA®-Thrombin), aPTT with SynthasIL® and STA®-C.K.Prest, and HTI were also determined within 1 hour on STA-R® on replicates of all NPP solutions.4. STA®- Fibrinogen-5 was diluted to the optimized [thrombin] (1.5 NIH/ml) on STA-R® and used as a thrombin reagent. Results1. The thrombin origin is a more important variable in comparison to the type of coagulometer. At [DA] of 30ng/ml and [thrombin] of 1.5 NIH/ml, the STA®-TT ranges from 56 sec to 74 sec according to the instrument, whereas on a same instrument, the TT varied of minimum 43 sec, depending on the thrombin origin.The optimized [thrombin] are the following : a) STA-R®, STA®-Thrombin : 1.5 NIH/ml, b) STA-R®, HemosIL® TT : 3.8 NIH/ml, c) ACLTOP®, STA®-Thrombin 3.8 NIH/ml, d) ACLTOP®, HemosIL® TT 5.0 NIH/ml, e) KC10®, STA®-Thrombin 1.5 NIH/ml, f) KC10®, HemosIL® TT 5.0 NIH/ml, g) CS2000i®, STA®-Thrombin 1.4 NIH/ml, h) CS2000i®, HemosIL® TT 3.8 NIH/ml.Except for STA®-Thrombin on STA-R®, the [optimized] is not the one recommended by the manufacturer. Figure 1 shows the results of optimized TT according to thrombin origin and coagulometer.2. Repeatability experiments showed that variability increased with the [DA] (i.e. STA®-Thrombin (STA-R®): coefficient of variation: 8.0% and 28.9% for 0ng/ml and 50ng/ml, respectively) and that the variability depends on the coagulometer and the reagent.3. Comparison of sensitivity and linearity of TT, aPTT and HTI are presented in Table 1. APTT is not sensitive enough in low [DA], whatever the reagent, whereas HTI is not suitable in [DA] lower than 50ng/ml.3. TT performed with fibrinogen reagent shows longer times in comparison to thrombin reagent (>120 sec vs 109 sec at 30ng/ml DA). [Display omitted] Table 1[Dabigatran] (ng/ml)TT (sec)aPTT (sec, SynthasIL®)aPTT (sec, STA®-C.K.Prest)HTI (ng/ml)017.930.331.20.0522.031.233.10.01031.532.434.50.02059.334.938.67.23082.936.039.610.340100.938.042.821.350120.039.344.227.4 ConclusionsTT may be more informative than aPTT and HTI to provide with guidance to carry out an urgent procedure or surgery for patients receiving DE. However, TT is affected by a lot of analytic variables that should be understood by laboratories. Each laboratory should optimize its TT procedure according to its combination coagulometer-reagent. Disclosures:No relevant conflicts of interest to declare.

Impact of Apixaban On a Large Panel of Routine or More Specific Coagulation Assays.

AbstractAbstract 2275 Introduction:Apixaban is direct factor-Xa inhibitor that reached the market for the prevention of venous thromboembolism in patients undergoing major orthopaedic surgery. It is also being evaluated in the reduction of recurrent ischemic events when added to antiplatelet therapy after an acute coronary syndrome and in the prevention of stroke in patients with non-valvular atrial fibrillation.Thanks to its predictable pharmacokinetic profile, biological monitoring is not required. Nevertheless, evaluation of plasma drug concentration may be valuable in specific situations such as recurrent thrombosis, bleedings, before urgent surgery, in case of bridging and in case of at least two risk factors among the following ones: drug interactions with caution, moderate renal impairment and moderate hepatic impairment; Monitoring may also be useful in infants, pregnant women or in extreme body weights, although no relevant data on drug levels associated with approximate therapeutic and harmful ranges are currently available. Material and Methods:Apixaban was spiked at increasing concentrations (0, 5, 10, 20, 50, 100, 200 and 500 ng/mL) in pooled citrated normal human platelet poor plasma (PPP) to measure Prothrombin Time (PT) and dilute PT with different thromboplastin, Thrombin Generation Assay (TGA) with different inducers and activity on different anti-Xa chromogenic assays. Activated Partial Thromboplastin Time with different reagents, Thrombin Time (TT), Ecarin Clotting Time (ECT) and Reptilase Time (RT), measurement of fibrinogen (Clauss method and PT-derived method) and antithrombin (anti-IIa and anti-Xa based chromogenic assays) were also tested. We also evaluated the impact of apixaban on assays used for the determination of lupus anticoagulant such as the DRVV-T.. (Screen and Confirm) as well as the PTT-LA.. and the Staclot-LA.. . Results and Discussion:As mentioned in previous studies, PT showed a weak sensitivity towards apixaban in comparison with the plasma range obtained in short pharmacokinetic studies. Indeed, the concentration needed to double the clotting time was 154 ng/mL with the most sensitive reagent while the mean Cmax obtained in a short PK study after one oral intake of 5 mg apixaban (dose given in atrial fibrillation) was 96 ng/mL. Therefore, the sensitivity of PT is not strong enough to allow accurate quantitative measurement of the plasma drug concentration (Table 1). Activated Partial Thromboplastin Time presented a better sensitivity but showed a plateau after 100 ng/mL reflecting the uselessness of this test for the quantification of apixaban. Thrombin Time, ECT and RT were logically not affected while DRVV-T.. showed a sensitivity of 205 ng/mL (Screen), which is once again not enough sensitive.On the opposite, chromogenic anti-Xa assays seemed to be very sensitive (Figure 2 and Table 1). Nevertheless, the relation was not always linear and some methodologies needed to be adapted to ensure a broader range of application.TGA (Figure 1) may be useful to assess the pharmacodynamics effects of apixaban on the coagulation process. Nevertheless, the turn around time and the lack of standardisation are currently limitations that restrict the use of this method.In the case of the exploration of an haemorrhagic event, specific tests such as RT, fibrinogen (Clauss and PT-derived method (dFib)), TT and clotting factor activity may be used. Apixaban did not interfere with these tests. Antithrombin determination if also of importance and chromogenic anti-IIa based assays should be used in face of patients treated with apixaban to avoid misdiagnosis since an overvaluation of 12% by 100 ng/mL was shown using one chromogenic anti-Xa based assay. Conclusion:PT may not be used as screening test to assess the risk of bleedings. A more specific and sensitive assay such as chromogenic anti-Xa assays using calibrators should be used to correctly assess the concentration of apixaban. Determination of lupus anticoagulant using DRVV-T.. and PTT-LA.. or Staclot LA.. as well as the determination of antithrombin using factor-Xa based chromogenic assays, were influenced by apixaban. Finally, standardization of the time between the last intake of apixaban and the sampling is mandatory.Figures: [Display omitted] [Display omitted] [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Thrombin Generation Assay Is Superior to Traditional Coagulometric and Chromogenic Assays to Monitor New Oral Anticoagulants

AbstractAbstract 1169 INTRODUCTION:New oral anticoagulants (NOACs) have been recently approved by the European Medicine Agency (EMA) and the Food and Drug Administration (FDA) for several indications. New oral anticoagulants include anti-IIa agent (dabigatran etexilate) and anti-Xa agents (rivaroxaban, apixaban and edoxaban). NOACs do not require monitoring nor frequent dose adjustment. However, searching for the optimal dose in the individual patient may be useful in some situations. Recent studies have shown that aPTT, HTI and ECT could be used to monitor dabigatran whereas PT and anti-Xa chromogenic assays could be used to monitor anti-Xa agents, while standardizing the time between the last intake of rivaroxaban and the sampling is mandatory. These tests only measure the initiation phase of the coagulation cascade. Thrombin generation assay (TGA) which measures the entire thrombin generation process could be used to better discriminate the inhibitory profile of the NOACs in patients. OBJECTIVES:To study the impact of dabigatran and rivaroxaban in patients by thrombin generation assay compared to other traditional coagulometric and chromogenic assays. MATERIALS AND METHODS:Five patients under dabigatran and 5 patients under rivaroxaban for atrial fibrillation were included in this study. Blood samples were taken at different intervals: at Ctrough, 2h and 3h after drug administration. The following tests were performed at each timepoint Rivaroxaban:Prothrombin Time (PT) using the following reagents: Triniclot PT Excel S.. and Innovin.., Thrombin Generation Assay (TGA) using PPP-Reagent and PPP-Reagent High, Biophen Direct Factor-Xa Inhibitor.. (DiXaI). Dabigatran: Activated Partial Thromboplastin Time (aPTT) using CK-Prest.. and Synthasil.., Hemoclot Thrombin Inhibitor.. (HTI) and Thrombin Generation Assay (TGA) using PPP-Reagent and PPP-Reagent Low. All the tests were calibrated by spiking rivaroxaban or dabigatran at increasing concentrations in pooled citrated normal human platelet poor plasma (PPP).RESULTS AND DISCUSSIONS: In vitroRivaroxabanThe Peak and mVRI were the most sensitive CAT parameters with a high sensitivity (Peak IC50 was 3ng/mL with PPP-Reagent Low and PPP-Reagent and 14ng/mL with PPP-Reagent High; mVRI IC50 was 1ng/mL with PPP-Reagent Low and PPP-Reagent and 3ng/mL with PPP-Reagent High) and both reagent showed a low variability (CV<1.0%).The PPP-reagent Low does not allow resolving TGA profiles with different rivaroxaban concentrations. Consequently, PPP-Reagent and PPP-Reagent High were only tested ex vivo.The low sensitivity of the lag time explains the lack of sensitivity of PT, even with the more sensitive reagent (i.e. Triniclot PT Excel S..). DabigatranDabigatran mainly delayed the initiation phase with a strong dose-dependent increase of lag time and Tmax and a slight dose-dependent decrease of Cmax and ETP.The concentration in dabigatran needed to double the lag time was 66 ng/mL and 70ng/mL with the PPP-Reagent and the PPP-Reagent Low respectively. The PPP-reagent High is less sensitive in comparison to PPP-Reagent and PPP-Reagent Low. Ex-VivoSimilar concentrations of rivarovaban or dabigatran in different patients provided with Biophen DiXaI.. and HTI are associated with differences in TGA profile (Figure 1 & 2 showing the dispersion of the different measurement). Therefore, anti-Xa tests and dilute thrombin time are not the most accurate tests to use to propose cut-off associated with a bleeding or thrombosis risk. This illustrates the interest to have tests able to evaluate the entire thrombin generation process to propose cut-off in one or more parameters associated with a bleeding or thrombosis risk. CONCLUSIONThrombin generation assay could be superior to traditional coagulometric and chromogenic assays to monitor new oral anticoagulants in terms of prediction of bleeding or thrombosis risk. [Display omitted] [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Laboratory Assays for Monitoring Rivaroxaban: In House in-Vitro Analysis and Review of Literature

Abstract 4320 Introduction:Rivaroxaban an oral direct Xa inhibitor has recently been approved for VTE prophylaxis. It doesn't require monitoring but in special circumstances like bleeds a monitoring tool will be useful. Also, it is prudent to know its effect on various routine coagulation tests. Currently, there is no commercially available method for monitoring of Rivaroxaban. Aim: We conducted an In house- in-vitro analysis to (a)determine the effects of rivaroxaban locally on a range of commercially available coagulation assays, lupus anticoaugulant, factor VIII level, thrombin generation time and (b) measure it by two anti-Xa assays. Materials: Rivaroxaban (Xeralto) tablet, strength-10 mg. Plasma samples: Citrated normal human platelet poor plasma procured and freezed at −70 degree centigrade, Baxter sterile water. Method:The tablet was dissolved over 72 hours in a litre of sterile water to make 10 ug/ml of solution. Despite stirring a fine particulate compound remained which settled on standing. Samples were vortexed prior to each assay. This stock solution was used to spike citrated normal human platelet-poor plasma (PPP) at increasing concentrations of rivaroxaban from 0.1,0.2, 0.3, 0.4, upto 0.5 ug/ml to see the effect on the following tests and to determine rivaroxaban concentration by two 1.anti-Xa methods. All method were run using standard protocols taken from manufacturers' instructions.Anti-Xa assay:a) BIOPHEN DiXaI kit for 2-stage chromogenic assay of Direct Xa inhibitors; Ref 221030 b) Rotachrome using 1:2 dilution and further using 1:10 and 1:20 dilutions; catalogue Number-00612.2.PT & INR (STAGO neoplastin CI Plus MNPT-13.2 s, ISI-1.30 s).3.aPTT (Triniclot aPTT reagent)4.LAC-dRVVT screening and confirm (STAGO).5.Factor VIII assay (Siemens- Factor VIII deficient plasma)6. TT (STAGO-Thrombin)7. Factor VIII assay- Helena SARP.8.TGT: Thrombinoscope 5 picomolar TF and standard CAT method.Values described for Xarelto/ Rivaroxaban on patients with thromboprophylaxis in orthopedic surgery are usually of about 0.1–0.2 ug /ml in plasma.Result & Discussion: Concentration dependent prolongation of PT was observed. We used a single reagent (STAGO neoplastin CI Plus) which was sensitive at 0.1ug/ml concentration. Samama et al found that the results of PT were variable with reagent and not corrected by conversion to INR.We found that aPTT at lower concentrations of Rivaroxaban was not significantly different. Hillarp et al used five different reagents and found a reagent dependent dose response of APTT to rivaroxaban.The effect on Thrombin Time was non concentration dependent with no prolongation of TT at lower levels.There was fall in FVIII level in concentration dependent manner with 35% fall at 0.5ug/ml. We did not come across any literature till date about FVIII levels in Rivaroxaban therapy and our conclusion emphasises the importance of drug history.The LAC by DRVVT test showed positive result at the lowest concentration with an ILR of 1.23 and a dose dependent prolongation of clotting time. Thus making thrombophilia screening difficult.Thrombin generation time showed dose dependent effect on all parameters with almost complete suppression of thrombin generation. The initiation and propagation phase of TGT was affected at the lowest concentration (Green et al).The two anti-Xa tests used for measuring the concentration of Rivaroxaban in spiked plasma showed that Biophen DiXAI method has a broad range of sensitivity (0.1–0.5ug/ml) with calibration curve almost identical to that of manufacturer's with an excellent R value through out.The Rotachrome method at the standard 1:2 dilution was unable to measure concentration beyond 0.2ug/ml and at 1:20 dilution could only measure upto 0.4ug/ml. Thus, this method needs modification to determine a broad range of concentration.There was a variable result with 0.5ug/ml concentration of Rivaroxaban despite repeated vortex with unclear cause. This could be due to instability or settling of non-dissolved particles of the sample.The calibration curves were linear throughout with an acceptable R-value but there was a progressive drop in assay over a period of time. Conclusion:Anti-Xa assay with Biophen DiXAI method is a promising candidate for measurement of concentration of Rivaroxaban in special circumstances. Our findings show a trend towards prolongation of global clotting assays. The tablet is not stable as a calibrator. Disclosures:Pathak: BIOPHEN-DIXAI: Research Funding. McRae:BIOPHEN-DIXAI: Research Funding.

Factor XII activation is essential to sustain the procoagulant effects of particulate matter

Particulate matter (PM) is a key component of ambient air pollution and has been associated with an increased risk of thrombotic events and mortality. The underlying mechanisms remain unclear. To study the mechanisms of PM-driven procoagulant activity in human plasma and to investigate mainly, the coagulation driven by ultrafine particles (UFPs; < 0.1 μm) in genetically modified mice. Thrombin generation in response to PM of different sizes was assessed in normal human platelet-poor plasma, as well as in plasmas deficient in the intrinsic pathway proteases factors XII (FXII) or XI (FXI). In addition, UFPs were intratracheally instilled in wild-type (WT) and FXII-deficient (FXII(-/-) ) mice and plasma thrombin generation was analyzed in plasma from treated mice at 4 and 20 h post-exposure. In normal human plasma, thrombin generation was enhanced in the presence of PM, whereas PM-driven thrombin formation was completely abolished in FXII- and FXI-deficient plasma. UFPs induced a transient increase in tissue factor (TF)-driven thrombin formation at 4 h post-instillation in WT mice compared with saline instillation. Intratracheal instillation of UFPs resulted in a procoagulant response in WT mice plasma at 20 h, whereas it was entirely suppressed in FXII(-/-) mice. Overall, the data suggest that PM promotes its early procoagulant actions mostly through the TF-driven extrinsic pathway of coagulation, whereas PM-driven long lasting thrombogenic effects are predominantly mediated via formation of activated FXII. Hence, FXII-driven thrombin formation may be relevant to an enhanced thrombotic susceptibility upon chronic exposure to PM in humans.

Comparison of Laboratory Assays to Measure Rivaroxaban and Dabigatran

AbstractAbstract 3332 Introduction:Recently, 2 oral anticoagulants have been marketed: dabigatran, a direct thrombin inhibitor, and rivaroxaban, a direct FXa inhibitor. Thanks to their safety and efficacy profiles, routine coagulation monitoring is generally not required. However, the most commonly reported adverse reactions with these drugs are bleedings. A sensitive laboratory assay might be valuable to measure the pharmacodynamics of these 2 drugs in different situations including risks of drug interaction (i.e. strong P-gp inhibitors), overdose or to measure patient compliance. Due to their different mechanism of action, the measurement of their anticoagulant level in plasma may require different types of clotting assays. Aims:The aim of the present study was to determine which coagulation assay(s) could be used to measure the impact of rivaroxaban and dabigatran among a range of routine or more specific tests. Methods:Both drugs were spiked at increasing concentrations in pooled citrated normal human platelet-poor plasma (PPP). The final concentrations were ranging from 10 nM to 2 μM for dabigatran and from 25 nM to 5 μM for rivaroxaban. Such concentrations covered the plasma range in patients during orthopedic surgery. The following routine clotting assays were performed with the spiked plasma according to the manufacturer's protocols: Prothrombin Time (PT) with Neoplastin CI+ and Neoplastin R on STA-R (Roche Diagnostics), with Innovin on BCS (Siemens) and with Recombiplastin on ACL-TOP (Instrumentation Laboratories); activated Partial Thromboplastin Time (aPTT) with CK-Prest, PTT-A and Cephascreen on STA-R, with Actin FS on BCS and with Synthasil on ACL TOP and Thrombin Time (TT) on STA-R. More specific tests included Prothrombinase-induced Clotting Time (PiCT, Pefakit®, Pentapharm) and Ecarin Clotting Time (ECT) on STA-R; dilute PT (dPT) and activated Clotting time (ACT) on KC10. Thrombin Generation Tests (TGT) were also performed with Calibrated Automated Thrombogram (CAT) using PPP or PPP LOW reagents (Thrombinoscope). Sensitivity of a coagulation assay was defined as the concentration required to double clotting time (2XCT).Results: Dabigatran.A concentration-dependent prolongation of PT, dPT and aPTT was observed. However, at high concentrations, sensitivity of aPTT decreased whereas sensitivity of PT increased. The results varied depending on the clotting reagent used. TT was too sensitive leading to high variability for concentrations higher than 250nM. PiCT showed a linear concentration coagulation time (CT) relationship until a plateau at 750nM. ECT showed a high sensitivity (2X CT ≈ 93 nM), a linear relationship in the whole concentration range and a very low variability (CV≤ 1,8%). ACT gave a similar profile to PT (Innovin) with a slightly higher sensitivity (2X CT ≈ 716 nM vs 842nM). Dabigatran induced a concentration-dependent delay and inhibition of the tissue factor-induced thrombin generation with 5 pM TF or 1 pM TF with 4 μM PL and 16.7 mM CaCl2. The drug strongly increased the lag time and Tmax whereas it slightly decreased the Cmax and ETP. The lag time was the most sensitive CAT parameter with a high sensitivity (2x lag time ≈ 145 nM) and a low variability (CV≤6%). Rivaroxaban.A concentration-dependent prolongation of PT, dPT and aPTT was observed. However, at high concentrations, sensitivity of aPTT increased. Sensitivity of dPT (2X CT ≈ 109 to 550 nM) and PT (2X CT ≈ 138 to 764 nM) were similar and superior to aPTT (2X CT ≈ 897 to 2050 nM). The results varied depending on the clotting reagent used. TT was insensitive to rivaroxaban until 2500nM. Both PiCT and ECT showed a low sensitivity (2X CT ≈ 2536 nM and 5750 nM, respectively). A concentration-dependent prolongation of ACT was observed until 2500nM (2X CT ≈ 2275 nM). Rivaroxaban induced a concentration-dependent reduction and delay of the TF-induced thrombin generation. The Cmax was more strongly decreased than the ETP whereas the Tmax was more prolonged than the lag time, showing a major influence on the amplification phase. The Cmax was the most sensitive CAT parameter with a high sensitivity (Cmax EC50 ≈ 50 nM) and a low variability (CV<1%). Conclusions:ECT and PT are the most appropriate tests to study the pharmacodynamic effects of dabigatran and rivaroxaban respectively, thanks to high sensitivity, linear relationship in the whole concentration range and low variability. Disclosures:No relevant conflicts of interest to declare.

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

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.

In vivo externalization of phosphatidylserine and phosphatidylethanolamine in the membrane bilayer and hypercoagulability by the lipid peroxidation of erythrocytes in rats.

Phospholipid distribution across erythrocyte membrane bilayer is asymmetrical. In normal erythrocytes, entire phosphatidylserine (PS) and most of the phosphatidylethanolamine (PE) is present on the cytoplasmic side of membrane bilayer, whereas phosphatidylcholine (PC) and sphingomyelin (SM) are predominantly present at the outer side of membrane bilayer. The present study was undertaken to determine whether membrane lipid peroxidation has any effect on the distribution of PS, PE, and PC across erythrocyte membrane bilayer in vivo in an animal model. Erythrocyte membrane lipid peroxidation was induced in rats by administering phenylhydrazine, an oxidant drug. Membrane phospholipid organization was determined by using bee venom phospholipase-A2 and indirectly by measuring clotting time on recalcification of normal human platelet-poor plasma in the presence of Russell's viper venom. Phenylhydrazine administration to rats caused significant membrane lipid peroxidation as measured by the accumulation of malonyldialdehyde (MDA), an end product of fatty acid peroxidation, as well as externalization of a significant portion of PS and PE from the inner to the outer side of membrane bilayer in erythrocytes. There was a significant positive correlation (r) between the amount of MDA accumulated in the erythrocytes and the movement of PS (r = 0.92) and PE (r = 0.96) from inner to the outer membrane bilayer and PC (r = 0.81) from outer to the inner membrane bilayer. Erythrocytes of phenylhydrazine-treated rats also showed significantly reduced clotting time. This reduction in clotting time had a significant positive correlation with MDA accumulation (r = 0.92) and PS externalization (r = 0.90). Both the effect of phenylhydrazine on erythrocyte membrane lipid peroxidation and alterations in phospholipid organization and coagulability were blocked when rats were simultaneously administered with vitamin E or C antioxidants.

The in Vitro Anticoagulant Activity of Human Plasma Lipids

SummaryThe present report describes the in vitro inhibition of prothrombin consumption by lipid extracted from normal human platelet-poor plasma. Pro-coagulant lipids and the test plasma lipids were dissolved in the same solvent and evaporated to dryness. The dried lipid mixture was emulsified in platelet-poor plasma, and the plasma clotted by recalcification. Inhibition of prothrombin consumption by plasma lipid was observed when the ratio of plasma lipid to procoagulant lipid equaled or exceeded 5:1. The inhibitory effect of plasma lipid on prothrombin consumption was not observed when procoagulant activity was provided by intact platelets or platelet granules.Human plasma lipids have been fractionated on silicic acid columns to permit identification of the plasma lipid component responsible for the inhibition of prothrombin consumption. The inhibitory activity is present in a lipid fraction which is 95% lecithin. Other plasma lipid components exhibit little or no inhibitory activity.