Improved Thrombin Generation Research Articles

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

BackgroundGlanzmann thrombasthenia (GT) is caused by an inherited defect of platelet αIIbβ3 integrin. Concizumab, a monoclonal antibody specific for tissue factor pathway inhibitor, abolishes its anticoagulant effect. ObjectivesTo evaluate the in vitro ability of concizumab to improve hemostasis in GT. MethodsThe 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. ResultsThe 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. ConclusionConcizumab 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.

An in Vitro Pilot Study of the Role of Factor IX As an Alternative to Bypassing Agents in the Treatment of Breakthrough Bleeds in Patients with Hemophilia a and Inhibitors Treated with Emicizumab

Introduction: Hemophilia A(HA) is an X linked disorder characterized by a deficiency in Factor VIII. 30% of patients with severe HA develop alloantibodies that inhibit the coagulant activity of FVIII. Emicizumab, a substitution therapy that bridges factor IX and X, is approved in patients with severe HA with and without inhibitors. Indeed, it is now the standard of care prophylactic therapy for patients with inhibitors. Breakthrough bleeds and perioperative management require the use of bypassing agents in addition to emicizumab, with recombinant VIIa deemed the safest of these options. Nevertheless there are still concerns about thrombotic risks and inability to monitor bypassing agents given to patients on emicizumab. Since the rate limiting step for FXa generation in the plasma of patients receiving emicizumab is the concentration of generated FIXa, we conducted an in vitro pilot study to evaluate the hemostatic effect of adding FIX to the plasma of patients on emicizumab prophylaxis. Methods: After informed consent was obtained, blood was collected in citrated tubes prefilled with 1.45mM of corn trypsin inhibitor(CTI) from 25 patients with HA receiving emicizumab prophylaxis. Aligning with previously published data, the added hemostatic effect of rFVIIa was studied in platelet rich plasma (PRP), while all other products were studied in platelet poor plasma (PPP). PRP was spiked with 0, 45, 90 and 270mg/kg of rFVIIa, and PPP was spiked with 0, 10, 20, 30 and 50 IU/kg of activated prothrombin complex concentrate (aPCC); 0, 25, 50 and 100 IU/kg of recombinant FIX(rFIX) and 0, 25, 50 and 100 IU/kg of plasma derived FIX(pdFIX). Thrombin generation was run on these spiked samples using the CAT method. Coagulation was triggered in PRP samples with tissue factor 1pM. PPP samples were activated using 1pM of TF and 1.5pM FIXa. Mean endogenous thrombin potential (ETP, area under the thrombin generation curve) values were compared between the different experiments and with samples obtained from controls. Baseline FIX concentrations were determined in these patients using bovine chromogenic assay. The mean plasma emicizumab concentration (+Standard Deviation) was 44.45 µg/mL (+15.83). Results: As shown in fig 1, ETP was significantly improved with increasing concentrations of aPCC, rFIX and pdFIX in PPP(p&amp;lt;0.005). Fig 2 shows that ETP normalized with the addition of rFVIIa 45mg/kg (p&amp;lt;0.0001) in most of the patients tested. Moreover, we did not see ETP above the upper limit of the normal range when FIX was added, while 50 IU/kg of aPCC and 270 mg/kg of rFVIIa clearly surpassed the upper limit of the normal range, suggesting a risk of hypercoagulability and thrombosis. FIX levels varied between 63.9% and 248.9% with a mean of 134.27%. ETP obtained after adding rFIX 100 IU/kg was significantly greater than that attained with pdFIX (p=0.027), that might be explained by the presence of the activated form of FIX in rFIX concentrates. No statistical significant difference between the doses of 50 IU/Kg of FIX and 100 IU/KG was observed (p&amp;gt;0.05), with a noted tendency to increased ETP with 100IU/KG dose. Conclusion: Our in vitro study demonstrates excess thrombin generation (and potential thrombosis risk) with higher concentrations of rFVIIa and aPCC that are used in the clinical care of patients. While addition of FIX at a range of doses appears to improve thrombin generation without exceeding the upper normal limits. These data suggest that FIX therapy may safely improve hemostasis in patients with HA and inhibitors receiving emicizumab prophylaxis. A clinical study looking at a starting dose of rFIX at 50 IU/KG for mild to moderate bleeds and 100 IU/Kg for more severe bleeds is warranted. In addition to the advantage of monitoring levels with FIX therapy, the longer half-life of FIX offers the potential advantage of controlling hemostasis with single dose administration. Future prospective clinical trials are needed to investigate the efficacy and safety of this therapy in this patient population.

Toward non-factor therapy in hemophilia: an antithrombin insensitive Gla-domainless factor Xa as tissue factor pathway inhibitor bait

BackgroundGla-domainless factor (F) Xa (GD-FXa) was proposed as a trap to endogenous anticoagulant tissue factor pathway inhibitor (TFPI) to restore thrombin generation in hemophilia. Using computational chemistry and experimental approaches, we previously showed that S195A GD-FXa also binds TFPI and restores ex vivo coagulation in plasma obtained from person(s) with hemophilia. MethodsTo design a GD-FXa variant with improved anti-TFPI affinity, we performed molecular dynamics simulations and identified suitable sites for mutagenesis. The calculations identified residues R150FXa and K96Fxa as cold-spots of interaction between GD-FXa and the K2 domain of TFPI. In the three-dimensional model, both residues face toward TFPI hydrophobic residues and are thus potential candidates for mutagenesis into hydrophobic residues to favor an improved protein–protein interaction. ResultsCatalytically inactive GD-FXa variants containing the S195A mutation and the additional mutations K96Y, R150I, R150G, R150F, and K96YR150F, were produced to experimentally confirm these computational hypotheses. Among these mutants, the R150FFXa and the K96YR150FFXa were slightly more effective than S195A GD-FXa in restoring coagulation in FVIII deficient plasmas. However, in surface plasmon resonance experiments, they showed TFPI binding affinities in the same range and acted similarly as S195A GD-FXa in FXa/TFPI competition assays. In contrast, the R150 mutants completely lost their interactions with antithrombin as observed in the surface plasmon resonance experiments. ConclusionsWe therefore conclude that their antithrombin resistance is responsible for their improved thrombin generation, through an extension of their half-lives.

Fitusiran Reaches People's with Hemophilia and Their Caregivers’ Treatment Expectations: Qualitative Semi-Structured Interviews of Participants of ATLAS-OLE Trial (Interim analysis)

Background: Current prophylaxis treatments for people with hemophilia A and B can be burdensome, involving frequent intravenous or subcutaneous doses in addition to on demand treatments due to breakthrough bleeds. Fitusiran is an, investigational, subcutaneously administered small interfering RNA therapy, meant to improve thrombin generation, restore hemostasis, and prevent bleeding in all people with hemophilia. Fitusiran has been evaluated in three phase 3 trials: two completed randomized phase 3 clinical trials (ATLAS-INH and ATLAS-A/B), and one phase 3 "switching” study (ATLAS-PPX). Individuals completing any of these 3 parent studies were eligible for the open-label extension study (ATLAS-OLE), which is currently ongoing. Objective: The aim of this interim analysis is to better understand patients’ and caregivers’ experiences with hemophilia A or B (with or without inhibitors), and its treatments, including fitusiran, and how this experience evolved over the trial period, including their perception and satisfaction with the therapy they received during the trials. Methods: Patients with hemophilia A or B (PwHA/B) with or without inhibitors, participating in the ATLAS-INH and ATLAS-A/B, and then ATLAS-OLE studies in the United States and India were invited to participate in semi-structured exit interviews to share their experiences. Caregivers of patients in the same studies were invited to participate in the interview if patients were unable (under 18 years of age, cognitively unable). A total of 24 individuals were invited to participate in a 60-minute telephone interview. Following a semi structured interview guide to ensure consistency across the interviews, participants were asked to describe their experiences with hemophilia and its treatment prior to entering any of the ATLAS trials. Next participants were asked about their perceptions of the impact of hemophilia and its treatment on their daily lives, their treatment expectations, and experiences with fitusiran during the ATLAS-OLE trial. Participants were asked to rate different aspects of hemophilia treatments (improving joint health, decreasing bleeds, protection from bleed for entire month, improving joint mobility, prevention of fear/anxiety of bleeds, minimize anxiety/stress in managing hemophilia and convenience) as 'moderately important', 'very important’ or 'extremely important'. Here we presented the data from the interim analysis conducted on the first 10 interviews, analyses of data collected during other interviews are in progress. Results: For this interim analysis, 8 PwHA/B and 2 caregivers were interviewed, the mean (SD) age was 24.6 (7.4) years. All participants rated improving joint health as extremely important; most participants rated decreasing bleeds (90%), protection from bleeds for an entire month (90%), improving joint mobility (90%), prevention of fear/anxiety of bleeds (80%) and minimize anxiety/stress in managing hemophilia (80%) as 'extremely important’ aspects in their hemophilia treatment (Figure 1). All participants reported improvements in these treatment aspects while under fitusiran prophylaxis with the following percentage of patients having rated "a lot of improvement": 100%, 100%, 90%, 100%, 90%, 80% and 80%, respectively (Figure 2). Majority of participants (n=9) preferred fitusiran prophylaxis over their previous hemophilia treatment and all reported being 'very satisfied’ with fitusiran treatment, especially with regards to its ability to prevent bleeds, durability of effects, and convenience. Among quotes recorded during the interviews, PwHA/B reported for example that "After I took fitusiran, I became like a normal person"; "...very confident because if I get the fitusiran I can say no bleed will happen"; "After I took fitusiran, I became like a normal person"; and "He is more free, He is more confident. That constant threat is not there... generally he is much happier now". Conclusions: Qualitative insights from this study provides encouraging and positive interim insights into the experiences of PwHA/B, with or without inhibitors, and their caregivers, with fitusiran prophylaxis. Fitusiran treatment demonstrated added value in PwHA/B, irrespective of inhibitor status, particularly resulting in improving joint health, decreasing bleeds, improving joint mobility, and an overall reduction of treatment and disease burden. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Update on platelet procoagulant mechanisms in health and in bleeding disorders.

Platelet procoagulant mechanisms are emerging to be complex and important to achieving haemostasis. The mechanisms include the release of procoagulant molecules from platelet storage granules, and strong agonist-induced expression of procoagulant phospholipids on the outer platelet membrane for tenase and prothrombinase assembly. The release of dense granule polyphosphate is important to platelet procoagulant function as it promotes the activation of factors XII, XI and V, inhibits tissue factor pathway inhibitor and fibrinolysis, and strengthens fibrin clots. Platelet procoagulant function also involves the release of partially activated factor V from platelets. Scott syndrome has provided important insights on the mechanisms that regulate procoagulant phospholipids expression on the external platelet membrane, which require strong agonist stimulation that increase cystolic calcium levels, mitochondrial calcium uptake, the loss of flippase function and activation of the transmembrane scramblase protein anoctamin 6. There have been advances in the methods used to directly and indirectly assess platelet procoagulant function in health and disease. Assessments of thrombin generation with platelet rich plasma samples has provided new insights on how platelet procoagulant function is altered in inherited platelet disorders, and how platelets influence the bleeding phenotype of a number of severe coagulation factor deficiencies. Several therapies, including desmopressin and recombinant factor VIIa, improve thrombin generation by platelets. There is growing interest in targeting platelet procoagulant function for therapeutic benefit. This review highlights recent advances in our understanding of platelet-dependent procoagulant mechanisms in health and in bleeding disorders.

Perioperative Management in Patients with Hemophilia Receiving Fitusiran, an Investigational RNAi Therapeutic Targeting Antithrombin for the Treatment of Hemophilia

Introduction: Hemophilia is a bleeding disorder characterized by ineffective clot formation due to insufficient thrombin generation. Fitusiran is a subcutaneously (SC) administered investigational RNA interference (RNAi) therapeutic targeting antithrombin (AT) as a means to improve thrombin generation (TG) and promote hemostasis in patients with hemophilia A or B with and without inhibitors. Previously reported data have shown that monthly administration of fitusiran led to dose dependent AT lowering, improved thrombin generation and decreased bleeding frequency. (Pasi et al. Blood . 2016.; Pasi et al. New Engl J Med . 2017.) Data from an open-label extension study demonstrated an encouraging safety and tolerability profile, including when used in combination with factor or bypassing agents to treat breakthrough bleeds. (Pasi et al. Res Pract Thromb Haemost . 2017.) The management of operative procedures while on novel, non-factor therapies for hemophilia, such as fitusiran, is of clinical interest. The purpose of this abstract is to describe details as reported by study investigators on the perioperative hemostatic management during dental/surgical procedures in patients with hemophilia receiving fitusiran in a clinical trial.Methods: The fitusiran Phase 1 study (NCT02035605) followed by the Phase 2 open-label extension (OLE) study (NCT02554773) included patients with hemophilia A or B with and without inhibitors. After the Phase 1 dose-escalation study, patients eligible to continue dosing in the Phase 2 OLE received monthly, fixed SC doses of fitusiran, 50 mg or 80 mg. Data on perioperative hemostatic treatment and hemostatic response were collected for patients undergoing dental/surgical procedures while AT was lowered on study.Results: Four patients, ages 22-36, with severe hemophilia A (2 with inhibitors) underwent 5 surgical procedures: endoscopic cholecystectomy and septoplasty; thoracotomy/partial lung segmentectomy; molar teeth extraction; premolar tooth extraction. Prior to the procedures, the AT level for each of these patients was <20% (range: 10.7 - 19%) relative to baseline. At the Investigators' discretion, perioperative hemostatic treatments (FVIII, rFVIIa, and/or aPCC) were administered for 4 of the 5 procedures. The duration of hemostatic treatment coverage varied depending on the type of procedure: 15 hours post-procedure (premolar tooth extraction), 7 days post-procedures (endoscopic cholecystectomy and septoplasty), and 13 days post-procedure (thoracotomy/partial lung segmentectomy). No thromboprophylaxis was used in any procedure. All procedures were rated by the respective investigator as resulting in minimal blood loss or blood loss similar to a patient without hemophilia. Further details on the perioperative treatment regimens and hemostatic responses will be presented.Conclusion: Successful perioperative hemostatic management of patients in the context of AT lowering with fitusiran has been observed; the number of dental/surgical procedures is limited and additional data are needed to further define appropriate perioperative hemostatic management plans. DisclosuresNegrier:Baxter: Research Funding; Inspiration: Research Funding; LFB: Honoraria, Speakers Bureau; Octapharma: Honoraria, Research Funding, Speakers Bureau; CSL Behring: Honoraria, Research Funding, Speakers Bureau; Baxalta: Honoraria, Speakers Bureau; Pfizer: Honoraria, Research Funding, Speakers Bureau; Alnylam: Research Funding; Bayer: Honoraria, Research Funding, Speakers Bureau; Novo Nordisk: Honoraria, Research Funding, Speakers Bureau; Biogen/SOBI: Honoraria, Research Funding, Speakers Bureau. Ragni:Alnylam: Consultancy, Honoraria, Research Funding; Biomarin: Consultancy, Honoraria, Research Funding; Bayer: Consultancy, Honoraria, Research Funding; MOGAM: Consultancy, Honoraria; Sangamo: Research Funding; Genentech/Roche: Research Funding; Bioverativ: Consultancy, Honoraria, Research Funding; NovoNordisk: Honoraria; SPARK: Research Funding; Shire: Consultancy, Honoraria, Research Funding. Georgiev:Alnylam: Consultancy. Van Nguyen:Alnylam: Employment, Equity Ownership. Madigan:Alnylam: Employment, Equity Ownership. Pasi:Bayer HealthCare; Biotest; Novo Nordisk; Pfizer Inc.; Roche: Speakers Bureau; Alnylam Pharmaceuticals, Inc; Biogen Idec Inc.; BioMarin Pharmaceutical Inc.; Octapharmal; Roche; Shire; SOBI: Consultancy; Pfizer, SOBI, Octapharma, Shire, Bayer, Alnylam, Biomarin, Biotest: Honoraria; Alnylam Pharmaceuticals; BioMarin Pharmaceutical Inc.; SOBI: Membership on an entity's Board of Directors or advisory committees; Roche, NovoNordisk, Pfizer: Other: paid instructor; BioMarin Pharmaceutical Inc.; Octapharma, Alnylam Pharmaceuticals, Bioverativ: Research Funding.

In silico Modeling of the Impact of Antithrombin Lowering on Thrombin Generation in Rare Bleeding Disorders

Introduction: Rare bleeding disorders (RBDs) arise from deficiencies in various clotting factors, such as factors V, VII, and X, which like hemophilia A or B, are often characterized by an inability to generate sufficient thrombin to prevent bleeds. However, compared to hemophilia, RBDs are less prevalent, more heterogeneous in phenotype, and less understood clinically. Furthermore, RBD patients, subsets of whom can suffer from frequent and severe bleeding episodes, often have limited treatment options. Fitusiran is an investigational RNAi therapeutic targeting antithrombin (AT), a major endogenous inhibitor of thrombin production, and is currently in Phase 3 trials for the treatment of hemophilia A or B, with or without inhibitors. Initial clinical studies with fitusiran have demonstrated that lowering AT resulted in improved thrombin generation and was correlated with reduced bleeding frequency in patients with hemophilia (Pasi et al. New Engl J Med . 2017.). Based on this mechanism of action, pharmacological lowering of AT may potentially have clinical applicability in RBDs. In this light, in silico kinetic models that predict the dynamic behavior of coagulation, including thrombin generation in plasma under a diverse set of experimental conditions, can provide a useful theoretical platform to test whether AT lowering may be effective in RBDs. In this abstract, we demonstrate the use of a computational model to predict the impact of AT lowering on thrombin generation in plasma with various degrees of deficiencies in Factors V, VII, and X.Methods: We implemented the model developed by Nayak et al. (Nayak et al. CPT Pharmacometrics Syst. Pharmacol. 2015.), which describes the coagulation cascade and simulates thrombin generation under various experimental conditions, using Matlab and the Simbiology Toolbox®. For each Factor deficiency (V, VII, X), we tested thrombin generation at factor levels of 0.1, 0.5, 1.0, 2.0, 5.0, and 10.0% of their mean plasma concentrations in normal healthy individuals as well as AT lowering of 0, 40, 60, 80, 90, and 99% of baseline levels. Peak thrombin values from each thrombin curve were computed and compared across experimental conditions. Patient heterogeneity was simulated by monte-carlo sampling of plasma factor concentrations based on known mean and variances reported in literature.Results: Consistent with published results in hemophilia A and B, the model predicted an increase in thrombin generation as AT was lowered in the setting of factor V, VII, and X deficiencies. AT lowering to 20% resulted in increased thrombin generation by 14, 4, and 3-fold for conditions of 1% factor V, VII, and X, respectively.Conclusions: Our results suggest that pharmacological AT lowering may serve as an effective strategy for certain RBDs. DisclosuresSridharan:Alnylam: Employment, Equity Ownership. Liu:Alnylam: Employment, Equity Ownership. Qian:Alnylam: Employment, Equity Ownership. Goel:Alnylam: Employment, Equity Ownership. Huang:Alnylam: Employment, Equity Ownership. Akinc:Alnylam: Employment, Equity Ownership.

Targeting of antithrombin in hemophilia A or B with investigational siRNA therapeutic fitusiran—Results of the phase 1 inhibitor cohort

BackgroundFitusiran, an investigational small interfering RNA therapy, reduces antithrombin production to rebalance hemostasis in people with hemophilia A or B, with or without inhibitors. ObjectivesTo evaluate the safety and efficacy of fitusiran treatment for people with moderate/severe hemophilia A or B with inhibitors. Patients/MethodsIn this open‐label phase 1, part D study, 17 males with hemophilia A or B with inhibitors received three once‐monthly subcutaneous injections of fitusiran 50 mg (n = 6) or 80 mg (n = 11); followed for up to 112 days. Endpoints included safety (primary), pharmacokinetics/pharmacodynamics (secondary), annualized bleeding rate, and patient‐reported outcomes (exploratory). ResultsThe most common adverse event was injection site erythema (n = 8). No thrombotic events were reported. At nadir, mean (standard error of the mean [SEM]) antithrombin activity decreased from baseline by 82.0% (2.2) and 87.4% (0.7) in the 50 mg and 80 mg groups, respectively. Antithrombin reduction was associated with increased thrombin generation. 11/17 (64.7%) participants had no bleeds during the observation period (mean [standard deviation] 69.4 [16.3] days). Mean (SEM) changes from baseline in Haemophilia Quality of Life Questionnaire for Adults total (−9.2 [2.9]) and physical health (−12.3 [3.9]) domain scores suggested clinically meaningful improvement. ConclusionsMonthly fitusiran was generally well tolerated, lowered antithrombin levels from baseline, and resulted in improved thrombin generation. These preliminary results suggest that monthly fitusiran treatment may reduce bleeding episodes and improve quality of life in participants with hemophilia A or B with inhibitors.

Thrombosis Complicating Non-Factor Therapy for Hemophilia

This is an exciting time in hemophilia treatment with the unprecedented development of novel non-factor therapies. These agents have re-balanced hemostasis in patients with hemophilia A and B, with and without inhibitors, tipping the balance toward hemostasis and improved thrombin generation. While there have been numerous publications about the beneficial hemostatic effects and significant bleed reduction possible with these novel non-factor agents, little has been written about the less well-recognized thrombotic complications. Yet, the latter underscores the fine balance between hemostasis and thrombosis and the fact these agents prevent but do not treat bleeds, requiring clotting factor requirement to treat acute bleeds. The purpose of this Commentary is to review thrombotic complications that have occurred with non-factor therapies, risk factors for thrombosis, potential mechanisms, and potential mitigation approaches.

Long-Term Durability, Safety and Efficacy of Fitusiran Prophylaxis in People with Hemophilia a or B, with or without Inhibitors - Results from the Phase II Study

Long-Term Durability, Safety and Efficacy of Fitusiran Prophylaxis in People with Hemophilia a or B, with or without Inhibitors - Results from the Phase II Study

Reducing Antithrombin in Plasma to Levels Observed in Fitusiran-Treated Patients Does Not Interfere with Coagulation Assays

Introduction: Fitusiran is being evaluated in phase 3 clinical studies as a once monthly subcutaneously administered RNA interference therapy designed to decrease endogenous expression of the anticoagulant antithrombin (AT). Reducing the anticoagulant potential of AT in the plasma of Hemophilia A and B patients, with or without inhibitors, restores the hemostatic balance and improves thrombin generation potential. Fitusiran and other non-Factor replacement molecules, such as emicizumab, use novel mechanisms to restore hemostasis. Consequently, some standard coagulation assays typically used to measure factor levels, such as the chromogenic, one-stage, and APTT assays have been shown to be affected by emicizumab. In order to assess the potential impact of fitusiran treatment on our ability to measure clotting factor activity in individuals with hemophilia A (HemA), we investigated the effect of decreased AT levels on standard coagulation laboratory assays. The following four assays were evaluated: one stage (OSA) and chromogenic (CSA) factor VIII (FVIII) activity assays, activated partial thromboplastin time (aPTT), and the prothrombin clotting time assay (PT). These assays are relevant to determine the FVIII plasma levels in patients dosed with FVIII replacement factors while treating potential breakthrough bleeds or undergoing surgery. Method and Results: Pooled human HemA plasma was used directly or after AT depletion using an affinity-based method to remove 95% AT activity, resulting in HemA plasma with 5% AT activity. Additional batches of HemA/AT levels were created by AT reconstitution to 10 and 20% AT plasma activity. Subsequently, the 4 HemA plasma pools (one with normal -100 %- AT and three with reduced levels of AT-5, 10, 20%) were prepared with FVIII spiked to 0, 5, 10, 20, 30, 50, and 100% . FVIII plasma activity was measured by OSA and CSA against Siemens normal standard plasma. Compared to FVIII plasma activity for all FVIII spikes in normal HemA plasma containing 100% AT, respective FVIII spikes in HemA/AT depleted plasma samples (5, 10, and 20% AT) did not show therapeutic significant effects of AT levels on measured FVIII activity by OSA and chromogenic FVIII activity assay. Additionally, we evaluated the effect of the various AT levels and FVIII spikes on clotting assays, aPTT and PT, among the HemA plasma with varying (5-100%) AT levels. AT level did not affect the aPTT for respective FVIII spikes in HemA and Hem/AT depleted plasma. Furthermore, no therapeutic relevant effect of ATIII and FVIII level was observed in PT values between HemA and Hem/AT depleted plasma. Conclusions: Our results demonstrate that a reduction of AT levels in plasma does not affect standard coagulation assays, suggesting these assays can be utilized to assess hemostasis and factor levels in individuals treated with fitusiran. The ability to monitor FVIII levels in patients treated with non-replacement therapies is important as these patients require factor to treat breakthrough bleeds and during some surgeries. Disclosures Wang: sanofi: Current Employment, Current equity holder in publicly-traded company. Kattula:Sanofi: Current Employment, Current equity holder in publicly-traded company. Ismail:Sanofi: Current Employment, Current equity holder in publicly-traded company. Leksa:Sanofi: Current Employment, Current equity holder in publicly-traded company. van Der Flier:sanofi: Current Employment, Current equity holder in publicly-traded company. Salas:sanofi: Current Employment, Current equity holder in publicly-traded company.

Thrombin generation and bleeding in cardiac surgery: a clinical narrative review

This narrative review discusses the role of thrombin generation in coagulation and bleeding in cardiac surgery, the laboratory methods for clinical detection of impaired thrombin generation, and the available hemostatic interventions that can be used to improve thrombin generation. Coagulopathy after cardiopulmonary bypass (CPB) is associated with excessive blood loss and adverse patient outcomes. Thrombin plays a crucial role in primary hemostasis, and impaired thrombin generation can be an important cause of post-CPB coagulopathy. Existing coagulation assays have significant limitations in assessing thrombin generation, but whole-blood assays designed to measure thrombin generation at the bed-side are under development. Until then, clinicians may need to institute therapy empirically for non-surgical bleeding in the setting of normal coagulation measures. Available therapies for impaired thrombin generation include administration of plasma, prothrombin complex concentrate, and bypassing agents (recombinant activated factor VII and factor eight inhibitor bypassing activity). In vitro experiments have explored the relative potency of these therapies, but clinical studies are lacking. The potential incorporation of thrombin generation assays into clinical practice and treatment algorithms for impaired thrombin generation must await further clinical development.

Thrombin generation in plasma of patients with haemophilia A and B with inhibitors: Effects of bypassing agents and antithrombin reduction

Antithrombin (AT) reduction has been shown to improve thrombin generation (TG) in haemophilia with or without inhibitors. As treatment with bypassing agents (BPAs) may be required in patients with breakthrough bleeding while receiving AT-lowering therapy, we assessed TG in platelet-poor plasma samples from haemophilia patients in the presence of BPA (recombinant activated factor VII [rFVIIa; 1.25 or 2.5 μg mL−1] or activated prothrombin complex concentrate [aPCC; 0.5 or 1 U mL−1]) and AT reduction (anti-AT antibody).Mean ± SEM baseline peak thrombin height was 19.9 ± 4.3 nM in plasma from haemophilia patients (n = 12) and 230.5 ± 9.8 nM in healthy males (n = 24). Reduced AT improved mean peak thrombin height in haemophilia patient plasma to 75.4 ± 17.4 nM. Spiking of 90% AT-reduced haemophilia patient plasma with 2.5 μg mL−1 rFVIIa or 1 U mL−1 aPCC increased the mean peak thrombin height to 82.5 ± 12 nM and 134.8 ± 18.7 nM, respectively.Peak thrombin levels did not exceed the range for healthy volunteers when plasma samples from haemophilia patients with in vitro AT reduction were treated with BPAs, suggesting the potential use of BPAs in conjunction with AT reduction. Further clinical investigations are needed to confirm the safety of this approach.

Targeting protease nexin-1, a natural anticoagulant serpin, to control bleeding and improve hemostasis in hemophilia

AbstractHemophilia A and B, diseases caused by the lack of factor VIII (FVIII) and factor IX (FIX) respectively, lead to insufficient thrombin production, and therefore to bleeding. New therapeutic strategies for hemophilia treatment that do not rely on clotting factor replacement, but imply the neutralization of natural anticoagulant proteins, have recently emerged. We propose an innovative approach consisting of targeting a natural and potent thrombin inhibitor, expressed by platelets, called protease nexin-1 (PN-1). By using the calibrated automated thrombin generation assay, we showed that a PN-1-neutralizing antibody could significantly shorten the thrombin burst in response to tissue factor in platelet-rich plasma (PRP) from patients with mild or moderate hemophilia. In contrast, in PRP from patients with severe hemophilia, PN-1 neutralization did not improve thrombin generation. However, after collagen-induced platelet activation, PN-1 deficiency in F8−/−mice or PN-1 blocking in patients with severe disease led to a significantly improved thrombin production in PRP, underlining the regulatory role of PN-1 released from platelet granules. In various bleeding models, F8−/−/PN-1−/− mice displayed significantly reduced blood loss and bleeding time compared with F8−/−mice. Moreover, platelet recruitment and fibrin(ogen) accumulation were significantly higher in F8−/−/PN-1−/− mice than in F8−/−mice in the ferric chloride-induced mesenteric vessel injury model. Thromboelastometry studies showed enhanced clot stability and lengthened clot lysis time in blood from F8−/−/PN-1−/− and from patients with hemophilia A incubated with a PN-1-neutralizing antibody compared with their respective controls. Our study thus provides proof of concept that PN-1 neutralization can be a novel approach for future clinical care in hemophilia.

Von Willebrand Factor Binding to Myosin Assists in Coagulation

Introduction: Von Willebrand factor (VWF) binds to platelets and collagen as a means of facilitating coagulation at sites of injury. Recent evidence has shown that myosin can serve as a surface for thrombin generation and binds to activated factors V and X (Deguchi et al, Blood 2016;128:1807-1878). We hypothesized that VWF could also bind myosin as a means of bringing factor VIII (FVIII) to sites of clot formation.Methods: Recombinant wild-type (WT) was transfected into HEK293T cells and supernatants collected for VWF to use in experiments. VWF variants containing point mutations at specific sites were constructed via site directed mutagenesis and expressed as above. Myosin from rabbit skeletal muscle was plated on maleic anhydride plates, recombinant VWF or plasma samples added and presence of VWF detected using anti-VWF monoclonal antibodies. Competition assays were performed with antibodies blocking various sites on VWF. FVIII activity was measured using a chromogenic substrate (Chromogenix Coatest). Thrombin generation was performed using a fluorogenic substrate from the Technothrombin TGA kit with either tissue factor +/- myosin or the RCL reagent +/- myosin added to platelet poor plasma from healthy controls.Results: WT VWF and human plasma VWF from healthy controls bound myosin, while plasma lacking VWF failed to demonstrate binding. Binding was multimer dependent, with a dose dependent decrease in binding seen with increasing loss of high molecular weight multimers. When myosin binding to VWF:Ag ratios were compared, a solution of ultra high molecular weight multimers demonstrated a mean ratio of 1.6, a solution of high molecular weight multimers a ratio of 1.4, a solution of medium molecular weight multimers a ratio of 0.4 and binding was undetectable with only low molecular weight multimers. A polyclonal anti-VWF antibody (DAKO) completely blocked VWF binding to myosin as did an antibody directed against the VWF A1 domain (AVW-3). Antibodies directed against other VWF sites including the N and C terminal ends failed to affect VWF binding to myosin. Since collagen IV also binds in this region, collagen IV was added to the assay as competition and also completely blocked VWF binding to myosin. VWF variants p.R1395A and p.R1399A showed undetectable binding, while a variant in the A3 domain (that abrogates type III collagen binding) showed normal binding to myosin (95% of WT). However, additional residues that affect VWF-collagen IV interactions were tested and had minimal effect on myosin binding, including p.1392A and p.1402A. Taken together, these results suggest a binding site for myosin in the VWF A1 domain similar but not identical to that of collagen IV.FVIII activity was detected when a VWF concentrate containing both VWF and FVIII was bound to myosin, with a dose-dependent increase in activity. However, no increase in FVIII activity was seen using recombinant FVIII alone in the absence of additional VWF. Because previous results from J. Griffin and coworkers showed a role for myosin in enhancing thrombin generation, we also looked at thrombin generation in platelet-poor plasma with and without myosin, but no difference was seen in the absence of additional phospholipids. Peak thrombin with myosin was 80 nM vs 62 nM without myosin (p=NS), and the area under the curve was 1022 vs 775 (p=NS). With additional phospholipids, there was a trend towards increased thrombin generation with myosin but the difference was again not statistically significant. Peak thrombin with myosin was 70 nM vs 64 nM without myosin (p=NS), and the area under the curve was 974 vs 867 (p=NS).Discussion: Myosin can also serve as a surface for VWF binding. This may help facilitate delivery of FVIII to sites of injury and improve thrombin generation, although in our hands myosin did not substantially increase thrombin generation in the presence of additional phospholipid. Unlike factor V, FVIII does not intrinsically interact with myosin to accelerate thrombin generation. However, myosin, similar to collagen, can increase local accumulation of VWF-FVIII and indirectly accelerate thrombin generation and clot formation. DisclosuresMontgomery:BCW: Patents & Royalties: GPIbM assay patent to the BloodCenter of Wisconsin.

The Tissue Factor Pathway Inhibitor Antibody, PF-06741086, Increases Thrombin Generation in Rare Bleeding Disorder and Von Willebrand Factor Deficient Plasmas

Hemophilia A and B are hereditary bleeding disorders that result from deficiencies in the intrinsic coagulation pathway leading to insufficient generation of Factor Xa (FXa) and thrombin to promote stable hemostasis. Coagulation defects are also observed in other inherited rare factor bleeding disorders. The extrinsic pathway of coagulation in these disorders cannot generate sufficient levels of FXa due to the regulation by Tissue Factor Pathway Inhibitor (TFPI). TFPI is a Kunitz-type serine protease inhibitor that negatively regulates thrombin generation by inhibiting the FXa/tissue factor (TF)/Factor VIIa (FVIIa) complex. PF-06741086, a fully human inhibitory monoclonal antibody, binds the Kunitz-2 domain and is currently under development as a potential prophylactic treatment to prevent bleeding episodes in hemophilia A and hemophilia B patients with and without inhibitors. The addition of PF-06741086 in vitro to donor plasma from both healthy normal volunteers and hemophilia patients promoted thrombin generation and restored hemostasis in vivo in murine hemophilia bleeding models. Pharmacological effects of PF-06741086 on thrombin generation were also observed in a healthy volunteer Phase 1 study. Other rare disease coagulopathies also result in the insufficient generation of thrombin. In this study, the potential of PF-06741086 to restore thrombin generation in rare disease plasma was explored.Thrombin generation was measured in citrated platelet poor Factor XI (FXI), Factor V (FV), FVII, von Willebrand Factor (vWF) deficient (Type 1, 2A, 2B and 3) congenital donor plasma following the in vitro addition of PF-06741086 (0, 1, 10 or 100 nM) or a human IgG1 antibody; initiated with 1 pM TF and 4 µM phospholipid. FXI, FV, and FVII donors had less than 1% coagulation factor activity. Non-hemophilic plasma from healthy donors alone was also included in the analysis. In FXI deficient plasmas, a concentration-dependent increase in peak thrombin and a shortening of the lag time was observed with the addition of PF-06741086 normalizing and restoring levels to those observed in the non-hemophilic plasma. A similar response was also observed in all of the vWF deficient plasmas. In one FVII deficient plasma, an increase in peak thrombin was observed at dose of 100 nM PF-06741086, however, the lag time (20 minutes) was significantly extended, relative to healthy volunteer non-hemophilic plasma. As expected, the addition of PF-06741086 to FV deficient plasma did not increase thrombin generation at any concentration.The in vitro addition of the TFPI antibody, PF-06741086, improved thrombin generation in selected coagulation factor deficient plasmas, including vWF deficiency, to the levels observed in normal plasma. This data suggestion that the inhibition of TFPI may promote hemostasis in rare bleeding disorders such as FXI deficiency and vWF deficiencies. DisclosuresRakhe:Pfizer: Employment. Patel-Hett:Pfizer: Employment. Bowley:Pfizer: Employment. Murphy:Pfizer: Employment. Pittman:Pfizer: Employment.

The Role of Antithrombin Lowering in Patients with Hemophilia: Hemostatic Control Pre- and Post-Fitusiran Dosing Interruption

IntroductionHemophilia A and hemophilia B are rare bleeding disorders characterized by insufficient thrombin generation due to deficiencies in factors VIII or IX, respectively. Standard treatment for hemophilia is currently based on replacement of the deficient factor with factor concentrate. This, unfortunately, subjects the patient to the risk of developing neutralizing antibodies, or inhibitors, against replacement factor VIII or IX. Individuals with inhibitors become refractory to standard replacement therapy, a serious complication for as many as one-third of patients with severe hemophilia A and a lower proportion of patients with hemophilia B. Fitusiran is a once-monthly subcutaneously administered investigational RNA interference therapeutic that targets antithrombin (AT) to improve thrombin generation (TG) and promote hemostasis in patients with hemophilia A or hemophilia B with or without inhibitors. In September 2017, fitusiran dosing in the Phase 2 open-label extension (OLE) study was temporarily suspended to investigate a case of fatal cerebral venous sinus thrombosis. Following this investigation, fitusiran dosing resumed in December 2017 with protocol amendments for bleed management dosing and safety monitoring. During the temporary dosing suspension, AT levels, TG, bleeding events, and frequency of factor replacement and bypassing agents were assessed.MethodsBefore the dosing suspension, 33 patients (hemophilia A=27, hemophilia B=6) were enrolled, of whom 28 patients continued treatment over 20 months in the Phase 2 OLE study, with a median of 11 months on study. As of June 2018, data collected monthly during the clinical hold included AT levels, TG, and description and management of treated bleeding events before and during the clinical hold, which were used to estimate annualized bleeding rates (ABRs) and bleeding rates per month.ResultsAT activity in patients previously receiving fitusiran demonstrated a progressive increase during the interruption of fitusiran dosing. Median %AT increased to >60% after a 5-month period compared with the last data point before dosing interruption. These data confirm our previous findings that discontinuation of fitusiran results in gradual recovery of AT activity over time. Subjects also demonstrated a concomitant steady decrease in TG during the interruption of fitusiran dosing, which occurred over a similar time course to that of AT recovery. Consistent with both the increase in AT activity and decrease in TG, preliminary analysis shows the median overall ABR increased from 1.43 events/year before the dosing interruption to 6.07 events/year during the dosing interruption.ConclusionsDuring a period of fitusiran dosing suspension, recovery of AT activity was accompanied by a decrease in TG and an increase in bleeding events. These observations provide support for the therapeutic hypothesis that AT activity lowering by fitusiran leads to an increase in TG and improved hemostasis. Phase 3 studies of fitusiran are ongoing. DisclosuresRagni:Bioverativ: Consultancy, Research Funding; MOGAM: Membership on an entity's Board of Directors or advisory committees; CSL Behring: Research Funding; SPARK: Consultancy, Research Funding; Shire: Research Funding; Alnylam: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novo Nordisk: Research Funding; Sangamo: Research Funding; Biomarin: Membership on an entity's Board of Directors or advisory committees, Research Funding. Georgiev:Alnylam: Consultancy. Lissitchkov:Novo Nordisk: Other: Investigator fees as a participant of the clinical trial. Austin:Pfizer: Research Funding. Chowdary:Novo Nordisk: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; CSL Behring: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Freeline: Consultancy; Bayer: Honoraria; Baxalta (Shire): Honoraria, Membership on an entity's Board of Directors or advisory committees; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Swedish Orphan Biovitrum AB (Sobi): Honoraria; Biogen: Honoraria, Membership on an entity's Board of Directors or advisory committees. Foster:Sanofi Genzyme: Employment. Yu:Sanofi Genzyme: Employment. Benson:Sanofi Genzyme: Employment. Madigan:Alnylam Pharmaceuticals Inc: Employment. Nguyen:Alnylam Pharmaceuticals Inc: Employment. Ali:Sanofi Genzyme: Employment. Kadam:Sanofi Genzyme: Employment. Jain:Sanofi Genzyme: Employment. Pasi:Octapharma: Honoraria; Catalyst Bio: Honoraria; Bayer: Speakers Bureau; Shire: Speakers Bureau; NovoNordisk: Speakers Bureau; Sobi: Honoraria; Apcintex: Honoraria; Biomarin: Honoraria, Research Funding; Pfizer: Speakers Bureau; Alnylam: Honoraria, Research Funding; Bioverativ: Honoraria, Research Funding.

An investigational RNAi therapeutic targeting antithrombin for the treatment of hemophilia A and B.

Fitusiran is an RNA interference therapeutic that targets antithrombin (AT) in the liver and interferes with AT translation by binding and degrading messenger RNA-AT, thereby silencing AT gene expression and preventing AT synthesis. In both preclinical and clinical studies, AT knockdown results in dose-dependent AT lowering when fitusiran is given weekly or monthly subcutaneously. In clinical trials, fitusiran dose escalation has resulted in improved thrombin generation and clinical hemostasis as measured by reduction in annualized bleed rate. Unlike currently licensed drugs, this improvement was not only in patients with hemophilia A but in also those with hemophilia B, with or without inhibitors. In dental and surgical procedures, fitusiran also provided perioperative hemostasis in association with AT lowering. Fitusiran is well tolerated, with minor local injection site reactions, but in one subject with severe hemophilia A, the concomitant use of daily high-dose factor VIII, inconsistent with trial guidance to avoid high, repeat doses of clotting factor, was associated with fatal thrombosis, suggesting the need for caution when using hemostatic agents in conjunction with fitusiran. Preclinical in vitro and in silico studies indicate improvement in thrombin generation in rare bleeding disorder plasmas, including in plasmas from patients with severe factors V, VII, and X deficiency, suggesting potential therapeutic benefit.

Four-factor prothrombin complex concentrate improves thrombin generation and prothrombin time in patients with bleeding complications related to rivaroxaban: a single-center pilot trial

BackgroundDirect oral anticoagulants (DOACs) pose a great challenge for physicians in life-threatening bleeding events. The aim of this study was to test the efficacy of reversing the DOAC rivaroxaban using four-factor PCC (prothrombin complex concentrate), a non-specific reversing agent.MethodsPatients with life-threatening bleeding events during rivaroxaban treatment were included and administered 25 U kg−1 of PCC. Blood samples were collected immediately prior to as well as after PCC treatment at predefined time intervals. The primary endpoint was defined as the difference in thrombin generation (TG) parameters ETP (endogenous thrombin potential) and Cmax (peak thrombin generation) prior to and ten minutes subsequent to PCC treatment.ResultsThirteen patients, of whom the majority suffered from intra-cranial haemorrhage (ICH) or subdural haemorrhage (SDH), were included and administered PCC. The results show that the ETP (TG) significantly (p = 0.001) improved by 68% and Cmax (TG) by 54% (p = 0.001) during PCC treatment. In addition, the Quick value (prothrombin time: QuickPT) significantly improved by 28% and the activated partial thromboplastin time (aPTT) was decreased by 7% ten minutes after PCC administration. Cmax was reduced at baseline, but not ETP, aPTT or QuickPT. Lag time until initiation (TG, tlag), thromboelastometry clotting time (CTEXTEM) and time to peak (TG, tmax) correlated best with measured rivaroxaban levels and were out of normal ranges at baseline, but did not improve after PCC administration. In 77% of the patients bleeding (ICH/SDH-progression) ceased following PCC administration. During the study three participants passed away due to other complications not related to PCC treatment. The possibility of thrombosis formation was also evaluated seven days after administering PCC and no thromboses were found.ConclusionsThis study shows that use of PCC improved ETP, Cmax, QuickPT and aPTT. However, of these parameters, only Cmax was reduced at the defined baseline. It can be concluded that CTEXTEM, tlag and tmax correlated best with the measured rivaroxaban levels. The study drug was found to be safe. Nonetheless, additional studies specifically targeting assessment of clinical endpoints should be performed to further confirm these findings.Clinical trial registrationEudraCT trial No. 2013–004484-31.

Coagulopathy in Zellweger spectrum disorders: a role for vitamin K

IntroductionZellweger spectrum disorders (ZSDs) are caused by an impairment of peroxisome biogenesis, resulting in multiple metabolic abnormalities. This leads to a range of symptoms, including hepatic dysfunction and coagulopathy. This study evaluated the incidence and severity of coagulopathy and the effect of vitamin K supplementation orally and IV in ZSD.MethodsData were retrospectively retrieved from the medical records of 30 ZSD patients to study coagulopathy and the effect of vitamin K orally on proteins induced by vitamin K absence (PIVKA-II) levels. Five patients from the cohort with a prolonged prothrombin time, low factor VII, and elevated PIVKA-II levels received 10 mg of vitamin K IV. Laboratory results, including thrombin generation, at baseline and 72 h after vitamin K administration were examined.ResultsIn the retrospective cohort, four patients (13.3%) experienced intracranial bleedings and 14 (46.7%) reported minor bleeding. No thrombotic events occurred. PIVKA-II levels decreased 38% after start of vitamin K therapy orally. In the five patients with a coagulopathy, despite treatment with oral administration of vitamin K, vitamin K IV caused an additional decrease (23%) of PIVKA-II levels and increased thrombin generation.ConclusionBleeding complications frequently occur in ZSD patients due to liver disease and vitamin K deficiency. Vitamin K deficiency is partly corrected by vitamin K supplementation orally, and vitamin K administered IV additionally improves vitamin K status, as shown by further decrease of PIVKA-II and improved thrombin generation.