FXa Inhibitors Research Articles

Characterisation of the pharmacokinetic and pharmacodynamic relationship of milvexian in total knee replacement patients

Abstract Background Milvexian, an oral factor XIa inhibitor, is currently being evaluated in patients at risk for thrombotic events due to atrial fibrillation, ischaemic stroke, or acute coronary syndrome. The exposure-response relationship between clinical efficacy and safety endpoints and milvexian pharmacokinetic (PK) metrics was recently submitted as a separate publication. Characterisation of the relationship between the coagulation pharmacodynamic (PD) markers versus milvexian PK metrics at the patient level has yet to be reported. Purpose This analysis characterised the relationship between the coagulation biomarkers, activated partial thromboplastin time (aPTT) and prothrombin time (PT), and milvexian systemic exposure in patients undergoing total knee replacement (TKR) evaluated in the AXIOMATIC-TKR study. Methods A population pharmacokinetic (PopPK) model was previously developed and characterised the PK of milvexian in TKR patients well. The PK/PD relationships between observed aPTT and PT versus PopPK model estimated time-matched milvexian plasma concentrations via nonlinear mixed effects modeling. The relationship between key clinical safety endpoints (e.g., any bleeding or clinically relevant non-major bleeding) and individual PD marker change (e.g., aPTT ratio to baseline) was also explored. Results The aPTT increased with increasing plasma concentration of milvexian over the range of doses evaluated in the study (25mg QD to 200mg BID). An Emax type of model reasonably characterised the PK/PD relationship between the aPTT and model predicted time-matched milvexian plasma concentration, which is consistent with previously observed relationships in healthy volunteers. No change was observed in PT with increased milvexian exposure. No obvious relationship was identified between any (including clinically relevant non-major) bleeding and aPTT ratio to baseline. Conclusions Increased milvexian exposure is directly correlated with aPTT prolongation over the range of doses evaluated in the TKR study. There was no obvious relationship between any (including clinically relevant non-major) bleeding and aPTT ratio. The PK/PD relationship of aPTT without a parallel relationship in PT distinguishes milvexian, a FXIa inhibitor, from previously studied FXa inhibitors. These analyses were a complementary component in dose selection for the milvexian Phase 3 program. Figure 1. PK/PD relationship between aPTT (left) and PT (right) and estimated time-matched plasma concentration (AXIOMATIC-TKR study)

3-Factor prothrombin complex concentrate versus 4-factor prothrombin complex concentrate for the reversal of oral factor Xa inhibitors.

Multiple agents exist for the reversal of oral Factor Xa inhibitor (FXa) associated bleeding, including Coagulation FXa Recombinant, Inactivated zhzo (andexanet alfa) and 4-factor prothrombin complex concentrate (4F-PCC). While classified as a 3F-PCC product, Profilnine contains up to 35IU of Factor VII (per 100IU of Factor IX) in addition to therapeutic levels of Factors II, IX, and X, and has demonstrated a similar impact on prothrombin time and blood product usage in non-warfarin related bleeding. This was a retrospective, multicenter study at four medical centers of adult patients who presented with major bleeding associated with oral FXa inhibitors and received either 4F-PCC (n = 64) or 3F-PCC (n = 61). The primary outcome was hemostatic effectiveness. Secondary outcomes included the incidence of thromboembolism, in-hospital mortality, and length of stay. The most common indication for reversal was intracranial bleeding. For the primary outcome, 84% of all patients were rated as effective with no difference noted between the groups (p = 0.81). No significant difference between groups was found in the multivariable analysis adjusting for baseline differences between groups including race, total body weight, type of bleeding, and the use of antiplatelet therapy. There was no difference in the length of stay, in-hospital mortality, or the incidence of thromboembolism between the groups. Overall, no significant differences were found in the effectiveness or safety of 4F-PCC and 3F-PCC use in the management of oral FXa inhibitor-associated bleeding. Further investigations are warranted to explore the use of 3F-PCC for this indication and its safety and effectiveness.

Pharmacodynamics of Rivaroxaban and Dabigatran in Adults with Diffuse Large B-Cell Lymphoma Receiving R-CHOP Immunochemotherapy.

Background/Objectives: Rivaroxaban and dabigatran are commonly used for thromboembolic disease management in active cancer patients. However, limited research explores the impact of concurrent chemotherapy on the pharmacodynamics of direct oral anticoagulants (DOAC). The aim of our study was to evaluate the impact of combined chemotherapy with rivaroxaban and dabigatran on the pharmacodynamics in patients with diffuse large B-cell lymphoma (DLBCL).; Methods: This was a prospective, pharmacodynamic study. Eligible subjects were ≥18 years old, diagnosed with DLBCL and initiating R-CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone) immunochemotherapy. The enrolled adults received either rivaroxaban (10 mg once daily) or dabigatran etixalate (110 mg twice daily). Plasma anti-factor Xa (FXa) in participants on rivaroxaban and diluted thrombin time (dTT) in participants on dabigatran were assessed over the dosing interval before and after R-CHOP administration. Pharmacodynamic parameters of rivaroxaban and dabigatran were determined using a non-compartmental analysis.; Results: Twenty-six adults participated, with twelve in the rivaroxaban group and fourteen in the dabigatran group. The mean age was 59 ± 14.4 years. In the rivaroxaban group, the AUEC of FXa inhibition showed no significant change after R-CHOP (mean difference 3.8 ng·h/mL, 95% confidence interval (CI) -155.4 to 163.0, p = 0.96). Similarly, in the dabigatran group, the AUEC of dTT remained unchanged post R-CHOP (mean difference 54.41 ng·h/mL, 95% CI -99.09 to 207.9 ng/mL, p = 0.46). However, the median time-to-peak dTT was significantly faster with R-CHOP (3 h, [min-max, 1.5-8] compared to without it (4 h, [min-max, 3-8], p = 0.04); Conclusions: Concurrent R-CHOP chemotherapy did not significantly impact FXa inhibition by rivaroxaban or dTT by dabigatran. The time-to-peak dTT was faster when dabigatran was administered with R-CHOP.

Monitoring the efficiency of reversal on anti-Xa direct oral anticoagulants using point-of-care viscoelastic testing

Bleeding events in patients receiving direct oral anticoagulation (DOAC) can be life-threatening even at therapeutic DOAC plasma concentrations, as anticoagulation impairs hemostasis and should therefore be identified immediately after hospital admission. The anticoagulatory effects of DOAC are typically not measurable in standard coagulation tests, such as PT or aPTT. Specific calibrated anti-FXa-tests allow specific drug monitoring, but they are too time-consuming for critical bleeding events and are commonly not available for 24 h/7 days in routine care. However, recent advances in point-of-care (POC) viscoelastic testing (VET) have shown a promising approach for rapid and quantitative detection of DOAC plasma concentrations using the Russell viper venom factor V activator (RVV for FXa-inhibitors) test or the ecarin clotting time (thrombin inhibitors). In acute bleeding situations, direct FXa inhibitors can be reversed by specific antidote andexanet alfa or hemostasis can be improved by prothrombin complex factor concentrates (PCCs). After reversal, confirmation of reversal efficacy is often requested, but no routine assays are currently available. Thus, the emergency management of bleeding DOAC patients is usually “blinded” with regard to reversal efficacy. POC VET laboratory assays might therefore also be helpful for measuring DOAC effects after reversal. We present a case series demonstrating the usefulness of RVV-clotting time post-DOAC reversal with andexanet alfa.

Impact of elevated direct factor Xa inhibitor plasma levels on perioperative blood loss in patients undergoing urgent surgery.

Data on the perioperative bleeding risk associated with elevated plasma levels of direct factor Xa inhibitors (FXa inhibitors) are limited. This study examines perioperative red blood cell (RBC) loss in patients undergoing urgent surgery with a residual FXa inhibitor level exceeding 100 mcg/L and without preoperative FXa inhibitor reversal. This retrospective analysis includes data from 32 patients who underwent urgent noncardiac surgery between 2018 and 2022. This study aims to analyze perioperative RBC loss in patients undergoing urgent surgery with a residual FXa inhibitor level exceeding 100 mcg/L and without preoperative FXa inhibitor antidote-based reversal or unspecific treatment with 4-factor prothrombin complex concentrate (PCC). All patients were managed using a watch-and-wait strategy. The last determination of FXa inhibitor plasma concentration prior to surgery showed a median of 245 mcg/L (IQR 144-345), with a median time interval of 3.8 h (IQR 2.4-7.2) before incision. Median RBC loss during surgery was 49 mL (IQR 0-253), 189 mL (IQR 104-217) until POD1 and 254 mL (IQR 58-265) until POD3. Only one patient required intraoperative treatment with 4-factor-PCC and none required reversal with andexanet alfa. Linear regression models found no significant influence of FXa inhibitor plasma levels on intraoperative RBC loss. Rivaroxaban was associated with higher RBC loss until postoperative Day 1 compared with apixaban. No thromboembolic events were observed. Despite markedly elevated plasma concentrations of residual direct FXa inhibitors, perioperative RBC loss was limited in patients undergoing urgent noncardiac surgery. The intraoperative watch-and-wait strategy with selective intraoperative FXa inhibitor reversal or treatment only when required appears to be an appropriate approach.

Treatment of atrial fibrillation and venous thromboembolism with factor Xa inhibitors in severely obese patients

BackgroundA paucity of data exists to support the use of factor (F)Xa inhibitors in severely obese patients with a weight of ≥150 kg or body mass index (BMI) of ≥50 kg/m2. ObjectivesThe purpose of this study was to evaluate whether FXa inhibitors are as safe and effective as warfarin for the treatment of atrial fibrillation (AF) and/or venous thromboembolism (VTE) in individuals with a BMI of ≥50 kg/m2 and/or weight of ≥150 kg. MethodsThis was a multicenter retrospective cohort study of severely obese adult patients with AF and/or VTE treated with a FXa inhibitor or warfarin. The primary effectiveness outcome was composite odds of stroke, systemic embolism, or VTE; the primary safety outcome was odds of major bleeding. Secondary outcomes included incidence of stroke or systemic embolism, VTE, major bleeding, clinically relevant nonmajor bleeding, all-cause mortality, change in anticoagulation, and total number of hospital encounters. Outcomes were assessed for 12 months following initiation of study drug. ResultsA total of 1736 patients were included. The mean weight and BMI of the overall cohort were 164.4 kg and 54.6 kg/m2, respectively. There was no difference in odds of stroke, systemic embolism or VTE (odds ratio, 1.005; 95% CI, 0.6-1.68), or major bleeding (odds ratio, 0.9; 95% CI, 0.47-1.7) between groups. ConclusionThese data suggest that apixaban and rivaroxaban are safe and effective alternatives to warfarin for the treatment of AF and/or VTE in individuals with a BMI of ≥50 kg/m2 and/or weight of ≥150 kg.

Chemical Synthesis and Structure-Activity Relationship Studies of the Coagulation Factor Xa Inhibitor Tick Anticoagulant Peptide from the Hematophagous Parasite Ornithodoros moubata.

Tick Anticoagulant Peptide (TAP), a 60-amino acid protein from the soft tick Ornithodoros moubata, inhibits activated coagulation factor X (fXa) with almost absolute specificity. Despite TAP and Bovine Pancreatic Trypsin Inhibitor (BPTI) (i.e., the prototype of the Kunitz-type protease inhibitors) sharing a similar 3D fold and disulphide bond topology, they have remarkably different amino acid sequence (only ~24% sequence identity), thermal stability, folding pathways, protease specificity, and even mechanism of protease inhibition. Here, fully active and correctly folded TAP was produced in reasonably high yields (~20%) by solid-phase peptide chemical synthesis and thoroughly characterised with respect to its chemical identity, disulphide pairing, folding kinetics, conformational dynamics, and fXa inhibition. The versatility of the chemical synthesis was exploited to perform structure-activity relationship studies on TAP by incorporating non-coded amino acids at positions 1 and 3 of the inhibitor. Using Hydrogen-Deuterium Exchange Mass Spectrometry, we found that TAP has a remarkably higher conformational flexibility compared to BPTI, and propose that these different dynamics could impact the different folding pathway and inhibition mechanisms of TAP and BPTI. Hence, the TAP/BPTI pair represents a nice example of divergent evolution, while the relative facility of TAP synthesis could represent a good starting point to design novel synthetic analogues with improved pharmacological profiles.

Preparation of an anticoagulant polyethersulfone membrane by immobilizing FXa inhibitors with a polydopamine coating

Anticoagulation treatment for patients with high bleeding risk during hemodialysis is challenging. Contact between the dialysis membrane and the blood leads to protein adsorption and activation of the coagulation cascade reaction. Activated coagulation Factor X (FXa) plays a central role in thrombogenesis, but anticoagulant modification of the dialysis membrane is rarely targeted at FXa. In this study, we constructed an anticoagulant membrane using the polydopamine coating method to graft FXa inhibitors (apixaban and rivaroxaban) on the membrane surface. Attenuated total reflection-Fourier transform infrared spectroscopy (ATR-FTIR), X-ray photoelectron spectroscopy (XPS), scanning electron microscopy (SEM), and atomic force microscopy (AFM) were used to characterize the membranes. The apixaban- and rivaroxaban-modified membranes showed lower water contact angles, decreased albumin protein adsorption, and suppressed platelet adhesion and activation compared to the unmodified PES membranes. Moreover, the modified membranes prolonged the blood clotting times in both the intrinsic and extrinsic coagulation pathways and inhibited FXa generation and complement activation, which suggested that the modified membrane enhanced biocompatibility and antithrombotic properties through the inhibition of FXa. Targeting FXa to design antithrombotic HD membranes or other blood contact materials might have great application potential.

Blood Lines: Intraspecific and Interspecific Variations in Anticoagulant Actions of Agkistrodon Viperid Venoms.

This study investigated the intraspecific and interspecific variability in the venom effects of Agkistrodon viperid snake species and subspecies (eleven venoms total) on plasma clotting times, fibrinogen levels, and fibrin clot strength. Significant delays in plasma clotting time were observed for A. conanti, A. contortrix mokasen, A. contortrix phaeogaster, A. howardgloydi, A. piscivorus leucostoma, and A. piscivorus piscivorus. Notably, the phylogenetically disjunct lineages A. conanti, A. contortrix mokasen, and A. howardgloydi exhibited the most potent anticoagulant effects, indicating the independent amplification of a basal trait. Inhibition assays with the activated clotting enzymes Factors XIa, IXa, Xa, and IIa (thrombin) revealed that FXa inhibition is another basal trait amplified independently on multiple occasions within the genus, but with A. howardgloydi, notably more potent than all others. Phospholipid degradation and zymogen destruction were identified as mechanisms underlying the variability in venom effects observed experimentally and in previous clinical reports. Thromboelastography demonstrated that the venoms did not clot fibrinogen directly but affected fibrin clot strength by damaging fibrinogen and that thrombin was subsequently only able to cleave into weak, unstable clots. The ability to activate Protein C, an endogenous anticoagulant enzyme, varied across species, with some venoms exceeding that of A. contortrix contortrix, which previously yielded the protein diagnostic agent Protac®. Phylogenetic analysis suggested that both fibrinogen degradation and Protein C activation were each amplified multiple times within the genus, albeit with negative correlation between these two modes of action. This study highlights the evolutionary, clinical, and biodiscovery implications of venom variability in the Agkistrodon species, underscoring their dynamic evolution, emphasising the need for tailored clinical approaches, and highlighting the potential for novel diagnostic and therapeutic developments inspired by the unique properties of snake venoms.

Factor XI inhibitors: a new option for the prevention and treatment of cancer-associated thrombosis

Venous thromboembolism (VTE) is a relatively common complication in cancer patients with potentially dire consequences. Anticoagulants are the mainstay of treatment of cancer-associated VTE. The anticoagulants most often used are low-molecular-weight heparin (LMWH) and direct oral factor (F) Xa inhibitors, which include apixaban, edoxaban, and rivaroxaban. Most guidelines recommend primary VTE prophylaxis with LMWH, apixaban, or rivaroxaban after abdominal or pelvic cancer surgery, or in high-risk ambulatory cancer patients. Both oral FXa inhibitors and LMWH have limitations. LMWH requires daily subcutaneous injections, and because of its renal clearance, its use may be problematic in patients with severe kidney disease. The risk of bleeding with oral FXa inhibitors may be higher than with LMWH in patients with intraluminal gastrointestinal or genitourinary cancers. Other problems with oral FXa inhibitors include potential drug-drug interactions and dosing issues in patients with thrombocytopenia or severe kidney or liver disease. Therefore, there remains a need for convenient and safer anticoagulants for VTE treatment in cancer patients. FXI has emerged as a potentially safer target for anticoagulants than FXa because FXI is essential for thrombosis, but mostly dispensable for hemostasis. This review summarizes the currently available therapeutic options for cancer-associated VTE, highlights knowledge gaps, and discusses the potential of FXI inhibitors to address key unmet clinical needs in this vulnerable patient population.

Activated factor X stimulates atrial endothelial cells and tissues to promote remodelling responses through AT1R/NADPH oxidases/SGLT1/2.

Atrial fibrillation (AF), the most common cardiac arrhythmia favouring ischemic stroke and heart failure involves left atrial remodelling, fibrosis and a complex interplay between cardiovascular risk factors. This study examined whether activated factor X (FXa) induces pro-remodelling and pro-fibrotic responses in atrial endothelial cells (AECs) and human atrial tissues and determined the underlying mechanisms. AECs collected from porcine hearts and human right atrial appendages (RAA) from patients undergoing heart surgery. Protein expression levels were assessed by Western blot and immunofluorescence staining, mRNA levels by RT-qPCR, formation of reactive oxygen species (ROS) and NO using fluorescent probes, thrombin and angiotensin II generation by specific assays, fibrosis by Sirius red staining and senescence by senescence-associated beta-galactosidase (SA-β-gal) activity. In AECs, FXa increased ROS formation, senescence (SA-β-gal activity, p53, p21), angiotensin II generation and the expression of pro-inflammatory (VCAM-1, MCP-1), pro-thrombotic (tissue factor), pro-fibrotic (TGF-β and collagen-1/3a) and pro-remodelling (MMP-2/9) markers whereas eNOS levels and NO formation were reduced. These effects were prevented by inhibitors of FXa but not thrombin, protease-activated receptors antagonists (PAR-1/2) and inhibitors of NADPH oxidases, ACE, AT1R, SGLT1/SGLT2. FXa also increased expression levels of ACE1, AT1R, SGLT1/2 proteins which were prevented by SGLT1/2 inhibitors. Human RAA showed tissue factor mRNA levels that correlated with markers of endothelial activation, pro-remodelling and pro-fibrotic responses and SGLT1/2 mRNA levels. They also showed protein expression levels of ACE1, AT1R, p22phox, SGLT1/2, and immunofluorescence signals of nitrotyrosine and SGLT1/2 colocalized with those of CD31. FXa increased oxidative stress levels which were prevented by inhibitors of the AT1R/NADPH oxidases/SGLT1/2 pathway. FXa promotes oxidative stress triggering premature endothelial senescence and dysfunction associated with pro-thrombotic, pro-remodelling and pro-fibrotic responses in AECs and human RAA involving the AT1R/NADPH oxidases/SGLT1/2 pro-oxidant pathway. Targeting this pathway may be of interest to prevent atrial remodelling and the progression of atrial fibrillation substrate.

Minimally modified human blood coagulation factor X to bypass direct factor Xa inhibitors

BackgroundDirect oral factor (F)Xa inhibitors are widely used as alternatives to conventional vitamin K antagonists in managing venous thromboembolism and nonvalvular atrial fibrillation. Unfortunately, bleeding-related adverse events remain a major concern in clinical practice. In case of bleeding or emergency surgery, rapid-onset reversal agents may be required to counteract the anticoagulant activity. ObjectivesThe ability of FXa variants to bypass the direct oral FXa inhibitors was assessed. MethodsHuman FXa variants were generated through substitution of phenylalanine 174 (F174) for either alanine, isoleucine, or serine. FXa variants were stably expressed in HEK293 cells and purified to homogeneity using ion-exchange chromatography. ResultsF174-substituted human FX variants demonstrated efficacy in restoring thrombin generation in plasma containing direct FXa inhibitors (apixaban, rivaroxaban, edoxaban). Their ability to bypass the anticoagulant effects stems from a significantly reduced sensitivity for the direct FXa inhibitors due to a decrease in binding affinity determined using molecular dynamics simulations and free energy computation. Furthermore, F174 modification resulted in a partial loss of inhibition by tissue factor pathway inhibitor, enhancing the procoagulant effect of F174-substituted FX. Consequently, the F174A- and F174S-substituted FX variants effectively counteracted the effects of 2 widely used anticoagulants, apixaban and rivaroxaban, in plasma of atrial fibrillation and venous thromboembolism patients. ConclusionThese human FX variants have the potential to serve as a rescue reversal strategy to overcome the effect of direct FXa inhibitors in case of life-threatening bleeding events or emergency surgical interventions.

Abstract 3095: Characterization Of Interactions Between Skeletal Muscle Myosin And Activated Factor XI

Background: We recently demonstrated that the procoagulant activity of skeletal muscle myosin (SkM) involved the binding of coagulation factor (F) XI to enhance FXI activation by thrombin. However, the impact of SkM on the enzymatic activities of activated FXI (FXIa) remains unclear. Aims: Determine the influence of SkM interactions on the expression of FXIa activities. Methods: Binding studies between FXIa and SkM were conducted using biolayer interferometry (BLI). Kallikrein, a FXIa homolog, served as a negative control. The interactions between SkM and FXIa were investigated through fluorescence spectroscopy using dansyl-labeled FXIa. Chromogenic assays for FXIIa were employed to measure FXII activation by FXIa. Tissue factor pathway inhibitor (TFPI) inactivation by FXIa was assessed by determining FXa inhibition by TFPI using FX chromogenic assays. Quantification of complement factor H (CFH) cleavage by FXIa as well as FV activation by FXIa were carried out using SDS PAGE and Coomassie blue staining. Results: BLI data showed that SkM exhibited a high binding affinity for FXIa with a dissociation constant of 1.6 nM, whereas SkM did not bind kallikrein. The addition of SkM induced a 9 nm shift in the emission spectrum of dansyl-labeled FXIa. The presence of SkM increased the rate of feedback FXII activation by FXIa but not by kallikrein. The addition of SkM inhibitors, trifluoperazine and blebbistatin, reduced the enhancement effect of SkM on the ability of FXIa to activate FXII. Furthermore, SkM increased the rate of enzymatic inactivation of TFPI by FXIa, the rate of cleavage of CFH by FXIa, and the rate of activation of FV by FXIa. Conclusions: SkM binds with high affinity to FXIa and serves as a co-factor to enhance multiple different enzymatic activities of FXIa. Future work is needed to examine the multiple procoagulant activities of SkM in the setting of hemostasis and acute trauma.

Ex Vivo Antiplatelet Effects of Oral Anticoagulants.

The impact of non-vitamin K antagonist oral anticoagulants (NOACs) on platelet function is still unclear. We conducted a comprehensive ex vivo study aimed at assessing the effect of the four currently marketed NOACs on platelet function. We incubated blood samples from healthy donors with concentrations of NOACs (50, 150 and 250 ng/mL), in the range of those achieved in the plasma of patients during therapy. We evaluated generation of thrombin; light transmittance platelet aggregation (LTA) in response to adenosine diphosphate (ADP), thrombin receptor-activating peptide (TRAP), human γ-thrombin (THR) and tissue factor (TF); generation of thromboxane (TX)B2; and expression of protease-activated receptor (PAR)-1 and P-selectin on the platelet surface. All NOACs concentration-dependently reduced thrombin generation compared with control. THR-induced LTA was suppressed by the addition of dabigatran at any concentration, while TF-induced LTA was reduced by factor-Xa inhibitors. ADP- and TRAP-induced LTA was not modified by NOACs. TXB2 generation was reduced by all NOACs, particularly at the highest concentrations. We found a concentration-dependent increase in PAR-1 expression after incubation with dabigatran, mainly at the highest concentrations, but not with FXa inhibitors; P-selectin expression was not changed by any drugs. Treatment with the NOACs is associated with measurable ex vivo changes in platelet function, arguing for antiplatelet effects beyond the well-known anticoagulant activities of these drugs. There are differences, however, among the NOACs, especially between dabigatran and the FXa inhibitors.

Andexanet Alfa (Ondexxya)


 CADTH reimbursement reviews are comprehensive assessments of the clinical effectiveness and cost-effectiveness, as well as patient and clinician perspectives, of a drug or drug class.
 The assessments inform non-binding recommendations that help guide the reimbursement decisions of Canada's federal, provincial, and territorial governments, with the exception of Quebec.
 This review assesses andexanet alfa (Ondexxya), 200 mg, powder for solution for infusion, IV infusion.
 Indication: For adult patients treated with FXa inhibitors (rivaroxaban or apixaban) when rapid reversal of anticoagulation is needed due to life-threatening or uncontrolled bleeding.

Interruption of perivascular and perirenal adipose tissue thromboinflammation rescues prediabetic cardioautonomic and renovascular deterioration.

The cardiovascular and renovascular complications of metabolic deterioration are associated with localized adipose tissue dysfunction. We have previously demonstrated that metabolic impairment delineated the heightened vulnerability of both the perivascular (PVAT) and perirenal adipose tissue (PRAT) depots to hypoxia and inflammation, predisposing to cardioautonomic, vascular and renal deterioration. Interventions either addressing underlying metabolic disturbances or halting adipose tissue dysfunction rescued the observed pathological and functional manifestations. Several lines of evidence implicate adipose tissue thromboinflammation, which entails the activation of the proinflammatory properties of the blood clotting cascade, in the pathogenesis of metabolic and cardiovascular diseases. Despite offering valuable tools to interrupt the thromboinflammatory cycle, there exists a significant knowledge gap regarding the potential pleiotropic effects of anticoagulant drugs on adipose inflammation and cardiovascular function. As such, a systemic investigation of the consequences of PVAT and PRAT thromboinflammation and its interruption in the context of metabolic disease has not been attempted. Here, using an established prediabetic rat model, we demonstrate that metabolic disturbances are associated with PVAT and PRAT thromboinflammation in addition to cardioautonomic, vascular and renal functional decline. Administration of rivaroxaban, a FXa inhibitor, reduced PVAT and PRAT thromboinflammation and ameliorated the cardioautonomic, vascular and renal deterioration associated with prediabetes. Our present work outlines the involvement of PVAT and PRAT thromboinflammation during early metabolic derangement and offers novel perspectives into targeting adipose tissue thrombo-inflammatory pathways for the management its complications in future translational efforts.

The use of andexanet alpha in the Polish setting: An interdisciplinary protocol. Expert consensus statement of the Polish Cardiac Society.

Andexanet alfa (AA) is a recombinant inactive analog of human activated factor X (FXa), effectively reversing the effects of its inhibitors - rivaroxaban and apixaban, which are available in Poland. The drug was approved for clinical use registration after the publication of the results of the ANNEXA-4 trial (Andexanet Alfa, a Novel Antidote to the Anticoagulation Effects of FXa Inhibitors 4), in which its efficacy in restoring hemostasis in life-threatening hemorrhages in patients receiving using the aforementioned anticoagulants was demonstrated. Hence, AA is now recommended for patients on apixaban or rivaroxaban therapy with massive and uncontrollable hemorrhages, including hemorrhagic strokes (HS) and gastrointestinal bleeding. Drug-specific chromogenic anti-Xa assays are generally best suited for estimating rivaroxaban and apixaban plasma levels, aside from direct assessment of their concentrations. The absence of anti-Xa activity, determined using these assays, allows us to rule out the presence of clinically relevant plasma concentrations of any FXa inhibitor. On the other hand, the dose of AA should not be modified based on the results of coagulation tests, as it depends solely on the time that elapsed since the last dose of FXa inhibitor and oon the dose and type of FXa inhibitor. AA is administered as an intravenous (i.v.) bolus, followed by an i.v. infusion of the drug. The maximum reversal of anti-Xa activity occurs within two minutes of the end of the bolus treatment, with the continuation of the continuous i.v. infusion allowing the effect to be maintained for up to two hours afterwards. Because anticoagulant activity can reappear after the infusion is completed, it is currently unclear at what point after AA administration FXa inhibitors or heparin should be re-administered. In Poland AA is starting to become available and its urgent need to administer it to patients with severe bleeding on apixaban or rivaroxaban.

Monitoring of Hemostatic Status and Treatment Response in Von Willebrand Disease Using a Microchip Flow Chamber Assay

Introduction: Von Willebrand factor (VWF) carries out its hemostatic roles by interacting with both platelets and FVIII, and its appropriate active multimeric conformation is strongly affected by blood flow shear. Our aim was to assess the hemostatic status and the response to treatments in VW disease (VWD) using a flow chamber-based thrombus formation analysis system (T-TAS ®, Zacros) that allows evaluation of both primary and secondary hemostasis in a scenario closer to in vivo conditions. Methods: Blood samples from patients diagnosed with VWD type 1 (n=3), type 2A (n=2), type 2B (n=1) and type 3 (n=8, two of them with inhibitory antibodies) were collected before and after in vivo administration of the prescribed treatment with desmopressin (DDAVP ®) or plasma-derived (pd)VWF/FVIII concentrates (Table-1). The effect of ex vivo administration of 100, 150 and 300 IU/dL of pdVWF/FVIII (Fanhdi ®, Grifols) was also evaluated. Thrombus formation analysis was performed according to the manufacturer's standardized protocols: Blood samples collected in tubes containing BAPA (inhibitor of FXa and thrombin) were loaded onto type-1 collagen-coated platelet (PL)-chips to assess platelet-dependent thrombus formation. Citrated blood samples were recalcified in presence of CTI and loaded onto collagen/tissue factor-coated atherome (AR)-chips to assess thrombus formation mediated by both platelet and coagulation activation. An optimized flow rate is automatically adjusted for each chip. Area under the flow-pressure curve (AUC) values were collected. Platelet count, fibrinogen levels, prothrombin time (PT), activated partial thromboplastin time (aPTT), aPTT ratio, antigenic VWF (VWF:Ag), VWF activity by recombinant GPIb binding assay (VWF:GPIbR) and FVIII activity (FVIII:C) were also analyzed. Results: All VWD patient samples tested, including type-1 patients, showed a deficient platelet function (PL_AUC) and a reduced coagulation-dependent thrombus formation (AR_AUC) prior to treatment (Table-1). VWD type-1 responded to desmopressin with a high increase of FVIII activity (FVIII:C >200%) and normalization of VWF activity (VWF:GPIbR >100%) and AUC levels. Treatment with pdVWF/FVIII increased VWF:GPIbR levels (>60% after treatment) in all cases, but failed to completely normalize the AUC values, which showed a slight increase (Table-1). A high correlation (r>0.8) was observed between plasma levels of VWF or FVIII activity and AUC values obtained both before and after treatment Ex vivo administration of pdVWF/FVIIIa resulted in a concentration-dependent increased of PL_ and AR_AUC, with values within the normal range at the highest dose tested (300 IU/dL, equivalent to 150 IU/kg) in all cases, except for VWD type-3 samples with inhibitor and for VWD type-2B sample with PL-chip assay (Figure-1). Conclusions: Monitoring treatment efficacy in VWD using a flow chamber-based thrombus formation analysis system could be a reliable and standardized method to assess the response to desmopressin and pdVWF/FVIII concentrates in patients with VWD, allowing an optimal personalized therapeutic dosing and a better prevention of bleeding episodes in these patients. Funding: Grifols; Zacros; Fundación Rodríguez Pascual; Instituto de Salud Carlos III (ISCIII) (PI22/01461), co-funded by the European Union.

Efficacy and Safety of Antithrombin Supplementation in Neonates and Infants on a Continuous Heparin Infusion

Introduction: Antithrombin (AT) is an inhibitor of several coagulation proteins, including FIIa and FXa. Heparins accelerate/potentiate the interaction between AT and these factors through a conformational change in AT. Plasma AT concentrations are low in neonates, lower in sick premature neonates, and reach adult levels by 6 to 12 months of age. The developmental differences in plasma AT levels are an important consideration when treating an infant with heparin, as the activity of heparin depends on the presence of AT as a cofactor. As a result, AT supplementation is often considered in this cohort, particularly in the setting of heparin resistance. Objective: The objective of this study was to describe the use of AT supplementation at Boston Children's Hospital (BCH), a quaternary care, free-standing children's hospital, and investigate the safety and efficacy of AT supplementation in infants on a continuous heparin infusion. Methods: This study was approved by the Institutional Review Board at BCH. Data were abstracted for infants who received AT supplementation while on a therapeutic heparin infusion in the neonatal, cardiac, and medical intensive care units between January 2016 and October 2022. Infants on extracorporeal membrane oxygenation and continuous renal replacement therapy were excluded. The primary outcome was attainment of therapeutic anticoagulation within 48 hours after an AT course, defined as at least one occurrence of a partial thromboplastin time (PTT) level between 60 and 85 seconds and/or anti-factor Xa (anti-FXa) activity between 0.35 and 0.7 units/mL. Clinically relevant bleeding was defined as moderate/clinically relevant non-major bleeding or severe/major bleeding, as determined by either the Neonatal Bleeding Assessment Tool for infants with post-menstrual age (PMA) < 44 weeks or the International Society on Thrombosis and Haemostasis criteria for infants with PMA > 44 weeks, respectively. Data were summarized using descriptive statistics. Results: Over the 7-year period, 50 infants receiving 61 courses of AT supplementation were included. 12 patients received multiple AT doses within a course, resulting in the administration of 75 total AT doses. The median gestational age was 38 weeks (range 28-41 weeks), with a mean birth weight of 3.1 kg (range 0.8-4.5 kg). Median postnatal age at the time of the first AT dose was 19 days (range 3-341 days). The majority of heparin courses were indicated for thrombosis (49%) and post-operative cardiac surgery (40%). Median baseline AT activity was 37% (range 18-61%). The primary outcome was achieved in 90% AT courses. Of these, 73% were therapeutic only by PTT levels, 9% only by anti-FXa activity, and 18% by both. The benefit of AT administration was not sustained, as only 39% PTT levels and anti-FXa activity measured 48 hours after the AT course remained therapeutic. 41 infants had an AT level measured within 6 hours after the dose, and 61% (25/41) achieved a level between 80 and 120% of the calculated target. AT levels decreased over 48 hours, with an estimated half-life of 49 hours. 21% of AT courses had an increase in bleeding severity, with the majority being minor bleeds. Clinically relevant bleeding occurred after AT administration in only 8% (4/50) subjects. The relationship between simultaneously measured PTT levels and anti-FXa activity was investigated in a secondary analysis. Of the 250 paired levels, only one pair was therapeutic for both values ( Figure 1). Other than the single pair, in all other samples with a therapeutic anti-FXa, the simultaneously measured PTT was supratherapeutic. Three of the four patients with clinically relevant bleeding had at least one paired PTT level and anti-FXa activity available on the day of bleeding. Out of the seven paired samples, 86% of moderate bleeding occurred when the PTT level was supratherapeutic and the anti-FXa activity was subtherapeutic. Discussion: AT supplementation in infants may briefly increase the therapeutic efficacy of heparin, dependent on the monitoring parameter used, as PTT levels and anti-FXa activity poorly correlate. Additionally, permitting the PTT to increase over a certain threshold may increase the risk of bleeding. Further studies are needed to provide recommendations on heparin infusion titrations, AT dosing, and the monitoring of PTT levels and/or anti-FXa activity in the neonatal/infant populations on a continuous heparin infusion.

HDAC Inhibitor CS014 Attenuates Thrombosis Alone and in Combination with Rivaroxaban without Increased Risk of Bleeding

Introduction: Targeting the human platelet for prevention of occlusive thrombotic events remains an active area of research due to the high incidence of myocardial infarction and stroke due to platelet-rich thrombotic clots. While, many patients taking FXa anticoagulant therapy require further inhibition of thrombosis, dual therapy including anticoagulant plus antiplatelet drugs result in an increased risk for bleeding. The current study sought to determine if a histone deacetylase (HDAC) inhibitor, either alone or in combination with the FXa inhibitor rivaroxaban, is able to effectively inhibit platelet and fibrin formation at the site of vascular injury without causing a significant increase in the bleeding risk in mice. Methods and Results: Thrombosis and bleeding models in the mouse were assessed in the presence of the HDAC inhibitors valproic acid (VPA) or the newly developed HDAC inhibitor, CS014. Both CS014 and VPA significantly, dose-dependently inhibit platelet accumulation and fibrin formation at the site of a vascular injury using the laser-induced cremaster thrombosis assay model. Further, to determine if CS014 has the potential to be used in conditions where an anticoagulant is already on board, mice were dosed for 5 days with rivaroxaban alone or in combination with CS014 and were assessed for prevention of laser-induced thrombosis in the cremaster arteriole. Both rivaroxaban and CS014 alone cause significant attenuation of clot formation. To determine if the combination results in an increased risk for bleeding, blood from mice treated with CS014 or rivaroxaban alone, or in combination was assessed for coagulation activity and clot strength using thromboelestography (TEG). Rivaroxaban was found to significantly delay onset of clotting in TEG whereas CS014 did not show any delay in coagulation or clotting with TEG. In combination CS014 was not observed to cause any additional delay in clotting time or decrease in maximal clot strength compared to rivaroxaban alone. Finally, a tail vein bleeding assay was conducted to determine if CS014 alone or in combination with rivaroxaban result in an increased risk for bleeding. Neither CS014 alone, rivaroxaban alone, or the combination of CS014 plus rivaroxaban resulted in increased bleeding time in the resected tail experiment. Conclusions: The data presented here suggests that while CS014 and rivaroxaban are each effective antithrombotic agents, they work well together to minimize clot formation without altering coagulation or clotting compared to rivaroxaban alone. Hence, CS014 has the potential to enrich the toolbox of anticoagulant therapies in patients with a high risk of thrombotic events.