Thrombin generation assay for hemostasis assessment in trans- latitudinal Arctic voyage

2021 ◽  
Author(s):  
Н.А. Воробьева ◽  
А.И. Воробьева ◽  
А.А. Марусий
Keyword(s):  
Thrombin Generation ◽  
Zero Point ◽  
Lag Phase ◽  
Time To Peak ◽  
Thrombin Generation Assay ◽  
Franz Josef Land ◽  
Arctic Sea ◽  
Background Data ◽  
In Trans ◽  
New Perspective

Введение. Существуют данные об активации системы гемостаза в виде формирования гиперкоагуляции при вахтовой работе в циркумполярных территориях. Новым перспективным методом, способным оценить как гипер-, так и гипокоагуляционные состояния, является тест генерации тромбина (ТГТ). Цель исследования: анализ динамики параметров ТГТ в условиях трансширотного морского арктического рейса. Материалы и методы. Выполнено проспективное клинико-лабораторное исследование во время трансширотной экспедиции «ТрансАрктика-2019». Отбор образцов плазмы для ТГТ у 52 членов экспедиции выполнен в нулевой точке (г. Архангельск, 64°33’ с. ш., 40°32’ в. д.) до выхода судна в рейс и в самой высокой точке экспедиции (остров Хейса, архипелаг Франца–Иосифа, 80°34’ с. ш., 57°41’ в. д.) на 18–20-е сутки арктического рейса. Параметры кинетики тромбина определены на анализаторе Ceveron- alpha с TGA-модулем (Technoclone, Австрия). Результаты. В высокой точке транширотного рейса время лаг-фазы (tLag) и время достижения пика тромбина (tPeak) были значимо меньше, чем в исходной нулевой точке, что указывало на состояние гиперкоагуляции. Значения эндогенного потенциала тромбина (AUC) у членов экспедиции были существенно больше в высокой точке экспедиции. Заключение. Нахождение в высоких широтах возможно связано с формированием состояния гиперкоагуляции. Background. Data are exist about hemostasis activation in the form of hypercoagulation during rotational work in circumpolar territories. Thrombin generation assay (TGA) is a new perspective method for assessing both hyper- and hypocoagulation. Objectives: to analyze the dynamics of TGA parameters in trans- latitudinal Arctic sea voyage. Patients/Methods. A prospective clinical and laboratory study was carried out in 52 expedition members during the trans- latitudinal expedition “TransArktika-2019”. Plasma samples for TGA were taken at the zero point (Arkhangelsk, 64°33’ N, 40°32’ E) prior to the ship’s voyage and at the highest point of the expedition (Heiss Island, Franz Josef Land, 80°34’ N, 57°41’ E) on 18–20 days of the voyage. Thrombin kinetic parameters were determined on a Ceveron-alpha analyzer with a TGA-module (Technoclone, Austria). Results. At the highest point of the trans-latitudinal voyage, the lag phase (tLag) and the time to peak thrombin (tPeak) were statistically less than at the initial zero point that pointed to hypercoagulation. The endogenous thrombin potential (AUC) values were significantly higher at the highest point. Conclusions. Location in high latitudes is possibly associated with the formation of hypercoagulation.

Factor VIII Efficacy and Tissue Factor Levels in Hemophilia A Patients Undergoing Total Knee Replacement.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 4029-4029
Author(s):  
Wolfgang Wegert ◽  
Manuela Krause ◽  
Inge Scharrer ◽  
Ulla Stumpf ◽  
Andreas Kurth ◽  
...  
Keyword(s):  
Total Knee Replacement ◽  
Plasma Levels ◽  
Thrombin Generation ◽  
Hemophilia A ◽  
Lag Time ◽  
Major Surgery ◽  
Plasma Samples ◽  
Time To Peak ◽  
Thrombin Generation Assay ◽  
Total Knee

Abstract The changes of tissue factor (TF) blood levels in patients undergoing major surgery has been reported presenting controversial data. Whether this TF is hemostatically active or if it interacts with other coagulation factors, e.g. FVIII, is still unclear, making thrombotic risk and complications assessment for even more difficult. We analyzed plasma samples from four male patients aged 27–55 with severe hemophilia A without inhibitory antibodies, undergoing total knee replacement, which all gave informed consent. Initial FVIII doses before intervention was 75–80 U/kg. Treatment following intervention was targeted at 100 % FVIII serum levels. None received heparin. No bleeding events occurred during the observation period. The samples were taken at these timepoints (TP): 1. before preoperative FVIII substitution, 2. at the time of first incision (intervention start), 3. at circulation arrest release + 90 s after prosthesis implantation, 4. final suture (intervention end), 5. 24 h and 6. 48 h after intervention to assay procedurally induced TF production. Coagulation analyses were carried out using a fluorometric thrombin generation assay (TGA) in platelet rich plasma (PRP), RoTEG (rotation thrombelastography) in whole blood and a TF ELISA for the plasma samples’ TF levels. Both clotting function tests were started using TF diluted 1:100.000 and calcium chloride 16,7 mM (final conc.). TGA parameters were ETP, PEAK (maximum thrombin generation velocity), TIME TO PEAK, LAG TIME. TGA parameters directly related to thrombin activity (ETP; PEAK) showed no change during the intervention, but a sharp decrease 24 h later with a partial recovery 48 h later. TGA time marks (TIME TO PEAK, LAG TIME) changed in an inverse way, except for the difference from LAG TIME and TIME TO PEAK, which shortened continously after circulation arrest removal. RoTEG was characterized using 4 parameters: clotting time (CT), clot formation time (CFT), maximum clot firmness (MCF) and clot formation rate (CFR). After preoperative FVIII substitution, CT decreased by 10 % and CFT by 50 % in 48 h. MCF stayed unchanged during the intervention and the following 24 h, but increased by 20 % at 48 h. CFR increased by 10 % during intervention, and by 20 % from 24 to 48 h. TF ELISA showed preoperative TF plasma levels from 11 to 271 pg/ml. After release of circulation arrest (TP 3) TF concentration increased sharply (4 times the initial value), which was not detectable in the samples taken at TPs 2 and 4. TF levels further increased at TPs 5 and 6 to 170 % and 317 % resp. Altogether, TF plasma levels elevated after major surgery seem to correspond to a potential risk factor for postoperative thrombosis, especially when elevation is induced after intervention. However, functional coagulation assays do not change uniformly, as the thrombin generation assay reflects no marked changes under intervention, but in the period after(24–48 h). Changes in the RoTEG whole blood clotting assay are not dramatic but indicate a thrombophilic shift in coagulation balance also pronouned at 24–48h, too. These results demonstrate that increased coagulability after orthopedic surgery detected using functional clotting assays correlates with increased TF levels, but further studies must be performed to prove this relation in healthy individuals.

Comparison of Thrombin Generation of Sickle Cell Patients in Microparticle Rich and Microparticle Poor Plasma Using Thrombin Generation Assay (TGA).

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2557-2557
Author(s):  
Nirmish Shah ◽  
Marilyn J. Telen ◽  
Thomas L. Ortel
Keyword(s):  
Steady State ◽  
Linear Regression ◽  
Regression Models ◽  
Thrombin Generation ◽  
Lag Phase ◽  
Linear Regression Models ◽  
Acceleration Phase ◽  
Thrombin Generation Assay ◽  
Free Plasma ◽  
D Dimer

Abstract Abstract 2557 Poster Board II-534 Introduction: The pathophysiology of sickle cell disease (SCD) is complex, with increasing evidence of abnormalities in nearly every component of hemostasis and a pronounced prothrombotic state. Many factors contribute to this prothrombotic state, including: 1) an increase in thrombin production and 2) an increase in circulating procoagulant microparticles. Currently little is known about procoagulant microparticles and their role in thrombin generation in SCD. We have therefore attempted to define the role of thrombin and procoagulant microparticles in SCD utilizing a thrombin generation assay (TGA) and to correlate the results with D-dimer levels, which are known to be abnormal in SCD. The use of TGA allows examination of lag phase, acceleration phase, peak thrombin, and endogenous thrombin potential (ETP). Patients and Methods: The study included a total of 20 HbSS adult and pediatric patients (mean age=22 years, range 3–35) who were admitted for vaso-occlusive crisis. Patients were excluded if they had been admitted within the last 2 weeks, were on chronic transfusion, or were on antiplatelet or anticoagulant medications. Blood was drawn within 36 hours of admission (crisis) and again following discharge, at least 2 weeks later (steady state). Nine of the enrolled patients were followed from inpatient to an outpatient follow up visit. All plasma was promptly separated and frozen, stored at −80C, thawed once and subsequently tested within 4 hours. For each patient, D-dimer and TGA were performed on platelet poor plasma (PPP). In addition, following initial thaw of PPP, samples were spun for 30 minutes at 30,000g to remove microparticles and isolate particle free plasma (PFP). TGA was also performed on PFP from both inpatient and outpatient samples. Results: The mean D-dimer during inpatient crisis (5358 ± 2052 ng/ml) was significantly higher than during outpatient steady state (1256 ± 298, p=0.042). In comparing crisis and steady state by TGA, there was a significant increase in mean ETP (1267 ± 56 nM vs 923 ± 231, p=0.032) and mean acceleration phase (5.14 ± 0.34 min vs 14.21 ± 6.75, p=0.038). Both lag phase (p=0.066) and peak thrombin (p=0.057) were not statistically different between crisis and steady states. Analysis of PPP and PFP by TGA revealed that all phases including ETP (p=0.070) and peak thrombin (p=0.080) were not statistically different between microparticle rich and poor plasma. Linear regression models for inpatient D-dimer versus age were also performed and a significant decrease in D-dimer was seen with increasing age (r2=0.20, p=0.044). This decreasing trend with age was also seen with outpatient D-dimer, although it did not reach significance (r2=0.21, p=0.076). There was no significant trend seen in linear regression models performed for TGA phases versus age. Linear regression models were performed for D-dimer with each phase of TGA and no significant correlation or trend was seen. Conclusions: Our initial results using TGA to evaluate the increase in hypercoagulability seen in patients with SCD during an acute crisis indicate that the expected increase is also seen with certain phases of the TGA, when compared to outpatient non-crisis state. ETP and acceleration phase were significantly elevated during crisis, indicating that the rate of thrombin production and total thrombin are abnormally increased during this time. Peak thrombin and lag phase also approached significance. In evaluating the significance of microparticles, we were unable to show a significant decrease in thrombin production in microparticle free plasma by use of the TGA. With inclusion of both pediatric and adult patients, it was also found that D-dimer significantly decreased with age, which is not consistent with other chronic disease states which exhibit a gradual increase with age. It is possible that a broader age range of patients may be needed to better determine this trend. Finally, comparison of D-dimer with each phase of TGA did not reveal a significant correlation. This may be due to other influencing factors on D-dimer which do not affect TGA. Thus, TGA appears to be a promising technique with which to assess thrombin generation and the hypercoagulable state in SCD; further studies of increased numbers of patients are needed to validate the use of TGA, and define the contribution of microparticles, in SCD. Disclosures: Shah: Thrasher Foundation: Research Funding.

Association rate constants rationalise the pharmacodynamics of apixaban and rivaroxaban

2015 ◽  
Vol 114 (07) ◽  
pp. 78-86 ◽  
Author(s):  
Georges Jourdi ◽  
Virginie Siguret ◽  
Anne Céline Martin ◽  
Jean-Louis Golmard ◽  
Anne Godier ◽  
...  
Keyword(s):  
Thrombin Generation ◽  
Factor Xa ◽  
Lag Time ◽  
Lower Sensitivity ◽  
Association Rate ◽  
Time To Peak ◽  
Thrombin Generation Assay ◽  
Direct Inhibitors ◽  
Association Rate Constants ◽  
Kinetics Of

SummaryRivaroxaban and apixaban are selective direct inhibitors of free and prothrombinase-bound factor Xa (FXa). Surprisingly prothrombin time (PT) is little sensitive to clinically relevant changes in drug concentration, especially with apixaban. To investigate this pharmacodynamic discrepancy we have compared the kinetics of FXa inhibition in strictly identical conditions (pH 7.48, 37 °C, 0.15 M). KI values of 0.74 ± 0.03 and 0.47 ± 0.02 nM and kon values of 7.3 ± 1.6 106 and 2.9 ± 0.6 107 M-1 s-1 were obtained for apixaban and rivaroxaban, respectively. To investigate if these constants rationalise the inhibitor pharmacodynamics, we used numerical integration to evaluate impact of FXa inhibition on thrombin generation assay (TGA) and PT. Simulation predicted that in TGA triggered with 20 pM tissue factor, 100 ng/ml apixaban or rivaroxaban increased 1.8– or 3.0-fold the lag time and 1.4– or 2.0-fold the time to peak, whilst decreasing 1.2– or 3.1-fold the maximum thrombin and 1.7– or 3.5-fold the endogenous thrombin potential. These numbers were consistent with those obtained through the corresponding TGA triggered in plasma spiked with apixaban or rivaroxaban. Simulated PT ratios were also consistent with the corresponding plasma PT: markedly less sensitive to apixaban than to rivaroxaban. Analogous differences in TGA and PT were obtained irrespective of the drug amount added. We concluded that kon values for FXa of apixaban and rivaroxaban rationalise the unexpected lower sensitivity of PT and TGA to the former.

Hypercoagulability in Pediatric Patients with Sickle Cell Disease and Correlation to Transcranial Doppler and Disease Severity

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 3221-3221
Author(s):  
Nirmish Shah ◽  
Jennifer Fowler ◽  
Thomas L. Ortel ◽  
Courtney Thornburg
Keyword(s):  
High Risk ◽  
Sickle Cell ◽  
Research Funding ◽  
Thrombin Generation ◽  
Lag Phase ◽  
Cell Disease ◽  
Moderate Risk ◽  
Time To Peak ◽  
Severity Scores ◽  
D Dimer

Abstract Abstract 3221 Children with sickle cell disease (SCD) are at high risk for thrombotic stroke. Transcranial doppler ultrasound (TCD) is utilized to predict children at highest risk. Anemia and and vessel occlusion are assocated with elevated TCD velocities and risk of stroke. There is increasing evidence that a prothrombotic state contributes to the complications of SCD including stroke. We hypothesized that increased thrombin generation and platelet activation are associated with increased TCD velocities. We conducted a cross-sectional cohort study of children with SCD at Duke University Medical Center. Children had been clinically well for at least 2 weeks, had not been transfused for at least 3 months, and had TCD performed at study enrollment (n= 28) or within the prior 6 months (n=7). TCD time-averaged mean velocities (TAMV) were calculated for each patient and used for analysis. Blood was collected at the time of study entry per study protocol. Thrombin generation was measured utilizing: D-dimer; thrombin antithrombin (TAT); and calibrated automated thrombography (CAT) which evaluates phases of thrombin production including lag phase, time to peak thrombin, endogenous thrombin potential (ETP), and peak thrombin. Platelet activation was assessed by measurement of soluble glycoprotein V (sGPV). Clinical data were abstracted from the medical record including confirmation of SS genotype, use of hydroxyurea, prior sickle cell-related clinical events, and hematological data including hemoglobin, platelets and white blood cell count. SCD severity scores were calculated to classify patients as either high risk or low/moderate risk (van den Tweel et al, 2010). Linear regression analyses were conducted to assess correlation. Children with high risk severity scores were compared to children with low/moderate risk severity scores. Statistical analysis was performed using Graphpad 5.0; p<0.05 was considered significant. We evaluated 35 patients (median age 10 years, range 3 – 17). Linear regression revealed a correlation between the TCD velocities for TAT (r2=0.147, p=0.03), ETP (r2=0.13, p=0.049), and peak thrombin (r2=0.12, p=0.05). There was no significant correlation between TCD velocity and D-dimer, lag phase or time to peak thrombin. There also was no correlation between TCD velocities and hemoglobin, platelets or sGPV. Patients taking hydroxyurea were found to have lower thrombin generation (D-dimer: 882 ± 93 ng/ml vs. 1679 ± 428, p<0.0001) and TAT (6.8 ± 0.67 ng/ml vs. 10.59 ± 2.22, p<0.0001). There were 8 (23%) patients classified as high risk and 27 (77%) classified as low/moderate risk. Comparison of comparison of D-dimer, TAT, and each phase of thrombin generation between patients with low/moderate severity scores and those classified as high severity revealed no significant differences. In summary, increased TAT, ETP, and peak thrombin levels correlated with increased TCD velocities. Markers of thrombin generation were lower in children taking hydroxyurea. The lag phase and time to peak thrombin which both reflect the ‘thrombin burst’, were not predictive of TCD velocities. In addition, hematological values and platelet activation did not correlate with TCD velocities. Further studies are warranted to understand the contribution of thrombin generation to stroke risk, to determine if markers of thrombin generation are independent markers of stroke risk, and determine if hydroxyurea can ameliorate this risk. Disclosures: Shah: Adventrx: Consultancy; Eisai: Research Funding. Off Label Use: Hydroxyurea for use in pediatric sickle cell disease. Ortel:Eisai: Research Funding; Glaxo SmithKline: Research Funding; Pfizer: Research Funding; Instrumentation Laboratory, Inc: Consultancy, Research Funding; Boehringer Ingelheim: Consultancy.

scholarly journals Mechanistic Studies of Thrombin Generation Assay to Evaluate Procoagulant Potential of Fitusiran

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 6-6
Author(s):  
Sravya Kattula ◽  
Ane Salvador ◽  
Shaobin Wang ◽  
Ayman Ismail ◽  
Arjan van der Flier ◽  
...  
Keyword(s):  
Complex Formation ◽  
Thrombin Generation ◽  
Active Role ◽  
Peak Height ◽  
Publicly Traded ◽  
Time To Peak ◽  
Thrombin Generation Assay ◽  
Hemostatic Activity ◽  
The Impact

Introduction: Fitusiran is a once-monthly subcutaneously administered RNA interference therapeutic targeting antithrombin III (AT) to rebalance hemostasis in patients with hemophilia A and B with or without inhibitors. As for other non-FVIII therapies in development, there is a need to better assess the procoagulant potential of fitusiran. Using thrombin generation assays (TGA), we studied the hemostatic activity in hemA plasma with low AT levels in the presence and absence of varying factor VIII (FVIII) levels to mimic conditions associated with breakthrough bleeds. To better understand the impact of AT depletion in the assay, we monitored multiple TGA parameters as well as the levels of several coagulation components potentially influencing the reactions. Methods: Pooled HemA plasma was applied to an AT affinity resin to remove AT. Residual AT activity was 5% and additional AT was spiked back to produce HemA plasmas with various amounts of AT (5, 10, 20 and 100%). FVIII was spiked into the 4 plasmas at 0, 5, 10, 20, 30, 50, and 100 IU/dL and all samples were analyzed by Calibrated Automated Thrombography (Stago). Thrombin generation parameters were calculated by thrombinoscope software.After the TGA reaction, samples were further analyzed by immunoblot for evaluation of thrombin-AT (TAT) and alpha2-macroglobulin-thrombin (a2M:T) complex formation, as well as remaining (pro)thrombin, AT and a2M levels . In parallel, a2M:T complex levels were assessed by ELISA capture of a2M followed by addition of thrombin substrate to directly measure the thrombin activity stemming from a2M-inhibited thrombin. . Results: TGA parameters for FVIII titrations in HemA plasmas with reduced AT were calculated and compared to the FVIII standard curves generated in HemA plasma with normal AT levels for each TGA parameter (lag time, time to peak, peak height, ETP and velocity index). As expected, a greater reduction of AT activity in hemA plasma correlated with increased thrombin generation compared to hemA plasma (with 100% AT). The apparent FVIII-like activity observed at low AT conditions in HemA plasma was dependent on the TGA parameter evaluated, with velocity index suggesting the lowest and ETP the highest FVIII-like activity. Furthermore, particularly at lower starting levels of AT, AT is depleted during the TGA reaction and leads to enhanced levels of a2M:T complex as observed by immunoblot or a2M:T chromocapture assays. Importantly, the a2M:T complex is capable of cleaving the assay substrate and contributing to the TGA signal. Typically, the a2M:T generated signal is calculated and substracted by the thrombinoscope software. However, under low AT conditions, increased levels of a2M-T lead to substrate consumption at later stages of the reaction, resulting in erroneous underestimation of the a2M:T activity that does not agree with direct measurement of a2M: T levels. These limitations need to be taken into consideration when interpreting TGA data under conditions of low AT activity. While this remains an area of investigation, parameters derived earlier in the reaction prior to AT depletion and significant levels of a2M:T complex formation may better correlate with the true hemostatic potential associated with AT inhibition. Conclusion: Our results show that TGA can be a valuable tool to measure the hemostatic potential associated with fitusiran therapy. We demonstrate that reduced levels of AT are associated with enhanced thrombin generation, but that activity comparison to FVIII varies significantly depending on the TGA parameter analyzed. In addition we show that under low AT conditions, AT can become depleted leading to a2M:T complex accumulation which may lead to overestimation of some TGA parameters. This overestimation is likely an artifact of the TGA method as it is a closed in vitro system that can be influenced by the depletion of components or accumulation of products. Our results suggest that TGA parameters calculated early in the reaction are less influenced by some of these limitations and may be better predictors of the true hemostatic activity of fitusiran. Furthermore, a2M may play a more active role during fitusiran treatment, ensuring free thrombin does not migrate outside of the site of injury. Disclosures Kattula: Sanofi: Current Employment, Current equity holder in publicly-traded company. Salvador:Sanofi: Current Employment, Current equity holder in publicly-traded company. Wang:Sanofi: Current Employment, Current equity holder in publicly-traded company. Ismail:Sanofi: Current Employment, Current equity holder in publicly-traded company. van der Flier:Sanofi: Current Employment, Current equity holder in publicly-traded company. Leksa:Sanofi: Current Employment, Current equity holder in publicly-traded company. Salas:Sanofi: Current Employment, Current equity holder in publicly-traded company.

Thrombin Generation Assay and by-Passing Therapies in Patients with Inhibitors,

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 3322-3322
Author(s):  
Kaan Kavakli ◽  
Yilmaz Ay ◽  
Deniz Yilmaz Karapinar ◽  
Can Balkan
Keyword(s):  
Thrombin Generation ◽  
Hemophilia A ◽  
Lag Time ◽  
Advisory Committees ◽  
Surgical Interventions ◽  
Time To Peak ◽  
Thrombin Generation Assay ◽  
Clinical Responses ◽  
Factor Therapy ◽  
Bleeding Episodes

Abstract Abstract 3322 No routine test is available for monitoring bypassing agent therapy in patients with inhibitors. General clotting tests such as PT, aPTT and factor activities do not reflect overall thrombin generation and are insensitive to hypercoagulation and hypocoagulation. In this study, we aimed to investigate the importance of thrombin generation assay (TGA) in monitoring hemostasis during factor supplementation in hemophilia patients with and without inhibitor. Study group consisted of 39 patients (27 hemophilia A, 8 hemophilia B, one vWD-3, two FVII def. and one FX def.). All patients were male. Seven out of 27 hemophilia A patients had an inhibitor. The mean age was 17±9.7 years (range: 3–55). A total of 59 (inhibitor positive=19) bleeding episodes and/or surgical interventions were evaluated. Acute bleeding episodes were classified as acute hemarthrosis (n=21), soft tissue hemorrhages (n=6), oral /nasal hemorrhages (n=7), hematuria (n=2) or GIT hemorrhages (n=3). Twenty major or minor surgical interventions were classified as radioisotope synovectomy (n=11), teeth extractions (n=2), circumcision (n=1), port-a-cath removal (n=1); hemorrhoid operation (n=1) or open synovectomy and arthroplasty n=4). rFVIIa and aPCC therapies were administered to inhibitor-positive patients. PPP was always prepared within 30 min of venipuncture. PT, aPTT and FVIII activities were measured in plasma from citrated blood samples taken before treatment of patients for bleeding or for surgery preparation. Before TGA test, aliquoted PPP was stored for 3 months at −80°C, and all samples were assessed together. Lab parameters were evaluated at basal levels and after 1 hour of factor therapy. These tests were repeated when clinical hemostasis was obtained within 24 hours of the post-op period. PT, aPTT, FVIII activities were assessed by Siemens kits with CA-1500 (Sysmex–Siemens). The Calibrated Automated Thrombogram (CAT; Thrombinoscope BV, Maastricht, The Netherlands) method was used for the TGA. No difference was found between 1th and 24th hour clinical responses after rFVIIa and aPCC treatment (P= 0.96) in hemophilic patients with inhibitor. Basal, first hour and 24th hour TGA lag time values were significantly shorter in hemophilic patients with inhibitor than in inhibitor-free patients (P= <.01). Basal TGA parameters (ETP, peak thrombin height, and time to peak values were significantly better in hemophilic patients without inhibitor than in those with inhibitor (P= <.01). No difference was found between the first hour and 24th hour TGA parameter values between hemophilic patients without/with inhibitor after factor therapy. TGA lag time values measured 24 hours post-event were significantly shorter in rFVIIa than aPCC treatment in hemophilic patients with inhibitor (P=.04). First hour ETP and first hour and 24 hour peak thrombin height were significantly higher in aPCC than rFVIIa treatment in hemophilic patients with inhibitor (P=.01). First hour TEG parameter (R, K and alpha angle degree) values were found significantly in aPCC (12 episodes)than rFVIIa treatment (7 episodes) (P=.03). First hour and 24th hour INR values were significantly shorter in rFVIIa than aPCC (P= <.01). No association was found between first hour clinical responses and TGA parameters after inhibitor bypassing agent treatment. In this study, before and after bleeding episode treatment/surgery prophylaxis, basal, 1st and 2–24th hours lag time values of inhibitor-positive patients were significantly smaller than inhibitor-negative patients. Basal TGA parameters such as ETP, peak thrombin height, and time to peak thrombin values of hemophilic patients without inhibitor were significantly better than those of hemophilic patients with inhibitor. When hemophilic patients with or without inhibitor were evaluated for accurate clinic responses at 24 hours after factor replacement treatment, the clinical response was significantly better in the inhibitor-negative group. As a conclusion, for monitoring hemostasis in inhibitor patients related severe bleeding and major operations, TGA test may be used for most rational using of by-passing therapies. Disclosures: Kavakli: NovoNordisk: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Baxter: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau.

scholarly journals Role of a thrombin generation assay in the prediction of infection severity

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Boaz Elad ◽  
Gilat Avraham ◽  
Naama Schwartz ◽  
Adi Elias ◽  
Mazen Elias
Keyword(s):  
Hospital Mortality ◽  
Thrombin Generation ◽  
Systemic Infection ◽  
Lag Time ◽  
Peak Height ◽  
Clinical Tool ◽  
Time To Peak ◽  
Thrombin Generation Assay ◽  
Failure Assessment

AbstractThrombin plays a central role in sepsis pathophysiology. The correlation of thrombin generation (TG) assays with infection severity and prognosis, and whether it can be used as a clinical tool, have been poorly explored and are the subjects of our research. We recruited 130 patients with systemic infection between 2016 and 2019. Patients were divided according to infection severity by using the sequential organ failure assessment (SOFA) and quickSOFA (qSOFA) scores. The hemostatic state was analyzed by Calibrated Automated Thrombogram. The primary end points were TG values and the secondary end point was in-hospital mortality. Patients with qSOFA ≥ 2 had a longer lag time (5.6 vs. 4.6 min) and time to peak (8 vs. 6.9 min) than those with lower scores (p = 0.014 and 0.01, respectively). SOFA ≥ 2 had a longer lag time (5.2 vs. 4.3 min), time to peak (7.5 vs. 6.7 min) and lower endogenous thrombin potential (ETP) (1834 vs. 2015 nM*min), p = 0.008, 0.019, and 0.048, respectively. Patients who died (11) had lower ETP (1648 vs. 1928 nM*min) and peak height (284 vs. 345 nM), p = 0.034 and 0.012, respectively. In conclusion TG assays may be a valuable tool in predicting infection severity and prognosis.

scholarly journals P121 The study of the hypercoagulable state in Crohn disease: Results of a prospective comparative study

2021 ◽  
Vol 15 (Supplement_1) ◽  
pp. S213-S214
Author(s):  
M Serghini ◽  
H Ben Youssef ◽  
A Laabidi ◽  
N Ben Mustapha ◽  
M Hafi ◽  
...  
Keyword(s):  
Comparative Study ◽  
Crohn Disease ◽  
Thrombin Generation ◽  
Hypercoagulable State ◽  
Time To Peak ◽  
Thrombin Generation Assay ◽  
Significant Difference ◽  
Inflammatory Phenotype ◽  
Prospective Comparative Study

Abstract Background The risk of thrombosis is increased in Crohn disease (CD). Abnormalities in hemostasis during CD have been evaluated in several studies but the results remain controversial. The thrombin generation assay (TGA) provides an overall assessment of hemostasis compared to traditional tests. The purpose of this work was to study the hypercoagulable state in patients with CD through TGA and to correlate it with the clinical and biological status of the disease. Methods This was a prospective comparative study, collating 50 patients diagnosed with CD and compared to 50 matched controls according with age and sex, between May 2016 and January 2020. TGA was effectuated in both groups. Data related to CD were collected. Parameters of lag time (min), time to peak (min), peak (nM), endogenous thrombin potential (ETP in nM.min) and velocity index (VI in nM/min) were analyzed. Laboratory parameters of inflammation, factor VIII and anti-coagulant factors were evaluated in patients. Results Median age of patients was 44.02 years (29 men and 21 women). The proportion of patients in remission was 66%. The mean levels of FVIII (285.1±175.2 vs 133.8±41.9; p=0.001), ProteinC (117±22.8 vs 103.2±13.6; p=0.003) and Antithrombin (97.1±16.9 vs 80.65±13.2; p=0.001) were significantly higher in patients with CD compared to controls. No significant difference was found for the rates of ProteinS. Thrombin generation was faster in patients with CD compared to controls (IV: 163.9±41.9 vs 138.2±67.4; p=0.04) and in cases of fistulizing or stenosing phenotype compared to the inflammatory phenotype. Thrombin generation was higher in patients: hospitalized compared to those seen as outpatients (ETP: 1515±406.6 vs 1248.2 + 380.8; p=0.05), treated with corticosteroids (ETP=1718.7 + 126.9) compared to the rest of the patients (p=0.03), with CRP&gt; 6 mg/L compared to those with normal CRP (ETP= 1515 + 384.8 vs 1245 + 348.2) (p=0.02) and with hyperfibrinogenemia level compared to those with normal fibrinogen (Peak = 351.6±74.1 vs 294.9 ±64.4) (p= 0.03). A significant positive correlation was noted between the parameters of TGT, CRP and fibrinogen. ETP value was significantly correlated with CRP (r=0.45; p=0.001) and fibrinogen (r=0.35; p=0.02). This allowed us to determine two biological scores correlating ETP and CRP (ETP=1384 + 2.9 x CRP), as well as fibrinogen level (ETP=1060.7 + 130.5 x Fibrinogen) Conclusion Thrombin generation appears to be more accelerated in patients with CD than in controls. This hypercoagulability is associated with hospitalization, stenosing and fistulizing phenotypes, elevated CRP and fibrinogen and corticosteroid therapy. The determination of ETP from CRP and fibrinogen would be a valuable tool for assessing the state of coagulability in these patients.

scholarly journals The in vitro addition of idarucizumab to plasma samples from patients increases thrombin generation

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Mojca Božič Mijovski ◽  
Rickard E. Malmström ◽  
Nina Vene ◽  
Jovan P. Antovic ◽  
Alenka Mavri
Keyword(s):  
Thrombin Generation ◽  
Ex Vivo ◽  
Lag Time ◽  
Plasma Samples ◽  
Blood Samples ◽  
Time To Peak ◽  
Thrombin Generation Assay ◽  
Coagulation Tests ◽  
Before And After

AbstractDabigatran interferes with many coagulation tests. To overcome this obstacle the use of idarucizumab as an in vitro antidote to dabigatran has been proposed. The aim of this study was to test the effect of idarucizumab as an in vitro antidote to dabigatran in ex vivo plasma samples from routine clinical patients examined by a thrombin generation assay (TGA). From 44 patients with atrial fibrillation five blood samples were collected. Thrombin generation was measured in all samples before and after the addition of idarucizumab. When idarucizumab was added to baseline plasma (no dabigatran), it caused a significantly shorter Lag Time and Time to Peak Thrombin, and a higher Peak Thrombin and Endogenous Thrombin Potential (ETP) of TGA. Similar results were obtained when idarucizumab was added to dabigatran-containing plasma, with TGA parameters comparable to baseline + idarucizumab plasma, but not to baseline plasma. In summary, our study showed that in vitro addition of idarucizumab to plasma samples from patients increases thrombin generation. The use of idarucizumab to neutralize dabigatran in patient plasma samples as well as the clinical relevance of in vitro increased thrombin generation induced by idarucizumab needs further investigation.

Antithrombin resistance rescues clotting defect of homozygous prothrombin-Tyr510N dysprothrombinemia

2021 ◽  
Author(s):  
Yeling Lu ◽  
Bruno O Villoutreix ◽  
Indranil Biswas ◽  
Qiulan Ding ◽  
Xuefeng Wang ◽  
...  
Keyword(s):  
Protein C ◽  
Thrombin Generation ◽  
Factor Xa ◽  
Peak Height ◽  
Prothrombin Activity ◽  
Time To Peak ◽  
Thrombin Generation Assay ◽  
Lower Reactivity ◽  
Specificity Constant ◽  
Abnormal Bleeding

A patient with hematuria in our clinic was diagnosed with urolithiasis. Analysis of the patient’s plasma clotting-time indicated that both APTT (52.6 s) and PT (19.4 s) are prolonged and prothrombin activity is reduced to 12.4% of normal, though the patient exhibited no abnormal bleeding phenotype and a prothrombin antigen level of 87.9%. Genetic analysis revealed the patient is homozygous for prothrombin Y510N mutation. We expressed and characterized the prothrombin-Y510N variant in appropriate coagulation assays and found that the specificity constant for activation of the mutant zymogen by factor Xa is impaired ~5-fold. Thrombin generation assay using patient’s plasma and prothrombin-deficient plasma supplemented with either wild-type or prothrombin-Y510N revealed that both peak height and time to peak for the prothrombin mutant are decreased however the endogenous thrombin generation potential is increased. Further analysis indicated that the thrombin mutant exhibits resistance to antithrombin and is inhibited by the serpin with ~12-fold slower rate constant. Protein C activation by thrombin-Y510N was also decreased ~10-fold, however, thrombomodulin overcame the catalytic defect. The Na+-concentration-dependence of the amidolytic activities revealed that the dissociation constant for the interaction of Na+ with the mutant has been elevated ~20-fold. These results suggest that Y510 (Y184a in chymotrypsin numbering) belongs to network of residues involved in binding Na+. A normal protein C activation by thrombin-Y510N suggests that thrombomodulin modulates the conformation of the Na+-binding loop of thrombin. The clotting defect of thrombin-Y510N appears to be compensated by its markedly lower reactivity with antithrombin, explaining patient’s normal hemostatic phenotype.

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