Thrombin Generation Assay Research Articles

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.

Pathophysiology and management of bleeding and thrombosis in patients with liver disease

Patients with liver disease often develop complex changes in their haemostatic system. Frequently observed changes include thrombocytopaenia and altered plasma levels of most of the proteins involved in haemostasis. Although liver disease was historically classified as a haemostasis‐related bleeding disorder, it has now been well established that the antihaemostatic changes that promote bleeding are compensated for by prohaemostatic changes. Conventional coagulation tests however do not accurately reflect these prohaemostatic changes, resulting in an underestimation of haemostatic potential. Novel coagulation tests, such as viscoelastic tests (VETs) and thrombin generation assays (TGAs) better reflect the net result of the haemostatic changes in patients with liver disease, and demonstrate a new, “rebalanced” haemostatic status. Although rebalanced, this haemostatic status is more fragile than in patients without liver disease. Patients with liver disease are therefore not only at risk of bleeding but also at risk of thrombosis. Notably, however, many haemostatic complications in liver disease are not related to the haemostatic failure. It is, therefore, crucial to identify the cause of the bleed or thrombotic complication in order to provide adequate treatment. In this paper, we will elaborate on the haemostatic changes that occur in liver disease, reflect on laboratory and clinical studies over the last few years, and explore the pathophysiologies of bleeding and thrombosis in this specific patient group.

Heterogeneous coagulant potential of emicizumab in neonatal factor VIII-deficient plasma.

Emicizumab prophylaxis reduces bleeding in hemophilia A (HA) patients. However, there are few data on emicizumab treatment in neonates with HA (neonate-HA), and the procoagulant effects of emicizumab in these patients are unknown. To investigate the coagulation activity of emicizumab in vitro in a plasma model of neonate-HA. Plasmas from 84 neonates with non-HA were enrolled. However, due to the limited plasma volumes in some cases, 50 plasmas were assigned to two different assay groups. To prepare the neonate-HA model, plasma was first preincubated with an antifactor (F) VIII A2 monoclonal antibody (mAb). After further incubation with emicizumab, global coagulation activity was measured: adjusted maximum coagulation velocity (Ad|min1|) in clot waveform analysis (CWA) and peak thrombin in thrombin generation assay (TGA). Because the addition of anti-FVIII mAb to 22 of 43 samples showed little decrease in Ad|min1|, the remaining 21 samples were analyzed by CWA. The addition of emicizumab increased Ad|min1| in 18 of the 19 cases (effective group) but not in the remaining 3 cases (noneffective group). Similarly, TGA found that emicizumab (effective group) improved peak thrombin in seven of the nine samples tested, but two cases did not respond (noneffective group). Although the effective group had lower levels of FX, there was no significant difference between the effective and noneffective groups in terms of FIX, protein S, protein C, antithrombin, and fibrinogen. The in vitro coagulant potentials of emicizumab in the neonate-HA model were more heterogeneous than those recorded in the adult-HA model.

Monitoring of heparin therapy beyond the anti-Xa activity assay: Evaluation of a thrombin generation assay.

Global coagulation assays may be of added value to the anti-Xa assay for monitoring heparin therapy. Unlike most testing methods, the thrombin generation assay (TGA) has the ability to assess the overall function of the hemostatic system, which provides information on the anticoagulation status of patients. We compared the TGA, measured with ST Genesia® STG-DrugScreen® reagent, with the anti-Xa assay for monitoring heparin therapy in inflammatory and non-inflammatory patients. We also determined reference values for STG-DrugScreen® thrombin generation (TG) parameters. Reference values were determined on 120healthy donors. Furthermore, a spiking experiment with unfractionated heparin (UFH) and low molecular weight heparin (LMWH) was performed, and samples of patients receiving UFH or LMWH were analyzed with ST Genesia® and the anti-Xa assay. High discrepancy between TG parameters and anti-Xa activity was observed for low LMWH anti-Xa levels. TG parameters were affected in 36/46 (time to peak) to 42/46 (peak height) patients during UFH therapy with sub-target anti-Xa activity levels. TGA seems insufficiently sensitive for low concentrations of LMWH. There may be an added value of the TGA for monitoring UFH in so-called heparin-resistant patients. Therefore, the TGA has the potential to be introduced as an additional tool for monitoring heparin therapy.

From the Editor’s Desk...

From the Editor’s Desk...

Thrombin in complex with dabigatran can still interact with PAR‐1 via exosite‐I and instigate loss of vascular integrity

BackgroundAtrial fibrillation (AF) can lead to the loss of microvascular integrity thereby enhancing AF progression. Mechanistically, the pro‐coagulant state that drives the risk of stroke in patients with AF may also play a causal role in microvascular loss. Direct oral anticoagulants (DOACs), the preferred anticoagulants for AF, can target factors upstream (factor Xa [FXa]) or downstream (thrombin) in the coagulation cascade and mediate differential vascular effects through interaction with protease‐activated receptors (PARs). ObjectiveTo investigate the potential effect of different DOACs on vascular integrity. MethodsTo model the impact of DOACs on vascular integrity, we utilized platelet‐free plasma in thrombin generation assays and endothelial barrier assays under identical experimental conditions. These multifactorial systems provide all coagulation factors and their respective natural inhibitors in physiological ratios in combination with the pro‐coagulant endothelial surface on which coagulation is initiated. Furthermore, the system provides pro‐ and anti‐barrier factors and monitoring both assays simultaneously permits coupling of thrombin kinetics to endothelial barrier dynamics. ResultsWe provide evidence that the anti‐FXa DOAC rivaroxaban and the anti‐thrombin DOAC dabigatran are efficient in blocking their target proteases. However, while rivaroxaban could preserve endothelial barrier function, dabigatran failed to protect endothelial integrity over time, which could be prevented in the presence of a custom‐made peptide that blocks thrombin’s exosite‐I. ConclusionsProteolytically inactive thrombin in complex with dabigatran evokes loss of barrier function that can be prevented by a protease‐activated receptor‐1 mimicking peptide blocking thrombin’s exosite‐I.

Recombinant porcine factor VIII corrects thrombin generation in vitro in plasma from patients with congenital hemophilia A and inhibitors

BackgroundNeutralizing factor VIII (FVIII) antibodies are a major complication in hemophilia A. Antihemophilic factor VIII (recombinant), porcine sequence (rpFVIII; susoctocog alfa; Baxalta US Inc., a Takeda company) has low cross‐reactivity to anti‐human FVIII antibodies and can provide functional FVIII activity in the presence of FVIII inhibitors. ObjectivesEvaluate in vitro thrombin generation and clot formation responses to rpFVIII in blood from patients with congenital hemophilia A. MethodsIn this multicenter study, blood was obtained for in vitro analyses that included human and porcine FVIII inhibitors, low <5 Bethesda units (BU)/ml or high ≥5 BU/ml titer (Nijmegen‐modified Bethesda assay); thrombin generation assay (TGA), clot viscoelasticity (thromboelastography), fibrin clot structure analysis (scanning electron microscopy), and epitope mapping. ResultsBlood samples were from 20 patients with congenital hemophilia A (FVIII activity <1%, mean [range] inhibitor titers: anti‐human FVIII, 14 [1–427] BU/ml [n = 13 high, n = 6 low, n = 1 data unavailable]); anti‐porcine FVIII, 12 (0–886) BU/ml (n = 11 high, n = 8 low, n = 1 data unavailable). Porcine inhibitor titer and TGA response measured by endogenous thrombin potential showed an inverse correlation (2.7–10.8 U/ml rpFVIII Spearman correlation coefficient: −0.594 to −0.773; p< 0.01). Clot structures in low anti‐porcine inhibitor titer plasmas were similar to those in noninhibitor plasma. ConclusionsRecombinant porcine factor VIII demonstrated a dose‐dependent correction of thrombin generation and clot formation in vitro, dependent on the anti‐porcine FVIII inhibitor titer. Procoagulant responses to rpFVIII occurred in plasma containing FVIII inhibitors.

Evaluation of novel coagulation and platelet function assays in patients with chronic kidney disease

BackgroundHemostasis evaluation in chronic kidney disease (CKD) is critical for optimal management of thrombotic and bleeding events. Standard coagulation screens are inadequate for predicting coagulopathy in CKD. ObjectiveTo evaluate hemostasis parameters in patients with different stages of CKD using novel coagulation assays. Patients/MethodsCross‐sectional study of 30 healthy controls (HC) and 120 CKD patients (10 Stage 2, 20 Stage 3, 20 Stage 4, 20 Stage 5 not requiring renal replacement therapy, 20 transplant, 10 newly started on hemodialysis [HD], 20 established on HD). Standard laboratory tests were performed in addition to rotational thromboelastometry (ROTEM), multiple electrode aggregometry (MEA), thrombin generation assays, D‐dimer, and markers of thrombogenesis (thrombin‐antithrombin [TAT]), fibrinolysis, and endothelial activation (intercellular adhesion molecule‐1 [ICAM‐1]). ResultsD‐dimer, TAT, and ICAM‐1 concentrations were significantly higher in patients with CKD than HC (P < .01). ROTEM maximum clot firmness was significantly higher in patients than in HC (P < .01). In CKD Stage 5 patients (pre‐HD and started HD) adenosine diphosphate and thrombin receptor activating peptide MEA tests were significantly lower than HC indicating platelet aggregation defect (P < .05). Multivariate analysis confirmed the direct effect of estimated glomerular filtration rate (eGFR) in the variance of ROTEM and MEA tests. Endogenous thrombin potential and peak thrombin were not statistically different between groups, but Stage 5 CKD patients had prolonged lag time (7.91 vs. 6.33, P < .001) and time to thrombin peak (10.8 vs. 9.5, P < .05) compared to HC. ConclusionsPatients with CKD exhibit features of concomitant hypercoagulability measured by ROTEM and platelet dysfunction measured with MEA. eGFR was an independent determinant of platelet dysfunction and hypercoagulability.

Purpura Fulminans Triggered by the Formation of Anti-Endothelial Autoantibodies in a Patient with Chronic Lymphocytic Leukemia

Purpura fulminans (PF), a life-threatening disorder characterized by cutaneous microvascular thrombosis with secondary hemorrhagic infarction, is a rare complication of sepsis and may also be caused by severe protein C or protein S deficiency. Here, we describe the case of a 58-year-old man who developed PF in close association with the onset of chronic lymphocytic leukemia (CLL). PF initially manifested with an ecchymosis of the right upper leg in the absence of sepsis or disseminated intravascular coagulation. PF was likely triggered by antiendothelial IgG autoantibodies functionally interfering with the anticoagulant protein C-protein S-thrombomodulin system on EA.hy926 endothelial cells in a modified thrombin generation assay. Although plasma exchange and immunosuppressive therapy with cyclophosphamide were temporarily effective, PF eventually progressed and the patient died from septic shock during treatment-associated neutropenia. In rare occasions, CLL may be associated with autoimmune-mediated PF requiring prompt diagnosis and aggressive multimodal therapy.

A correlation of thrombin generation assay and clot waveform analysis in patients on warfarin

ABSTRACT Objectives Thrombin generation assays and activated partial thromboplastin time (aPTT)-based clot waveform analysis (CWA), are some examples of global coagulation assays. Each modality evaluates different aspects of the clot forming process to globally define haemostasis with exclusive measurement parameters. Data on CWA are emerging, but its performance against other haemostatic assays is yet to be ascertained. This study evaluates the correlation between aPTT-based CWA and CAT parameters across a range of INR in warfarin-treated patients. Patients/Methods A prospective study consisting of patients on warfarin anticoagulation with varying INR levels. CWA and CAT were performed for the study subjects. Results 54 samples were included covering an INR range from 1.33–6.89, with a mean of 4.31 +/- 1.13. For CAT parameters, endogenous thrombin potential (ETP) and peak thrombin were assessed. Both unadjusted and adjusted (adjusted for final plateau transmittance) aPTT-based CWA were evaluated for parameters min1 (maximum velocity), min2 (maximum acceleration) and max2 (maximum deceleration). Peak thrombin showed significant correlation with all CWA parameters (min1: r = 0.435, P<0.001; min2: r = 0.485, P<0.001; max2: r = 0.578, P<0.001; adjusted min1: r = 0.734, P<0.001, adjusted min2: r = 0.693, P<0.001; adjusted max2: r = 0.751, P<0.001). ETP correlated significantly with all CWA parameters except unadjusted min1 (min1: r = 0.235, P = 0.087; min2: r = 0.326, P = 0.016; max2: r = 0.437, P<0.001; adjusted min1: r = 0.610, P<0.001, adjusted min2: r = 0.563, P<0.001; adjusted max2: r = 0.642, P<0.001). Conclusion We demonstrated a modest correlation between CAT and CWA parameters. Adjusted CWA improved this correlation. These findings provide additional understanding of CWA and it’s role in the evaluation of global haemostatic function.

Routine and Advanced Laboratory Tests for Hemostasis Disorders in COVID-19 Patients: A Prospective Cohort Study.

Background: Thrombosis is frequent during COVID-19 disease, and thus, identifying predictive factors of hemostasis associated with a poor prognosis is of interest. The objective was to explore coagulation disorders as early predictors of worsening critical conditions in the intensive care unit (ICU) using routine and more advanced explorations. Materials: Blood samples within 24 h of ICU admission for viscoelastic point-of-care testing, (VET), advanced laboratory tests: absolute immature platelet count (A-IPC), von Willebrand-GPIb activity (vWF-GpIb), prothrombin fragments 1 + 2 (F1 + 2), and the thrombin generation assay (TGA) were used. An association with worse outcomes was explored using univariable and multivariable analyses. Worsening was defined as death or the need for organ support. Results: An amount of 85 patients with 33 in critical condition were included. A-IPC were lower in worsening patients (9.6 [6.4–12.5] vs. 12.3 [8.3–20.7], p = 0.02) while fibrinogen (6.9 [6.1–7.7] vs. 6.2 [5.4–6.9], p = 0.03), vWF-GpIb (286 [265–389] vs. 268 [216–326], p = 0.03) and F1 + 2 (226 [151–578] vs. 155 [129–248], p = 0.01) were higher. There was no difference observed for D-dimer, TGA or VET. SAPS-II and A-IPC were independently associated with worsening (OR = 1.11 [1.06–1.17] and OR = 0.47 [0.25–0.76] respectively). The association of a SAPS-II ≥ 33 and an A-IPC ≤ 12.6 G/L predicted the worsening of patients (sensitivity 58%, specificity 89%). Conclusions: Immature platelets are early predictors of worsening in severe COVID-19 patients, suggesting a key role of thrombopoiesis in the adaption of an organism to SARS-CoV-2 infection.

Reversing direct factor Xa or thrombin inhibitors: Factor V addition to prothrombin complex concentrate is beneficial in vitro

BackgroundProthrombin complex concentrate (PCC) is a human plasma‐derived mixture of partially purified vitamin K‐dependent coagulation factors (VKCF). Current therapeutic indication is treatment and perioperative prophylaxis of bleeding in acquired VKCF deficiency. Off‐label uses include treatment of direct factor Xa‐ or thrombin inhibitor‐associated bleeds, treatment of trauma‐induced coagulopathy, and hemorrhagic complications in patients with liver disease. ObjectiveConsidering PCC as a general prohemostatic drug, we argued that its clinical efficacy can benefit from supplementation with coagulation factors that are absent in the current PCC formulation. In this study, we focused on factor V. MethodsWe mimicked a coagulopathy in vitro by spiking whole blood or derived plasma with the direct oral anticoagulants (DOAC) rivaroxaban or dabigatran. We studied DOAC reversal by PCC and factor V concentrate (FVC) using a thrombin generation assay, thromboelastography, fibrin generation clot lysis test, and microfluidic thrombus formation under flow. ResultsIn DOAC‐treated plasma, PCC increased the amount of thrombin generated. The addition of FVC alone or in combination with PCC caused a partial correction of the thrombin generation lag time and clotting time. In DOAC‐treated whole blood, the combination of PCC and FVC synergistically improved clotting time under static conditions, whereas complete correction of fibrin formation was observed under flow. Clot strength and clot resistance toward tissue plasminogen activator‐induced lysis were both increased with PCC and further enhanced by additional FVC. ConclusionOur in vitro study demonstrates a beneficial effect of the combined use of PCC and FVC in DOAC reversal.

A modified, optimized kinetic photometric thrombin generation assay components to measure the potency of thrombin in plasma

The purpose of the present study was to improve, optimize and modification, a kinetic photometric assay of Factor IIa activity with regard to blood sample types, calibration, analytic corrections, and activation reagents, in order to establish a rapid routine, less time consuming, and suitable method for the evaluation of large numbers of clinical samples. Our modified method was based on the continuous monitoring of the Factor IIa released from the reacting substance, in the presence of a specific chromogenic peptide substrate (S2238). The optimization of pH and of the concentrations of substrate, activator (CaCl2), EDTA, and tissue factor was carried out as well as blood sample types (Platelet-rich and platelet-poor plasma samples) so as to ensure the best compromise between reliability, specificity, and sensitivity, However, the Factor IIa activity was compared in the plasma of different donors. In conclusion, our modified and optimized FIIa generation assay is a rapid and simple method for the diagnosis and monitoring of different clinical cases, at the same time it can be easily adapted to clinical chemistry analyzers.

The influence of corn trypsin inhibitor on the contribution of coagulation determinants to the Technoclone Thrombin Generation Assay (TGA) and the Calibrated Automated Thrombogram (CAT).

In thrombin generation (TG) assays, regarded as global coagulation tests, contact activation is considered a major problem which can be eliminated by adding Corn Trypsin Inhibitor (CTI). In previous studies, however, venous thrombosis risk prediction using TG assays did not improve after CTI addition. However, it is unknown whether CTI addition could help to detect subtle but relevant nuances in determinants of TG, making the assay more suitable to detect disturbances in the coagulation system. This study’s objective was to assess whether the addition of CTI is associated with a broader contribution of individual coagulation factors to the total amount of thrombin formed in Calibrated Automated Thrombogram (CAT) and Technoclone Thrombin Generation Assay (TGA). Thrombin generation was measured in 326 healthy individuals from THE VTE study at very low tissue factor concentrations, with and without addition of CTI prior to blood sampling. The influence of several coagulation factors on total amount of thrombin formed, i.e. area under the curve (AUC) or endogenous thrombin potential (ETP), was analysed using multiple linear regression with standardisation of all values resulting in Z-scores with 95% confidence intervals (95%CI). Association between coagulation factors and TG changed minimally after addition of CTI. Largest changes after CTI addition were found for following factors: for CAT: free protein S (from 0.00 (95%CI -0.12 to 0.12) to -0.29 (95%CI -0.43 to -0.15)) and protein S (from -0.05 (95%CI -0.18 to 0.08) to -0.21 (95%CI -0.37 to -0.05)); for TGA: antithrombin (from -0.11 (-0.23 to 0.02) to -0.19 (-0.30 to -0.07)), factor VIII (from 0.15 (0.03 to 0.27) to 0.24 (0.13 to 0.36)) and fibrinogen (from 0.12 (-0.01 to 0.26) to 0.19 (0.06 to 0.32)). In conclusion, there is no clear trend towards a broader contribution of coagulation factors in samples handled with CTI compared with those handled without CTI.

A Prospective Observational Study on Multiplate®-, ROTEM®- and Thrombin Generation Examinations Before and Early After Implantation of a Left Ventricular Assist Device (LVAD).

BackgroundHeart failure patients are frequently on coagulation-active medications before LVAD implantation and perioperative bleeding is a frequent complication after left ventricular assist device (LVAD) implantation. The role of point-of-care coagulation tests in assessing bleeding risk for LVAD implantation and the early postoperative time course of these tests is not well established.MethodsWe prospectively enrolled 25 patients with terminal heart failure undergoing LVAD implantation. Study related TRAP-, ASPI- and ADP- tests of Multiplate® platelet aggregometry, ROTEM® rotational thromboelastometry (INTEM, EXTEM, FIBTEM), thrombin generation assay and conventional laboratory studies were measured at 11 predefined time-points during the first 21 postoperative days. We examined if preoperative TRAP-, ASPI-, ADP- and ROTEM values are correlated with estimated total blood loss (primary outcome parameter) during the first 21 days after LVAD implantation and compared the baseline values of these measurements between patients with a bleeding event to those without. We performed Spearman's correlation and non-parametric tests for paired and non-paired comparisons.Results7 out of 25 (28%) patients experienced a bleeding event of which 4 required surgical revision. Of the preoperatively performed measurements the TRAP test [Spearman's Rho (ρ) = −0.5, p = 0.01], INTEM CFT (ρ = 0.72, p < 0.001), INTEM alpha (−0.7, p < 0.001), EXTEM MCF (ρ = −0.63; p < 0.001), EXTEM alpha (ρ = −0.67; p < 0.001), FIBTEM MCF (ρ = −0.41; p = 0.042), Fibrinogen (Clauss) (ρ = −0.5; p = 0.011), Anti-thrombin activity (ρ = −0.49; p = 0.013) and platelet count (ρ = −0.42; p = 0.034) were significantly correlated to total blood loss. Patients undergoing a surgical bleeding revision had significantly reduced values in TRAP—[31.5 IQR (17.25–43.5U) vs. 69 IQR (52.5–87U); p = 0.004], ASPI—[16.5 IQR (5.5–35.7U) vs. 39 IQR (24.5–62.5U); p = 0.038], ADP—[30 IQR (22–69U) vs. 12.5 IQR (8.7–21.5U); p = 0.01], EXTEM MCF—[63 IQR (57.7–63.7) vs. 67 IQR (65–75.5); p = 0.019] and EXTEM alpha [74 IQR (68.75–74) vs. 79 IQR (78–80.5); p = 0.002] values before LVAD implantation.ConclusionMultiplate® and ROTEM® measurements before LVAD implantation may identify LVAD candidates with platelet dysfunction and alterations of the primary hemostasis and could guide anesthetists and intensive care practitioners in bleeding risk stratification and in the perioperative clinical management.

Active FXI Can Independently Predict Ischemic Stroke in Anticoagulated Atrial Fibrillation Patients: A Cohort Study.

Atrial fibrillation (AF) is associated with a prothrombotic state. Presence of active tissue factor (TF), activated factor IX (FIXa) and FXIa in circulating blood contributes to thrombosis. We investigated a prognostic value of these factors in AF patients. In this cohort study, 284 AF patients (aged 63.3 ± 8.8 years) treated with oral anticoagulants were enrolled. Plasma levels of active coagulation factors were evaluated using thrombin generation assay. Concentrations of fibrinogen, D-dimer, interleukin-6 (IL-6), and endothelial damage markers, including von Willebrand factor (VWF) and soluble (s)E-selectin, were also measured. Ischemic stroke and cardiovascular death, analyzed separately or as a composite endpoint, were recorded during a mean follow-up of 47 months. Cerebrovascular events were observed in 20 patients (1.8%/year) who had at baseline higher fibrinogen, D-dimer, and VWF levels. Active TF and FXIa at enrollment were detectable in 12 (60%) and 15 (75%) patients who experienced ischemic stroke during follow-up. The composite endpoint observed in 23 patients (2.1%/year) was associated with increased concentrations of the above laboratory variables, along with 26% higher IL-6 levels. sE-selectin did not differ between the studied groups. On multivariable regression analysis, advanced age, anticoagulation discontinuation, and detectable FXIa, but not active TF, independently predicted the composite endpoint. No associations of FIXa with the study endpoints were observed. FXIa present in circulating blood is associated with increased risk of ischemic stroke and cardiovascular death in anticoagulated AF patients during long-term follow-up. FXIa inhibition could be useful in cardiovascular prevention in AF beyond the current oral anticoagulation.

Report of surgeries, their outcome and the thrombin generation assay in patients with Factor XI deficiency: A retrospective single-centre study.

In patients with FXI deficiency, the risk of surgery-related bleeding is poorly correlated with plasma FXI activity (FXI:C); the latter can therefore not be used as a reliable predictor of bleeding in surgeries. The aim of this retrospective study was to determine whether thrombin generation assay (TGA) could be used to evaluate the risk of surgery-related bleeding in FXI-deficient patients. TGA parameters were compared to FXI:C values, haemostatic treatments and surgical outcomes. All patients followed at the haemophilia treatment care centre (Lyon, France) with a FXI:C<50IU/dL, and for whom a baseline TGA was performed between January 2014 and December 2019, were included. Among the 175 surgeries reported herein in 49 patients, FXI concentrates were used for 11 (6%) surgeries and fresh frozen plasma was used for five (3%) surgeries; these surgeries were performed in patients with two or three impaired TGA parameters. No haemostatic treatment was prescribed for 119 (68%) surgeries. A surgery-related bleeding occurred in 12 patients during 21 (12%) surgeries. Thrombin generation was significantly reduced or delayed in patients who reported surgery related-bleeding. Among the 34 (68%) surgeries performed without haemostatic treatment in patients with three impaired TGA parameters, a surgery-related bleeding was reported in 44% of cases (15 surgeries out of 34). The present study confirmed that TGA is an interesting laboratory test in FXI deficiency, for determining the bleeding risk and guiding the haemostatic management of surgeries, while taking into account the surgical bleeding risk and the history of bleeding.

Hemostatic efficacy of marstacimab alone or in combination with bypassing agents in hemophilia plasmas and a mouse bleeding model

BackgroundPatients with hemophilia have deficiencies in intrinsic coagulation factors and can develop inhibitors that limit the effectiveness of replacement coagulation factors. Marstacimab, a human monoclonal antibody, binds and inhibits the human tissue factor pathway inhibitor. Marstacimab is currently under development as a potential prophylactic treatment to prevent bleeding episodes in patients with hemophilia A and B. ObjectiveTo assess the effects of marstacimab alone or in combination with the bypassing agent recombinant factor FVIIa (rFVIIa) or activated prothrombin complex concentrate (aPCC) on thrombin generation and bleeding. MethodsMarstacimab and/or rFVIIa or aPCC were added to hemophilic A or B plasma or nonhemophilic plasma in vitro. Hemostatic activity was measured using the thrombin generation assay. In vivo effects were assessed using a mouse acute bleeding model. Male hemophilia A mice were dosed with marstacimab plus aPCC before tail clip; blood loss was quantified by measuring hemoglobin. ResultsMarstacimab plus rFVIIa or aPCC slightly increased peak thrombin levels compared with either agent alone. This increase was within the reported range for nonhemophilic plasma and did not exceed levels observed in nonhemophilic plasma treated with marstacimab alone. Hemophilia A mice that received 200 U/kg aPCC had significantly reduced bleeding (62%) compared with vehicle‐treated mice (p < 0.05), and marstacimab plus aPCC reduced bleeding by 83.3% compared with vehicle (p= 0.0009). ConclusionsMarstacimab alone or with bypassing agents increased hemostasis in hemophilia plasma without generating excessive thrombin. The hemostatic activity of marstacimab plus aPCC was confirmed in hemophilia A mice.

Targeted SERPIN (TaSER): A dual‐action antithrombotic agent that targets platelets for SERPIN delivery

BackgroundOcclusive thrombi are not homogeneous in composition. The core of a thrombus is rich in activated platelets and fibrin while the outer shell contains resting platelets. This core is inaccessible to plasma proteins. We produced a fusion protein (targeted SERPIN–TaSER), consisting of a function‐blocking VHH against glycoprotein Ibα (GPIbα) and a thrombin‐inhibiting serine protease inhibitor (SERPIN; α1‐antitrypsin 355AIAR358) to interfere with platelet‐driven thrombin formation. AimTo evaluate the antithrombotic properties of TaSER. MethodsBesides TaSER, we generated three analogous control variants with either a wild‐type antitrypsin subunit, a non‐targeting control VHH, or their combination. We investigated TaSER and controls in protease activity assays, (platelet‐dependent) thrombin generation assays, and by western blotting. The effects of TaSER on platelet activation and von Willebrand factor (VWF) binding were studied by fluorescence‐activated cell sorting, in agglutination studies, and in ATP secretion experiments. We studied the influence of TaSER in whole blood (1) on platelet adhesion on VWF, (2) aggregate formation on collagen, and (3) thrombus formation (after recalcification) on collagen and tissue factor. ResultsTaSER binds platelets and inhibits thrombin activity on the platelet surface. It blocks VWF binding and disassembles platelet agglutinates. TaSER delays tissue factor‐triggered thrombin generation and ATP secretion in platelet‐rich plasma in a targeted manner. In flow studies, TaSER interferes with platelet adhesion and aggregate formation due to GPIbα blockade and limits thrombus formation due to targeted inhibition of platelet‐dependent thrombin activity. ConclusionThe synergy between the individual properties of TaSER makes it a highly effective antithrombotic agent with possible clinical implications.

In Vitro Apixaban Removal By CytoSorb Whole Blood Adsorber: An Experimental Study

The use of unopposed oral anticoagulants while undergoing cardiothoracic surgery is associated with severe bleeding and increased morbidity. The aim of this experimental study was to examine if the apixaban concentration in reconstituted blood could be reduced in an in vitro setup by the use of CytoSorb whole blood adsorber, and to study how this affected global coagulation assays. An experimental study performed in a laboratory. An in vitro setup with reconstituted whole blood. Reconstituted whole blood spiked with apixaban circulated in an in vitro circuit with the CytoSorb 300 mL device connected. Blood samples were drawn at 0, 5, 15, 30, 60, and 120 minutes of adsorption. The apixaban concentration was measured at each time point. In addition, the global coagulation assays, thromboelastometry clotting time and thrombin generation, were performed, and the results were compared with baseline values obtained before spiking blood with apixaban. After 30 minutes of adsorption, the mean apixaban concentration was reduced from 414.3 (±69.1) ng/mL to 33 (±11.4) ng/mL. Thrombin generation showed maximum effect after 60 minutes, and the thromboelastometry clotting time was close to baseline values after 120 minutes. In this in vitro study, apixaban concentrations were effectively reduced, and the clotting time and thrombin generation assays were normalized by the use of CytoSorb whole blood adsorber.