Tissue Factor Concentration Research Articles

Substrate delivery mechanism and the role of membrane curvature in factor X activation by extrinsic tenase

Membrane-bound enzyme complex of extrinsic tenase (VIIa/TF) is believed to be the primary activator of blood clotting in vivo. This complex (where factor VIIa (FVIIa) is a catalytically active part and tissue factor (TF) is its essential cofactor) activates its primary substrate factor X (FX) leading to factor Xa (FXa) (‘a’ stands for ‘activated’). Both FX and FXa are able to bind to phospholipid membrane and, therefore, are distributed between solution and membrane surface. As a result, two possible mechanisms of substrate delivery to the extrinsic tenase exist: via lateral diffusion on the membrane surface or directly from the solution. Determination of the predominant pathway of substrate delivery is an important key to understanding the precise reaction mechanism. Here we construct a mechanism-driven computational model of FX activation by extrinsic tenase on the surface of phospholipid vesicles of different size. We show that experimentally observed dependence of the tenase activity on the phospholipid concentration could be obtained only if the substrate (FX) is membrane-bound. For correct experimental data description it is also necessary to take into account the dependence of FX/FXa membrane binding parameters (equilibrium dissociation constant and the number of phospholipid molecules per bound FX/FXa) on the membrane curvature. The model predicts that small vesicles promote activation of FX by the extrinsic tenase significantly better than large vesicles (with the same overall phospholipid, factors VIIa, X and TF concentrations in the solution).

Interindividual Variability and Normal Ranges of Whole Blood and Plasma Thrombin Generation.

Assays measuring thrombin generation (TG) in plasma increasingly gained attention in the field of thrombosis and hemostasis. Adaptation of the method enabled the measurement of TG in whole blood (WB). Despite their potential, TG assays did not reach the stage of universal clinical application, partly because of the absence of normal ranges. Our study aimed to accurately determine normal ranges and interindividual variability of TG and correlate results with coagulation factor levels, sex, and oral contraceptive usage. The study protocol was evaluated by the local medical ethical board. In total, 129 healthy volunteers gave full informed consent. Normal ranges of TG in platelet-poor plasma (PPP), platelet-rich plasma (PRP), and WB were determined according to CLSI guidelines. Our study is the first to measure normal ranges of TG in PPP, PRP, and WB in a large healthy cohort. Significant correlations were found between TG in plasma and WB. Interindividual variability of TG in WB was comparable to that of plasma. Oral contraceptive use increased TG in PPP, PRP, and WB. The inhibitory effect of thrombomodulin on TG was significantly lower in females than in males. This effect was more pronounced upon oral contraceptive use. Primary clotting factor determinants for TG parameters depended on the tissue factor concentration, but were similar in WB and plasma. Establishing normal ranges for TG brings us 1 step closer to clinical use. Good correlations between plasma and WB (including clotting factor determinants for TG) suggest that WB TG can be reliably used in clinic.

The role of the tissue factor and its inhibitor in the development of subclinical atherosclerosis in people living with HIV.

IntroductionHIV infection is associated with an increased risk of cardiovascular disease in connection with atherosclerosis and thromboembolic complications. The pathogenesis of atherosclerosis is still unclear in this group of patients. Studies on pathogenesis of atherosclerosis in the general population emphasize the role of the extrinsic pathway of blood coagulation, particularly the tissue factor (TF) and tissue factor pathway inhibitor (TFPI). The effect of persistent activation of the immune system on enhanced expression of TF on the surface of monocytes in subjects infected with HIV is known to be correlated with the level of HIV RNA in blood serum.Study aimThe aim of this study was to evaluate the concentration of TF and its inhibitor TFPI in blood plasma, the impact of traditional and non-traditional cardiovascular risk factors on their concentration and the impact of both markers of haemostasis on the severity of subclinical atherosclerosis as assessed by the intima-media measurement of the carotid artery in HIV infected patients.MaterialsThe study included 121 HIV-infected people with known clinical, immunological and virological status. The control group consisted of 42 healthy individuals, selected in terms of age and sex.Results and conclusionsHigher concentrations of TF occurred in HIV-infected patients with a low current plasma HIV RNA level, nadir CD4+ T-cell count and longer duration of cumulative antiretroviral treatment. In multivariate analysis, it was the length of cumulative NRTI treatment that impacted on the concentration of TF. The determinants of cardiovascular disease (CVD) risk factors and inflammatory markers did not show any effect on the concentrations of TF.The TFPI level in HIV-infected patients was significantly higher than in the control group and was negatively correlated with the current level of HIV RNA and nadir CD4+ T-cell count, being higher in patients subjected to antiretroviral treatment. It was shown that the higher the cardiovascular risk and the higher the levels of total cholesterol, low-density lipoprotein cholesterol (LDL) and non-high-density lipoprotein cholesterol (non-HDL), the higher the concentrations of TFPI observed. The levels of TF and TFPI were positively correlated with carotid intima media thickness (cIMT); in the multivariate analysis, TF, non-HDL cholesterol and lifetime smoking (pack-years) independently affected the growth of cIMT. A similar effect on cIMT was demonstrated by TFPI.

Effect of electro-acupuncture at Zusanli acupoint on blood coagulation during intestinal ischemia-reperfusion in rats

Objective To evaluate the effect of electro-acupuncture (EA) at Zusanli (ST36) acupoint on blood coagulation during intestinal ischemia-reperfusion (I/R) in rats. Methods Forty healthy male Sprague-Dawley rats, aged 6-8 months, weighing 250-300 g, were divided into 5 groups (n=8 each) using a random number table: sham operation group (group S), intestinal I/R group (group I/R), EA at Zusanli acupoint group (group EA), EA at non-acupoint group (group NE) and α7 nicotinic acetylcholine receptor antagonist α-bungarotoxin (α-BGT) group (group α-BGT). Intestinal I/R was induced by clamping the superior mesenteric artery for 45 min followed by 120 min of reperfusion.Bilateral Zusanli acupoints were stimulated with an electric stimulator (frequency 3 Hz, voltage 2-4 V, wave length 2 ms) for 30 min starting from the time point immediately after beginning of ischemia in group EA, while EA was performed at the points 5 mm lateral to the bilateral Zusanli instead in group NE.In group α-BGT, α-BGT 1 μg/kg was intraperitoneally injected at 45 min before ischemia, and the other treatments were similar to those previously described in group EA.Blood samples were collected from the abdominal aorta at 120 min of reperfusion for determination of the concentrations of tumor necrosis factor alpha (TNFα), tissue factor (TF), antithrombin (AT), tissue plasminogen activator (tPA), fiber plasminogen activator inhibitor-1 (PAI-1) and D-dimer in plasma (by enzyme-linked immunosorbent assay) and platelet count (PLT). The animals were sacrificed after blood sampling, the distal ileum specimens were removed for examination of the pathological changes with a light microscope, and the damage to the intestinal mucous membrane was assessed and scored according to Chiu. Results Compared with group S, the concentrations of plasma TNFα, TF, tPA, PAI-1 and D-dimer were significantly increased, and the plasma AT concentration and PLT were decreased in I/R, NE and α-BGT groups, the concentrations of plasma TNFα and TF were significantly increased, and the plasma AT concentration was decreased in group EA, and Chiu′s scores were significantly increased in I/R, EA, NE and α-BGT groups (P 0.05). Compared with group EA, the concentrations of plasma TNFα, TF, tPA, PAI-1 and D-dimer were significantly increased, the plasma AT concentration and PLT were decreased, and Chiu′s scores were increased in group NE (P<0.05). Conclusion EA at Zusanli acupoint can improve blood coagulation during intestinal I/R in rats, and the mechanism is related to activating the cholinergic anti-inflammatory pathway. Key words: Electroacupuncture; POINT ST36 (ZUSANLI); Reperfusion injury; Intestines; Blood coagulation

Significant inhibition of the triggering mechanism of the extrinsic system of blood coagulation in rats by thymus peptides thymulin, thymosin alpha 1 and thymosin beta 4

INTRODUCTION : The role of the thymus gland and the effects of thymic peptides on the coagulation status are relatively insufficiently studied. Recent data indicate controversial and not persuasive results due to in­vestigation of single hemostasis parameters and application of various pathological models. AIM : The present study aimed to elucidate the influence of thymus peptides thymulin, thymosin alpha 1 and thymosin beta 4 applied s.c. on intact rats, on key triggering factors of blood coagulation via the extrin­sic pathway: plasma concentration of tissue factor (TF); concentration and activity of free TFPI; and the ac­tivity of plasma clotting factor VII, as well as on prothrombin time (PT) - an integral clinical parameter of the intrinsic pathway. MATERIALS AND METHODS : The study was performed on 52 male Wistar strain rats, weighing 200-220 g and bred under standard conditions at dark/light cycle 12/12 h. The rats were divided into four equal groups (n=13) and injected s.c. once daily in the time interval 08.00-09.00 h in the course of three consecutive days as follows: 1st group (controls) - with saline (the solvent of the peptides); 2nd group - with thymulin (Sigma Aldrich, 0.4 mg/kg b.m.); 3rd group - with thymosin alpha 1 (Sigma Aldrich, 0.3 mg/kg b.m.); and the 4th group - with thymosin beta 4 (Sigma Aldrich, 0.3 mg/kg b.m.). RESULTS : Results indicate that thymulin and thymosin alpha 1 expressively decrease TF and increase TFPI concentrations. All three thymic peptides reduce FVIIa activity, increase TFPI activity, and significantly elongate PT. CONCLUSIONS : Thymus peptides thymulin, thymosin alpha 1 and thymosin beta 4 significantly suppress blood coagulation through the extrinsic pathway and generate a distinct tendency for hypocoagulability.

Correlation between factor (F)XIa, FIXa and tissue factor and trauma severity.

It has been observed that trauma patients often display elevated procoagulant activity that could be caused, in part, by tissue factor (TF). We previously observed that trauma patients with thermal, blunt, and penetrating injuries have active FIXa and FXIa in their plasma. In the current study, we evaluated the effect of injury severity, with or without accompanying shock, on the frequency and concentration of TF, FIXa, and FXIa in plasma from trauma patients. Eighty trauma patients were enrolled and divided equally into four groups based on their Injury Severity Score and base deficit:Blood was collected at a 0 time-point (first blood draw upon arrival at hospital) and citrate plasma was prepared, frozen, and stored at -80 °C. FXIa, FIXa, and TF activity assays were based on a response of thrombin generation to corresponding monoclonal inhibitory antibodies. The frequency and median concentrations of TF were relatively low in non-severe injury groups (17.5% and 0 pM, respectively) but were higher in those with severe injury (65% and 0.5 pM, respectively). Although FXIa was observed in 91% of samples and was high across all four groups, median concentrations were highest (by approximately fourfold) in groups with shock. FIXa was observed in 80% of plasma samples and concentrations varied in a relatively narrow range between all four groups. No endogenous activity was observed in plasma from healthy individuals. (1) Frequency and concentration of TF is higher in patients with a higher trauma severity. (2) Concentration of FXIa is higher in patients with shock. (3) For the first time reported, the vast majority of plasma samples from trauma patients contain active FIXa and FXIa. Prognostic/epidemiological study, level II.

Effect of circulating tissue factor on hypercoagulability in type 2 diabetes mellitusstudied by rheometry and dielectricblood coagulometry.

Background:Hypercoagulability in type 2 diabetes mellitus (T2DM) patients increases their risk of cardiovascular diseases.Objective:The aim of this work was to investigate the hypercoagulation mechanism in T2DM patients in terms of circulating tissue factor (TF).Methods:Whole blood coagulation tests by damped oscillation rheometry and dielectric blood coagulometry (DBCM) were performed.Results:The average coagulation time was significantly shorter for T2DM patients than for healthy controls. In vitro addition of either anti-TF or anti-activated factor VII (FVIIa) antibody to hypercoagulable blood samples prolonged coagulation times for one group of patients, while coagulation times remained short for another group. The levels of circulating TF were estimated in the former group by measuring the coagulation times for blood samples from healthy subjects with addition of various concentrations of TF and comparing them with the coagulation times for the group. The results indicated that the levels of circulating TF were on the order of subpicomolar at most.Conclusions:Circulating TF is at least partially responsible for a hypercoagulable group of T2DM patients, while an abnormality in the intrinsic coagulation pathway probably occurs in the other group.

Whole blood coagulation assays ROTEM and T-TAS to monitor dabigatran treatment

BackgroundA rapid and reliable assessment of the dabigatran effect is desirable in dabigatran treated patients with uncontrolled bleeding or before acute surgery. ObjectiveTo evaluate how the viscoelastic point-of-care test Rotational thromboelastometry (ROTEM) and Total Thrombus-formation system (T-TAS), which studies thrombus formation under flowing conditions, correlate with dabigatran concentrations in patients with atrial fibrillation (AF). MethodROTEM using the reagents In-tem, Ex-tem, Fib-tem or low tissue factor concentration (TF), and T-TAS with the AR-chip (shear rate 600s−1, representing flow in large arteries) were investigated in whole blood samples. Plasma concentrations were determined by mass spectrometry (LC-MS/MS) at trough and post-dose in 30 patients on dabigatran 150mg BID. ResultsMedian plasma dabigatran concentrations at trough were 86ng/mL (29–150) and post-dose (2.8h after ingestion) 175ng/mL (67–490). The ROTEM clotting time (CT) correlated strongly with dabigatran concentrations when activated with the reagents Ex-tem (r=0.92, p<0.01) and Fib-tem (r=0.93, p<0.01), while with In-tem and low TF the correlation was weaker (r=0.72 and r=0.36, p<0.01). There were significant but weaker correlations also between dabigatran concentrations and T-TAS variables (r-values 0.39–0.41, p<0.01), aPTT (r=0.70, p<0.01) and PT-INR (r=0.43, p<0.01) respectively. ConclusionsROTEM Ex-tem and Fib-tem CT shows a strong correlation with dabigatran concentrations in real-life AF-patients, and results are obtained within minutes. This could make ROTEM useful in acute situations. T-TAS detect differences in hemostasis caused by dabigatran, but the relationships to plasma concentrations of dabigatran are weaker than for ROTEM CT with the settings used in this study.

In vitro activation of coagulation by human neutrophil DNA and histone proteins but not neutrophil extracellular traps

AbstractNETosis is a physiologic process in which neutrophils release their nuclear material in the form of neutrophil extracellular traps (NETs). NETs have been reported to directly promote thrombosis in animal models. Although the effects of purified NET components including DNA, histone proteins, and neutrophil enzymes on coagulation have been characterized, the mechanism by which intact NETs promote thrombosis is largely unknown. In this study, human neutrophils were stimulated to produce NETs in platelet-free plasma (PFP) or in buffer using phorbol myristate actetate or calcium ionophore. DNA and histone proteins were also separately purified from normal human neutrophils and used to reconstitute chromatin using a salt-gradient dialysis method. Neutrophil stimulation resulted in robust NET release. In recalcified PFP, purified DNA triggered contact-dependent thrombin generation (TG) and amplified TG initiated by low concentrations of tissue factor. Similarly, in a buffer milieu, DNA initiated the contact pathway and amplified thrombin-dependent factor XI activation. Recombinant human histones H3 and H4 triggered TG in recalcified human plasma in a platelet-dependent manner. In contrast, neither intact NETs, reconstituted chromatin, individual nucleosome particles, nor octameric core histones reproduced any of these procoagulant effects. We conclude that unlike DNA or individual histone proteins, human intact NETs do not directly initiate or amplify coagulation in vitro. This difference is likely explained by the complex histone-histone and histone-DNA interactions within the nucleosome unit and higher-order supercoiled chromatin leading to neutralization of the negative charges on polyanionic DNA and modification of the binding properties of individual histone proteins.

Topical application of recombinant activated factor VII during cesarean delivery for placenta previa

During cesarean delivery in patients with placenta previa, hemorrhaging after removal of the placenta is often challenging. In this condition, the extraordinarily high concentration of tissue factor at the placenta site may constitute a principle of treatment as it activates coagulation very effectively. The presumption, however, is that tissue factor is bound to activated factor VII. We hypothesized that topical application of recombinant activated factor VII at the placenta site reduces bleeding without affecting intravascular coagulation. We included 5 cases with planned cesarean delivery for placenta previa. After removal of the placenta, the surgeon applied a swab soaked in recombinant activated factor VII containing saline (1 mg in 246 mL) to the placenta site for 2 minutes; this treatment was repeated once if the bleeding did not decrease sufficiently. We documented the treatment on video recordings and measured blood loss. Furthermore, we determined hemoglobin concentration, platelet count, international normalized ratio, activated partial thrombin time, fibrinogen (functional), factor VII:clot, and thrombin generation in peripheral blood prior to and 15minutes after removal of the placenta. We also tested these blood coagulation variables in 5 women with cesarean delivery planned for other reasons. Mann-Whitney test was used for unpaired data. In all 5 cases, the uterotomy was closed under practically dry conditions and the median blood loss was 490 (range 300-800) mL. There were no adverse effects of recombinant activated factor VII and we did not measure factor VII to enter the circulation. Neither did we observe changes in thrombin generation, fibrinogen, activated partial thrombin time, international normalized ratio, and platelet count in the peripheral circulation (all P values >.20). This study indicates that in patients with placenta previa, topical recombinant activated factor VII may diminish bleeding from the placenta site without initiation of systemic coagulation.

Salivary tissue factor concentration and activity in patients with oral lichen planus

PurposeThis study aimed to evaluate the tissue factor (TF) concentration and activity in patients with oral lichen planus (OLP) under oxidative stress. MethodsTwenty patients with OLP were selected from the patients who were referred for treatment to the Marmara University, Faculty of Dentistry, Oral and Maxillofacial Radiology Department. Twenty healthy subjects from faculty staff and their family members were selected for the study. Salivary TF concentration and TF activity; total oxidant capacity; total antioxidant capacity; total thiol, malondialdehyde and glutathione concentrations; and oxidative stress index were measured in saliva samples. ResultsSalivary total oxidant capacity, oxidative stress index and malondialdehyde and TF concentration and activity were significantly increased in the patient group compared to those in the control group. Total thiol and glutathione concentration and total antioxidant capacity were significantly decreased in the patient group. ConclusionImpaired oxidant-antioxidant balance and inflammatory features of OLP might cause an increase in the salivary TF concentration and activity. As TF factor plays a critical role in inflammation progress, the use of an antioxidant agent in OLP may decrease the salivary TF concentration by decreasing oxidative stress. The findings of this study might represent a novel approach to OLP monitoring and treatment in terms of TF as the measurement of TF is easy and cost-effective.

Activated Factors XI and IX and Tissue Factor in Patients with Advanced Cancer

Background.Cancer patients often display procoagulant activity and are at a risk for developing venous thromboembolism (VTE). Multiple mechanisms may explain this procoagulant state, such as tumor cell expression of tissue factor (TF), host tissue response to tumor formation and also contributions from comorbid factors and treatment. We previously observed elevated levels of traditional biomarkers of coagulation (such as D-dimer, CRP, etc.) in patients with advanced cancer on therapy enrolled in a randomized trial comparing rosuvastatin versus placebo. In this study, we evaluated the frequency and concentration of endogenous plasma TF, Factor (F)XIa and FIXa in the cancer patient population analyzed for traditional coagulation biomarkers. The choice of new analytes was based on previous studies in which these 3 proteins were detected and quantitated in patients with cardiovascular diseases, inflammation, and trauma - often present over the course of several days to even weeks. By our knowledge, there are no published studies of the presence of FXIa and/or FIXa in cancer patients.Methods.Thirty-seven adult cancer patients receiving systemic therapy were originally enrolled in a randomized crossover trial comparing effects of rosuvastatin versus placebo on biomarkers of VTE. Inclusion criteria included locally-advanced or metastatic cancer, estimated survival time of greater than 6 months, and anticipated duration of therapy of at least 3 months. Patients on antithrombotic or statin therapy were excluded. Blood was collected on up to 4 occasions; at the start and end of each of 2 treatment periods corresponding to 2 cycles of systemic therapy with a 3-4 week washout period. Citrate plasma was prepared, frozen and stored at -80C. The assay, performed in previously unthawed and contact-pathway inhibited plasma, is based on a response of the lag phase of thrombin generation to corresponding monoclonal inhibitory antibodies (αFXIa-2, αFIXa-91 and αTF-5; all at 0.1 mg/mL). Concentrations of TF, FXIa and FIXa were calculated from corresponding calibration curves built by titrating purified proteins into multi-donor pooled plasma from healthy individuals.Results.A total of 116 blood samples were collected (not all patients were able to complete their regime) and analyzed for endogenous levels of TF, FXIa and FIXa (see table). Overall, rosuvastatin treatment did not have any significant impact on the levels and frequency of these 3 proteins. For the samples obtained from each patient’s first blood draw (n = 37) there was a weak correlation between FXIa and FIXa levels (R = 0.37, P = 0.02) and a moderate correlation between FXIa and TF levels (R = 0.57, P < 0.001), while no correlation between FIXa and TF was observed (R = 0.02, P = 0.91).Conclusions.1) A portion of patients with advanced cancer had active TF in their plasma while the majority, for the first time reported, had active FXIa and FIXa; 2) No pronounced differences were observed between the frequency and concentrations of these proteins at baseline versus later time-points; 3) There was a weak correlation between FXIa and FIXa and a moderate correlation between FXIa and TF, suggesting the TF-pathway is driving the majority of FXIa generation, although contribution from the contact pathway cannot be excluded; 4) Rosuvastatin treatment did not lead to any significant changes in the levels of TF, FXIa and/or FIXa. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Andexanet Alfa, a Universal Antidote Under Development for Factor Xa Inhibitors, Reverses Rivaroxaban-Induced Inhibition of Thrombin Generation Initiated By the Intrinsic Coagulation Pathway Independent of TFPI

Background: Andexanet alfa (AnXa) is a modified, recombinant human fXa molecule being developed to reverse the anticoagulant activity of fXa inhibitors in patients during episodes of major bleeding. As a modified fXa, AnXa retained high binding affinity to fXa inhibitors and had no significant interaction with major plasma coagulation proteins, except for tissue factor pathway inhibitor (TFPI), an endogenous fXa inhibitor with sub-nanomolar affinity to both fXa and AnXa. In previous in vitro and clinical studies in healthy volunteers, AnXa has shown dose-dependent and complete reversal of both direct and indirect fXa inhibitors in tissue factor (TF)-initiated thrombin generation (TG). To delineate the contribution of AnXa-TFPI interactions to TG, we compared rivaroxaban (Riva)-induced inhibition of TG initiated via the extrinsic pathway (TF) versus the intrinsic pathway (non-TF). The differential effect of AnXa on TG and clot formation via the two pathways was further studied in the context of recombinant tissue plasminogen activator (rtPA)-induced fibrinolysis using thromboelastography (TEG).Methods: TF-initiated TG in human plasma was measured using a calibrated automated thrombogram (CAT) and the PPP-reagent (5 pM TF). Non-TF-initiated TG was measured using CAT and Actin FS, an aPTT reagent. Anti-fXa activity was measured using a modified anti-fXa chromogenic assay with reagents from the Coamatic Heparin kit (DiaPharma). Riva was used as the standard. Pooled human plasma was spiked with Riva (0 - 2 µM) or Riva (1 µM)+AnXa (0 - 4 µM), and TG and anti-fXa activity were measured. Clot formation with or without AnXa was measured in plasma using a TEG 5000 analyzer. The functional fibrinogen (TF) and Kaolin reagents (both from Haemonetics) were used according to the manufacturer’s instruction. For low TF-initiated clot formation, the PPP-reagent (5 pM TF) was diluted in phospholipid (4.0 µM) followed by measuring the TEG profiles with 0.25, 0.5 and 1.0 pM TF.Results: The potential contribution of AnXa-TFPI interaction to TG was studied under similar conditions using TF or Actin FS in human plasma containing AnXa, Riva or Riva+AnXa. AnXa alone had minimal effect on the endogenous thrombin potential (ETP) in either assay. AnXa was able to fully reverse Riva-induced anticoagulation in the Actin FS TG assay, independent of AnXa-TFPI interaction that primarily modulates TF activity. The modulation of TF activity could be assessed by correlating ETP vs. the anti-fXa activity in samples containing Riva or Riva+AnXa. Riva dose-dependently inhibited TF-initiated TG as anti-fXa activity increased. At similar anti-fXa levels (i.e., similar free Riva concentration), Riva+AnXa had higher ETP than Riva alone. As expected, this difference was not observed in the Actin FS TG assay.To further investigate the role of AnXa-TFPI interaction on coagulation and fibrinolytic pathways, the profile of clot formation was studied in human plasma using TEG without Riva. AnXa (4 µM) had no effect on the TEG parameters in the kaolin assay or the functional fibrinogen assay containing high TF, with or without rtPA (150 ng/mL). When low TF (0.25, 0.5, and 1.0 pM) was used to initiate clot formation in the absence of rtPA, AnXa reduced the TEG-R parameter (lag time equivalent to clotting time), but had no effect on maximum amplitude (MA). The fibrin clot formed under each condition (±AnXa) was lysed slowly at low rtPA (75 ng/mL, ~1 nM), resulting in well-segregated processes of coagulation and fibrinolysis. However, rtPA at 150 ng/mL (~2 nM) dramatically changed the lysis profiles that overlapped the fibrin formation, possibly due to the higher rtPA activity ratio, relative to the major plasma inhibitor PAI-1 (<1 nM). With the optimal rtPA, fibrin clot formed at each TF concentration (±AnXa) was compensated by the fibrinolytic activity of rtPA resulting in an increased fibrin degradation product.Conclusions: In the absence of a fXa inhibitor, AnXa had minimal effect on TF or Actin FS-initiated TG with no direct interaction or effect on rtPA function. AnXa dose-dependently and completely reversed Riva-induced inhibition of TG initiated by either the intrinsic or extrinsic pathway, but had different effect on ETP due to the AnXa-TFPI interaction that mainly modulates TF function. AnXa-TFPI interaction may enhance TF-initiated TG at low TF conditions with increased fibrin degradation product in the presence of rtPA. DisclosuresLu:Portola Pharmaceuticals, Inc.: Employment. Lin:Portola Pharmaceuticals, Inc.: Employment. Curnutte:3-V Biosciences: Equity Ownership; Sea Lane Biotechnologies: Consultancy; Portola Pharmaceuticals: Employment, Equity Ownership, Patents & Royalties, Research Funding. Conley:Portola Pharmaceuticals, Inc.: Employment.

Shift to Anti-Fibrinolysis on the Administration of L-Asparaginase (L-Asp) during Induction Therapy for Pediatric Acute Lymphoblastic Leukemia (ALL)

Introduction: Thromboembolism is a serious complication associated with ALL. The use of central venous catheter and treatment protocols involving corticosteroids and L-Asp is assumed as important thrombogenic factors at the induction phase. In particular, L-Asp has profound effects on hepatic synthesis of pro-, anti-coagulant and fibrinolytic factors. In this study, we hypothesized that change of coagulation and fibrinolytic function contributes to hyper-coagulation condition during the induction phase with L-Asp. In order to clarify this, we evaluated the dynamic change in coagulation and fibrinolysis by simultaneous measurement of both thrombin and plasmin generation assay (T/P-GA). Patients: Twenty-seven pediatric patients with newly diagnosed ALL were enrolled from Aug. 2014 to Oct. 2015 at 3 hospitals in Japan. All cases had no thrombotic predisposition. Eighteen cases (66.7%) (BCP-ALL; n=17, T-ALL; n=1) received Berlin-Frankfürt-Münster (BFM)-95 oriented induction therapy included prednisolone (and dexamethasone for T-ALL), vincristine, daunorubicin, and E.coli L-Asp (a total of 8 doses of 5,000 U/m2). The others (BCP-ALL; n=8, T-ALL; n=1) received Japan Association of childhood Leukemia Study (JACLS) ALL02 oriented induction therapy included prednisolone, dexamethasone, vincristine, daunorubicin, cyclophosphamide and E.coli L-Asp (a total of 6 doses of 6,000 U/m2). Methods: The individual hemostatic parameters were monitored by fibrinogen (Fbg), FDP, AT, TAT and PIC. Additionally, the global functions of coagulation and fibrinolysis were evaluated using T/P-GA established by our group [Matsumoto et al. TH 2013]. This assay was initiated by the addition of a mixture of optimized concentrations of tissue factor and tissue-type plasminogen activator. Thrombin and plasmin generation were monitored simultaneously using individual fluorescent substrates in separate microtiter wells. Standard curves were set using purified alpha-thrombin and plasmin. Patients' plasmas were collected at the following points, T0; pre-phase of L-Asp, T1; intermittent phase of L-Asp, T2; post-phase of L-Asp, and T3; post-induction phase. Endogenous potentials of thrombin generation (T-EP) for coagulant activity and plasmin peak levels (P-Peak) of plasmin generation for fibrinolytic activity were selected as parameters for evaluation in this study. A ratio of T-EP and P-Peak of patients' plasmas to those of control normal plasma were calculated. Results: All cases obtained first remission, and none of them developed coagulopathy. Six cases received FFP transfusion for low Fbg level, whilst 21 cases received AT supplement for low AT level. Fbg showed a median of 170, 99.0, 99.0 and 328 mg/dl at T0, T1, T2 and T3, respectively, whilst the other individual parameters showed relatively unchanged. T-EP revealed a median of 1,126, 1,059, 1,175, 1,343 and 1,132 nM, whilst P-Peak showed a median of 6.67, 4.54, 4.12, 5.50 and 5.77 nM for T0, T1, T2, T3 and control plasma, respectively, indicating the elevated T-EP ratios and reduced P-Peak ratios (Fig. 1). The most significant difference in both ratios demonstrated a median of 1.5-fold (range, 1.0 to 2.6) at T2, consistent with the lowest Fbg levels. The FFP transfusion group showed significantly lower T-EP ratios than non-transfusion group at T1 (a median of 0.87 vs. 1.01, P=0.041) and T2 (a median of 0.96 vs. 1.07, P=0.009), whilst P-Peak ratios revealed no significant changes. The AT supplement group showed no significant changes of both ratios. Conclusion: The results from decreased Fbg and unchanged FDP might reveal the hepatic synthesis disorder of Fbg, whilst the results from T/P-GA showed that their hemostatic dynamics appear likely to be thrombotic tendency, since their coagulation state was hyper-coagulation and anti-fibrinolysis at post-phase of L-Asp. These results suggest that the impaired balance of coagulation and fibrinolysis due to L-Asp therapy might play an important role of a thrombotic complication at induction phase. On the other hand both conventional FFP transfusion and AT supplement therapy might not dramatically repair this unbalance state. A further research would be required to examine the role of coagulant and fibrinolytic function using T/P-GA in the pathogenesis of coagulopathy associated with L-Asp therapy in order to establish the optimal supportive therapy. [Display omitted] DisclosuresNogami:F. Hoffmann-La Roche Ltd.: Honoraria, Membership on an entity’s Board of Directors or advisory committees; Sysmex Corporation: Patents & Royalties, Research Funding; Chugai Pharmaceutical Co., Ltd.: Honoraria, Membership on an entity’s Board of Directors or advisory committees, Patents & Royalties, Research Funding. Matsumoto:Sysmex Corporation: Patents & Royalties, Research Funding; Chugai Pharmaceutical Co., Ltd.: Patents & Royalties, Research Funding. Shima:Chugai Pharmaceutical Co., Ltd.: Consultancy, Honoraria, Membership on an entity’s Board of Directors or advisory committees, Patents & Royalties, Research Funding; F. Hoffmann-La Roche Ltd.: Consultancy, Honoraria, Membership on an entity’s Board of Directors or advisory committees; Sysmex Corporation: Patents & Royalties, Research Funding.

A Novel Clot Waveform Analysis to Measure the Plasma Coagulation Potency in the Presence of Anti-Fixa/Fx Bispecific Antibody, Emicizumab

Emicizumab (also termed ACE910) is a humanized anti-factor (F)IXa/FX bispecific antibody with FVIIIa cofactor function. A clinical phase 3 study was initiated in 2015 for hemophilia A patients (HA-pts) with FVIII inhibitors. Since emicizumab, unlike FVIII, does not require activation by thrombin, its APTT-shortening effect is much greater than that of FVIII. Thus, APTT, a conventional assay to assess whole coagulation potency, would have limited utility in emicizumab-administered HA-pts, because emicizumab would mask the effect of residual FVIII or a FVIII agent on APTT. Clot waveform analysis (CWA) can provide multidimensional coagulation potencies by monitoring the process of plasma clot formation with an automated coagulation analyzer. We considered the possibility, therefore, that this assay system would overcome the above issue on APTT. In this study, we aimed to optimize concentrations of tissue factor (TF) and ellagic acid (Elg) in a trigger reagent for CWA as well as CWA parameters to provide precise evaluation of coagulation potency even in the presence of emicizumab with neither masking nor being masked by FVIII or bypassing agents. First, we determined an optimal concentration of TF/Elg trigger reagent. Various concentrations (10, 30, 100, and 300 μg/mL) of emicizumab were spiked into commercially available FVIII-deficient plasmas (George King) for testing. Recombinant (r)FVIII (Kogenate FS; Bayer)-spiked samples were also tested as a reference. PT reagent (under development; Sysmex) and APTT reagent (Thrombocheck APTT-SLA; Sysmex), used as a source of TF and Elg, respectively, were mixed in various ratios. The optimized mixture ratio (PT:APTT:buffer=1:15:135) was chosen to ensure that the maximum coagulation velocity (|min1|) in the presence of emicizumab would be in agreement with the animal study-based estimated conversion rate "0.2-0.4 IU/dL of equivalent FVIII per 1 μg/mL of emicizumab" (Muto. J Thromb Haemost. 2014). When evaluating several lots of FVIII-deficient plasmas, however, we observed large variations in transmittance depending on fibrinogen concentration of each plasma, which resulted in large variations of |min1| between donor plasmas. To decrease the bias due to fibrinogen concentration, % transmittance of clot waveform (CW) was adjusted to 100% and 0% at the pre- and post-coagulation phase, respectively. By using |min1| from the adjusted CW (adjusted-|min1|), we successfully reduced the inter-donor variations and chose it as a main parameter. Next, we evaluated adjusted-|min1| using plasmas from HA-pts without inhibitors (severe; n=2, moderate; n=2) and HA-pts with inhibitors (<10 BU/mL; n=2, >10 BU/mL; n=2) by adding emicizumab (30, 100, and 300 μg/mL) in vitro. After the addition of emicizumab, concentration-dependent increases in adjusted-|min1| were observed in all plasmas with rather small individual variations. Finally, we examined whether adjusted-|min1| reflected the effects of FVIII or bypassing agent that was added to plasmas containing emicizumab. Additive effects of a plasma-derived FVIII agent (CROSS EIGHT M; Japan Blood Products Organization), a rFVIII agent (ADVATE; Baxalta) and activated prothrombin complex concentrate (FEIBA; Baxalta) were confirmed by the increase of adjusted-|min1|. As for rFVIIa agent (NovoSeven; Novo Nordisk), its additive effect on adjusted-|min1| was not clear enough in this assay condition, but its additive effects were confirmed by the clot time. In conclusion, we established the Elg/TF-triggered CWA assay condition and parameters for measuring coagulation potency in plasmas from HA-pts even in the presence of emicizumab and a FVIII/bypassing agent without masking each other. DisclosuresNogami:Sysmex Corporation: Patents & Royalties, Research Funding; Chugai Pharmaceutical Co., Ltd.: Honoraria, Membership on an entity’s Board of Directors or advisory committees, Patents & Royalties, Research Funding; F. Hoffmann-La Roche Ltd.: Honoraria, Membership on an entity’s Board of Directors or advisory committees. Matsumoto:Sysmex Corporation: Patents & Royalties, Research Funding; Chugai Pharmaceutical Co., Ltd.: Patents & Royalties, Research Funding. Tabuchi:Sysmex Corporation: Employment, Patents & Royalties; Chugai Pharmaceutical Co., Ltd.: Patents & Royalties. Soeda:Sysmex Corporation: Patents & Royalties; Chugai Pharmaceutical Co., Ltd.: Employment, Patents & Royalties. Arai:Sysmex Corporation: Employment. Kitazawa:Chugai Pharmaceutical Co.: Employment, Equity Ownership, Patents & Royalties; Sysmex Corporation: Patents & Royalties. Takaoka:Sysmex Corporation: Employment. Hattori:Chugai Pharmaceutical Co.: Employment, Equity Ownership, Patents & Royalties. Shima:F. Hoffmann-La Roche Ltd.: Consultancy, Honoraria, Membership on an entity’s Board of Directors or advisory committees; Chugai Pharmaceutical Co., Ltd.: Consultancy, Honoraria, Membership on an entity’s Board of Directors or advisory committees, Patents & Royalties, Research Funding; Sysmex Corporation: Patents & Royalties, Research Funding.

Activation of Coagulation and Fibrinolysis in Acute Respiratory Distress Syndrome: A Prospective Pilot Study.

Coagulation and fibrinolysis remain sparsely addressed with regards to acute respiratory distress syndrome (ARDS). We hypothesized that ARDS development might be associated with changes in plasma coagulation and fibrinolysis. Our aim was to investigate the relationships between ARDS diagnosis and plasma concentrations of tissue factor (TF), tissue plasminogen activator (t-PA), and plasminogen activator inhibitor-1 (PAI-1) in mechanically ventilated patients at increased risk of developing ARDS. We performed an ethically approved prospective observational pilot study. Inclusion criteria were patients with PaO2/FiO2 < 300 mmHg admitted to the intensive care unit (ICU) for mechanical ventilation for 24 h, or more, because of one or more disease conditions associated with increased risk of developing ARDS. Exclusion criteria were age below 18 years; cardiac disease. We sampled plasma prospectively and compared patients who developed ARDS with those who did not using descriptive statistics and chi-square analysis of baseline demographical and clinical data. We also analyzed plasma concentrations of TF, t-PA, and PAI-1 at inclusion (T0) and on third (T3) and seventh day (T7) of the ICU stay with non-parametric statistics inclusive their sensitivity and specificity associated with the development of ARDS using receiver operating characteristic curve analysis. Statistical significance: p < 0.05. Of 24 patients at risk, 6 developed mild ARDS and 4 of each moderate or severe ARDS, respectively, 3 ± 2 (mean ± SD) days after inclusion. Median plasma concentrations of TF and PAI-1 were significantly higher at T7 in patients with ARDS, as compared to non-ARDS. Simultaneously, we found moderate correlations between plasma concentrations of TF and PAI-1, TF and PaO2/FiO2, and positive end-expiratory pressure and TF. TF plasma concentration was associated with ARDS with 71% sensitivity and 100% specificity, a cut off level of 145 pg/ml and AUC 0.78, p = 0.02. PAI-1 displayed 64% sensitivity and 100% specificity with a cut off concentration of 117.5 pg/ml and AUC 0.77, p = 0.02. t-PA did not change significantly during the observation time. This pilot study showed that increased plasma concentrations of TF and PAI-1 might support ARDS diagnoses in mechanically ventilated patients after 7 days in ICU.

Activation of the tissue factor-dependent extrinsic pathway and its relation to JAK2 V617F mutation status in patients with essential thrombocythemia.

Thrombotic complications may occur in 7.6-29.4% of patients with essential thrombocythemia. According to the cellular theory, tissue factor (TF) activating extrinsic blood coagulation pathway is essential for the activation of blood clotting. The aim of the study was to evaluate the activation of the TF-dependent extrinsic pathway in patients with essential thrombocythemia, depending on the presence or absence of the Janus kinase 2 (JAK2) V617F mutation. The study included 74 newly diagnosed patients (F/M: 47/27; mean age 61 years) with essential thrombocythemia (Tefferi and Vardiman, Leukemia 2008; 22(1):14-22). Patients were diagnosed in the Department of Clinical Hematology and Hematological Malignancies University Hospital No. 2, Bydgoszcz, Poland. The control group consisted of 30 healthy volunteers (F/M: 17/13; mean age 49 years). The concentration and activity of TF and TF pathway inhibitor (TFPI) were measured using ELISA method. In patients with essential thrombocythemia, we observed a higher concentration of TF [median (Me) = 686.90 vs 164.28 pg/ml] and over 10-fold higher activity of TF (Me = 46.05 vs 4.01 pmol/l) when compared with the control group. We also reported significantly higher activity of TFPI compared with the control group (Me = 1.93 vs 1.78 U/ml). Moreover, a concentration of TFPI was significantly lower in patients with essential thrombocythemia with JAK2 V617F mutation as compared with patients without the mutation (Me = 1.90 vs 2.16 U/ml; P = 0.039639). Increased TF activity and concentration is responsible for higher procoagulant potential in patients with essential thrombocythemia. Reduced activity of TFPI in patients with essential thrombocythemia with JAK2 V617F mutation indicates an increased prothrombotic risk in this group of patients.

Reduction of coagulability state one year after bariatric surgery

BackgroundObese patients are in a hypercoagulable state relative to normal-weight patients. Low-grade inflammation may be a key factor for this condition. ObjectivesOur study aimed to compare the coagulability state of morbidly obese patients before and 1 year after bariatric surgery (BS) using the Thrombin Generation (TG) test, a validated method to assess coagulation in vitro. SettingUniversity hospital. MethodsAll patients undergoing BS between September 1, 2014 and April 30, 2015 were eligible for this prospective study (n = 42). Two distinct reagents were used for TG initiation based on the tissue factor concentration (Reagents LOW and HIGH). The main outcomes were endogenous thrombin potential (ETP) and peak height of TG. The rate of follow-up after one year was 97%. ResultsOne year after surgery, %weight loss was 32.5±8.4%; CRP decreased from 9.0 (3.7–12.9) to 1.1 (0.3–2.8) mg/mL (P<.001) and fibrinogen from 4.2±.8 to 3.5±.8 g/L (P<.001). The ETP (%) decreased from (108.0 (95.0–117.0) to 78.0 (71.0–98.0) (P<.001) (LOW reagent) and from 113.0 (103.0–134.0) to 96.0 (86.0–107.0) (P<.001) (HIGH reagent). Peak height (%) decreased from (117.0 (92.0–139.0) to 82.0 (70.0–111.0) (P = .003) (LOW reagent) and from 106.0 (96.0–118.0) to 97.0 (87.8–105.2) (P = .003) (HIGH reagent). ConclusionOur study shows a significant reduction in TG potential one year after BS in morbidly obese patients. Reduction of low grade inflammation may be one of the underlying mechanisms.

Reduced fibrinolytic resistance in patients with factor XI deficiency. Evidence of a thrombin‐independent impairment of the thrombin‐activatable fibrinolysis inhibitor pathway

Essentials Plasma of factor XI-deficient patients (FXI-dp) displays enhanced fibrinolysis. We investigated the role of thrombin activatable fibrinolysis inhibitor (TAFI) in 18 FXI-dp. FXI-dp generated less activated TAFI (TAFIa) on clotting challenge and were resistant to TAFIa. TAFI activation and TAFIa resistance correlated with bleeding score and bleeding phenotype. Background Factor XI (FXI) deficiency, a rare disorder with unpredictable bleeding, has been associated with reduced fibrinolytic resistance as a result of abnormal fibrin density. Objective We investigated the involvement of thrombin-activatable fibrinolysis inhibitor (TAFI) in the increased lysability of FXI-deficient (FXI-def) clots and the role of thrombin. Patients/Methods Eighteen patients with FXI deficiency (1-58%) and 17 matched controls were investigated for fibrinolytic resistance to t-PA, thrombin generation, TAFI activation and response to TAFIa. Results When clotting was induced by 0.5 pm tissue factor (TF), FXI-def plasmas displayed less thrombin and TAFIa generation and shorter lysis time than controls. A 100-fold higher TF concentration (to bypass FXI) abolished the difference in thrombin generation but not in lysis time between patients and controls. Normalization of FXI levels by a FXI concentrate increased thrombin generation but had no effect on the lysis time of FXI-def plasma. Moreover, when clots were induced by purified thrombin and high concentrations of FXa inhibitor, FXI-def plasma still generated less TAFIa and displayed a shorter lysis time than controls. Finally, upon TAFIa addition, the lysis time of FXI-def plasma was prolonged significantly less than that of control plasma, suggesting a TAFIa resistance. TAFIa generation and TAFIa resistance were correlated with the bleeding score, displaying a considerable capacity to discriminate between patients with and without bleeding. Conclusions TAFI pathway impairment, largely caused by a hitherto unknown TAFIa resistance, appears to be one main cause of decreased fibrinolytic resistance in FXI deficiency and might be clinically useful for assessing the bleeding risk of FXI-def patients.

Haemostatic factors do not account for worse outcomes from ischaemic stroke in patients with higher C-reactive protein concentrations.

Background Although the role of microparticles was recently implicated in stroke pathophysiology, the association between microparticles and inflammation is still not fully understood. The aim of this cohort study of 66 patients was to assess a relation between haemostatic factors, C-reactive protein and clinical outcome of ischaemic stroke. Methods Plasma microparticles procoagulant activity, concentrations of tissue factor-bearing microparticles, tissue factor and tissue factor pathway inhibitor in ischaemic stroke patients were determined with enzyme-linked immunosorbent assays at the time of initial diagnosis, along with serum C-reactive protein concentrations. Patients were divided into two groups depending on their C-reactive protein concentrations (C-reactive protein <3 mg/L; n = 28 vs. C-reactive protein ≥3 mg/L; n = 38). The analysed clinical outcome measures included the National Institutes of Health Stroke Scale and the Barthel Index. Results The two C-reactive protein groups did not differ significantly in terms of microparticles procoagulant activities, tissue factor-bearing microparticles, tissue factor and tissue factor pathway inhibitor concentrations. A significant correlation was observed between tissue factor pathway inhibitor and National Institutes of Health Stroke Scale score at admission ( R = 0.3, P = 0.03). Patients with C-reactive protein ≥3 mg/L presented with significantly higher National Institutes of Health Stroke Scale scores (median, 9.00 vs. 5.50, P = 0.002) and lower Barthel Index scores (median, 20.00 vs. 65.00, P = 0.002) than individuals with C-reactive protein <3 mg/L. The C-reactive protein concentrations correlatedpositively with National Institutes of Health Stroke Scale scores ( R = 0.3, P = 0.02) and inversely with Barthel Index scores ( R = - 0.4, P = 0.002). Conclusions Altogether, these findings imply that haemostatic parameters (microparticles, tissue factor-bearing microparticles, tissue factor, tissue factor pathway inhibitor) do not account for elevated C-reactive protein concentrations in ischaemic stroke patients.