Thrombin Generation Studies Research Articles

Elevated Thrombin Generation Biomarkers and Their Relevance with Decreased Thrombin Generation Potential in Patients with Pulmonary Embolism

Background: Pulmonary embolism (PE) is a catastrophic complication in patients with venous thrombosis and is the third leading cardiovascular cause of death after myocardial infraction and stroke. Inflammation, hemostatic dysregulation, and generation of thrombotic mediators contribute to the pathogenesis of PE. Persistent thrombin generation contributes to the overall pathology of PE. The purpose of this study is to investigate endogenous thrombin potential (ETP) in PE patients and demonstrate its relationship with thrombin generation biomarkers, such as prothrombin fragment 1+2 (F1+2), thrombin anti-thrombin complex (TAT) and D-Dimer. Methods: Citrated blood samples from 201 patients with confirmed PE were collected under an IRB approved protocol. Fifty normal human plasma (NHP) samples were used as a reference. Thrombin generation studies were carried out by using fluorogenic substrate method (calibrated automated thrombogram, Diagnostica Stago; Paris, France). Thrombin generation parameters such as peak thrombin, lag time and endogenous thrombin potential (ETP) were compiled. Plasma samples were analyzed for such biomarkers as prothrombin fragment 1+2 (F1+2), thrombin anti-thrombin complex (TAT), and D-Dimer by using commercially available Sandwich ELISA methods. Results were compiled as mean ± SD and applicable statistical analysis was performed by using GraphPad Prism software. Result: PE patients exhibited a wide variation in thrombin generation parameters. ETP in PE patients decreased to 549.2±508.4 nM*min compared to NHP 701.8±214.6 nM*min, thrombin levels in PE patients also decreased to 83.7±80.1 nM compared to NHP 158.5±31.5 nM, while lag time increased 5.4±12.7 mins to 2.6±0.6 mins compared to NHP. Biomarker analysis showed elevated levels of F1+2 (972.3±1508.0 vs 294.2±105.0 pmol/L, 3.3-fold), TAT (34.9±54.3 vs 2.3±1.1ug/L, 15.3-fold), and D-Dimer (6720.0±4810.0 vs 182.6±205.8 ug/ml, 36.8-fold) compared to NHP. Endogenous thrombin potential levels demonstrate moderate and positive correlations with F1+2 (r = 0.22) while weak correlations were noted with TAT (r = 0.14) and D-Dimer (r = 0.05). F1+2 possess strong correlation with TAT (r = 0.55) and moderate for D-Dimer (r = 0.21). TAT also showed strong correlation with D-Dimer (r = 0.45). Thrombin generation parameters and biomarker levels based on PE severity has been shown in figure 1. Marked increase in thrombin generation biomarker levels were noted with increase PE severity. However, for the thrombin generation parameters, there is no differences were noted between the low grade and sub-massive PE, while the ETP and peak thrombin levels decreased, and lag time increased for the massive PE. Conclusion: These studies suggest that persistent thrombin generation in PE patients results in the consumption of prothrombin complex. However, such biomarkers of thrombin generation as F1+2, TAT and D-Dimer are formed, leading to higher circulating levels. Simultaneous measurement of thrombin generation biomarkers and thrombin generation parameters may be useful to determine the pathogenesis of thrombotic complications and optimization of treatment options. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Endogenous Thrombin Generation Potential in Patients with Pulmonary Embolism and Its Relevance to Thrombo-Inflammatory Biomarkers

Background: Pulmonary embolism (PE) is a catastrophic outcome in patients with venous thrombosis and third leading cardiovascular cause of death after myocardial infraction and stroke, estimates around 100,000 deaths annually.Inflammation, hemostatic dysregulation including fibrinolytic imbalance, endothelial compromise, and generation of thrombotic mediators, along with cellular defects and hemodynamic derangement contribute to the pathogenesis of PE.Endogenous thrombin generation contributes to the overall pathology of PE. The purpose of this study is to investigate endogenous thrombin potential (ETP) and the circulating biomarkers of fibrinolytic, inflammatory, and endothelial dysfunction in PE samples. Methods: Citrated blood samples from 400 patients with confirmed PE were collected at Loyola University Medical Center and Gottlieb Memorial Hospital under an IRB approved protocol. Normal human plasma (NHP) comprised of 25 male and 25 female samples were obtained from a commercial source. Thrombin generation studies were carried out using a commercially available a kinetic system. Endogenous GAG's contents were measured by fluorogenic quenching method. Thrombin generation parameters such as peak thrombin, lag time and area under the curve (AUC) were compiled. Plasma samples were retrospectively analyzed for biomarkers, including D-Dimer, PAI-1, tPA, TAFIa, vWF, CRP, IL6, TFPI and VEGF using commercially available sandwich ELISA methods. Results were compiled as mean ± SD and analyzed for significance and correlation by using GraphPad Prism software. Result: On a cumulative basis, PE patients showed a wide variation in thrombin generation parameters (Table 1). Biomarker analysis showed elevated levels of D-Dimer (37.05-fold), CRP (35.34-folds), IL6 (21.62-folds), tPA (4.69-folds), PAI-1a (3.95-folds), VEGF (2.38-folds), TFPI (2.17-folds), GAG's (1.92-folds), vWF (1.89-folds), and TAFIa (1.21-folds). Peak thrombin levels exhibited varying degree of positive and negative correlations with various biomarkers as illustrated in figure 1. Thrombin levels showed significant negative correlations with tPA, endogenous GAG's & TFPI, while positively correlated with VEGF. D-dimer showed positive correlations with CRP & IL6. PAI-1 demonstrated positive correlations with tPA, IL6, and endogenous GAG's. tPA showed negative correlation with TAFI, while positive correlations with endogenous GAG's, and TFPI. vWF showed positive correlations CRP, IL6, and endogenous GAG's. CRP showed positive correlations with IL6. Endogenous GAG's showed positive correlations with TFPI while TFPI showed negative correlation with VEGF. Conclusion: PE patients exhibit a wide variation in endogenous thrombin generation parameters. Overall, peak thrombin and ETP were decreased, whereas the lag time was increased. The increase in thrombo-inflammatory markers is suggestive of upregulation of thrombogenesis, and inflammatory processes in these patients. Thus, the profiling of thrombo-inflammatory biomarkers along with thrombin generation profile may provide additional information on the pathogenesis and severity of PE. However, biomarkers of hemostatic activation, vascular dysfunction and inflammation were increased. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Bio‐equivalence of Potency Adjusted Approved Heparin Solutions Compared to a Newly Developed Heparin Solution

IntroductionPorcine mucosal heparin solutions with a defined potency adjusted concentrations are widely used for the anticoagulation in medical, surgical and interventional indications. Recently, Sintetica has developed a 20.000 IU (48 ml, 416 IU/ml) solution. The active pharmaceutical ingredient used is a porcine mucosal origin with a specific activity of 194 U/mg. The purpose of this investigation is to compare the Sintetica heparin solution with other commercially available potency adjusted heparin solutions such as Fresenius, Roche, Hospira and Medefil heparins to demonstrate their bio‐equivalence in various biological assays.MethodCommercially available heparin solutions were obtained from Sintetica, Roche, Fresenius Kabi, Hospira and Medefil. USP reference standard was obtained from US Pharmacopeia. USP compliant anti‐Xa and anti‐IIa method based on chromogenic substrate assays were obtained from Aniara Diagnostics (Westchester, OH). Thrombin generation studies were carried out using a fluorokinetic method (Diagnostico France). The quantification of glycosaminoglycan (GAG) content was made by using a fluorescence probe (Red Probe, Munster, Germany). The clot based assays and amidolytic assays were carried out on ACL Elite analyzer. HIT mediated platelet aggregation studies were carried out using HIT antibodies from clinically diagnosed patient serum. All solutions were initially diluted to a working concentration of 100 USP U/ml in sterile saline. Working concentrations were prepared at 10 and 1 U/ml. For the potency measurements calibration curves were prepared with the USP standard for cross‐referencing. NHP was supplemented with each of these heparins in 0 to 1 U/ml concentrations. All other tests were carried out according to standard operating procedures.ResultsIn the USP compliant anti‐Xa and anti‐IIa assays all standardized solutions provided comparable results. Similarly in the clot‐based assays such as the aPTT, thrombin time, prothrombinase‐induced clotting time, all solutions produced comparable anticoagulant effects. Similarly in the thrombin generation assays all products produced comparable inhibitory responses of thrombin generation in terms of peak thrombin, area‐under‐the‐curve and the lag time. The HIT antibody mediated platelet aggregation was similar with all solutions. Total GAG content as measured by Red Probe was also comparable with calculated amounts of 0.520 mg/100U for Sintetica, 0.540 mg/100U for Fresenius, 0.510 mg/100U for Hospira, 0.540 mg/100U for Medefil and 0.530 mg/100Us for Roche’s product.ConclusionThese result demonstrate that commercially available potency adjusted heparin solutions and the developed Sintetica heparin solution exhibit comparable anticoagulant and anti‐protease effects. Based on these results the Sintetica product is similar to the commercially available clinically approved potency adjusted heparin solutions. Furthermore these results suggest that these solutions are interchangeable for clinical use.

Abstract 110: C1 Esterase Inhibitor Functions As An Anticoagulant In Human And Murine Venous Thrombosis

Factor (F) XII and FXI play a central role in venous thrombus formation in preclinical disease models and contributes to venous thromboembolism (VTE) in humans. Agents targeting FXIa have been developed and effectively prevent VTE in humans. C1 esterase inhibitor (C1INH) is a serine protease inhibitor that inhibits several proteases, including FXII and FXI. In this study, we investigated the anticoagulant activity of C1INH in humans and mice. First, plasma C1INH levels were determined in a population-based nested case-control study consisting of 405 VTE patients and 829 age- and sex-matched controls derived from the Tromsø Study. Participants with plasma C1INH levels in the highest quartile had a significantly lower risk of VTE (odds ratio [OR] of 0.68, confidence interval 0.49-0.96, P<0.05), unprovoked VTE (OR 0.59, confidence interval 0.39-0.89) and pulmonary embolism (OR 0.57, confidence interval 0.34-0.92) compared to participants with C1INH levels in the lowest quartile after adjustment for age and sex. Secondly, plasma-based thrombin generation studies were conducted to assess the anticoagulant function of C1INH. Supplementation of normal human pooled plasma with exogenous human purified C1INH (0.2 and 0.4mg/ml) significantly inhibited thrombin generation initiated with silica (P<0.05) and significantly prolonged the activated partial thromboplastin assay clotting time (P<0.001). Thirdly, administration of a clinically approved human purified C1INH product to mice significantly reduced thrombus weight (P<0.05) in the murine inferior vena cava stenosis model of venous thrombosis. Studies with C1 inhibitor deficient mice and substrate selective C1INH variants are currently ongoing. Our results indicate that C1INH serves as an endogenous anticoagulant with higher plasma levels of C1INH associated with a decreased future risk of VTE. Further, exogenous C1INH effectively inhibits thrombin generation in vitro and venous thrombus formation in a mouse model. Our findings suggest that C1INH may prevent thrombosis caused by activation of the intrinsic coagulation pathway.

Comparative Studies on a Branded Recombinant Factor VIIa and Its Generic Version to Demonstrate Bioequivalence

Background: Recombinant factor VIIa (NovoSeven® Copenhagen, Denmark) is used in the management of bleeding in patients with hemophilia. A generic biosimilar version of NovoSeven® is also developed for the same indication (AryoSeven™, AryoGen, Tehran-Iran). These generic versions of rFVIIa are claimed to be biosimilar to the Novoseven. The purpose of this study is to compare several batches of generic versions of rFVIIa namely, Aryoseven with multiple commercially available batches of branded Novoseven to determine bioavailability.Methods: Four commercially available random lots of Novoseven were obtained through US and European sources and compared to 5 clinical batches of Aryoseven obtained from Aryogen (Tehran, Iran). These drugs were diluted with saline to make a stock solution of 1mg/ml. Further dilutions were made to obtain working concentrations of 100, 10, 1 and 0.1 µg/ml. Molecular profile using surface enhanced laser desorption ionization (SELDI), gel electrophoretic profile (GEP), immunoblotting studies, and procoagulant activity as measured by clot based method were carried out. Thrombin generation studies were carried out after supplementing with NHP at 0-1.0 ug/ml. The pharmacokinetic profile of one representative batch of Novoseven and Aryoseven was studied in primates after a 50 ug/ml bolus IV in terms of factor VIIa levels.Results: The SELDI mass spectrometric profile of all rFVIIa preparations were comparable. The GEP and immunoblotting studies of the two groups of agents showed a comparable profile with distinct peaks at 50 KDa and 29 KDa. In the clot based functional assays performed in the citrated whole blood and retrieved plasma the two batches of FVIIa produced comparable procoagulant effects in the PT and aPTT assays. In the 0.25 - 1.0 µg/ml range in comparison to the control with saline supplementation, the aPTT did not exhibit any significant shortening whereas there was a sharpening of PT values in the whole blood which was identical in both the AryoSeven™ and NovoSeven® preparations. In the retrieved plasma both products produced comparable effect on the PT and aPTT assays. In platelet rich plasma (PRP) and platelet poor plasma (PPP) AryoSeven and NovoSeven at graded concentration of 0.125 to 1 µg/ml did not produce any measurable change in both PT and aPTT assays. The correction studies carried out in the FVII deficient plasma at a concentration range of 1 to 0.01 µg/ml did not reveal any differences between 2 groups of drugs. The relative correction was comparable in all concentrations for the 2 tissue factor reagents namely Innovin and Recomboplastin 2G. The results were indistinguishable for the 2 groups of drugs. The effect of NovoSeven® and AryoSeven™ on the reversal of anticoagulation in orally anticoagulated patients' were similar in relative correction of the plasma INR for the 2 groups of FVIIa preparations in the range of 0.03 to 1 µg/ml. In the thrombin generation studies both the Novoseven and Aryoseven produced similar effects as measured by increased thrombin generation. In the primate pharmacokinetic studies both groups exhibited similar pharmacokinetic profile.Conclusions: These results demonstrate that four batches of Novoseven and 5 individual clinical batches of Aryoseven were found to be biosimilar. The pharmacokinetic profile of the Novoseven and Aryoseven were studied in primates measuring the factor VIIa level for 6 hours and were found to be comparable. Thus, the generic Aryoseven is biosimilar to branded Novoseven and warrants further validation studies. DisclosuresNo relevant conflicts of interest to declare.

Inhibition of Thrombin and Its Generation By Recombinant Thrombomodulin and Activated Protein C in Whole Blood and Plasma Based Systems

Introduction:Recombinant thrombomodulin (rTM) and activated protein C (APC) are important modulators of coagulation and fibrinolysis. Both agents have been developed for various clinical indications. Beside their direct effect on coagulation, these agents also modulate other components of hemostatic processes to mediate their pharmacologic actions such as the direct inhibition of proteases and their generation.Aim:The aim of this study is to compare the differential inhibition of thrombin and its generation by rTM (ART-123, Asahi Kasei, Tokyo, Japan) and a plasma derived APC (Hematologic Technologies, Burlington, VT, USA).Methods:The inhibitory effects of APC and rTM on thrombin were measured in a plasma based and fluorogenic substrate assays. In the whole blood studies, native blood from healthy volunteers (n=10) was supplemented with rTM and APC at concentrations levels of 0-1.0 ug/ml. Thrombin generation was triggered by adding diluted APTT activator (Triniclot) and the reaction was stopped after 3 minutes. Thrombin generation markers such as F1.2 and TAT were measured using commercially available ELISA methods. In the citrated plasma studies both agents were supplemented at 0-1.0 ug/ml. Thrombin generation studies were carried out using a flouremtric assay (TGA, Techniclone, Vienna, Austria). Lag time peak thrombin generation and area under the curve (AUC) were measured. The IC50 values for each of the agents was calculated.Results:APC did not have any direct inhibitory effects on thrombin in both the clotting and amidolytic assays. However, rTM produced a concentration dependent inhibition of thrombin in both the clot based and amidolytic assays. In the whole blood systems both the APC and rTM produced a concentration dependent inhibition of thrombin generation markers such as F1.2 and TAT. APC produced a slightly stronger inhibition of stronger inhibition of TG as measured by F1.2, IC50 = 0.55 ug/ml compared with thrombomodulin, IC50 = 0.70 ug/ml. In the plasma based system the IC50 for peak thrombin inhibition was 0.46 ug/ml for APC and 0.97 ug/ml for rTM. Additional differences in the lag phase and AUC profiles were also noted. Conclusions: Beside the direct anticoagulant effects both the APC and rTM are capable of inhibiting the TF mediated thrombin generated in whole blood and plasma based systems. APC exhibits slightly stronger inhibitory effects in TG assays in both plasma and whole blood. .Conclusions:In contrast to APC, rTM produces direct inhibitory effects on thrombin in both the amidolytic and clot based assays. Both APC and rTM are capable of inhibiting the TF mediated thrombin generation in whole blood and plasma based systems. APC exhibits slightly stronger inhibitory effects in thrombin generation assays in both plasma and whole blood. These studies suggest that modulation of thrombin and other proteases contribute to the clinical effects of both rTM and APC. DisclosuresTanaka:Asahi Kasei Pharma America Corporation: Employment. Tsuruta:Asahi Kasei Pharma America Corporation: Employment.

Thrombin Generation in Chronic Liver Diseases-A Pilot Study.

The knowledge about coagulation disorders in patients with chronic liver disease changed in the last decade. The aim of this study was to analyze the parameters of thrombin generation in patients with chronic liver disease, as they are the most appropriate biomarkers to explore coagulation. (1) Background: The knowledge about coagulation disorders in patients with chronic liver disease changed in the last decade. The study of thrombin generation in patients with chronic liver disease provides a much more accurate assessment of the coagulation cascade; (2) Methods: This study is a prospective observational pilot study on hospitalized patients with chronic liver diseases that analyzed thrombin generation performed from their platelet-poor plasma versus that of control subjects. We analyzed a group of 59 patients with chronic liver disease and 62 control subjects; (3) Results: Thrombin generation was lower in hepatitis and cirrhosis patients compared to controls and decreases as the disease progressed. Lag time was higher in ethanolic etiology compared to the control group. Peak thrombin and endogenous thrombin potential were shorter in all etiologies when compared to the control group. The velocity index was significantly lower in HCV hepatopathies, ethanolic, and mixed etiology when compared with normal individuals; (4) Conclusions: Given the variability of thrombin generation in patients with chronic liver disease, its assay could serve to identify patients with high thrombotic and hemorrhagic risk and establish personalized conduct toward them.

Study of thrombin generation in healthy children

Exploring coagulation in newborns and children is a challenge due to the low levels of both procoagulant factors and inhibitors. Conventional coagulation tests might be inadequate to explore all of these changes. The aim of the study is to evaluate the thrombin generation assay, a global test to explore coagulation, in a pediatric population (n=586) compared to an adult population (n=166). The thrombin generation assays were performed using Calibrated Automated Thrombography with two different tissue factor concentrations (1 and 5 pM), with and without thrombomodulin (TM). In the absence of TM, the endogenous thrombin potential (ETP) is significantly lower in the pediatric population, reflecting the decrease in procoagulant factors. In the presence of TM, ETP values in pediatric subjects are within the reference range of adult values. The thrombin generation assay demonstrates that coagulation balance is maintained in the pediatric population.

Assay Dependent Reversal of the Oral and Parenteral Anti-Xa Agents By Andexanet Alfa

Introduction: Andexanet alfa (AA, Portola Pharmaceuticals, San Francisco, USA) is an approved reversal agent for the control of potential bleeding associated with apixaban and rivaroxaban. Beside the oral anti-Xa agents, parenteral forms of the inhibitors of factor Xa such as otamixaban (Sanofi Aventis, Paris, France) and DX9065a (Mitsubishi Pharmaceuticals, Tokyo, Japan) have also been developed. These agents represent synthetic organo-mimerics with comparable selectivity and inhibitory profile to the currently available oral anti-Xa agents. Parenteral anti-Xa agents are considered for clinical development. Andexanet alfa is a broad-spectrum neutralizing agent for anti-Xa drugs including heparin and heparino-mimerics. We hypothesized that andexanet alfa may also reverse the effects of such parenteral anti-Xa agents as otamixaban and DX9065a. This study is designed to compare the neutralization profile of andexanet alfa for apixaban and rivaroxaban with otamixaban and DX9065a in various laboratory assays. Materials and Method: Apixaban, rivaroxaban, otamixaban and DX9065a were commercially obtained in powdered form and diluted in 0.9 % sodium chloride to make stock solution of 1.0 mg/ml. Andexanet alfa was obtained from the hospital pharmacy. Drugs were supplemented in plasma in the concentration range of 0.0 - 1.0 ug/ml. Individual aliquots of samples were supplemented with either saline or andexanet alfa at a final concentration of 100 ug/ml. Factor Xa activity was measured by using an amidolytic method. For clotting profile, prothrombin time (PT) and activated partial thromboplastin time were measured. Thrombin generation studies were carried out using a calibrated automated thrombogram (CAT, Diagnostica Stago, Paris, France). Such parameters as peak thrombin (PT), area under the curve (AUC) and lag time (LT) were measured. The inhibitory effects of each of these agents towards factor Xa were calculated and their reversal by andexanet alfa was determined. Results were compiled as mean SD of 3 individual determination. Result: Both the oral and parenteral anti-Xa agents produced a concentration dependent inhibition of factor-Xa with the IC50 values ranging from 0.17 - 1.1 ug/ml in control group. Supplementation of andexanet alfa at 100 ug/ml resulted in the neutralization of the anti-Xa activities of these agents with the IC50 values ranging from 0.22 - 1.1 ug/ml. Andexanet alfa did not produce any reversal of the anti-Xa activities of DX9065a. In the thrombin generation studies, Apixaban, rivaroxaban and otamixaban produced strong concentration dependent inhibition of thrombin formation. However, DX9065a produced relatively weaker anti-Xa effects. The IC50 values varied with apixaban (0.08 ug/ml), rivaroxaban (0.22 ug/ml), otamixaban (0.6 ug/ml) and DX9065a (>2.5 ug/ml). In the clot-based prothrombin time assay all agents produced a concentration dependent prolongation of PT in the range of 0 - 1 ug/ml. Andexanet alfa at 100 ug/ml produced a complete neutralization of apixaban, rivaroxaban and otamixaban, whereas it partially neutralized the anticoagulant effects of DX9065a in this assay. The parenteral anticoagulants otamixaban and DX9065a produced a much stronger anticoagulant effects in the aPTT assay in comparison to both apixaban and rivaroxaban. Andexanet alfa at 100 ug/ml effectively neutralized the anticoagulant effects of otamixaban in comparison to Apixaban and rivaroxaban. Whereas DX9065a were not neutralized. Table 1 shows the composite results for the neutralization of oral and parenteral anti-Xa agents at 0.5 ug/ml by andexanet alfa at 100 ug/ml. Conclusion: Our results suggest that andexanet alfa is capable of neutralizing the effects of potent parenteral anti-Xa agents such as otamixaban in an assay dependent fashion. The data also points to the varying inhibitory effects of anti-Xa agents which are differentially neutralized by andexanet alfa. These results also underscore that the in-vitro anti-Xa potency of both the oral and parenteral anti-Xa agents does not fully reflect their inhibitory effects on the overall coagulation process. Nevertheless, andexanet alfa may be a useful agent in the neutralization of parenteral anti-Xa agents. Disclosures No relevant conflicts of interest to declare.

ENDOGENOUS THROMBIN GENERATION POTENTIAL IN PATIENTS WITH PULMONARY EMBOLISM AND ITS RELEVANCE TO THROMBOTIC AND INFLAMMATORY BIOMARKERS

SESSION TITLE: Pulmonary Vascular Disease Posters SESSION TYPE: Original Investigation Posters PRESENTED ON: October 18-21, 2020 PURPOSE: Pulmonary embolism (PE) is a catastrophic outcome in patients with venous thrombosis. In the United States around 900,000 people (1 to 2 per 1,000) patients are affected each year with the mortality rate ranging from 10 – 30 %. Inflammation, hemostatic dysregulation and vascular complications contribute to the pathogenesis of PE. Endogenous thrombin generation contributes to the overall pathology of PE. The purpose of this study is to investigate endogenous thrombin potential (ETP) and the circulating biomarkers of inflammation, hemostatic activation and endothelial dysfunction in plasma samples obtained from the patients at the onset of PE. METHODS: Citrated blood samples from 78 patients with confirmed PE were collected at Loyola University Medical Center and Gottlieb Memorial Hospital under an IRB approved protocol. Normal human plasma (NHP) comprised of 25 male and 25 female samples were obtained from a commercial source (George King Biomedical, Overland park, KS.) Thrombin generation studies were carried out using a commercially available a kinetic system (Diagnostica Stago, Paris, France). Thrombin generation parameters such as peak thrombin, lag time and area under the curve (AUC) were compiled. Frozen samples were retrospectively analyzed for biomarkers, including D-Dimer, PAI-1, TNFa, IL6, IL1b, VEGF and TFPI using commercially available sandwich ELISA methods. Results were compiled as mean ± SD and analyzed for significance and correlation. Applicable statistical analysis was carried out by using GraphPad Prism software. RESULTS: On a cumulative basis, PE patients showed a wide variation in thrombin generation parameters. Peak thrombin levels (91.1 nM vs 160.0 nM) and AUC (554.7 nM*min vs 700.9 nM*min) were decreased in PE patients while lag time was increased (6.41 min vs 2.7 min). Biomarkers analysis showed elevated levels of D-Dimer (7521.3 ng/ml) in comparison to NHP (192.8 ng/ml), PAI (67.3 ng/ml) in comparison to NHP (10.5 ng/ml), TNF-a (3.2 pg/ml) in comparison to NHP (1.9 pg/ml), IL6 (99.9 pg/ml) in comparison to NHP (1.24 pg/ml), IL1b (1.6 pg/ml) in comparison to NHP (0.79 pg/ml) and TFPI (139.1ng/ml) in comparison to NHP (62.3 ng/ml) while and VEGF (31.24 pg/ml) levels were decreased in comparison to NHP (45.5 pg/ml. The biomarker levels did not show any correlation with peak thrombin levels with the exception of VEGF and TFPI. Samples with higher thrombin generation (>150 nM) showed marked elevation of all of the biomarkers studied. CONCLUSIONS: PE patients exhibit a wide variation in endogenous thrombin generation parameters. However, biomarkers of hemostatic activation, vascular dysfunction and inflammation were increased. CLINICAL IMPLICATIONS: Measurement of thrombin generation parameters along with the biomarkers of inflammation and endothelial dysfunctions may be useful in the overall management of PE patients. DISCLOSURES: No relevant relationships by Emily Bontekoe, source=Web Response No relevant relationships by Yevgeniy Brailovsky, source=Web Response No relevant relationships by Amir Darki, source=Web Response No relevant relationships by Jawed Fareed, source=Web Response No relevant relationships by ambar Farooqui, source=Web Response No relevant relationships by Debra Hoppensteadt, source=Web Response No relevant relationships by Omer Iqbal, source=Web Response No relevant relationships by Fakiha Siddiqui, source=Web Response

Decreased Thrombin Generation Potential in Lymphoma Patients is associated with Increased D‐dimer, CRP, vWF and TNF‐α. Interrelationship between Thrombogenesis and Inflammation

IntroductionInflammation and other vascular factors contribute to the pathogenesis of thrombotic complications in lymphoma patients. Biomarkers of hemostatic activation, vascular dysfunction and inflammation are upregulated in lymphoma. It has been reported that thrombin generation and inflammatory biomarkers are increased in cancer while the thrombin generation potential is decreased. This study compares the thrombin generation potential and its relevance to the generation of various biomarkers of hemostatic activation process and inflammatory responses.Materials and MethodCitrated blood samples from 90 patients with confirmed diagnosis of non‐Hodgkin lymphoma (NHL), Hodgkin lymphoma (HL) and Chronic lymphocytic leukemia/Small lymphocytic lymphoma (CLL/SLL) were collected from University of Belgrade Clinic, Belgrade, Serbia. 50 samples of normal human plasma (NHP) was obtained from George King Biomedical (Overland park, KS). Thrombin generation studies were carried out using a commercially available a kinetic system. Thrombin generation parameters such as peak thrombin (PT), lag time (LT) and area under the curve (AUC) were compiled. Plasma levels of D‐Dimer, CRP, vWF and TNF‐α were measured by using commercially available Sandwich ELISA assays. Results were compiled in terms of mean ± SD. Correlation analysis were carried out by using Prism, Graphpad.ResultsOn a cumulative basis, lymphoma patients showed an increase in lag time (2.9 ± 1.17) in comparison to the NHP (2.7± 0.61). The peak thrombin levels was decreased (119.0 ± 46.2) in the lymphoma patients in comparison to NHP (159.7 ± 31.1). The AUC was decreased (670.2 ± 290.01) in the lymphoma patients in comparison to NHP (700.9 ± 198.0). When the lymphoma patients were sub grouped, peak thrombin levels were similar in HL (129.9), NHL (114.7) and CLL (127.4) and were decreased compared to NHP (159.7). However the AUC was increased in the HL (769), decreased in NHL (642.4) and was comparable for CLL (715.4) compared to NHP (700.9). Lymphoma patients also exhibited elevated levels of D‐Dimer (878 ± 1205 ng/ml) in comparison to NHP (280 ng/ml), CRP (15.2 ± 26.6 ug/ml) in comparison to NHP (0.9 ug/ml), vWF (271.8 ± 189.7 %) in comparison to NHP (122%) and TNF‐α (16.4 ± 20.64 pg/ml) in comparison to NHP (4.2 pg/ml). In the HL group D‐Dimer and CRP correlated well with AUC and PT, in this group vWF also showed a good correlation with PT. In the NHL group, CRP correlated well with LT.ConclusionLymphoma patients represent a heterogenous group in which both the hypercoagulable state and inflammatory responses simultaneously occur as evident by increase in D‐Dimer and inflammatory biomarkers. The decrease in thrombin generation potential in these patients may be due to the consumption of prothrombin and other coagulation factors. Wide variations in circulating levels of various biomarkers and thrombin generation parameters between various lymphoma groups are noted. Profiling of thrombin generation and inflammatory biomarkers may be helpful in thrombotic risk stratification of lymphoma patients and their anti‐thrombotic management.

Differential Augmentation of Thrombin Generation by Andexanet Alfa in Lymphoma Patients

IntroductionThe prevalence of thrombosis in lymphoma patients is reportedly high and ranges from 3–10%. Vascular malfunction and inflammatory processes further contribute to the thrombotic activation processes in these patients. Andexanet alfa is an antidote for factor Xa inhibitors and its usage has been reported with thrombotic complications. This study was designed to compare effect of andexanet alfa (AA) on the thrombin generation potential and its relevance to the generation of thrombin.Materials and MethodCitrated blood samples from 78 patients with confirmed diagnosis of non‐Hodgkin lymphoma (NHL), Hodgkin lymphoma (HL) and Chronic lymphocytic leukemia/Small lymphocytic lymphoma (CLL/SLL) were collected from the Clinic of Hematology Unit, University of Belgrade, Belgrade, Serbia. 50 samples of normal human plasma (NHP) was obtained from George King Biomedical (Overland park, KS). NHP was prepared for referencing purposes. Individual samples were supplemented with andexanet alfa at 100 ug/ml. Thrombin generation studies were carried out using a commercially available a kinetic fluorogenic substrate method (calibrated automated thrombogram; CAT). Thrombin generation parameters such as peak thrombin (PT), lag time (LT) and area under the curve (AUC) were compiled. Results were compiled in terms of mean ±SD.ResultsOn a cumulative basis, lymphoma patients showed an increase in LT (2.9 1.15) in comparison to NHP (2.06 .02) which decreases with AA (1.94 0.1). The PT levels were decreased (120.34 44.3) in comparison to NHP (159.20 4.8) and increases with AA (153.70 37.2). The AUC was also decreased (668.85 284.3) in comparison to NHP (756.46 34.5) which increases with AA (758.32 226.4). When the lymphoma patients were sub‐grouped, peak thrombin levels were similar in HL (128.16), NHL (113.99) and CLL (129.77) were decreased compared to normal human plasma (159.2) which increases with AA, in HL (180.75), NHL (154.16) and CLL (142.46) compared to AA control (153.7). The AUC was increased in the HL (769), decreased in NHL (642.4) and was comparable for CLL (715.4) compared to normal human plasma (756.46). However, with AA the AUC values increases for HL (938.31) and decreases for NHL (744.52) and CLL (725.26) in comparison with AA control (758.32). Variations in lag time were noted in the three groups.ConclusionLymphoma patients represent a heterogenous group of patients in which both the hypercoagulable state and inflammatory responses simultaneously occur. Thrombin is constantly generated in some of these patients leading to the formation of fibrin. The observed decrease in thrombin generation potential in these patients may be due to the consumption of prothrombin and other coagulation factors. Differential increase in thrombin generation in post andexxa supplemented samples suggest that the use of this agent may potentially be associated with thrombotic complications.

Study of thrombin generation in three populations of patients on oral anticoagulants: Bleeding with reversion, haemorrhagic accidents without reversion or haemorrhagic accident

Bleeding under oral anticoagulant is a frequent reason for consultation in the emergency department. The aim of our study was to compare thrombin generation (TG) parameters in 3 populations under oral anticoagulant: severe bleeding patients who received procoagulant factors, patients with non-severe bleeding without reversion and anticoagulated patients without bleeding events. We conducted a prospective, single-center study of TG in patients under oral anticoagulant (VKA, Direct Oral Anticoagulants (DOACs): rivaroxaban, dabigatran, apixaban). The study was performed on platelet-poor plasma collected on arrival (V1) before reversal therapy, then 30 min (V2), 6 h (V3) and 24 h (V4) after reversal. A total of 307 patients were included in this study with 98 severe bleeding reversed, 95 patients with clinical relevant bleeding but non-reversed and 108 patients without bleeding. For V1, VKA patients with reversed bleeding had a significant decrease in the main TG parameters compared to patients without bleeding ( P = 0.017). Moreover at V1, the main parameters of TG were significantly decreased for VKA compared to DOACs ( P < 0.001) and between anti-IIa compared to anti-Xa ( P < 0.001). At visits V2, V3 and V4, reversal therapy restored a normocoagulable state for VKA patients with a significant increase in key TG parameters between V1 and V2-V3-V4 ( P < 0.001). For DOACs reversed by PCC or PCCactivated, a significant hypercoagulability state was demonstrated on the TG whereas a normal coagulation and no hypercoagulable state was found for those under dabigatran reversed by the idarucizumab. Patients on VKA regained a comparable coagulation state as healthy subjects after reversal therapy, whereas patients treated with DOACs and reversed with PCC or PCCa induced a hypercoagulable state. Probably, this hypercoagulable state could be minimized by reducing the doses of PCC or PCCa, or be circumvented by the use of specific antidotes.

Decreased Thrombin Generation Potential in Lymphoma Patients Is Associated with Increased D-Dimer, CRP, Vwf and TNF-α. Interrelationship between Thrombogenesis and Inflammation

Introduction: The prevalence of thrombosis in lymphoma patients is reportedly high and ranges from 3 - 10%, and further increased at advanced stages of the disease especially in hgNHL. Inflammation and other vascular factors contribute to the pathogenesis of these thrombotic complications. Biomarkers of hemostatic activation, vascular dysfunction and inflammation are elevated in lymphoma. We have previously reported that thrombin generation biomarkers such as D-Dimer, thrombin anti-thrombin complex (TAT) and prothrombin fragment (F1.2) along with inflammatory biomarkers are increased in cancer patients. Interestingly, despite an increase in thrombin generation markers, thrombin generation potential in these patients is decreased. This study was designed to compare the thrombin generation potential and its relevance to the generation of various biomarkers of hemostatic activation process and inflammatory responses. Methods: Citrated blood samples from 90 patients with confirmed diagnosis of non-Hodgkin lymphoma (NHL), Hodgkin lymphoma (HL) and Chronic lymphocytic leukemia/Small lymphocytic lymphoma (CLL/SLL) were collected from the Clinic of Hematology Unit, University of Belgrade, Belgrade, Serbia. 50 samples of normal human plasma (NHP) was obtained from George King Biomedical (Overland park, KS). NHP was prepared for referencing purposes. Thrombin generation studies were carried out using a commercially available a kinetic fluorogenic substrate method (calibrated automated thrombogram; CAT). Thrombin generation parameters such as peak thrombin (PT), lag time (LT) and area under the curve (AUC) were compiled. Plasma levels of D-Dimer, CRP, vWF and TNF- α were measured by using commercially available Sandwich ELISA assays. Results were compiled in terms of mean ± SD. Correlation analysis carried out by using Prism, Graphpad. Results: On a cumulative basis, lymphoma patients showed an increase in lag time (2.9 1.17) in comparison to the NHP (2.7). The peak thrombin levels was decreased (119.0 46.2) in the lymphoma patients in comparison to NHP (159.7 31.1). The AUC was decreased (670.2 290.01) in the lymphoma patients in comparison to NHP (700.9 ). When the lymphoma patients were sub grouped, peak thrombin levels were similar in HL (129.9), NHL (114.7) and CLL (127.4) and were decreased compared to NHP (159.7). However the AUC was increased in the HL (769), decreased in NHL (642.4) and was comparable for CLL (715.4) compared to NHP (700.9). Variations in lag time were noted in the three groups. The lymphoma patients also exhibited elevated levels of D-Dimer (878 1205 ng/ml) in comparison to NHP (280 ng/ml), CRP (15.2 26.6 ug/ml) in comparison to NHP (0.9 ug/ml), vWF (271.8 189.7 %) in comparison to NHP (122%) and TNF-α (16.4 20.64 pg/ml) in comparison to NHP (4.2 pg/ml). Interestingly, when the lymphoma group were subdivided into various groups, the D-Dimer level was highest in HL following the order HL&gt;NHL&gt;CLL. CRP and vWF levels were highest in HL and comparable in both NHL and CLL. Wide scatter in TNF-α data was noted with CLL exhibiting highest levels with the ranked order CLL&gt;NHL&gt;HL. The composite data on thrombin generation parameters and biomarkers is shown on the table 1A. In the HL group D-Dimer and CRP correlated well with AUC and PT, in this group vWF also showed a good correlation with PT. In the NHL group, CRP correlated well with LT. In the CLL group none of the biomarkers correlated well with the thrombin generation parameters. On a cumulative basis, D-Dimer, CRP, vWF and TNF-α had no significant relevance to thrombin generation parameters. The composite regression coefficience data is shown on table 1B. Conclusion: Lymphoma patients represent a heterogenous group in which both the hypercoagulable state and inflammatory responses simultaneously occur as evident by increase in D-Dimer and inflammatory biomarkers. While the overall thrombin generation potential in the plasma of these patients is decreased, thrombin is constantly generated in some of these patient leading to the formation of fibrin. The observed decrease in thrombin generation potential in these patients may be due to the consumption of prothrombin and other coagulation factors. Wide variations among these lymphoma groups are noted. Profiling of thrombin generation and inflammatory biomarkers may be helpful in thrombotic risk stratification of lymphoma patients and their anti-thrombotic management. Disclosures No relevant conflicts of interest to declare.

Factor Xa Inhibitory Profile of Apixaban, Betrixaban, Edoxaban, and Rivaroxaban Does Not Fully Reflect Their Biologic Spectrum.

The currently available oral anti-Xa agents are claimed to produce their anticoagulant and antithrombotic effects solely by the inhibition of factor Xa. This study profiled various anti-Xa drugs in routinely used laboratory assays to demonstrate that their effects are not solely related to the anti-Xa activities. Apixaban, betrixaban, edoxaban, and rivaroxaban were obtained commercially. Native and citrated whole blood was used for the activated clotting time (ACT) and thromboelastography (TEG). Citrated plasma was used for monitoring the prothrombin time (PT), activated partial thromboplastin time (aPTT), Heptest, and prothrombinase-induced clotting time (PiCT) tests. An amidolytic method was used for the determination of anti-Xa effects. Thrombin-induced fibrinokinetics was monitored optically. Thrombin generation studies were carried out using the calibrated automated thrombogram. All of the anti-Xa agents produced concentration- and assay-dependent effects. In the ACT at 2.5 μg/mL and TEG at 1.0 μg/mL, edoxaban exhibited the strongest anticoagulation effect. In the PiCT, PT, and aPTT assay at 1 μg/mL, edoxaban showed stronger effects than other agents. The half maximal inhibitory concentration of these agents for the inhibition of factor Xa ranged from 340 to >1000 ng/mL. In the thrombin generation inhibition assay, apixaban showed the strongest activity. In the fibrinokinetics, different anti-Xa agents produced varying degrees of inhibition. These results demonstrate that the measured anti-Xa activity alone does not fully reflect the overall biologic spectrum of these agents.

Decreased Thrombin Generation Potential Is Associated with Increased Thrombin Generation Markers in Sepsis Associated Coagulopathy

Introduction: Sepsis associated coagulopathy (SAC) is commonly seen in patients which eventually leads to dysfunctional hemostasis and disseminated intravascular coagulation (DIC). Thrombin generation plays an important role in the overall pathophysiology of this process. Previous studies have reported an increase in thrombin generation markers such as thrombin antithrombin complex (TAT) and prothrombin fragment (F1.2). (Hoppensteadt et al. Clin Appl Thromb Hemost. 2014 Mar;20(2):129-35). Sepsis eventually results in consumption coagulopathy in which some of the clotting factors are consumed. The purpose of this study is to determine the thrombin generation potential of baseline blood samples obtained from sepsis associated coagulopathy patients and demonstrate their relevance to thrombin generation markers.Materials and Methods: Baseline citrated blood samples were prospectively collected from 49 patients with sepsis and suspected DIC. DIC scores were determined according to the ISTH scoring system (PTINR, fibrinogen, D-dimer and platelet count). Healthy control (n=50) represented citrated plasma obtained from a commercial supplier (George King Biomediacl, Overland Park, KS). Thrombin generation studies were carried out using a commercially available florigenic substrate methods with thrombin calibrator and PPP reagent (calibrated automated thrombogram; CAT). Such parameters as peak thrombin, lag time and area under the curve were compiled. TAT and F1.2 were measured using commercially available ELISA methods (Seimens, Indianpolis, IN). Functional antithrombin levels were measured using a chromogenic substrate method. All results were calculated in terms of mean ± SD. Applicable statistical methods were used to correlate the thrombin generation parameters with thrombin generation markers and antithrombin.Results: The peak thrombin levels were lower (82±40nM) in the SAC patients in comparison to higher levels observed in the normal plasma (133±10nM). The AUC was lower (561 ±280) in the SAC group in comparison to the normal (624±18). The SAC group showed much longer lag time (4.1±2.1) in comparison to the normal (2.1 ± 2.2). Wide variations in the results were observed in these parameters in the SAC group. The compsoite data is compiled in the table. the F1.2 levels in the DIC group were much higher (570±48 pmol) in comparison to the normal (210±25 pmol). The TAT levels also increased in the SAC group (27.9 ±5.1 ng/ml) in comparison to the normal (2.8 ±0.8 ng/ml). The functional antithrombin levels were decreased in the SAC group (64 ±11%). No correlation was observed between thrombin generation parameters and thrombin generation markers.Conclusion: These results validate the earlier observations that thrombin generation such as F1.2 and TAT are decreased in patients with SAC. However thrombin generation parameters are significantly elevated in this group in comparison to normals. This may be due to the consumption of prothrombin due to the activation of the coagulation system. The SAC group also showed wide variations in both the thrombin generation parameters and the F1.2 and TAT. These variations may be due to the differences in the endogenous pathophysiology state in the SAC patients. The decreased functional AT levels observed in the SAC group are due to the formation of the complex between generated thrombin and antithrombin. Simultaneous profiling of thrombin generation and thrombin generation markers may be helpful in the risk stratification of these patients. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Thrombin Generation Impairments in Platelet Function Disorders

Many platelet function disorders (PFD) present as uncharacterized disorders that impair aggregation responses to multiple agonists and/or cause non-syndromic dense granule deficiency (DGD). We postulated that some of these commonly encountered PFD might also impair the ability of platelets to support thrombin generation, given that an important subgroup are caused by mutations in transcription factors, such as RUNX1, that impair multiple aspects of platelet function. Accordingly, we initiated a study of thrombin generation (TG) in a prospective cohort of individuals presenting with an uncharacterized PFD. In addition, we reassessed how Quebec platelet disorder (QPD, previously called Factor V Quebec) affects coagulation as this disorder (which is caused by a duplication mutation of PLAU) triggers intraplatelet (but not systemic) plasmin generation and proteolysis of platelet but not plasma factor V (FV), and the normal plasma FV might potentially compensate for the loss of platelet FV in QPD.The study was conducted with written informed consent of participants and with the approval of the Hamilton Integrated Research Ethics Board and the Research Ethics Board of Centre Hospitalier Universitaire Sainte Justine. Participants included: 1) five individuals with QPD; 2) eighteen individuals presenting with an uncharacterized PFD and confirmed reduced maximal aggregation responses to ≥2 agonists and/or confirmed DGD, including five that had a pathogenic RUNX1 mutation; and 3) eighteen similarly-aged general population controls. TG was assessed by the calibrated automated thrombogram (CAT) procedure on a Fluoroskan (Thermo Fisher Scientific AG, Reinach, Switzerland) using Thrombinoscope software (Synapse BV, Maastricht, The Netherlands), manufacturer- and ISTH-recommended protocols and reagents for testing platelet poor plasma (PPP) and platelet rich plasma (PRP). Endpoints included: endogenous thrombin potential (ETP, nM·min), peak thrombin concentration (nM), time-to-peak (min) and lag time (min). Platelet lysate and plasma FV concentrations were determined by enzyme-linked immunoassay. Data were analyzed using Mann-Whitney tests with Bonferroni correction for multiple comparisons.All TGA endpoints for PPP were comparable for controls and participants with PFD, including QPD (p values ≥0.10). In TGA with PRP, most PFD participants had findings similar to controls, however, the subgroups with pathogenic RUNX1 mutations or QPD had significantly reduced ETP and peak thrombin concentration compared to controls (data as median [range]: ETP: controls: 1860 [1530-2630]; RUNX1 mutation subgroup: 1520 [805-1640], p=0.004; QPD: 1370 [981-2010], p=0.01; peak thrombin concentration: controls: 111 [65-152]; RUNX1 mutations: 66 [32-93], p=0.006; QPD: 59 [41-91], p=0.005). Plasma FV levels were similar in all subjects (µg/ml PPP, median [range]: controls: 7.7 [6.3-10.7]; QPD: 8.4 [7.0-10.2], p=0.33; other PFD: 7.5 [5.4-12], p=0.74) and showed no significant association to TG endpoints for PPP or PRP (p values ≥0.14). Only QPD subjects had platelet FV deficiency (µg FV/mg platelet protein, median [range]: controls: 0.89 [0.63-1.54]; QPD: 0.35 [0.18-0.46], p<0.001; other PFD: 0.83 [0.47-1.75], p=0.48; RUNX1 mutation subgroup: 0.73 [0.50-1.53], p=0.41). In QPD, but not other participants, platelet FV showed a significant association to ETP (R2=0.81, p=0.04) and peak TG endpoints (R2=0.88, p=0.01).Our study illustrates that platelet-dependent thrombin generation, evaluated by CAT, is abnormal in QPD but normal in many uncharacterized PFD with defective aggregation and/or DGD except for the important subgroup with pathogenic RUNX1 mutations, which impair TG but do not reduce platelet FV (unlike QPD). In QPD, the platelet-dependent TG defect shows a unique relationship to the platelet FV deficiency, which suggests that the normal levels of plasma FV are insufficient to compensate for the platelet procoagulant abnormalities in this PFD. DisclosuresNo relevant conflicts of interest to declare.

Alterations in Fibrin Structure in Patients with Liver Diseases.

The hemostatic balance in patients with liver diseases is relatively well preserved due to concomitant alterations in pro- and antihemostatic pathways. Thrombin generation studies support the notion of hemostatic competence in liver diseases, but in such tests alterations in fibrinogen level and function are not taken into account. We have recently studied structural and functional properties of the fibrin clot in patients with liver diseases. Although we have confirmed previous findings that hypersialylation of the fibrinogen molecule in patients with liver diseases contributes to a defective fibrinogen-to-fibrin conversion, we have found that once the clot has been formed, it has a thrombogenic nature as assessed by permeability assays. These thrombogenic properties of the fibrin clot in cirrhosis relate to incompletely characterized intrinsic changes in the fibrinogen molecule, which may include oxidation and hypersialylation. In addition, in patients with nonalcoholic fatty liver disease thrombogenic properties of the fibrin clot are not only due to liver disease but also to obesity and the metabolic syndrome. During liver transplantation, the clot normalizes and becomes increasingly permeable, and the functional properties of the fibrin clot are markedly normalized by fibrinogen concentrate, when added to plasma samples in vitro. These new insights in the functional properties of the fibrin clot in patients with liver diseases facilitate a more rational approach to treatment and prevention of both bleeding and thrombotic complications.

A Generic Version of Recombinant FVIIa Is Similar to the Branded Product (NovoSeven)

Background: Commercially available recombinant factor VIIa (Novoseven) is widely used in the management of hemophilia patients with inhibitors. Recently several generic versions of recombinant VIIa (rFVIIa) have become available. The generic versions of rFVIIa are claimed to be biosimilar to the barnded Novoseven (Novo Nordisk, Copenhagen, Denmark). The purpose of this study is to compare the US and European Novoseven products with a generic version of rFVIIa namely, Aryoseven (Aryogen, Tehran, Iran).Methods: Four commercially available random lots of Novoseven were obtained from the US and European sources. Four different batches of Aryoseven were obtained from Aryogen. All individual rFVIIa preparations were diluted to obtain working concentrations of 100, 10, 1 and 0.1 ug/ml. Protein content (Lowry's method), molecular profile using surface enhanced laser desorption ionization (SELDI), gel electrophoretic profile (GEP), factor VII related antigen level (FVII:Ag), factor VII correction studies in depleted plasma and thrombin generation (TG) studies were carried out. In addition, VIIa/tissue factor mediated thrombin generation studies were carried out in various prothrombin complex concentrates such as Beriplex and Prothromplex.Results: The protein content and SELDI mass spectrophotometric profile of all 4 rFVIIa preparations were comparable. There was no differences in the Novoseven obtained from the US and European sources. The GEP of the two groups of agents showed a comparable profile with distinct peaks at 50 KDa and 25 KDa. The FVII related antigen levels were also comparable in the Novoseven and Aryoseven preparations. Supplementation of both the Novoseven and Aryoseven preparations at 10 and 100 ug/ml resulted in a comparable correction of the factor VII deficient plasma as measured by PT(INR). Thrombin generation was comparable in the branded and generic product.Conclusions: These results demonstrate that the US and European Aryoseven are comparable. Four batches of Novoseven and 4 individual clinical batches of Aryoseven were found to be comparable. When the US purchased Novoseven preparation was compared with the European Novoseven product, no differences were noted. Thus, the generic Aryoseven is biosimilar to barnded Novoseven and warrant in vivo validation studies. DisclosuresNo relevant conflicts of interest to declare.

Inverted Erythrocyte Membranes Are a Novel Platform for Expressing Phosphatidylserine and Investigating the Antiphospholipid Syndrome

Background: The antiphospholipid (aPL) syndrome (APS) is an acquired thrombophilic disorder in which autoantibodies target proteins bound to anionic phospholipids; the mechanisms involved have not yet been clearly established. Anionic phospholipids are normally present in the internal leaflets of cytoplasmic membranes and become expressed after cell injury or death. Current diagnosis of APS is based on a panel of non-mechanistic "criteria" tests, which consist of the paradoxical lupus anticoagulant (LA) and immunoassays for autoantibodies binding to anionic phospholipids (cardiolipin) or β2glycoprotein I. Also, there has been difficulty in identifying biologically relevant and robust phospholipid platforms for analyzing aPL-membrane interactions. We therefore decided upon a novel path by: 1) developing a cell-based model consisting of inverted erythrocyte membranes (iEMs) and 2) developing novel ways of detecting aPL-iEM interactions.Materials & Methods: iEMs were prepared from erythrocytes using an established protocol, suspended in 20 mOsm Tris buffer and used fresh or stored at -80°C. iEMs were incubated with normal or patient plasmas for a minimum of 30 minutes and washed by centrifugation at 15,000g. Binding of IgG to iEMs was investigated by goat anti-human IgG-Alexa 633 (GαH IgG-Alexa 633) in flow cytometry. PS exposure on membranes was confirmed by microscopy with annexin A5 (A5)-Oregon green and A5-Alexa 647 binding in flow cytometry. Bright field images of aggregated membranes were obtained to visually assess aggregation. The potential of iEMs to serve as phospholipids necessary for coagulation was assessed through calibrated automated thrombinography.Results: Incubation of iEMs with normal and aPL plasmas resulted in increased IgG binding to the iEMs, as determined by flow cytometry (figure 1A). The IgG staining was more pronounced with aPL compared to normal plasmas (mean fluorescence ± standard deviation; aPL (n = 3): 10004 ± 5416 versus healthy (n = 3): 4055 ± 1680). When studying these samples with bright field microscopy aggregate formation of iEMs was observed. The aggregates induced by aPL plasma were more numerous and larger than those forming in plasma of healthy donors (figure 2A-B). Amplified agglutination was observed in the presence of broad spectrum anti-hIgG/anti-hC3d reagent (figure 2C-D).Fluorescence microscopy of iEMs incubated with A5-OG confirmed that a population of iEMs was exposing PS. Thrombin generation (TG) studies established that iEMs contribute to coagulation reactions by providing a source of anionic phospholipids. TG increased dose-dependently with higher levels of iEMs until a plateau concentration was reached. Additionally, A5 resistance was investigated by means of flow cytometry. The aPL plasmas induced a decrease in A5 binding to iEMs compared to normal plasma and the control conditions (figure 1B), consistent with the functional A5 resistance assay.Discussion: We have developed a novel model for a more physiological approach to phospholipid based assays in APS using iEMs as a source of anionic phospholipids. They are easily prepared by hypotonic lysis of erythrocytes and can be frozen for storage until use.We have thus far demonstrated that a higher level of iEM aggregation is induced by aPL samples compared to healthy plasmas. Moreover, iEMs expose PS and dose-dependently accelerate TG by functioning as a platform for coagulation. Previous findings on A5 resistance induced by aPL plasma are confirmed with this iEM model.Conclusion: IEMs offer a novel and robust biologically-derived platform; they express PS, promote coagulation, bind aPL antibodies and open different approaches to investigate APS. [Display omitted] [Display omitted] DisclosuresDe Laat:Synapse bv: Employment.