Thromb Haemost Research Articles

The Effect of Concizumab Prophylaxis on Target Joints, Resolution and Joint Bleeds in Patients With Hemophilia A or B With or Without Inhibitors in Phase 3 Clinical Trials

Background: Concizumab is a recombinant anti-tissue factor pathway inhibitor (TFPI) monoclonal antibody under development as a once-daily subcutaneous prophylactic treatment for hemophilia patients with and without inhibitors, and has the potential to be one of the first subcutaneous treatment options for hemophilia B. Recurring joint bleeds may cause hemophilic arthropathy and reduce the quality of life of patients with hemophilia (van Vulpen LFD et al. Haemophilia. 2018; 24 Suppl 6:44-49). Results on target joints, their resolution, and joint bleeds in patients with hemophilia (hemophilia A or B with inhibitors [HAwI or HBwI] or without inhibitors [HA or HB]) from the prospective, multicenter, open-label explorer7 (NCT04083781) and explorer8 (NCT04082429) phase 3 concizumab clinical trials at the 56-week cut-off are presented. Aims: To assess and communicate the effect of longer term (56 weeks) concizumab prophylaxis (PPX) on target joints, their resolution, and joint bleeds in patients with hemophilia A or B with or without inhibitors. Methods: After informed consent/ethics committee approval were obtained, patients in both explorer7 and explorer8 trials were exposed to no PPX (arm 1) or concizumab PPX (arms 2-4) based on their treatment regimen before the trial. After the main part of the trial, all patients (arms 1-4) could continue in the extension part of the trial receiving concizumab for up to 136 weeks. Upon treatment restart following a treatment pause due to thromboembolic events, patients received a 1.0 mg/kg concizumab loading dose on Day 1, followed by an initial 0.20 mg/kg daily dose starting on Day 2, with potential adjustment to 0.15 or 0.25 mg/kg based on measured plasma concizumab concentration after week 4. The data presented here are from the 56-week cut-off that was defined as when all patients had completed the visit after 56 weeks or permanently discontinued treatment. Annualized bleeding rate (ABR) of joint bleeds was evaluated for all patients. Target joints at baseline by age group and target joint resolutions during the trial were assessed separately for this abstract. Target joints were defined as 3 or more spontaneous bleeds into a single joint within a consecutive 6-month period, and target joints were deemed resolved when there have been ≤2 bleeding episodes in the joint during the previous 12 months of exposure (Blanchette VS et al. J Thromb Haemost. 2014;12(11):1935-1939). Results: A total of 144 patients with HA or HB and a total of 127 patients with HAwI or HBwI were exposed to concizumab PPX at the 56-week cut-off. Upon treatment restart, 144 patients with HA or HB and 112 patients with HAwI or HBwI were included in the studies and were allocated to the new concizumab PPX dosing regimen, of which the majority were adults (HA + HB: 105; HAwI + HBwI: 72), some were adolescents (12-17 years) (HA + HB: 36; HAwI + HBwI: 40), and a few were elderly/very elderly (65-84 years) (HA + HB: 3; HAwI + HBwI: 0). Patients who had at least one target joint at baseline (HA + HB: adults [45/108], adolescents [18/36]; HAwI + HBwI: adults [35/72], adolescents [20/40]) mostly reported target joints in the ankle, elbow, and knee, and a few in the hip and shoulder, as presented per age group in Table 1. Of these patients who were exposed to concizumab PPX for at least 12 months, 49 patients with HA or HB reported a total of 80 target joints at baseline, of which 69 (86.3%) were resolved at the 56-week cut-off, and 49 patients with HAwI or HBwI reported 85 target joints at baseline, of which 78 (91.8%) were resolved at the 56-week cut-off. Most target joints were resolved within 12 months after treatment start (median [25th; 75th percentile])( 12.0 months [12.0; 12.0]), both for patients with HA or HB and HAwI or HBwI. The median ABRs for treated spontaneous and traumatic target joint bleeding episodes were 0.0 for patients with HA or HB and HAwI or HBwI, and the median ABRs for treated spontaneous and traumatic joint bleeding episodes were 1.3 for patients with HA or HB and 0.0 for patients with HAwI or HBwI at the 56-week cut-off, as presented in Table 2. Conclusions: Once-daily, subcutaneous concizumab prophylaxis resolved 86.3% of target joints in patients with HA or HB, and 91.8% of target joints in patients with HAwI or HBwI, most patients within 12.0 months. The median ABRs for treated spontaneous and traumatic target joint bleeding episodes at 56 weeks for both patients with HA or HB and HAwI or HBwI were 0.0.

Characterization and Quantification of Von Willebrand Factor Proteoforms in Von Willebrand Disease Patients and Healthy Controls: Insights from the Win-Study

Background: Von Willebrand disease (VWD) is a commonly inherited bleeding disorder which is characterized by either low (type 1) or absent (type 3) levels of Von Willebrand factor (VWF) or by the occurrence of dysfunctional VWF proteoforms (type 2) in plasma. The complexity of diagnosing and managing VWD arises from limited understanding of the diverse VWF proteoforms present in both healthy individuals as well as in VWD patients. Objective: The aim of this study was to investigate the plasma proteome and to characterize VWF proteoforms in Von Willebrand disease (VWD) patients, that were enrolled in the Willebrand in the Netherlands (WiN) 1 study, compared with healthy controls. Method: We selected type 1, type 2A, type 2M, type 2N and type 3 patients from the WiN cohort for which extensive laboratory- and genotype data were available. Plasma from 64 VWD patients and ten healthy controls was analyzed using a combination of discovery- and targeted mass spectrometry-based approaches. Results: Both discovery-based as well as targeted quantitative proteomics analysis revealed significant variation in VWF levels across the selected VWD patient group, which was in line with reported clinical laboratory data. Elevated VWF-propeptide/VWF ratios were observed in plasma samples from a range of VWD patients, with notable increases observed in VWD type 2A, B, and M, indicating accelerated clearance of these VWF proteoforms. Discovery-based proteomics revealed a strong VWF-F8 relationship in all VWD subtypes and healthy controls, except for type 2N VWD. We were able to identify peptides corresponding with common polymorphisms ( e.g. p.Thr798Ala, p.Gln852Arg, p.Thr1381Ala), for which the observed peptide distribution was in line with the minor allele frequency reported by the Broad institute. In addition, we identified peptides corresponding with 16 pathogenic VWF variants (e.g. p.Arg1306Trp, p. Phe1293Leu, p.Cys1190Arg, p.Arg1374His or p.Tyr1584Cys) of which p.Cys1190Arg wild-type and variant peptide aligned with the zygosity at DNA level (Figure X). Based on spike-in with stable isotope-labeled peptides, the variant-to-wildtype ratios varied among VWD patients with distinct pathogenic variants, ranging from 0.5-2.5 in type 2M (p.Phe1293Leu) and type 1 patients (p.Tyr1584Cys), <1 in type 2A patients (p.Arg1374His), to >8 in type 2B patients (p.Arg1306Trp), suggesting distinct stoichiometry of VWF variants among these patients (Figure 2). Conclusion: Our study highlights the feasibility of VWF proteoform identification and quantification from plasma and suggest a potential role in unravelling their contribution to the biological variations and clinical diversity observed in VWD. 1 De Wee et al., Determinants of bleeding phenotype in adult patients with moderate or severe von Willebrand disease. Thromb Haemost. 2012 Oct;108(4):683-92.

Contribution of the Residues 488-490 on the Factor (F)VIII A2 Domain and the Residue 2239 on the FVIII C2 Domain for the Interaction between FVIII and Protein S

Factor (F)VIII functions as a cofactor in the Xase complex responsible for phospholipid (PL) surface-dependent conversion of FX to activated FX (FXa) by FIXa. Protein S (PS), a vitamin K-dependent anticoagulant protein, functions as a cofactor of activated protein C (APC) that inactivates FVIIIa and FVa. We have previously reported a new regulatory mechanism on coagulation that PS directly impaired the Xase complex by competing the FVIIIa-FIXa interaction (Takeyama, Br J Haematol. 2008), and identified at least two PS-interactive sites that were the 488-490 residues on FVIII A2 domain (Takeyama, Thromb Haemost. 2009) and the 2239 residue on FVIII C2 domain (Furukawa, ASH 2016). In the present study, to investigate the contribution of binding sites on FVIII A2 and C2 domains for the interaction between FVIII and PS, three recombinant B-domainless FVIII mutants substituted to alanine were expressed by BHK cell, such as the mutant of the 488-490 residues (S488A/R489A/R490A), the mutant of the 2239 residue (K2239A), and the mutant of both residues (S488A/R489A/R490A/K2239A), subsequently compared interaction with PS of the three mutants with wild-type (wt-) FVIII. First, we performed surface plasmon resonance-based assays to compare the direct binding of each FVIII mutant to PS. When PS was immobilized on the sensor chip and bound to the wt-FVIII and each FVIII mutant as an analyte, the K d value of wt-FVIII was 3.5 nM, while those of S488A/R489A/R490A, K2239A, and S488A/R489A/R490A/K2239A mutants were 43.7 nM, 6.7 nM, and 44.7 nM, respectively. These results demonstrated that the residues 488-490 and 2239 contributed to PS-interactive sites as described in the previous reports. Next, the effect of PS on the directly reduction of FVIIIa function was evaluated using the FXa generation assay with the three FVIII mutants and compared with wt-FVIII. After each FVIII mutant (1 nM) was reacted with PS (0-500 nM) in the presence of PL vesicles (20 µM) for 30 min, FVIII was activated by thrombin (30 nM), followed by the reaction with FIXa (40 nM) and FX (300 nM) for 1 min. Generated FXa in wt-FVIII was reduced in the presence of PS dose-dependently and was reduced to 7.7% at 125 nM of PS, compared to that in the absence of PS. However, generated FXa in K2239A, S488A/R489A/R490A, and S488A/R489A/R490A/K2239A were mildly reduced to 23.1 %, 23.6 %, and 29.2 % even at 500 nM of PS, respectively, compared to those in the absence of PS. Finally, to investigate the contribution of these FVIII and PS binding sites when PS plays as a cofactor of APC in FVIIIa inactivation, FXa generation assays were measured in each FVIII mutant in the presence of PS (150 nM) and APC (0-10 nM). In the presence of PS, generated FXa in wt-FVIII and each FVIII mutant were decreased in APC dose-dependently. At the 10 nM of APC, generated FXa in the wt-FVIII and K2239A were reduced to 48.1 % and 40.6 %, respectively, in comparison to the absence of APC, while generated FXa in S488A/R489A/R490A and S488A/R489A/R490A/K2239A mutants were mildly reduced to 65.8 % and 69.0 %, respectively. These findings suggested that the contribution of the 488-490 residues on the FVIII A2 domain was particularly great for the FVIII-PS interaction between the two PS binding sites identified on the FVIII A2 and C2 domains. In addition, the coexistence of the K2239A mutation on the FVIII C2 domain with S488A/R489A/R490A mutation further reduced the direct FVIII-PS interaction, suggesting that K2239A mutation further reduced the inhibitory effect of FVIII function by PS. On the other hand, mainly not the 2239 residue on the FVIII C2 domain but the 488-490 residues on the FVIII A2 domain were thought to affect the PS function as a cofactor in FVIIIa inactivation by APC. Overall, our data indicated that the 488-490 residues on the FVIII A2 domain were key residues for the FVIII-PS interaction including FVIIIa inactivation by APC, and the 2239 residue affected partially for only the FVIII-PS direct interaction.

Terbutaline Modulates Macrophage Homeostasis and Macrophage-T-Cell Interactions in Immune Thrombocytopenia

Background: Increased platelet destruction by macrophage phagocytosis contributes to immune thrombocytopenia (ITP). Neural immune modulation in autoimmune diseases has been given considerable interest. Our preliminary studies showed a decreased sympathetic innervation in the bone marrow and spleen of ITP mice and that abnormal T cell immunity caused by sympathetic modulation promoted the progression of ITP ( Blood Advances 2019; J Thromb Haemost, 2023). Moreover, previous studies indicated a potential interaction between T cells and macrophages ( Immunity, 2017). However, the regulation of spleen sympathetic denervation on macrophage function and the interaction between splenic macrophages and T cells under the sympathetic modulation in ITP remain unknown. Methods: Whole-tissue immunolabeling and 3D imaging of the human spleen were performed to compare the sympathetic distribution between ITP with splenectomy and non-ITP with traumatic splenectomy. RNA sequencing was used to detect the transcriptional changes and metabolomics and seahorse extracellular flux analyses were carried out to determine the metabolic reprogramming in splenic macrophages of ITP mice. The expression of cell surface markers of macrophages and T cells and cytokines was assessed by flow cytometry, western blot, and enzyme-linked immunosorbent assay. Chemical sympathectomy with 6-hydroxydopamine (6-OHDA), a β2-AR agonist terbutaline, and a β2-AR antagonist ICI 118,551 were used to detect the sympathetic modulation. Genetic lineage tracing was used to compare serum norepinephrine (NE) and its origin in the spleen of WT mice with or without chemical sympathectomy. Results: A panicle-shaped sympathetic neural architecture was revealed in the spleen of both ITP and non-ITP patients. The distribution of sympathetic nerves in the spleen and the level of serum NE were both significantly decreased in ITP patients. Immunofluorescence staining revealed an anatomical colocalization between sympathetic nerves, macrophages, and T cells in the spleen, suggesting potential sympathetic modulation of macrophages and a potential interaction between splenic macrophages and T cells. RNA-sequencing data of splenic macrophages in ITP mice compared with wild type (WT) mice revealed higher expression of M1-specific genes, including IL1b, Tnf and IL6, and lower expression of M2-specific genes, including IL10 and Arg1, indicating an M1 polarization tendency. In splenic macrophages isolated from ITP mice, we observed an enrichment of glycolysis pathway genes compared to WT mice, including Hk2, Pkm2and Ldha. An increase in the extracellular acidification rate and a decrease in the oxygen consumption rate were also found in splenic macrophages of ITP mice, indicating a switch to glycolysis. To investigate the interaction between splenic macrophages and T cells under sympathetic modulation in ITP, we measured the expression of cytokines that might have an influence on macrophage polarization and T cell differentiation and observed upregulation of interleukin (IL)-12, IL-18 and interferon (IFN)-γ. We also used macrophages from ITP mice and WT mice as antigen-presenting cells for naïve CD4 + T cells, which were interrogated for IFN-γ production following stimulation with OVA 323-339. We observed increased IFN-γ production and upregulation of IL-12 and IL-18 in response to macrophages from ITP mice, and we also observed diminished production of IL-12, IL-18 and IFN-γ when pretreated with aIL-12 mAb and aIL-18 mAb. Moreover, chemical sympathectomy with 6-OHDA was carried out in WT mice and significantly decreased serum NE and platelet counts at D7 and D14 were observed. Increased expression of M1-specific markers and upregulation of IL-12, IL-18 and IFN-γ were also determined. The β2-AR agonist terbutaline was shown to decrease the expression of proinflammatory factors and inhibit M1 polarization, and its effects were reversed by the β2-AR antagonist ICI 118,551. Conclusions: Splenic sympathetic denervation-mediated proinflammatory M1 polarization, metabolic alterations, and proinflammatory interactions between macrophages and T cells were involved in ITP pathogenesis. The β2-AR agonist terbutaline was demonstrated to restore the homeostasis of splenic macrophages, indicating a novel potential ITP treatment strategy.

Proposal of New Diagnostic Criteria for Primary Immune Thrombocytopenia

Primary immune thrombocytopenia (ITP) is an autoimmune disorder characterized by isolated thrombocytopenia due to accelerated platelet destruction and impaired platelet production. Diagnosis of ITP is still challenging because ITP has been diagnosed by exclusion. Exclusion of thrombocytopenia due to bone marrow failure is especially important in Japan because of high prevalence of aplastic anemia compared to Western countries. Hence, we propose new diagnostic criteria involving the measurement of plasma thrombopoietin (TPO) levels and percentage of immature platelet fraction (RP% or IPF%) as shown in Fig. 1. These biomarkers reflect the pathology of ITP, in which shortened platelet lifespan associated with increased platelet destruction and relatively maintained platelet production. Diagnostic criteria is as follows: 1. ALL of the following *1 ・Thrombocytopenia (＜100,000/μL) ・Absence of anemia (excluding anemia due to bleeding and/or iron deficiency) ・Normal leukocyte count (but may present with mild abnormal leukocyte count) ・No morphological evidence of dysplasia in any blood cell type in a blood smear 2. Normal or slightly increased plasma TPO level (<cutoff) 3. Elevated percentage of immature platelet fraction (RP% or IPF%; > upper limit of normal) 4. Absence of other conditions that potentially cause thrombocytopenia including secondary ITP ⋆2 ● A diagnosis of ITP is made if conditions 1 - 4 above are all met (ITP) ● Cases in which criterion 2 or 3 is not met or unavailable are defined as “possible ITP, pITP”. A diagnosis of ITP can be made if the patient is a glycoprotein (GP)-specific platelet-antibody positive, increased GP-specific platelet antibody-producing B cells, or typical clinical course. (Footnotes) *1 Bone marrow examination is strongly recommended to rule out other diseases if peripheral blood demonstrates any of the following findings: leukocyte count <3,000 or ≥10,000/μL, MCV ≥110, neutrophils <30% or lymphocytes ≥50%, presence of immature leukocytes *2 Includes drug-related thrombocytopenia, radiation injury, aplastic anemia, myelodysplastic syndrome (MDS), paroxysmal nocturnal hemoglobinuria (PNH), systemic lupus erythematosus (SLE), leukemia, malignant lymphoma, bone marrow metastasis of cancer cells, disseminated intravascular coagulation (DIC) , thrombotic thrombocytopenic purpura (TTP), hypersplenism, megaloblastic anemia, sepsis, infections (tuberculosis, etc.), sarcoidosis, and huge hemangioma such as Kasabach-Merritt syndrome. Congenital diseases involving thrombocytopenia include Bernard-Soulier syndrome, Wiskott-Aldrich syndrome, MYH9-related disease and Upshaw-Schulman syndrome. Sensitivity and specificity analysis of this criteria was performed by using data of previously performed prospective study (Kuwana et al. J Thromb Haemost 2006). In this study plasma TPO level were measured by R&D Systems ELISA Kit and RP% were measured by flow cytometry. 112 thrombocytopenic patients who were initially suspected ITP (final diagnosis: ITP 88, aplastic anemia 11, MDS 10, others 3) were included in the study. The sensitivity and specificity of “ITP” who met all 4 conditions of the new criteria were 53.4% (47/88) and 95.8% (23/24), respectively. If cases of diagnosed as “ITP” or “pITP” by the new criteria were considered as ITP, sensitivity and specificity were 94.3% (83/88) and 75.0% (18/24), respectively. More importantly, these criteria exclude aplastic anemia completely from true ITP. For clinical diagnostic use, new CLEIA-based TPO measurement kit (MBL, Japan) has recently been developed in Japan. However, it is still crucial to carefully differentiate ITP from other thrombocytopenic diseases before applying these biomarkers. These new criteria enable us to clearly differentiate ITP from aplastic anemia and other forms of hypoplastic thrombocytopenia, and can be highly useful in clinical practice for avoiding unnecessary bone marrow examination as well as for appropriate selection of treatments.

Factor VIII Use in the Treatment of Breakthrough Bleeds in People with Moderate or Mild Hemophilia A without Factor VIII Inhibitors Receiving Emicizumab Prophylaxis: The Phase III HAVEN 6 Experience

Background: Emicizumab, a bispecific monoclonal antibody that bridges activated factor (F) IX and FX, substitutes for the function of deficient FVIII in people with hemophilia A (HA). Data from the Phase III HAVEN 6 trial (NCT04158648) show a favorable safety profile and efficacy of emicizumab in people with moderate or mild hemophilia A without FVIII inhibitors (Négrier et al. Lancet Haematol 2023). This analysis characterizes the dose and frequency of FVIII and tranexamic acid used for treatment of breakthrough bleeds in people with moderate or mild HA treated with emicizumab during HAVEN 6. Methods: HAVEN 6 is a multicenter, open-label, single-arm, Phase III study designed to assess safety and efficacy of emicizumab in people with moderate or mild HA. The full study design has been published (Négrier et al.). Eligible participants included people of all ages with moderate or mild HA without inhibitors, who warranted prophylaxis as assessed by the investigator. Participants received a standard loading dose of emicizumab 3 mg/kg once weekly for 4 weeks, then a maintenance dose of 1.5 mg/kg once weekly, 3 mg/kg every 2 weeks or 6 mg/kg every 4 weeks. The lowest FVIII dose required to achieve hemostasis was recommended for breakthrough bleeds. Self-reported bleeds were defined as per the International Society on Thrombosis and Haemostasis (Blanchette et al. J Thromb Haemost 2014); treated bleeds excluded surgical bleeds. Tranexamic acid use in the 24 hours before a surgical procedure was excluded from this analysis. Inferential statistical analyses were not conducted. Results: At the data cut-off (October 30, 2021), 72 participants were enrolled; 51 (70.8%) had moderate HA and 21 (29.2%) had mild HA. Median (range) follow-up was 55.6 (8.7, 89.9) weeks.At baseline, 18 (35.2%) participants with moderate HA and 6 (28.5%) with mild HA had ≥1 target joint ( Table 1). The mean (SD) number of bleeds in the 24 weeks prior to the study was 2.7 (3.1) for those with moderate HA and 9.3 (23.7) for those with mild HA. During the HAVEN 6 study, 48 (66.7%) participants did not experience a treated bleed. Sixteen (31.4%) of the 51 participants with moderate HA and 8 (38.1%) of the 21 participants with mild HA received on-demand FVIII to treat a breakthrough bleed. All participants who experienced a bleed remained on emicizumab, regardless of additional FVIII usage. Of the 71 treated bleeds, 57 (80.3%) were in those with moderate HA and 14 (19.7%) were in those with mild HA ( Table 2). A total of 56 (78.9%) of the 71 treated bleeds were traumatic and 15 (21.1%) were spontaneous. Four participants experienced ≥6 treated bleeds during the study; 28 (90.3%) of the 31 bleeds experienced by these participants were traumatic. Overall, the median (range) number of FVIII infusions per treated bleed was 2 (1, 47) for participants with moderate HA and 1 (1, 6) for those with mild HA. Of the 71 treated bleeds, 54 (76.1%) required 1-2 FVIII infusions. Two participants, both aged 13 years and with moderate HA, had >9 infusions for a bleed. One participant had 19 infusions for a traumatic joint bleed on the finger/thumb. The other participant experienced two traumatic bleeds in the thigh/knee, treated with a total of 47 infusions. The same participant experienced a spontaneous thoracic bleed treated with 19 infusions, a traumatic joint bleed on the left ankle treated with 18 infusions, and a traumatic joint bleed in the finger/thumb treated with 13 infusions. The median (range) cumulative FVIII dose per bleed was 67.1 (31.5, 170.0) IU/kg for those with moderate HA and 47.0 (40.1, 50.5) IU/kg for those with mild HA. The mean (SD) annualized number of infusions per participant was 4.1 (17.2) for those with moderate HA and 2.0 (4.7) for those with mild HA. Seven (13.7%) people with moderate HA and 1 (4.8%) person with mild HA received tranexamic acid, ranging in duration from 1 to 7 days. Tranexamic acid was given for indications including fall with soft tissue bleed, nose bleeds, prophylaxis for dental procedure, and other. No adverse events, including thrombotic events, occurred with concomitant use of emicizumab and tranexamic acid. No desmopressin usage was recorded. Conclusions: Reported bleeds were mainly traumatic and resolved after 1-2 doses of FVIII . No safety concerns were observed with concomitant use of emicizumab and FVIII and/or tranexamic acid. These data may inform breakthrough bleed treatment in people with moderate or mild HA receiving emicizumab.

Heterobivalent Peptide Agonists Comprising Covalently Linked Protease Activated Receptor 1 and Protease Activated Receptor 3 Peptides Mimic the Cytoprotective Cell Signaling Activities of Activated Protein C

Introduction: Activated protein C (APC) exhibits both anticoagulant and cytoprotective cell signaling actions, including anti-inflammatory and anti-apoptotic activities and endothelial barrier stabilization. Extensive in vitro and in vivo data for APC's cell signaling benefits identified requirements for two G-protein coupled receptors (GPCR), protease-activated receptor (PAR) PAR1 and PAR3 which are cleaved by APC at the noncanonical sites (R46 and R41, respectively). Agonism of PARs following proteolysis of the N-terminal tail is driven by the newly revealed N-terminus and related synthetic peptides, e.g., for APC, PAR1 peptides (P1) beginning with N47 or PAR3 peptides (P3) beginning with G42. We found that the non-canonical P1-47-66 and P3-42-65 peptides mimic APC in that each is anti-inflammatory by reducing NLRP3-driven caspase-1 activity in human THP-1 cells (Healy LD et al, J Thromb Haemost 2021;19:269-280). Remarkably, these two peptides act synergistically to attenuate activity of caspase-1 at low concentrations where either peptide alone has little activity. Thus, we undertook studies of P1 peptides that were covalently linked to P3 peptides for ability to mimic APC's anti-inflammatory and endothelial barrier stabilization actions. Methods: Monocytes (THP-1 cells) were differentiated into macrophages using PMA, then pretreated with APC or peptides prior to stimulation with lipopolysaccharide and adenosine triphosphate to induce caspase-1 whose activity was measured using a luminescent caspase-1 substrate. Endothelial barrier stability was monitored for transendothelial electrical resistance (TEER) using an electric cell-substrate sensing system (iCelligence apparatus) when cultured human EAhy926 or murine aortic endothelial cells (EC) were exposed to thrombin treatment (0.25 nM) plus or minus treatment with P1 and/or P3 peptides or G10-related peptides. Synthetic P1 and P3 sequence-derived peptides of various compositions were made, and bivalent P1:P3 peptides were made using oligo-Gly-peptide linkers containing from 4 to 14 Gly residues. Results: Using THP-1 cell anti-inflammatory assays for various peptides, studies showed that P1-47-55 was as potent as P1-47-66 and that the N-terminal N47 was essential. In anti-inflammatory activity assays, some P3 peptides shorter than P3-42-65 (e.g., P3-44-54, P3-51-65, and P3-51-59) were as active as P3-42-65. Remarkably, this is the first example for any PAR-agonist peptide that does not require a native-like sequence with an intact free alpha-amino N-terminus to agonize the PAR response. For bivalent P1:P3 studies, we covalently linked P3-47-55 to P3-51-65 using oligo-Gly linkers with 4-14 Gly residues. In anti-inflammatory activity assays, the maximal activity was seen for a 10-Gly linker. The bivalent peptide comprising P1-47-55-[Gly(10 residues)]-P3-51-65, designated as the G10 peptide, was much more potent than any P1 peptide or P3 peptide in anti-inflammatory THP-1 cell assays in dose-response studies. In TEER studies of thrombin-challenged EAhy926 EC where APC markedly attenuates thrombin-induced endothelial barrier dysfunction, P1-47-55 and the G10 peptide mimicked APC's protective actions. In dose-response studies, the G10 peptide was much more potent than the P1-47-55 peptide or any combination of P1-47-55 with a P3 peptide. The P3 peptides alone were very weak. In TEER studies of thrombin-challenged murine aortic EC where murine APC markedly attenuates thrombin-induced endothelial barrier dysfunction, the G10 peptide comprising murine PAR sequences mimicked murine APC's protective actions. Conclusions: Covalent linkage of a PAR1-derived peptide agonist to a PAR3-derived peptide agonist using a 10-Gly-residue linker provided the first heterobivalent PAR peptide GPCR agonist. This heterobivalent G10 peptide potently mimicked APC's cytoprotective activities of anti-inflammation and endothelial barrier stabilization. Molecular mechanisms for G10 likely involve both orthosteric and allosteric effects for PAR1 and PAR3 which can form heterodimers. Given APC's beneficial cell signaling effects in many preclinical injury models and given the increasing usage of peptide-based drugs, studies of the G10 peptide for beneficial APC-mimicking effects may help to identify important directions for future translational research.

Povetacicept (ALPN-303), a Potent Dual BAFF/APRIL Antagonist in Development for the Treatment of Autoimmune Cytopenias, Suppresses Disease in a Mouse Model of Autoimmune Hemolytic Anemia

Introduction: B cell activating factor (BAFF) and a proliferation inducing ligand (APRIL) play key roles in B cell development, survival, and differentiation into antibody-secreting cells (ASC). Given their importance in ASC survival and antibody production, inhibition of BAFF and/or APRIL (particularly in combination) is a promising approach for the treatment of a variety of autoimmune or autoantibody-related diseases including autoimmune cytopenias like warm autoimmune hemolytic anemia (wAIHA), immune thrombocytopenia (ITP), and cold agglutinin disease (CAD). Significantly higher levels of both BAFF and APRIL have been observed in the serum of patients with AIHA and ITP compared to healthy subjects, and polymorphisms in BAFF and TACI have been associated with ITP [Abdel-Hamid et al., Am J Med, 2011; Xu et al., Int J Hematol., 2015; Emmerich, et al., Br J Haematol, 2007; Peng, et al., J Thromb Haemost, 2017; Yu, et al., Blood Adv, 2021]. Belimumab, an anti-BAFF antibody, has demonstrated encouraging efficacy in the treatment of ITP associated with systemic lupus erythematosus [De Marchi, et al., Clin Exp Rheumatol, 2017] and in combination with rituximab in patients with ITP [Mahévas, et al., Haematologica, 2021]. Currently, there are no BAFF and/or APRIL inhibitors approved to treat AIHA or ITP. Povetacicept (ALPN-303) is an engineered, potent dual BAFF/APRIL antagonist. We previously evaluated the impact of BAFF/APRIL co-inhibition preclinically in the accelerated HODxOTII transgenic mouse model of AIHA, in which povetacicept treatment significantly suppressed plasma cells in the spleen and bone marrow, reduced pathogenic anti-RBC autoantibodies, and significantly increased hematocrit [Dillon et al., ASH 2022 abstract #3763]. Here, we have evaluated povetacicept in a second preclinical model of AIHA, a rat red blood cell (RRBC)-to-mouse model of erythrocyte autoimmunity. Methods: In the RRBC model, C57BL/6NJ mice were immunized weekly with 2 x 10 8 Wistar rat RBC for 10 weeks, leading to development of anti-mouse RBC autoantibodies. Groups of mice were treated twice weekly via intraperitoneal injection with povetacicept or a matched Fc isotype control. Analyses included anti-RRBC alloantibodies and autoantibodies bound to mouse erythrocytes (measured via direct anti-globulin test [DAT]). While significant anemia does not occur in this model, serum lactate dehydrogenase (LDH) was also measured as an index measure of subclinical hemolysis. Immune cell subset analysis of splenocytes by flow cytometry was also performed. Results: As compared to Fc control, povetacicept treatment significantly reduced levels of anti-RRBC and DAT of IgM and IgG isotypes and significantly limited serum LDH elevations at various timepoints during the study ( Figure 1). Furthermore, at 10 weeks, numbers of T follicular helper (Tfh) cells, total B cells, germinal center (GC) B cells, and plasma cells were significantly lower in spleens of povetacicept-treated mice ( Figure 2). Conclusions: Povetacicept effectively reduced pathogenic autoantibodies and key B cell populations (e.g., ASC) and limited serum LDH elevations in this mouse model of erythrocyte autoimmunity. These data suggest that povetacicept may reduce multiple pathogenic inflammatory processes including autoantibody production and may be an effective treatment for AIHA and other autoimmune cytopenias. Povetacicept is currently being evaluated in an open-label study (RUBY-4; NCT05757570) of adults with autoimmune cytopenias (ITP, AIHA, and CAD).

Phase II Study of Danazol with Plasma Exchange and Corticosteroids for the Treatment of Thrombotic Thrombocytopenic Purpura

Background: Ahn and colleagues reported a decrease in plasma exchange (PE)/plasma infusion requirement with danazol in seven cases of thrombotic thrombocytopenic purpura (TTP) (Ahn Blood 2006;108(11):1057a). We reported an increase in platelet count (PLT) within 24 hours with danazol in two of three cases of human immunodeficiency virus-associated TTP resistant to PE (Torri Transfusion 2010;50:86a). In 2009, we initiated a phase II study of danazol with PE and corticosteroids (CS) for the treatment of TTP. This trial was approved by the institutional review board and was registered at ClinicalTrials.gov (ID# NCT00953771). We report the final results. Aims: To determine if danazol reduces number of PE's, time to remission (TR), and length of stay (LOS) when administered with standard therapy for the initial treatment of TTP. Methods: Experimental Group (EG): Patients with TTP (PLT < 100,000/µL and microangiopathy) receive daily PE, prednisone 1 mg/kg, and danazol 600 mg. Following remission (PLT > 150,000/µL for three consecutive days), PE is tapered to every other day for one week then stopped. Then prednisone is tapered weekly over four weeks then stopped. After one week danazol is tapered by 200 mg weekly then stopped. Historical controls (HC): Patients treated for TTP at St. Luke's-Roosevelt Hospital Center with PE with or without CS from 2000 to 2007 (n=20). Relapse definition: PLT < 100,000/µL with microangiopathy > 30 days after remission. Total number of PE's, TR, LOS, and continuous response rates are compared between the 2 groups. Accrual target: 16 patients to detect a 40% decrease in the number of PE's with 80% power and a level of significance of 0.05.The study was terminated in 2018 due to low accrual. Results: Nine patients were enrolled. Mean number of PE's (days) was 14.8 (EG) and 16.1 (HC), mean TR (days) was 11.4 (EG) and 11 (HC), and mean LOS (days) was 27.3 (EG) and 24.9 (HC), p > 0.05. Four patients in HC had a total of 6 relapses at our institution at a median of 28.25 months, range 2.75 to 97 months after the initial event. One patient in EG relapsed 43 months after study enrollment. One patient in EG died from an unrelated cause. None of the additional seven patients in EG have relapsed during a median follow-up of 49 months, range 12 to 125 months. One episode of pulmonary edema and one episode of hypotension occurred in EG, for which treatment was not discontinued. Discussion: Our study shows that danazol does not reduce PE requirement, TR, or LOS during treatment of TTP, but may prevent relapse. This pattern of response is similar to that which has been observed with rituximab. In a phase 2 study of rituximab in conjunction with PE and CS for the treatment of TTP (Scully Blood 2011), in which results were compared with historical controls who did not receive rituximab, median number of PE's (days) and median LOS (days) were 16.5 and 16.5 in the rituximab group versus 18.0 and 20 in the historical control group, p=ns. Ten percent of patients relapsed at a median of 27 months in the rituximab group whereas 57% of patients relapsed at a median of 18 months in the historical control group. Of interest, the cost of rituximab 375 mg/m 2 weekly for 4 weeks as given would have been approximately $ 25,366.50. The cost of danazol in our protocol would have been approximately $ 2196.00. Caplacizumab is a humanized monoclonal antibody fragment that inhibits von Willebrand factor binding to platelet glycoprotein lb-IX-V. In a phase 3 study (Scully N Engl J Med 2019), caplacizumab in conjunction with PE and CS for the treatment of TTP led to reduced PE's, time to normalization of platelet counts, and LOS, but increased relapses in comparison with placebo. In 2020, the International Society on Thrombosis and Haemostasis strongly recommended adding CS to PE for the treatment of TTP and conditionally recommended adding rituximab and caplacizumab to PE and CS (Zheng J Thromb Haemost 2020). Conclusion: Danazol in conjunction with PE and CS may be a low-cost option to prevent relapses of TTP.

Three-Year Interim Analysis Results of the AOZORA Study to Evaluate the Long-Term Safety and Joint Health in Children with Hemophilia A without Factor VIII Inhibitors Receiving Emicizumab

Background: Recurrent joint bleeding from a young age in people with hemophilia A (PwHA) may lead to hemophilic arthropathy, resulting in limited movement and chronic pain. Emicizumab is a bispecific monoclonal antibody that bridges activated factor (F) IX and FX to substitute for activated FVIII and provides stable plasma concentrations and maintenance of trough levels within the therapeutic range. Limitations in long-term joint health preservation have been reported with FVIII prophylaxis (Warren et al. Blood Adv 2020); it is expected that emicizumab may overcome these limitations, as joint health improvements have been demonstrated in PwHA aged 12-39 years (Kiialainen et al. Haemophilia 2022). Here, we report the 3-year interim analysis of the ongoing, open-label, Phase IV AOZORA study (JapicCTI-194701), analyzing long-term safety and joint health in pediatric PwHA without FVIII inhibitors receiving emicizumab (Shima et al. BMJ Open 2022). Methods: Eligible participants were aged <12 years with severe hemophilia A (HA) without FVIII inhibitors. Participants entered AOZORA as emicizumab-naïve or following the Phase III HOHOEMI study (Shima et al. Haemophilia 2019). Primary endpoints include adverse events (AEs), and joint health and function assessed by magnetic resonance imaging (MRI) and the Hemophilia Joint Health Score (HJHS) version 2.1. MRI evaluation uses the International Prophylaxis Study Group (IPSG) MRI scale; a positive IPSG score indicates pathological changes in the knee and/or ankle joints. Exploratory endpoints include annualized bleed rate (ABR) for treated bleeds and treated joint bleeds. All participants will receive emicizumab for 6 years from their first dose until study end. Results: Data cut-off was the last day of Week 145 for individual participants. Ten participants entered from HOHOEMI and 20 participants were newly enrolled. The median (range) age was 4.2 (0.7-11.1) years, all participants were male, and 27 (93.1%) participants had been treated with prior FVIII prophylaxis ( Table 1). One participant had 1 target joint (knee) at Week 1. In AOZORA, 5 (16.7%) participants experienced 7 emicizumab-related AEs: 6 injection site reactions in 5 participants and 1 case of anemia in 1 participant. Seven serious AEs, reported as unrelated to emicizumab, occurred in 5 (16.7%) participants. No thrombotic events or thrombotic microangiopathies occurred. Twenty-nine participants at Week 1 and 26 participants at Week 145 were assessed using the IPSG MRI scale. At Week 145, MRI scores remained at 0 for 11 (42.3%) of the 26 participants. Of 116 joints assessed at Week 1, 28 (24.1%) had soft tissue changes (effusion/hemarthrosis, synovial hypertrophy and/or hemosiderin deposition; Table 2). At Week 145, 104 joints were assessed and 33 (31.7%) of those had soft tissue changes. Of 10 (8.6%) joints with synovial hypertrophy and hemosiderin at Week 1, all had improved at Week 145 and synovial hypertrophy and hemosiderin were no longer present in 9 joints. One new joint developed synovial hypertrophy and hemosiderin at Week 145. The rest of the soft tissue changes were effusion/hemarthrosis, present in 21 (18.1%) joints at Week 1 and 31 (29.8%) at Week 145. Thirty participants at Week 1 and 27 participants at Week 145 were assessed using the HJHS v2.1. Eighteen (66.7%) participants' HJHS scores remained at 0 from Week 1 to Week 145 and 1 (3.7%) participant's remained at 4; 6 (22.2%) participants' scores improved; 2 (7.4%) participants' scores worsened. Mean (standard deviation) HJHS total score improved from 0.90 (1.79) at Week 1 to 0.44 (1.05) at Week 145. The mean (95% confidence interval [CI]) ABR for treated bleeds was 3.7 (0.94-9.80) prior to emicizumab and 0.7 (0.01-5.10) while receiving emicizumab. The mean (95% CI) ABR for treated joint bleeds was 0.4 (0.00-4.55) prior to emicizumab and 0.2 (0.00-4.04) while receiving emicizumab. Conclusions: This 3-year interim analysis of the AOZORA study confirmed the safety profile of emicizumab. The proportion of joints with effusion/hemarthrosis increased by Week 145. However, all joints with synovial hypertrophy and hemosiderin, which are strongly associated with the progression of arthropathy (Gualtierotti et al. J Thromb Haemost 2021), improved by Week 145. These results suggest that emicizumab is effective in maintaining and improving joint health in pediatric PwHA.

Ddxof

An algorithm helps reverse anticoagulation for intracranial hemorrhageFigure: An Algorithm for Reversing Anticoagulation for Intracranial HemorrhageA 65-year-old man with a history of hypertension, diabetes mellitus, and atrial fibrillation presented with a posterior scalp hematoma and altered mental status after a mechanical fall. The patient's family reported that he was taking apixaban, and his last dose had been four hours before arrival. A physical examination revealed a GCS of 13, a blood pressure of 175/99 mm Hg, and asymmetric pupils. The patient was taken to CT, where head imaging revealed a left-sided subdural hematoma with a midline shift and developing uncal herniation. A nicardipine infusion was initiated and the head of the bed was elevated. Andexanet alfa was not available, so an infusion of 4-factor prothrombin complex concentrate was initiated. The patient was taken emergently to the operating room by neurosurgery for a craniotomy and hematoma evacuation. Discontinue Anticoagulation Patients may require blood pressure control including antihypertensive infusions (goal SBP <140). Avoid reversal for intracranial hemorrhage associated with cerebral venous thrombosis. Use cautiously in patients with concomitant life-threatening ischemia, thrombosis, or severe DIC. Vitamin K Antagonists (Ex. Warfarin) Initial Dose: A fixed dose of 4F-PCC 1500 to 2000 units can be given as an initial dose with repeat dosing based on INR measurement 15 minutes after completion of the infusion. Follow local institution guidelines if available. Monitoring and Repeat Dosing: Give vitamin K if the INR is ≥1.4 at 12 hours (Chest. 2008;133[6 Suppl]:160S.) Consider repeat PCC dosing based on INR, though with increased DIC and thrombotic risk, it is recommended to correct further with FFP if INR remains ≥1.4. (J Thromb Haemost. 2008;6[4]:622; https://bit.ly/3ZDvpdU.) Direct Factor Xa Inhibitors (Ex. Rivaroxaban, Apixaban) Activated charcoal may be effective for up to six hours for apixaban (https://bit.ly/41PvXPJ) and eight hours for rivaroxaban (https://bit.ly/3L2mQFm). Andexanet Alfa Regimens (Stroke. 2021;52[6]:2096; https://bit.ly/3STy01c; J Am Coll Emerg Physicians Open. 2022;3[2]:e12655; https://bit.ly/3IYilZH.) Low-dose: rivaroxaban <10 mg, apixaban <5 mg, edoxaban <30 mg or eight or more hours since last dose High-dose: If greater than the thresholds above or dose/timing unknown Pentasaccharides (Ex. Fondaparinux) Use high-dose Andexanet alfa regimen (Nat Med. 2013;19[4]:446). Direct Thrombin Inhibitors (Ex. Dabigatran) Monitoring and Repeat Dosing: If the patient has ongoing significant bleeding after treatment, consider redosing idarucizumab or hemodialysis. Alternative Regimens: If idarucizumab is not available, aPCC (50-80 units/kg), 4F-PCC, or 3F-PCC (50 units/kg) can be used in order of preference. Unfractionated Heparin Dosing: Heparin units are based on estimated active agent (half-life 1-2 hours). Protamine sulfate 1 mg/100 units IV, maximum dose 50 mg Alternatively, give fixed dose of 25 to 50 mg. Monitoring and Repeat Dosing: If aPTT is persistently elevated, repeat 0.5 mg/100 units. Low-Molecular Weight Heparin (Clin Pharmacokinet. 2003;42[12]:1043.) Reversal is not indicated if more than three to five half-lives have passed since administration: Enoxaparin mean half-life: Four to five hours Dalteparin mean half-life: 2.8 hours Nadroparin mean half-life: 3.7 hours If bleeding persists or the patient has or renal insufficiency, repeat dose 0.5 mg/1 mg enoxaparin or 0.5 mg/100 anti-Xa units. Share this article on Twitter and Facebook. Access the links in EMN by reading this on our website: www.EM-News.com. Comments? Write to us at [email protected].

The American Psychiatric Association Practice Guideline for the Treatment of Patients With Eating Disorders.

The American Psychiatric Association Practice Guideline for the Treatment of Patients With Eating Disorders.

SOD2 in platelets: with age comes responsibility

SOD2 in platelets: with age comes responsibility

Von Willebrand factor: from figurant to main character in the scene of inflammation

von Willebrand factor: from figurant to main character in the scene of inflammation

Thrombosis in hereditary hemorrhagic telangiectasia

Thrombosis in hereditary hemorrhagic telangiectasia

Natural history of ‘silent’ atrial fibrillation from subclinical to asymptomatic: State of the art and need for research

In the last 30 years clinical research in the atrial fibrillation (AF) field has seen a great advance both in terms of productivity and heterogeneity of areas that developed significant new knowledge and evidence. Indeed, it is very easy to verify how in the last 3 decades the number of articles regarding AF registered in MEDLINE has increased exponentially overtime. This large amount of evidence led to significant changes in the guidelines, with an improvement of the overall quality of recommendations and an increase in the high-quality recommendations (despite being still generally low) [ [1] Boriani G. Vitolo M. Lane D.A. Potpara T.S. Lip G.Y. Beyond the 2020 guidelines on atrial fibrillation of the European society of cardiology. Eur J Intern Med. 2021; 86: 1-11https://doi.org/10.1016/j.ejim.2021.01.006 Google Scholar , [2] Barnett A.S. Lewis W.R. Field M.E. Fonarow G.C. Gersh B.J. Page R.L. et al. Quality of evidence underlying the American Heart Association/American College of Cardiology/Heart Rhythm Society Guidelines on the management of atrial fibrillation. JAMA Cardiol. 2017; 2: 319https://doi.org/10.1001/jamacardio.2016.4936 Google Scholar ]. Several areas about AF clinical management are now largely assessed and established, as the need for oral anticoagulant (OAC) prescription, the preference of the non-vitamin K antagonist oral anticoagulants over vitamin k antagonist ones, as well as the assessment of thromboembolic and bleeding risks [ [3] Imberti J.F. Mei D.A. Vitolo M. Bonini N. Proietti M. Potpara T. et al. Comparing atrial fibrillation guidelines: focus on stroke prevention, bleeding risk assessment and oral anticoagulant recommendations. Eur J Intern Med. 2022; 101: 1-7https://doi.org/10.1016/j.ejim.2022.04.023 Google Scholar , [4] Hindricks G. Potpara T. Dagres N. Arbelo E. Bax J.J. Blomström-Lundqvist C. et al. 2020 ESC Guidelines for the diagnosis and management of atrial fibrillation developed in collaboration with the European Association for Cardio-Thoracic Surgery (EACTS). Eur Heart J. 2021; 42: 373-498https://doi.org/10.1093/eurheartj/ehaa612 Google Scholar ]. Also, the need for implementation of integrated care, emerged as the main AF management strategy in the last guidelines, is now largely assessed and evidence based [ 4 Hindricks G. Potpara T. Dagres N. Arbelo E. Bax J.J. Blomström-Lundqvist C. et al. 2020 ESC Guidelines for the diagnosis and management of atrial fibrillation developed in collaboration with the European Association for Cardio-Thoracic Surgery (EACTS). Eur Heart J. 2021; 42: 373-498https://doi.org/10.1093/eurheartj/ehaa612 Google Scholar , 5 Romiti G.F. Pastori D. Rivera-Caravaca J.M. Ding W.Y. Gue Y.X. Menichelli D. et al. Adherence to the “atrial fibrillation better care” pathway in patients with atrial fibrillation: impact on clinical outcomes-a systematic review and meta-analysis of 285,000 patients. Thromb Haemost. 2022; 122: 406-414https://doi.org/10.1055/a-1515-9630 Google Scholar , 6 Romiti G.F. Proietti M. Vitolo M. Bonini N. Fawzy A.M. Ding W.Y. et al. Clinical complexity and impact of the ABC (Atrial fibrillation Better Care) pathway in patients with atrial fibrillation: a report from the ESC-EHRA EURObservational Research Programme in AF General Long-Term Registry. BMC Med. 2022; 20: 326https://doi.org/10.1186/s12916-022-02526-7 Google Scholar ].

Endothelial Cell Activation Enhances Thromboinflammation in Vitt

Aim: Vaccine induced thrombosis thrombocytopenia (VITT) syndrome is a rare complication of ChAdOx1 vaccination for COVID19. VITT shares similarities with heparin induced thrombocytopenia (HIT). In both VITT and HIT, patients develop antibodies against platelet factor 4 (PF4) which promote platelet activation and thrombosis. Involvement of the endothelium in the pathogenic process has been described for HIT. Binding of HIT antibodies to PF4/glycosaminoglycan complexes on the surface of endothelial cells leads to their activation and increased procoagulant activity (Madeeva, J Thromb Haemost, 2016). However, the contribution of the endothelium to VITT has not been described. Here, we evaluate the effect of VITT plasma or serum on endothelial cell activation and thromboinflammation using an endothelialized biochip. Method: Serum and plasma samples were received from patients fulfilling clinical criteria of VITT including venous and/or arterial thrombosis, thrombocytopenia <150 x 10^3/uL, D-dimer >5-fold of upper limit of normal within 4-42 days after 1st dose of ChAdOx1 vaccine (termed "Clinical VITT") and from healthy, age and sex matched, ChAdOx1 vaccinated individuals within the same timeframe from vaccination (termed "Vaccine controls"). PF4 ELISA and VITT functional assays were performed on all samples (Lee, Blood Adv, 2022). Human umbilical vein endothelial cells (HUVECs) were treated with heat inactivated serum or plasma in the presence of peptide GPRP. Polydimethylsiloxane microfluidic straight channels were seeded with HUVECs (Endochip) and treated with VITT or control serum and compared with TNF alpha treatment. The endothelial surface expression of intercellular adhesion molecule 1 (ICAM-1), vascular cell adhesion molecule 1 (VCAM-1), P-selectin, tissue factor (TF) and von Willebrand factor (vWF) were measured by immunostaining in the presence or absence of native platelet factor 4 (PF4). In some experiments HUVECs were treated with ChAdOx1 vaccine or vehicle for 48-120h. Thromboinflammation was measured by fluorescence area of antibody-stained platelets, neutrophils and fibrin on the Endochip after perfusion of recalcified blood from HIT antibody-reactive healthy volunteers (Morel-Kopp, J Thromb Haemost, 2016) at a shear rate of 100s-1. Results: Samples from 10 patients presenting with clinically confirmed VITT were paired with samples from 10 vaccinated individuals. Clinical VITT samples were separated into negative and positive for both PF4 ELISA and VITT functional assay (defined as VITT ELISA positive and VITT ELISA negative, Panel A). Endothelial cell surface expression of P-selectin was increased after 12 h incubation with 25% v/v VITT serum or plasma compared with samples from AZ vaccinated non-VITT individuals (median integrated intensity/cell 8065 vs 3515 a.u, p=0.03). The expression of TF was significantly increased by addition of PF4 to the VITT plasma (median integrated intensity/cell 42038 vs 14881 a.u., p=0.01 by Mann Whitney test). ChAdOx1 treatment of HUVECs at 1:1000 dilution induced the expression of spike protein but did not significantly enhance thromboinflammation compared with media, as assessed by the perfusion of whole blood through the Endochip. Treatment of the Endochip with VITT serum, without the addition of PF4, significantly increased the adhesion of platelets compared with control serum (median 2.2-fold, p=0.002), neutrophils (2.5-fold, p=0.003) and fibrin (5.4-fold, p=0.002 by Wilcoxon paired samples test (Panel B). There was no difference in the thromboinflammatory response between ELISA negative and positive group (Panel B). Conclusion: Endothelial inflammation contributes to the prothrombotic tendency in VITT in an in vitro model of thromboinflammation. VITT serum induces the endothelial expression of P-selectin and TF. Endothelial inflammation may explain thrombotic events in cerebral or splanchnic veins of vaccinated individuals without detectable anti-PF4 antibodies by ELISA or platelet-based VITT functional assays. Endothelialised biochips have diagnostic potential for detecting immune prothrombotic tendencies. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Quantitative Estimation of the In Vivo Equivalent Factor VIII Activity of NXT007, an Emicizumab-Based, Next-Generation, Activated Factor VIII-Mimetic Bispecific Antibody, from Nonclinical Data

INTRODUCTION: NXT007 is a bispecific antibody (BsAb) that bridges activated factor IX (FIXa) and factor X (FX), thereby mimicking the cofactor function of activated factor VIII, and was discovered by engineering the first-in-class drug of emicizumab. Nonclinical investigations suggested the potential of NXT007 to provide a supra-emicizumab and even non-hemophilic level of coagulation activity to patients with hemophilia A [1,2]. Meanwhile, quantitative characterization and comparison of the in vivo hemostatic activity of NXT007 with factor VIII (FVIII) and emicizumab remained to be addressed. This research aimed to quantitatively estimate the in vivo hemostatic activity of NXT007 in terms of relative potency to FVIII ("equivalent FVIII activity") by leveraging and analyzing previously published nonclinical data with model-based approaches. METHODS: Simulation of factor IX (FIX)-BsAb-FX ternary complex formation in human plasma using a binding kinetic model was employed for predicting the in vivo equivalent FVIII activity of NXT007, as previously investigated to associate the ternary complex concentration with in vivo equivalent FVIII activity in a non-human primate model of acquired hemophilia A for emicizumab [3]. Dissociation constants against human FIX and FX, which were determined by a surface plasmon resonance analysis in buffer solution, were used for simulating the equilibrium binding of the BsAbs with these antigens as functions of their plasma concentrations. The conversion factor from ternary complex concentration to in vivo equivalent FVIII activity for NXT007 was assumed to be equivalent to that for emicizumab, given their comparable effects for increasing the turnover rate of FIXa-catalyzed FX activation on top of their similar affinities to FIX/FIXa. To support the validity of this assumption, in vitro equivalent FVIII activities of NXT007 and emicizumab were estimated and compared. Peak height data from in vitro activated factor XI (FXIa)- or tissue factor (TF)-triggered thrombin generation (TG) assays, in which FVIII-deficient human plasma was spiked with vehicle, FVIII (10.0 to 150 IU/dL), NXT007 (0.0300 to 3000 μg/mL), or emicizumab (0.0300 to 3000 μg/mL), were used for developing pharmacodynamic models to describe the concentration-response relationships of the three active drugs. The developed models were then transformed to derive the corresponding in vitro equivalent FVIII activities of NXT007 and emicizumab as functions of their plasma concentrations. RESULTS: The simulated relationships between plasma concentration and equivalent FVIII activity by the ternary complex, FXIa-triggered TG, and TF-triggered TG approaches for NXT007 and emicizumab were all bell-shaped, peaking at 109, 63.7, and 127 IU/dL with 53.1, 130, and 82.8 μg/mL, respectively, for NXT007 and at 28.2, 19.0, and 48.2 IU/dL with 265, 452, and 476 μg/mL, respectively, for emicizumab (Figure). With higher peak equivalent FVIII activities achieved with lower plasma concentrations for NXT007 than emicizumab, the interrelationship among the simulated relationships between plasma concentration and equivalent FVIII activity by the three approaches appeared consistent between NXT007 and emicizumab, as characterized by the same order of peak equivalent FVIII activity, i.e., TF-triggered TG > ternary complex > FXIa-triggered TG. This consistency may suggest comparable coagulation activities per ternary complex between NXT007 and emicizumab, supporting the validity of assuming an equivalent conversion factor from ternary complex concentration to in vivo equivalent FVIII activity for NXT007 and emicizumab. By the ternary complex approach, the minimum plasma concentrations of NXT007 to achieve in vivo equivalent FVIII activities of 15 (anticipated therapeutic level with emicizumab), 40 (threshold for hemophilia A), and 100 (typical normal) IU/dL were predicted to be 2.50, 7.26, and 30.2 μg/mL, respectively (Figure). CONCLUSIONS: Model-based approaches supported application of the ternary complex approach for predicting the in vivo equivalent FVIII activity of NXT007. Findings from this research provided a basis for designing a phase I/II clinical study of NXT007 that is currently ongoing (NXTAGE; JapicCTI-194919). REFERENCES: 1. Res Pract Thromb Haemost 2020;4 Suppl 1:579. 2. Blood 2020;136 Suppl 1:19. 3. Thromb Haemost 2017;117:1348-57. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

A Real-World Population Pharmacokinetic (PK) Study of Two Recombinant Factor VIII (rFVIII) Concentrates in Boys with Severe Hemophilia a without Inhibitors

Introduction To personalize long-term prophylaxis in boys with hemophilia A with a severe bleeding phenotype, the individual's pharmacokinetic (PK) profile must be established. Traditional FVIII PK assessment has required a 2-3-day washout, which can place boys at risk of bleeding, and up to 11 post-infusion blood samples collected over a 48h period (Lee et al., ISTH guidelines, 2001). Population PK models with Bayesian forecasting are currently available that permit the use of a no-washout PK protocol with sparse sampling, which is clinically practical and less demanding for patients and their families. Our previous experience with a no-washout, single-clinic visit, reverse 2-sampling time point (24 and 3h) PK protocol established that this practical, patient-friendly PK protocol was sufficiently accurate for generating clinically useful individual PK profiles in patients receiving a standard half-life (SHL) FVIII concentrate [antihemophilic factor (recombinant) plasma/albumin-free method (Advate®)] for long-term prophylaxis (Blanchette et al., Thromb Haemost, 2021). Aim To determine the accuracy of a clinically practical single-clinic visit, reverse 2-sampling time point population PK protocol for generating individual PK profiles [clearance (Cl) and terminal half-life (t1/2)] in boys with severe hemophilia A without inhibitors receiving either an SHL [antihemophilic factor (recombinant) BAY 81-8973 (Kovaltry®)] or an extended half-life (EHL) FVIII concentrate [antihemophilic factor (recombinant) PEGylated BAY 94-9027 (Jivi®)] for long-term prophylaxis. Methods In this multicenter observational study, participants had their first infusion given at home. Of a total of 14 boys with hemophilia A without inhibitors receiving long-term prophylaxis, 5 receiving a SHL completed a single clinic visit, reverse 2-point (24 and 3h) and a multiple clinic visit 6-point (baseline, 1, 3, 8, 24 and 48h) PK sampling protocol; and 4 boys completed a SHL single clinic visit reverse 2-point (48 and 3h) and a 2 clinic visit reverse 3-point (48, 3 and 24h) PK sampling protocol. 5 boys receiving EHL completed single clinic visit, reverse 2-point (72 and 3h) and 2-clinic visit 3-point (72, 3 and 48h) PK sampling protocols and they had multiple spot samplings (n=12) at various post-infusion times to evaluate the precision of their predicted PK profiles based on their 2-point PK sampling protocol. One-stage (OS) and chromogenic (CHR) FVIII:C and von Willebrand factor antigen (VWF:Ag) assays were performed in a central reference coagulation laboratory in Kingston, Ontario. PK parameters (Cl and t1/2) were estimated using the WAPPS-Hemo population PK program. The median of the % difference between the 2-sampling time point PK protocol compared to the 6- and 3- sampling time point PK protocols is reported. Results 14 boys with hemophilia A (FVIII:C <2%; median age: 14.5 yrs, range: 5-19 yrs) participated in the study. The 2-point PK protocol for both SHL and EHL showed a higher Cl and lower t1/2 when compared to either a 6-sampling time point (for SHL) or a 3-sampling time point (for either SHL or EHL) PK assessment (Table 1). The difference was greatest for the 2-point (24 and 3h) vs the 6-point (Table 1). In the case of the 2-point (48 and 3h for SHL and 72 and 3h for EHL) vs 3-point (an additional 24h time point for SHL and 48h for EHL) PK parameter estimates, the 2-point protocol gave values that were all within 6% of those obtained with the 3-sampling time point PK (Table 1). The OS and CHR assays gave similar estimations for Cl and t1/2 with the exception of t1/2 when comparing the 6-point to the 2-point PK protocol (Table 1). Evaluation of the 2-sampling time point PK protocol through spot sampling showed approximately 66% and 81% of FVIII:C values falling within the 95% prediction limits for the OS and CHR assays, respectively (n=12 for EHL OS; n=11 for EHL CHR). There was a strong association between the observed FVIII:C levels from the spot sampling and their predicted values from the 2-point PK for EHL (R2=94% and 71% for the OS and CHR assays, respectively) (Fig. 1). Conclusion The reverse 2-point, single clinic visit, PK protocol (24/48 or 72h and 3h) is a practical approach to generate PK parameters that provide clinically useful information for guiding personalized prophylaxis regimens for boys receiving SHL or EHL for long-term prophylaxis. Funding Supported by Bayer (Investigator-Initiated Research grant) Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

A Randomized, Placebo-Controlled, Phase 1 Study in Healthy Adult Volunteers of the Safety, Tolerability, Pharmacokinetics, and Pharmacodynamics of ALPN303, a Potent Dual BAFF/APRIL Antagonist for the Treatment of Autoimmune Cytopenias

Background B cell activating factor (BAFF) and a proliferation-inducing ligand (APRIL) are tumor necrosis factor superfamily (TNFSF) members that bind transmembrane activator and calcium-modulating cyclophilin ligand interactor (TACI), B-cell maturation antigen, and/or BAFF receptor (BAFF-R) on B cells and together support B-cell development, survival, and differentiation into antibody-secreting cells (ASC). In preclinical studies, co-neutralization of BAFF and APRIL can directly suppress ASC and reduce circulating immunoglobulins. Autoimmune hemolytic anemia (AIHA) and immune thrombocytopenic purpura (ITP) are diseases characterized by autoantibodies directed against red blood cells and platelets, respectively. Significantly higher levels of both BAFF and APRIL have been observed in the serum of patients with AIHA and ITP compared to healthy subjects, and polymorphisms in BAFF and TACI have been associated with ITP [Abdel-Hamid, et al., Am J Med, 2011; Emmerich, et al., Br J Haematol, 2007; Peng, et al., J Thromb Haemost, 2017; Yu, et al., Blood Adv, 2021]. Currently, there are no B cell targeting agents approved to treat AIHA or ITP. Rituximab, an anti-CD20 therapeutic which depletes CD20+ B cells, and thereby only indirectly reduces autoantibody production, has demonstrated some efficacy in both ITP [Neunert, et al., Blood, 2019] and AIHA [Jäger, et al., Blood Rev, 2019], though it is not approved for use in either indication. Belimumab, an anti-BAFF neutralizing antibody (Ab), has demonstrated encouraging efficacy in the treatment of ITP associated with systemic lupus erythematosus (SLE) [De Marchi, et al., Clin Exp Rheumatol, 2017] and also in combination with rituximab in patients with ITP [Mahévas, et al., Haematologica, 2021], but only had a modest impact on hematological manifestations in SLE patients [Manzi, et al., Ann Rheum Dis, 2012]. To address the shortcomings of existing B-cell-targeting agents, we have developed ALPN-303, an Fc fusion of an engineered TACI variant TNFRSF domain, which substantially improves upon the ligand affinity liabilities of wild type (WT) TACI, resulting in highly potent dual BAFF/APRIL inhibition superior to WT TACI-Fc, or to BAFF- or APRIL-specific monoclonal Abs. In preclinical studies, ALPN-303 demonstrates enhanced pharmacokinetic (PK) and immunomodulatory properties vs. WT TACI-Fc, which may translate to lower and/or less frequent doses in humans. ALPN-303 also suppresses autoantibodies and nephritis in mouse models of lupus. Taken together, these observations suggest that a potent dual BAFF/APRIL antagonist such as ALPN-303, by lowering pathogenic autoantibody levels, may be particularly beneficial in the treatment of antibody-related hematologic diseases, such as autoimmune cytopenias. Methods In this first-in-human study (NCT05034484), healthy adult volunteers have been enrolled in single ascending dose cohorts of intravenously (IV) or subcutaneously (SC) administered ALPN-303. For each IV cohort, the first 2 sentinel subjects are randomized 1:1 to receive ALPN-303 or placebo, followed by the remaining 4 subjects randomized 3:1 to receive ALPN-303 or placebo. SC cohorts are randomized 4:2 to receive a single SC dose of ALPN-303 or placebo. All subjects are followed to assess safety, PK, and pharmacodynamic (PD) effects including impacts on circulating immunoglobulins (Ig), and peripheral B-cell populations. Results ALPN-303 has been well tolerated in all cohorts evaluated to date and exhibits dose-dependent PK and expected PD effects, including dose-dependent reductions in circulating CD38+ (CD19+CD27+IgD-) ASC (Figure 1) and in serum Ig (Figure 2). To date, there have been no serious adverse events, and no events of cytokine release syndrome. Dose escalation will be complete by the time of the meeting; the presentation will include all available safety, PK, and PD data. Conclusions In this phase 1 study, ALPN-303 demonstrates acceptable preliminary safety and tolerability and exhibits dose-dependent PK and expected PD effects on circulating Ig and B-cell populations. These findings support future clinical development of ALPN-303 in patients with antibody-related diseases, including autoimmune cytopenias. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal