Direct oral anticoagulants in cirrhosis: Rationale and current evidence.

Cirrhosis is a major health concern worldwide with a complex pathophysiology affecting various biological systems, including all aspects of haemostasis. Bleeding risk is mainly driven by portal hypertension, but in end-stage liver disease it is further increased by alterations in haemostatic components, including platelet function, coagulation, and fibrinolysis. Concurrently, patients with cirrhosis are prone to venous thromboembolic events (VTE) because of the altered haemostatic balance, in particular an increase in thrombin generation. In patients with cirrhosis, vitamin K antagonists (VKA) and low molecular weight heparins (LMWH) are currently the standard of care for VTE prevention, with VKA also being standard of care for stroke prevention in those with atrial fibrillation. However, direct oral anticoagulants (DOAC) could have specific advantages in this patient population. Clinical experience suggests that DOAC are a safe and possibly more effective alternative to traditional anticoagulants for the treatment of VTE in patients with compensated cirrhosis. In addition, emerging data suggest that primary prophylactic treatment with anticoagulants may improve clinical outcomes in patients with cirrhosis by reducing the risk of hepatic decompensation. The selection of the most appropriate DOAC remains to be clarified. This review focuses on the rationale for the use of DOAC in patients with cirrhosis, the specific effects of the different DOAC (as assessed by in vitro and in vivo pharmacokinetic and pharmacodynamic studies), as well as clinical outcomes in patients with cirrhosis on DOAC.

Clot lysis time and thrombin generation in patients undergoing transcatheter aortic valve implantation.

BACKGROUND: Aortic valve stenosis (AS) is the most prevalent valvular heart disease and is associated with a significant increase in mortality. AS has been shown to be linked with numerous coagulation system abnormalities, including increased fibrin deposition on the stenotic aortic valves. Transcatheter aortic valve implantation (TAVI) is the primary treatment method for patients at high surgical risk. OBJECTIVES: The aim of the study was to assess the impact of treating severe AS with TAVI on thrombin generation and clot lysis time (CLT). METHODS: We studied 135 symptomatic AS patients recommended for TAVI by the local Heart Team. All measurements were performed before and 5-7 days after TAVI. Alongside clinical assessment and echocardiographic analysis, we assessed clot lysis time (CLT) and thrombin generation parameters, including lag time, peak thrombin generation, time to peak thrombin generation (ttPeak), and endogenous thrombin potential (ETP). RESULTS: 70 patients were included in the final analysis. After TAVI, there was a significant 9% reduction in CLT despite a 12% increase in fibrinogen concentration. We observed significant increase in lag time and ttPeak (20% and 12%, respectively), and 13% decrease in peak thrombin concentration compared to pre-procedural levels. Multivariable linear regression analysis demonstrated that baseline CLT and C-reactive protein (CRP) levels were independent predictors of significant reduction in mean aortic gradient, defined as TAVI procedure success. CONCLUSIONS: CLT and peak thrombin concentration decreased, while Lag time and ttPeak increased significantly after TAVI. Multivariable linear regression analysis demonstrated CLT and CRP levels as independent predictors of achieving a reduction in mean aortic gradient, defining TAVI procedure success.

Predelivery Haemostatic Biomarkers in Women with Non-Severe Postpartum Haemorrhage.

Background: Postpartum haemorrhage (PPH) is a frequent complication of childbirth that is difficult to predict. Predelivery coagulation biomarkers may help to guide preventive strategies. Our objective was to evaluate the association of predelivery haemostatic biomarkers with non-severe PPH. Methods: A nested case-control study was conducted within the « Study of Biological Determinants of Bleeding Postpartum ¼ in order to compare different haemostatic biomarkers in plasma from pregnant women with non-severe PPH (cases) and controls without PPH matched for age, body mass index, term, and mode of delivery. Blood was collected at entry in the delivery room. Global haemostatic assays (thrombin generation assay (TGA) and plasmin generation assay (PGA)) were then performed on freshly thawed aliquots of platelet-poor plasma. Results: A total of 370 pregnant women (185 cases and 185 controls) were included. Median [interquartile range] predelivery platelet count was lower in PPH cases than in controls (217 [181-259] versus 242 [196-280] G/L). TGA and PGA parameters were similar between cases and controls. In a subset analysis of vaginal deliveries (n = 144), median predelivery TGA thrombin peak was lower, and median predelivery PGA lag phase was longer in cases compared to controls. In multivariable analysis, only predelivery platelet count was independently associated with non-severe PPH. Conclusions: Predelivery platelet count is associated with non-severe PPH. Differences in other haemostatic parameters are tenuous, questioning their usefulness in predicting non-severe PPH.

Global Hemostatic Methods to Tailor Treatment With Bypassing Agents in Hemophilia A With Inhibitors- A Single-Center, Pilot Study.

For patients with hemophilia A and high-titer inhibitors treated with bypassing agents there are no reliable methods to assess treatment effect. We investigated the utility of global hemostatic methods in assessing treatment with bypassing agents (rFVIIa or activated prothrombin complex [aPCC]). All patients with hemophilia A and inhibitors followed at the Coagulation Unit or the Pediatric Coagulation Unit at Karolinska University Hospital aged 6 years and above were eligible for this noninterventional study. Baseline plasma samples were spiked with bypassing agents in increasing concentrations (aPCC 50 U/kg, 100 U/kg, 150 U/kg, and rFVIIa 90 µg/kg and 270 µg/kg) in vitro. For patients treated with factor concentrates or bypassing agents follow-up samples were collected (in vivo tests). The samples were analyzed using overall hemostatic potential (OHP), and calibrated automated thrombogram, Calibrated Automated Thrombogram (CAT). Nine patients with hemophilia A with inhibitors were included. Spiking with rFVIIa normalized the coagulation potential in 6/8 samples, in 3 only with high dose. Only one sample did not improve adequately after spiking with aPCC. The improvement in hemostasis was reliably shown by both CAT and OHP. The baseline potential was, however, more often measurable by OHP compared to CAT. Factor concentrate had been administered to 5 patients normalizing the hemostatic potential in vivo in 2 (without spiking). The hemostatic improvement induced by spiking with rFVIIa or aPCC is shown by OHP and CAT, but the results have to be evaluated in larger cohorts.

Elevated plasma factor XI is associated with postthrombotic syndrome.

INTRODUCTION: Postthrombotic syndrome (PTS), a common complication of deep vein thrombosis (DVT), is largely inflammatory by nature with contribution of prothrombotic mechanisms. The role of factor (F)XI in PTS has not been explored yet. We investigated whether elevated FXI is associated with PTS occurrence. MATERIALS AND METHODS: We enrolled 180 consecutive patients (aged 43 ± 13 years) with first-ever DVT. After 3 months FXI levels were measured, along with inflammatory markers, thrombin generation, plasma clot permeability (Ks), clot lysis time (CLT), and fibrinolysis proteins. We assessed PTS using the Villalta score and recorded symptomatic venous thromboembolism (VTE) at a 1-year and venous ulcers at a median 53 months follow-up. RESULTS: Baseline median FXI was 102 % [IQR 92-113 %] and showed positive association with Villalta score (R = 0.474, P < 0.001). Patients with PTS (n = 48, 26.7 %) had 16.1 % higher FXI (P < 0.001) and FXI ≥120 % occurred more often in PTS patients (odds ratio [OR] 5.55, 95 % confidence interval [CI] 2.28-13.47). There were associations of baseline FXI with Ks and CLT along with thrombin activatable fibrinolysis inhibitor (TAFI) activity, C-reactive protein, and interleukin-6, but not with fibrinogen, or thrombin generation. After age adjustment higher FXI was independently associated with PTS occurrence (OR per 1 % 1.06, 95 % CI 1.02-1.09) and VTE recurrence (OR 1.03, 95 % CI 1.01-1.06). At long-term follow-up, patients with venous ulcers had 13.6 % higher baseline FXI (P = 0.002). CONCLUSIONS: Elevated FXI in association with inflammation and prothrombotic fibrin clot properties may contribute to the development of PTS following DVT.

Comparative analysis of thrombin generation platforms for patients with coagulation factor deficiencies: A comprehensive assessment.

INTRODUCTION: Thrombin generation assays (TGAs) assess the overall functionality of the hemostatic system and thereby provide a reflection of the hemostatic capacity of patients with disorders in this system. Currently, four (semi-)automated TGA platforms are available: the Calibrated Automated Thrombogram, Nijmegen Hemostasis Assay, ST Genesia and Ceveron s100. In this study, we compared their performance for detecting patients with congenital single coagulation factor deficiencies. MATERIALS AND METHODS: Pooled patient samples, healthy control samples and normal pooled plasma were tested on all four platforms, using the available reagents that vary in tissue factor and phospholipid concentrations. The TGA parameters selected for analysis were peak height and thrombin potential. Results were normalized by using the calculated mean of healthy controls and a correction for between-run variation. Outcomes were presented as relative values, with the mean of healthy controls standardized to 100 %. RESULTS: Across all platforms and reagents used, thrombin potentials and peak heights of samples with coagulation factor deficiencies were lower than those of healthy controls. Reagents designed for bleeding tendencies yielded the lowest values on all platforms (relative median peak height 19-32 %, relative median thrombin potential 19-45 %). Samples representing more severe coagulation factor deficiencies generally exhibited lower relative peak heights and thrombin potentials. CONCLUSIONS: Thrombin generation assays prove effective in differentiating single coagulation factor deficient samples from healthy controls, with modest discrepancies observed between the platforms. Reagents designed for assessing bleeding tendencies, featuring the lowest tissue factor and phospholipid concentrations, emerged as the most suitable option for detecting coagulation factor deficiencies.

Targeting tissue factor pathway inhibitor with concizumab to improve hemostasis in patients with Glanzmann thrombasthenia: an in vitro study.

BACKGROUND: Glanzmann thrombasthenia (GT) is caused by an inherited defect of platelet αIIbß3 integrin. Concizumab, a monoclonal antibody specific for tissue factor pathway inhibitor, abolishes its anticoagulant effect. OBJECTIVES: To evaluate the in vitro ability of concizumab to improve hemostasis in GT. METHODS: The effects of concizumab were evaluated in whole blood or platelet-rich plasma from GT patients (n = 5-9) using a thrombin generation assay, rotational thromboelastometry (ROTEM), a global fibrinolytic capacity assay, and a flow chamber assay (Total Thrombus formation Analysis System). Washed platelets (WPs) and 20 nM recombinant activated factor VII (rFVIIa) were included for comparison. RESULTS: The lag time in the thrombin generation assay was significantly longer (+85%; P < .0001) in GT patients than in controls. WPs, rFVIIa, and concizumab each significantly improved thrombin generation profiles. The ROTEM clotting time (CT) was significantly longer in GT patients than in controls (677 seconds vs 523 seconds; P = .03). However, CT improved after adding WPs, rFVIIa, or concizumab. Under flow, occlusive thrombi were present in all healthy controls after 10 minutes, whereas platelet-fibrin depositions were not seen in GT patients. Subocclusive or occlusive thrombi formed when GT blood was mixed with WPs, rFVIIa, or concizumab. Clots in GT platelet-rich plasma were more susceptible to fibrinolysis and were improved by WPs, rFVIIa, or concizumab. CONCLUSION: Concizumab enhanced thrombin generation, decreased the ROTEM CT, improved thrombus formation under flow, and reduced clot lysis. Our results demonstrate the potential of concizumab for subcutaneous prophylaxis in GT patients.

Global coagulation assays detect an early prothrombotic state in children with acute lymphoblastic leukemia.

BACKGROUND: Pediatric patients with acute lymphoblastic leukemia (ALL) are at highest risk of venous thromboembolism during the induction therapy (IT). These events are not predictable by conventional coagulation assays. OBJECTIVES: To investigate the utility of global coagulation assays (GCAs) for assessing the hemostatic state in children with ALL during IT. METHODS: We included children with ALL (n = 15) and healthy controls (n = 15). Analyses were performed at different time points during IT of the AIEOP-BFM protocols. In addition to prothrombotic biomarkers, natural anticoagulant proteins, and in vivo thrombin generation (TG) markers, ex vivo TG was measured using the gold standard calibrated automated thrombogram method, automated ST Genesia, and thrombodynamics analyzer (TD). The latter also provided measurement of fibrin clot formation. RESULTS: Different from conventional coagulation assays and in vivo TG markers, ex vivo GCAs detected increasing prothrombotic changes during IT. Particularly, TG measured with TD as expressed by endogenous thrombin potential was already significantly elevated at days 8 to 12 (P < .01) and continued to increase during IT compared with prior to beginning treatment, indicating a very early shift toward a procoagulant state. A similar pattern was observed for the rate of fibrin clot formation (stationary rate of clot growth: P < .01 at days 8-12). Remarkably, in patients developing thrombotic complications (n = 5), both GCAs, ST Genesia and TD, showed a significantly higher endogenous thrombin potential very early (already at days 8-12, P < .05), well before clinical manifestation. CONCLUSION: GCAs capture prothrombotic changes early during IT in ALL pediatric patients. If confirmed, this approach will allow tailoring thromboprophylaxis in children with ALL at highest risk for venous thromboembolism.

A small-molecule hemostatic agent for the reversal of direct oral anticoagulant-induced bleeding.

Background: The bleeding risk associated with direct oral anticoagulants (DOACs) remains a major concern, and rapid reversal of anticoagulant activity may be required. Although specific and nonspecific hemostatic biotherapies are available, there is a need for small-molecule DOAC reversal agents that are simple and cost-effective to produce, store, and administer. Objectives: To identify and characterize a small molecule with procoagulant activity as a DOAC reversal agent. Methods: We sought to identify a small procoagulant molecule by screening a chemical library with a plasma clotting assay. The selected molecule was assessed for its procoagulant properties and its ability to reverse the effects of the DOACs in a thrombin generation assay. Its activity as a DOAC reversal agent was also evaluated in a tail-clip bleeding assay in mice. Results: The hemostatic molecule (HeMo) dose-dependently promoted thrombin generation in plasma, with dose values effective in producing half-maximum response ranging between 3 and 5 µM, depending on the thrombin generation assay parameter considered. HeMo also restored impaired thrombin generation in DOAC-spiked plasma and reversed DOAC activity in the mouse bleeding model. HeMo significantly reduced apixaban-induced bleeding from 709 to 65 µL (vs 43 µL in controls; P < .01) and dabigatran-induced bleeding from 989 to 155 µL (vs 126 µL in controls; P < .01). Conclusion: HeMo is a small-molecule procoagulant that can counterbalance hemostatic disruption by a thrombin inhibitor (dabigatran) or factor Xa inhibitors (apixaban and rivaroxaban). The compound's effective clot formation and versatility make it a possible option for managing the inherent hemorrhagic risk during DOAC therapy.

Extracellular histones: a unifying mechanism driving platelet-dependent extracellular vesicle release and thrombus formation in COVID-19.

BACKGROUND: COVID-19 can cause profound inflammation and coagulopathy, and while many mechanisms have been proposed, there is no known common pathway leading to a prothrombotic state. OBJECTIVES: From the beginning of the COVID-19 pandemic, elevated levels of extracellular histones have been found in plasma of patients infected with SARS-CoV-2. We hypothesized that platelet activation triggered by extracellular histones might represent a unifying mechanism leading to increased thrombin generation and thrombosis. METHODS: We utilized blood samples collected from an early clinical trial of hospitalized COVID-19 patients (NCT04360824) and recruited healthy subjects as controls. Using plasma samples, we measured the procoagulant and prothrombotic potential of circulating extracellular histones and extracellular vesicles (EVs). Platelet prothrombotic activity was assessed via thrombin generation potential and platelet thrombus growth. Circulating EVs were assessed for thrombin generation potential in vitro in plasma and enhancement of thrombotic susceptibility in vivo in mice. RESULTS: Compared with controls, COVID-19 patients had elevated plasma levels of citrullinated histone H3, cell-free DNA, nucleosomes, and EVs. Plasma from COVID-19 patients promoted platelet activation, platelet-dependent thrombin generation, thrombus growth under venous shear stress, and release of platelet-derived EVs. These prothrombotic effects of COVID-19 plasma were inhibited by an RNA aptamer that neutralizes both free and DNA-bound histones. EVs isolated from COVID-19 plasma enhanced thrombin generation in vitro and potentiated venous thrombosis in mice in vivo. CONCLUSION: We conclude that extracellular histones and procoagulant EVs drive the prothrombotic state in COVID-19 and that histone-targeted therapy may prove beneficial.

Testosterone therapy does not affect coagulation in male hypogonadism: a longitudinal study based on thrombin generation.

CONTEXT: Testosterone therapy has been variably associated with increased thrombotic risk but investigations of global coagulation in this setting are lacking. OBJECTIVE: To compare global coagulation of hypogonadal men before (T0) and 6 months after (T1) starting testosterone replacement therapy (TRT), and healthy controls. DESIGN: Observational prospective cohort study. SETTING: Two tertiary endocrinological ambulatory care centers. PATIENTS: Thirty-eight men with hypogonadism (mean age 55, SD 13) and 38 age-matched healthy controls. INTERVENTIONS: Thrombin generation assay (TGA) was performed at T0 and T1 in hypogonadal men and in controls. TGA is an in vitro procedure based on the continuous registration of thrombin generation and decay under conditions mimicking the process that occurs in vivo. MAIN OUTCOME MEASURES: The following TGA parameters were recorded: lag-time; thrombin-peak concentration; time-to-reach the peak, velocity index and endogenous thrombin potential (ETP), the latter representing the total amount of thrombin generated under the driving forces of procoagulants opposed by the anticoagulants. PC, antithrombin, factor (F)VIII, and fibrinogen were assessed. RESULTS: No changes of TGA parameters were observed between T0 and T1. Hypogonadal men displayed significantly higher ETP, fibrinogen, and significantly lower antithrombin levels both at T0 and T1 compared to controls. Thrombin-peak of hypogonadal men was significantly higher than controls at T0 but not at T1. ETP and antithrombin were correlated with testosterone levels. CONCLUSIONS: Hypogonadal men display a procoagulant imbalance detected by increased thrombin generation. Short-term TRT does not worsen global coagulation, suggesting that the treatment can be safely prescribed to men diagnosed with hypogonadism.

Cryptogenic ischemic stroke in cardiac transthyretin amyloidosis and sinus rhythm: a case report.

Cardiac amyloidosis is a group of diseases characterized by the deposition of amyloid fibers in cardiac tissue. Two forms are mainly reported: light chain (AL) and transthyretin (ATTR) amyloidosis. Among the complications of transthyretin amyloidosis there are thrombotic events and, to a lesser extent, hemorrhagic events. The latter are likely caused by perivascular amyloid deposition resulting in capillary fragility, in addition to INR lability during anticoagulant therapy. The onset of thrombotic events may be caused by the high prevalence of atrial fibrillation (AF), mechanical cardiac dysfunction and atrial myopathy observed in patients with transthyretin amyloidosis. It remains unclear why thromboembolic events occur even in patients with sinus rhythm or adequate anticoagulation, though a hypercoagulable state or underlying inflammation may be involved. We report a case of cryptogenic ischemic stroke in an 86-year-old woman with transthyretin amyloidosis and sinus rhythm. Traditional coagulation tests, whole blood rotational thromboelastometry and impedance aggregometry did not show a hypercoagulable state. The thrombin generation assay did not reveal a prothrombotic state. However, the study of extracellular vesicles highlighted underlying immune-mediated endothelial damage likely responsible for the thrombotic diathesis. It could be hypothesized that inflammation plays a role in the hypercoagulability of patients with transthyretin amyloidosis. Larger prospective studies are needed to validate our hypothesis.

Longitudinal assessment of coagulation potential before, during, and following an in vitro fertilization cycle.

INTRODUCTION: The association between estrogen and hypercoagulability is well-established but little is known about coagulation dynamics during IVF. Our goal was to measure coagulation potential prior to, during, and following an IVF cycle and to investigate differences by conception outcome. MATERIALS AND METHODS: Patients undergoing IVF with fresh embryo transfer at a single academic center using oral contraceptive pills for cycle batching underwent evaluation of thrombin generation using the calibrated automated thrombogram at multiple points during the IVF cycle. Multiple thrombin generation parameters were compared across timepoints and by IVF cycle outcome using ANOVA repeated measures analysis. RESULTS: Of the 17 patients included, 11 conceived. There was a significant increase in peak and total thrombin generation in the entire cohort between the pre-treatment natural follicular phase and following a short course of oral contraceptive pills used for cycle batching. Further increase in these parameters was seen at the time of oocyte retrieval. In the pre-treatment natural follicular phase, patients who conceived had lower peak thrombin generation. There were changes throughout the cycle for factors II, V, VIII, X, XI, XII, antithrombin, and tissue factor pathway inhibitor. Only Factor XI was distinguishable by conception status; values were lower at all visits in patients who conceived. CONCLUSION: Increases in coagulation potential are seen in patients undergoing IVF following a short course of oral contraceptive pills for cycle batching and continue during controlled ovarian hyperstimulation. Those who conceived were seen to have lower peak thrombin generation in the pre-treatment natural follicular phase.

Multicenter evaluation of the hemostatic activity of emicizumab in patients with severe hemophilia A.

BACKGROUND: Emicizumab has been approved for the prophylaxis of patients with hemophilia A with or without inhibitors. However, spontaneous and trauma-induced breakthrough bleeds have been reported in patients on emicizumab prophylaxis, and no laboratory assay has been validated to evaluate the hemostatic activity of emicizumab. OBJECTIVES: The thrombin generation assay (TGA) could be a surrogate marker of the hemostatic efficacy of emicizumab. The correlation between TGA and the methods used to measure emicizumab blood concentration was evaluated in this study. METHODS: TGA was modified by the use of a trigger reagent combining a very low concentration of tissue factor and activated factor (F)XI. Emicizumab quantification was performed by 3 methods: the modified 1-step FVIII assay and 2 methods based on liquid chromatography and mass spectrometry (LC-MS). RESULTS: Using tissue factor/activated FXI-triggered TGA and platelet-poor plasma, a relationship was observed between the area under the thrombin generation curve (endogenous thrombin potential [ETP]) and the clinical response of patients to emicizumab. The ultrastructure of fibrin clots was consistent with ETP results and showed that emicizumab had a hemostatic activity equivalent to 20 to 30 IU/dL of FVIII. Finally, pharmacokinetic/pharmacodynamic analyses showed no correlation between ETP and LC-MS nor with modified 1-stage FVIII assay, but a statistically significant correlation between the LC-MS methods and the time-to-peak results of the TGA. CONCLUSION: Using a modified TGA, this study showed that patients who experienced breakthrough bleeds while on emicizumab had a lower thrombin-generating capacity compared with others with good clinical response to emicizumab.

Blood clot properties, thrombin generation, and platelet activation in patients with dysglycemia and established atherosclerotic cardiovascular disease: The CASCARA study.

BACKGROUND: There is a strong link between coronary artery disease (CAD), type 2 diabetes (T2D) on one hand, and altered fibrin clot properties, including increased clot density, and unfavorable fibrin clot structure on the other. T2D-related changes in fibrin clots can increase cardiovascular (CV) disease risk, including future CV events. We aimed to assess fibrin clot properties, thrombin generation, and platelet activation in CAD patients with prediabetes (PD) or T2D, compared to CAD patients without glycemic disorders. METHODS: We allocated patients to three groups: 1) Those with angiographically established CAD but without glycemic abnormalities (CAD group); 2) individuals with PD and established CAD (CAD+PD group); and 3) patients with T2D and CAD (CAD+T2D group). We conducted comparisons across these groups for thrombin generation, fibrin clot permeability, fibrin clot lysis, and platelet activation. RESULTS: The final analysis included 116 eligible patients: 1) CAD group (n = 31); 2) CAD+PD (n = 42); and 3) CAD+T2D (n = 43). The CAD+T2D patients enrolled had well-controlled T2D (median HbA1c level of 5.90%; IQR: 5.7%-6.3%). We found no significant differences in thrombin generation, fibrin clot properties, or platelet activation markers across the three analyzed groups (all P-values >0.20). However, elevated interleukin-6 (IL-6) levels were noted in both the highest and lowest glucose concentration quartiles. Additionally, a substantial increase in endogenous thrombin potential (ETP) was observed in patients in the highest glycated hemoglobin quintile. CONCLUSIONS: Individuals with established CAD and concomitant PD or well-controlled T2D exhibited comparable fibrin clot phenotypes, thrombin generation potential, and platelet activation when compared to CAD patients without dysglycemia.

Relationship between plasma tissue Factor Pathway Inhibitor (TFPI) levels, thrombin generation and clinical risk of bleeding in patients with severe haemophilia A or B.

INTRODUCTION: Bleeding severity in severe haemophilic patients, with low thrombin generation (TG) capacity, can vary widely between patients, possibly reflecting differences in tissue factor pathway inhibitor (TFPI) level. AIM: To compare free TFPI (fTFPI) levels in patients with severe haemophilia A (sHA) and severe haemophilia B (sHB) and to investigate in these patients as a whole the relationships between bleeding and TG potential, between TG potential and fTFPI level and between fTFPI level and bleeding tendency. METHODS: Data on bleeding episodes retrospectively recorded during follow-up visits over 5-10 years were collected and used to calculate the annualised joint bleeding rate (AJBR). fTFPI levels and basal TG parameters were determined in platelet-poor plasma (PPP) and platelet-rich plasma (PRP) using calibrated automated tomography (CAT). RESULTS: Mean fTFPI levels did not differ significantly between sHA (n = 34) and sHB (n = 19) patients. Mean values of endogenous thrombin potential (ETP) and thrombin peak (peak) in PPP and PRP were two-fold higher when fTFPI levels < 9.4 versus > 14.3 ng/mL. In patients treated on demand, ETP and peak in PRP were doubled when AJBR was ≤ 4.9 $ \le 4.9$ , AJBR being halved in patients with a low fTFPI level (9.4 ng/mL). In patients on factor prophylaxis, no association was found between TG parameters and either fTFPI level or AJBR. CONCLUSION: In patients treated on demand, bleeding tendency was influenced by fTFPI levels, which in turn affected basal TG potential. In patients on prophylaxis, bleeding tendency is probably determined primarily by the intensity of this treatment.

Exploring the effects of Factor Xa inhibitors on thrombin generation in people with haemophilia.

Optimal management of cardiovascular disease (CVD) in people with haemophilia (PWH) is a growing issue, given the continuing improvement in life expectancy among PWH. The evolving treatment paradigms targeting higher trough levels and the advent of non-factor replacement therapies (NFRT) means much of the 'protection' PWH were thought to have against CVD may be lost. There is a paucity of evidence regarding the safety of using anticoagulants in PWH. We designed a study assessing the thrombin generation (TG) of PWH of different severities and treatments, compared to non-haemophilia patients receiving a Factor Xa (FXa) inhibitor (apixaban or rivaroxaban), healthy controls, and assessing TG parameters of adding FXa inhibitor to the plasma of PWH receiving emicizumab prophylaxis. In total, 40 patients were included. TG was initiated with 5pM tissue factor (TF) using the calibrated automated thrombinoscope. Compared to those with mild haemophilia, patients receiving a FXa inhibitor had higher endogenous thrombin potential (ETP) (1278.42 vs 1831.36) and velocity index (40.71 vs 112.56), but both had a similar peak height (154.0 vs 262.63) and time to peak (both 5.83). People with severe haemophilia receiving emicizumab had significantly improved TG parameters compared to those not receiving emicizumab - ETP 1678.11 vs 809.96 and peak height 233.8 vs 92.05; however, when FXa inhibitor was added their TG parameters deteriorated to the severe haemophilia range (ETP 1179.60 and peak height 103.05). TG may provide additional useful information regarding the use of anticoagulants in PWH.

Andexanet Alfa Neutralizes the Anticoagulant Effects of Unfractionated Heparin of Bovine, Ovine and Porcine Origin Almost as Protamine Sulfate.

INTRODUCTION: Andexanet alfa (AA) - zhzo, recombinant coagulation factor Xa, is an approved antidote for oral Xa inhibitors (apixaban and rivaroxaban). Unfractionated heparin (UFH) is commonly used for therapeutic, interventional, and surgical indications. Protamine sulfate (PrSO4) is frequently used to neutralize UFH. This study aimed to investigate the comparative neutralization profiles of AA and PrSO4 for heparins of bovine, ovine, and porcine origin. MATERIALS AND METHODS: The neutralization effect of PrSO4 at 25 µg/ml and AA at 100 µg/ml was studied on an approximate surgical/interventional concentration of heparin by supplementing whole blood with each of the heparins at 25 µg/ml. For the clotting profile (activated partial thromboplastin time: aPTT), amidolytic (anti-Xa and anti-IIa), and thrombin generation assay each of the heparin were supplemented from -10-0.62 µg/ml. RESULTS: In the whole blood ACT studies, all three heparins produced strong anti-coagulant effects (400-450 seconds) compared to saline (130-150 seconds). Both AA and PrSO4 almost fully neutralized the anti-coagulant effects of heparins (140-160 seconds). Both antidotes completely reversed the anticoagulant effects of all three heparins in the aPTT and thrombin generation assay. However, PrSO4 was more effective in neutralizing the anti-Xa, and anti-IIa effects than AA, which only partially neutralized these effects. CONCLUSION: Andexanet alfa at 100 µg/ml effectively neutralizes the therapeutic and surgical/interventional concentrations of heparins in in-vitro settings. While differences in the anti-Xa, and anti-IIa effects between heparins were noted, anti-coagulant effect of these agents in the aPTT assay were comparable. A similar neutralization profile was observed in the ACT and thrombin generation assays by both agents.

Antithrombin supplementation attenuates heparin resistance in plasma spiked with Gla-domainless factor Xa S195A in vitro.

BACKGROUND: Andexanet alfa is a Gla-domainless mutant (S195A) factor Xa (GDXa) approved for acute reversal of oral factor Xa inhibitors. Cardiac surgery patients exposed to andexanet before cardiopulmonary bypass often exhibit severe heparin resistance. There is a paucity of data on the effectiveness and optimal dosage of antithrombin use in this setting. The objective of this study was to evaluate the in vitro effect of increased heparin with antithrombin levels on attenuating heparin resistance induced by GDXa. METHODS: Heparinised normal pooled plasma and cardiopulmonary bypass plasma were spiked with GDXa 4 µM. Tissue factor-activated thrombin generation was used to assess heparin reversal effects of GDXa and restoration of anticoagulation with additional heparin with and without antithrombin. Serum thrombin-antithrombin complex, antithrombin activity, and tissue factor pathway inhibitor were also measured in tissue factor-activated, recalcified cardiopulmonary bypass plasma spiked with GDXa. RESULTS: In normal pooled plasma, GDXa-induced heparin reversal was mitigated by maintaining a high heparin concentration (12 U ml-1) and supplementing antithrombin (1.5-4.5 µM) based on peak and velocity of thrombin generation. Heparin reversal by GDXa was also demonstrated in cardiopulmonary bypass plasma, but supplementing both heparin (8 U ml-1) and antithrombin (3 µM) attenuated GDXa-induced changes in peak and velocity of thrombin generation by 72.5% and 72.2%, respectively. High heparin and antithrombin levels attenuated thrombin-antithrombin complex formation in tissue factor-activated, GDXa-spiked cardiopulmonary bypass plasma by 85.7%, but tissue factor pathway inhibitor remained depleted compared with control cardiopulmonary bypass plasma. CONCLUSIONS: Simultaneous supplementation of heparin and antithrombin mitigate GDXa-induced heparin resistance by compensating for the loss of tissue factor pathway inhibitor.