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

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

FRONTIER1: a partially randomized phase 2 study assessing the safety, pharmacokinetics, and pharmacodynamics of Mim8, a factor VIIIa mimetic.

BACKGROUND: Mim8 (denecimig) is a factor VIII (FVIII) mimetic bispecific antibody in development for the treatment of hemophilia. Data from the phase 1 part of FRONTIER1 (EudraCT: 2019-000465-20, NCT04204408, and NN7769-4513) suggested that Mim8 was well tolerated in healthy participants and exhibited pharmacokinetic (PK) properties consistent with dose proportionality. OBJECTIVES: The partially randomized, phase 2, multiple ascending dose (MAD) part of FRONTIER1 aimed to evaluate the safety, PK, pharmacodynamics (PD), and exploratory efficacy of Mim8 in participants with hemophilia A with or without FVIII inhibitors. METHODS: The MAD part of FRONTIER1 consisted of 42 participants, assigned to 5 cohorts, with participants in cohorts 3 and 4 randomized 1:1 to dosing weekly or every 4 weeks, respectively. Four of the 42 participants (9.5%) had FVIII inhibitors prior to study enrolment. The primary endpoint was treatment-emergent adverse events (TEAEs). PK and PD were evaluated by Mim8 plasma concentration and thrombin generation, respectively. Exploratory efficacy was assessed via the number of treated bleeds. Safety and PD parameters were also evaluated from an exploratory cohort treated with emicizumab. RESULTS: Mim8 was well tolerated, with 1 serious TEAE (anxiety-related chest pain) deemed unrelated to Mim8. There was no dose dependency on the number, causality, type, or severity of TEAEs. PK/PD properties supported weekly to monthly dosing approaches, and few participants experienced treated bleeds beyond the lowest dose cohort (1 in cohorts 2 and 3, and 3 in cohort 5). CONCLUSION: These data support the continued clinical development of Mim8, and FRONTIER1 has proceeded onto an extension phase.

A multicentric consortium study demonstrates that dimethylarginine dimethylaminohydrolase 2 is not a dimethylarginine dimethylaminohydrolase.

Dimethylarginine dimethylaminohydrolase 1 (DDAH1) protects against cardiovascular disease by metabolising the risk factor asymmetric dimethylarginine (ADMA). However, the question whether the second DDAH isoform, DDAH2, directly metabolises ADMA has remained unanswered. Consequently, it is still unclear if DDAH2 may be a potential target for ADMA-lowering therapies or if drug development efforts should focus on DDAH2's known physiological functions in mitochondrial fission, angiogenesis, vascular remodelling, insulin secretion, and immune responses. Here, an international consortium of research groups set out to address this question using in silico, in vitro, cell culture, and murine models. The findings uniformly demonstrate that DDAH2 is incapable of metabolising ADMA, thus resolving a 20-year controversy and providing a starting point for the investigation of alternative, ADMA-independent functions of DDAH2.

Characterization of bleeding symptoms in Ehlers-Danlos syndrome.

BACKGROUND: Easy bruising is included as a major or minor criterion for the classification of multiple types of Ehlers-Danlos syndrome (EDS). Despite a longstanding recognition of the association between EDS and bleeding, we still lack a definitive understanding of the frequency, severity, and types of bleeding complications in patients with EDS. OBJECTIVES: To evaluate hemorrhagic symptoms using the International Society of Thrombosis and Haemostasis bleeding assessment tool (ISTH-BAT) in a cohort of patients with defined types of EDS. METHODS: We utilized the ISTH-BAT to characterize hemorrhagic symptoms and their severity in a cohort of 52 patients with classical, classical-like, hypermobile, or vascular EDS and a matched group of 52 healthy control subjects. RESULTS: The mean ISTH-BAT score was 0.1 for healthy subjects and 9.1 for patients with EDS (p < .0001). An abnormal ISTH-BAT score was observed in 32 of 52 (62%) patients with EDS and 0 of 52 healthy controls (p < .0001). The most frequent bleeding symptoms were bruising, muscle hematomas, menorrhagia, epistaxis, bleeding from the oral cavity, and bleeding after tooth extraction. Menorrhagia that was life-threatening or required surgery was reported in 7 of 52 (14%) patients with EDS. CONCLUSION: Patients with multiple types of EDS exhibit a wide range of bleeding symptoms ranging from mild to life-threatening episodes.

Association of low serum albumin with venous thrombosis in pediatric patients.

OBJECTIVES: The incidence of venous thromboembolism (VTE) in children is increasing, attributed in part to increased utilization of central venous catheters (CVCs). Children with protein losing disorders (PLDs) and low serum albumin may have an increased incidence of thrombosis. We sought to determine the prevalence of PLDs and hypoalbuminemia at the time of diagnosis of VTE in pediatric patients and its relationship to central venous catheters. METHODS: We performed a single institution retrospective study of 65 consecutive hospitalized pediatric patients with an acute VTE. Data collected included clinical diagnoses, type of thrombosis, presence or absence of a CVC, and serum albumin level, if available. RESULTS: Of 65 patients with acute VTE, 51 % (33/65) had catheter-related thrombosis (CRT), including 71 % (19/27) of patients <12 years of age and 37 % (14/38) of patients aged 12 to 23 (P = 0.008). Eleven VTEs occurred in patients with a diagnosis of a PLD; of these, ten (91 %) were CRT and one (9 %) was a non-CRT (P = 0.003). Serum albumin levels obtained within four days of diagnosis of VTE were available for 38 patients. An albumin level below the lower limit of the age-adjusted normal reference range was documented in 27/38 (71 %) patients with VTE compared to 1011/3028 (33 %) of all pediatric patients admitted to the hospital during a two-year period (P < 0.0001). Albumin levels were low in 19/22 (86 %) patients with CRT compared with 8/16 (50 %) patients with non-CRT (P = 0.019). CONCLUSION: Low serum albumin levels are highly prevalent among pediatric patients with VTE, especially in those patients with CRT.

Engineering viral genomics and nano-liposomes in microfluidic platforms for patient-specific analysis of SARS-CoV-2 variants.

New variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are continuing to spread globally, contributing to the persistence of the COVID-19 pandemic. Increasing resources have been focused on developing vaccines and therapeutics that target the Spike glycoprotein of SARS-CoV-2. Recent advances in microfluidics have the potential to recapitulate viral infection in the organ-specific platforms, known as organ-on-a-chip (OoC), in which binding of SARS-CoV-2 Spike protein to the angiotensin-converting enzyme 2 (ACE2) of the host cells occurs. As the COVID-19 pandemic lingers, there remains an unmet need to screen emerging mutations, to predict viral transmissibility and pathogenicity, and to assess the strength of neutralizing antibodies following vaccination or reinfection. Conventional detection of SARS-CoV-2 variants relies on two-dimensional (2-D) cell culture methods, whereas simulating the micro-environment requires three-dimensional (3-D) systems. To this end, analyzing SARS-CoV-2-mediated pathogenicity via microfluidic platforms minimizes the experimental cost, duration, and optimization needed for animal studies, and obviates the ethical concerns associated with the use of primates. In this context, this review highlights the state-of-the-art strategy to engineer the nano-liposomes that can be conjugated with SARS-CoV-2 Spike mutations or genomic sequences in the microfluidic platforms; thereby, allowing for screening the rising SARS-CoV-2 variants and predicting COVID-19-associated coagulation. Furthermore, introducing viral genomics to the patient-specific blood accelerates the discovery of therapeutic targets in the face of evolving viral variants, including B1.1.7 (Alpha), B.1.351 (Beta), B.1.617.2 (Delta), c.37 (Lambda), and B.1.1.529 (Omicron). Thus, engineering nano-liposomes to encapsulate SARS-CoV-2 viral genomic sequences enables rapid detection of SARS-CoV-2 variants in the long COVID-19 era.

Myeloid Cell PKM2 Deletion Enhances Efferocytosis and Reduces Atherosclerosis.

BACKGROUND: The glycolytic enzyme PKM2 (pyruvate kinase muscle 2) is upregulated in monocytes/macrophages of patients with atherosclerotic coronary artery disease. However, the role of cell type-specific PKM2 in the setting of atherosclerosis remains to be defined. We determined whether myeloid cell-specific PKM2 regulates efferocytosis and atherosclerosis. METHODS: We generated myeloid cell-specific PKM2-/- mice on Ldlr (low-density lipoprotein receptor)-deficient background (PKM2mye-KOLdlr-/-). Controls were littermate PKM2WTLdlr-/- mice. Susceptibility to atherosclerosis was evaluated in whole aortae and cross sections of the aortic sinus in male and female mice fed a high-fat Western diet for 14 weeks, starting at 8 weeks. RESULTS: PKM2 was upregulated in macrophages of Ldlr-/- mice fed a high-fat Western diet compared with chow diet. Myeloid cell-specific deletion of PKM2 led to a significant reduction in lesions in the whole aorta and aortic sinus despite high cholesterol and triglyceride levels. Furthermore, we found decreased macrophage content in the lesions of myeloid cell-specific PKM2-/- mice associated with decreased MCP-1 (monocyte chemoattractant protein 1) levels in plasma, reduced transmigration of macrophages in response to MCP-1, and impaired glycolytic rate. Macrophages isolated from myeloid-specific PKM2-/- mice fed the Western diet exhibited reduced expression of proinflammatory genes, including MCP-1, IL (interleukin)-1ß, and IL-12. Myeloid cell-specific PKM2-/- mice exhibited reduced apoptosis concomitant with enhanced macrophage efferocytosis and upregulation of LRP (LDLR-related protein)-1 in macrophages in vitro and atherosclerotic lesions in vivo. Silencing LRP-1 in PKM2-deficient macrophages restored inflammatory gene expression and reduced efferocytosis. As a therapeutic intervention, inhibiting PKM2 nuclear translocation using a small molecule reduced glycolytic rate, enhanced efferocytosis, and reduced atherosclerosis in Ldlr-/- mice. CONCLUSIONS: Genetic deletion of PKM2 in myeloid cells or limiting its nuclear translocation reduces atherosclerosis by suppressing inflammation and enhancing efferocytosis.

Turoctocog alfa pegol (N8-GP) in severe hemophilia A: Long-term safety and efficacy in previously treated patients of all ages in the pathfinder8 study.

Background: N8-GP (turoctocog alfa pegol; Esperoct) is a glycoPEGylated human recombinant factor VIII (FVIII). Objectives: Pathfinder8 (NCT01480180) was a phase 3, multinational, open-label, nonrandomized trial to investigate the long-term safety and efficacy of N8-GP in people of all ages with severe hemophilia A previously treated with N8-GP. Patients/Method: Patients were recruited from the completed phase 3 pathfinder2 and pathfinder5 trials to receive intravenous N8-GP prophylaxis for up to 104 weeks, administered every 7 days, twice weekly, or three times weekly. Primary and secondary end points were the number of adverse events (AEs) reported and efficacy of treatment, respectively. Results: Overall, 160 patients were exposed to N8-GP for a mean of 179 exposure days and 681 calendar days (≈1.9 years) per patient. In total, 119 patients experienced 510 AEs, corresponding to a rate of 1.71 AEs per patient-year of exposure; 97.5% of AEs were mild or moderate in severity, and no AEs led to withdrawal. No patients developed FVIII inhibitors during the trial. The Poisson estimate of mean annualized bleeding rate for all bleeds (excluding surgery) and across all regimens was 1.10 (median, 0.00), and for spontaneous bleeds was 0.61 (median, 0.00). Most (55.6%) patients experienced no bleeds that required FVIII treatment (excluding perioperative bleeds). The estimated hemostatic success rate for the treatment of 322 bleeding episodes (excluding surgery) was 95.8%, including missing values as failure. Conclusions: Long-term prophylactic use of N8-GP appeared safe and efficacious across all age groups in people with severe hemophilia A previously treated with N8-GP.

Postoperative bleeding complications in patients with hemophilia undergoing major orthopedic surgery: A prospective multicenter observational study.

BACKGROUND: Persons with hemophilia (PWH) are at risk for chronic hemophilic arthropathy (HA). Joint replacement surgery may be used to relieve intractable pain and/or restore joint function. OBJECTIVES: This multicenter, prospective, observational cohort study evaluated the rate of bleeding during the postoperative period after total hip (THA) or knee arthroplasty (TKA). PATIENTS/METHODS: We included PWH of any severity ≥18 years of age who were undergoing THA or TKA. Clinical decisions were made at the discretion of the treating physician according to local standards of care. Clinical data were prospectively recorded. Major bleeding was defined as bleeding in a critical site, bleeding that resulted in either a 2 g/dl or greater decrease in hemoglobin during any 24-h period, or transfusion of two or more units of packed red blood cells. RESULTS: One hundred thirty-one procedures (98 TKA and 33 THA) were performed, 39 (29.8%) of which were complicated by major bleeding, including 46% of THA and 25% of TKA. The risk of major bleeding was increased in THA compared to TKA (OR 2.50, p = .05), and by the presence of an inhibitor (OR 4.29, p = .04), increased BMI (OR 4.49 and 6.09 for overweight and obese, respectively, compared to normal BMI, each p < .01), and non-use of an antifibrinolytic medication (OR 3.00, p = .03). Neither continuous clotting factor infusion (versus bolus infusion) nor pharmacologic thromboprophylaxis were associated with bleeding risk. CONCLUSIONS: The bleeding risk remains substantial after THA and TKA in PWH, despite factor replacement. Use of antifibrinolytic medications is associated with decreased risk.

Thrombotic potential during pediatric acute lymphoblastic leukemia induction: Role of cell-free DNA.

BACKGROUND: Thromboembolism affects up to 30% of children undergoing treatment for acute lymphoblastic leukemia (ALL). Increased thrombin generation has been reported in ALL, but the mechanisms remain elusive. OBJECTIVE: We aimed to show that extracellular traps and cell-free DNA (cfDNA) promote thrombin generation in pediatric ALL. METHODS: In a longitudinal single-center study, we recruited 17 consecutive pediatric ALL patients. Serial blood samples were collected at diagnosis and weekly during the 4-week induction phase of antileukemic chemotherapy. Healthy children (n = 14) and children with deep vein thrombosis (DVT; n = 7) or sepsis (n = 5) were recruited as negative and positive controls, respectively. In plasma, we measured endogenous thrombin generation potential (ETP) and components of extracellular traps, including cfDNA. RESULTS: In patients with ALL, ETP was increased at baseline and remained significantly elevated throughout the induction therapy. Plasma levels of cfDNA were increased at baseline and during the first 3 weeks of induction therapy. The extent of enhancement of ETP and plasma cfDNA in patients with ALL was similar to that seen in patients with DVT or sepsis. Treatment of plasma with DNase 1 lowered ETP in patients with ALL at each time point but did not affect ETP in healthy controls. CONCLUSION: We conclude that childhood ALL is associated with a prothrombotic milieu at the time of diagnosis that continues during induction chemotherapy, and cfDNA contributes to increased thrombogenic potential.

Standard prophylactic versus intermediate dose enoxaparin in adults with severe COVID-19: A multi-center, open-label, randomized controlled trial.

BACKGROUND: Coronavirus disease 2019 (COVID-19) is associated with coagulopathy but the optimal prophylactic anticoagulation therapy remains uncertain and may depend on COVID-19 severity. OBJECTIVE: To compare outcomes in hospitalized adults with severe COVID-19 treated with standard prophylactic versus intermediate dose enoxaparin. METHODS: We conducted a multi-center, open-label, randomized controlled trial comparing standard prophylactic dose versus intermediate dose enoxaparin in adults who were hospitalized with COVID-19 and admitted to an intensive care unit (ICU) and/or had laboratory evidence of coagulopathy. Patients were randomly assigned in a 1:1 ratio to receive standard prophylactic dose enoxaparin or intermediate weight-adjusted dose enoxaparin. The primary outcome was all-cause mortality at 30 days. Secondary outcomes included arterial or venous thromboembolism and major bleeding. RESULTS: A total of 176 patients (99 males and 77 females) underwent randomization. In the intention-to-treat population, all-cause mortality at 30 days was 15% for intermediate dose enoxaparin and 21% for standard prophylactic dose enoxaparin (odds ratio, 0.66; 95% confidence interval, 0.30-1.45; P = .31 by Chi-square test). Unadjusted Cox proportional hazards modeling demonstrated no significant difference in mortality between intermediate and standard dose enoxaparin (hazard ratio, 0.67; 95% confidence interval, 0.33-1.37; P = .28). Arterial or venous thrombosis occurred in 13% of patients assigned to intermediate dose enoxaparin and 9% of patients assigned to standard dose enoxaparin. Major bleeding occurred in 2% of patients in each arm. CONCLUSION: In hospitalized adults with severe COVID-19, standard prophylactic dose and intermediate dose enoxaparin did not differ significantly in preventing death or thrombosis at 30 days.

Smooth Muscle Cell-Specific PKM2 (Pyruvate Kinase Muscle 2) Promotes Smooth Muscle Cell Phenotypic Switching and Neointimal Hyperplasia.

[Figure: see text].

The metabolic enzyme pyruvate kinase M2 regulates platelet function and arterial thrombosis.

Very little is known about the role of metabolic regulatory mechanisms in platelet activation and thrombosis. Dimeric pyruvate kinase M2 (PKM2) is a crucial regulator of aerobic glycolysis that facilitates the production of lactate and metabolic reprogramming. Herein, we report that limiting PKM2 dimer formation, using the small molecule inhibitor ML265, negatively regulates lactate production and glucose uptake in human and murine stimulated platelets. Furthermore, limiting PKM2 dimer formation reduced agonist-induced platelet activation, aggregation, clot retraction, and thrombus formation under arterial shear stress in vitro in both human and murine platelets. Mechanistically, limiting PKM2 dimerization downregulated phosphatidylinositol 3-kinase (PI3K)-mediated protein kinase B or serine/threonine-specific protein kinase (Akt)/glycogen synthase kinase 3 (GSK3) signaling in human and murine platelets. To provide further evidence for the role of PKM2 in platelet function, we generated a megakaryocyte or platelet-specific PKM2-/- mutant strain (PKM2fl/flPF4Cre+). Platelet-specific PKM2-deficient mice exhibited impaired agonist-induced platelet activation, aggregation, clot retraction, and PI3K-mediated Akt/GSK3 signaling and were less susceptible to arterial thrombosis in FeCl3 injury-induced carotid- and laser injury-induced mesenteric artery thrombosis models, without altering hemostasis. Wild-type mice treated with ML265 were less susceptible to arterial thrombosis with unaltered tail bleeding times. These findings reveal a major role for PKM2 in coordinating multiple aspects of platelet function, from metabolism to cellular signaling to thrombosis, and implicate PKM2 as a potential target for antithrombotic therapeutic intervention.

Turoctocog alfa pegol provides effective management for major and minor surgical procedures in patients across all age groups with severe haemophilia A: Full data set from the pathfinder 3 and 5 phase III trials.

INTRODUCTION: Turoctocog alfa pegol is a glycoPEGylated recombinant factor VIII (FVIII) with an extended half-life developed for prophylaxis, treatment of bleeds and perioperative management in patients with haemophilia A. AIM: Evaluate the efficacy and safety of turoctocog alfa pegol treatment for major and minor surgeries in the pathfinder 3 and 5 phase III trials. METHODS: Adults/adolescents aged ≥12 years with severe haemophilia A (FVIII <1%) received perioperative turoctocog alfa pegol treatment planned to achieve FVIII activity levels >80% during major surgery (pathfinder 3). The primary end point was haemostatic efficacy during surgery; secondary end points were blood loss, haemostatic effect postsurgery, consumption, transfusions, safety and health economics. Children (0-11 years) undergoing minor surgeries received 20-75 IU/kg turoctocog alfa pegol at Investigator's discretion (pathfinder 5). RESULTS: pathfinder 3 included 35 patients undergoing 49 major surgeries. Haemostasis was successful in 47/49 (95.9%) surgeries; two had moderate haemostatic responses. Median (mean) blood loss during major surgery was 75 (322.6) mL. Four bleeds were reported postsurgery; three were successfully treated with turoctocog alfa pegol (one was not evaluated). On the day of surgery, overall mean (median) dose was 75.5 (74.5) IU/kg and mean (median) number of doses was 1.7 (2.0). Five procedures required 11 transfusions on the day of surgery or days 1-6. No safety concerns or inhibitors were identified. Forty-five minor surgeries in 23 children were performed without complications. CONCLUSION: Turoctocog alfa pegol was effective for perioperative haemostatic management of major and minor surgeries in patients across age groups with severe haemophilia A.

Targeting Myeloid-Specific Integrin α9ß1 Improves Short- and Long-Term Stroke Outcomes in Murine Models With Preexisting Comorbidities by Limiting Thrombosis and Inflammation.

RATIONALE: Currently, there is no effective intervention available that can reduce brain damage following reperfusion. Clinical studies suggest a positive correlation between the increased influx of neutrophils and severity of brain injury following reperfusion. Integrin α9ß1 is highly expressed on activated neutrophils and contributes to stable adhesion, but its role in stroke outcome has not been demonstrated to date. OBJECTIVE: We sought to determine the mechanistic role of myeloid-specific α9ß1 in the progression of ischemic stroke in murine models with preexisting comorbidities. METHODS AND RESULTS: We generated novel myeloid-specific α9-deficient (α9-/-) wild type (α9fl/flLysMCre+/-), hyperlipidemic (α9fl/flLysMCre+/-Apoe-/-), and aged (bone marrow chimeric) mice to evaluate stroke outcome. Susceptibility to ischemia/reperfusion injury was evaluated at 1, 7, and 28 days following reperfusion in 2 models of experimental stroke: filament and embolic. We found that peripheral neutrophils displayed elevated α9 expression following stroke. Irrespective of sex, genetic deletion of α9 in myeloid cells improved short- and long-term stroke outcomes in the wild type, hyperlipidemic, and aged mice. Improved stroke outcome and enhanced survival in myeloid-specific α9-/- mice was because of marked decrease in cerebral thromboinflammatory response as evidenced by reduced fibrin, platelet thrombi, neutrophil, NETosis, and decreased phospho-NF-κB (nuclear factor-κB), TNF (tumor necrosis factor)-α, and IL (interleukin)-1ß levels. α9-/- mice were less susceptible to FeCl3 injury-induced carotid artery thrombosis that was concomitant with improved regional cerebral blood flow following stroke as revealed by laser speckle imaging. Mechanistically, fibronectin containing extra domain A, a ligand for integrin α9, partially contributed to α9-mediated stroke exacerbation. Infusion of a specific anti-integrin α9 inhibitor into hyperlipidemic mice following reperfusion significantly reduced infarct volume and improved short- and long-term functional outcomes up to 28 days. CONCLUSIONS: We provide genetic and pharmacological evidence for the first time that targeting myeloid-specific integrin α9ß1 improves short- and long-term functional outcomes in stroke models with preexisting comorbidities by limiting cerebral thrombosis and inflammation.

Memantine Protects From Exacerbation of Ischemic Stroke and Blood Brain Barrier Disruption in Mild But Not Severe Hyperhomocysteinemia.

Background Hyperhomocysteinemia is a risk factor for ischemic stroke; however, a targeted treatment strategy is lacking partly because of limited understanding of the causal role of homocysteine in cerebrovascular pathogenesis. Methods and Results In a genetic model of cystathionine beta synthase (CBS) deficiency, we tested the hypothesis that elevation in plasma total homocysteine exacerbates cerebrovascular injury and that memantine, a N-methyl-D-aspartate receptor antagonist, is protective. Mild or severe elevation in plasma total homocysteine was observed in Cbs+/- (6.1±0.3 µmol/L) or Cbs-/- (309±18 µmol/L) mice versus Cbs+/+ (3.1±0.6 µmol/L) mice. Surprisingly, Cbs-/- and Cbs+/- mice exhibited similar increases in cerebral infarct size following middle cerebral artery ischemia/reperfusion injury, despite the much higher total homocysteine levels in Cbs-/- mice. Likewise, disruption of the blood brain barrier was observed in both Cbs+/- and Cbs-/- mice. Administration of the N-methyl-D-aspartate receptor antagonist memantine protected Cbs+/- but not Cbs-/- mice from cerebral infarction and blood brain barrier disruption. Our data suggest that the differential effect of memantine in Cbs+/- versus Cbs-/- mice may be related to changes in expression of N-methyl-D-aspartate receptor subunits. Cbs-/-, but not Cbs+/- mice had increased expression of NR2B subunit, which is known to be relatively insensitive to homocysteine. Conclusions These data provide experimental evidence that even a mild increase in plasma total homocysteine can exacerbate cerebrovascular injury and suggest that N-methyl-D-aspartate receptor antagonism may represent a strategy to prevent reperfusion injury after acute ischemic stroke in patients with mild hyperhomocysteinemia.

Smooth muscle cell-specific fibronectin-EDA mediates phenotypic switching and neointimal hyperplasia.

Fibronectin-splice variant containing extra domain A (Fn-EDA) is associated with smooth muscle cells (SMCs) following vascular injury. The role of SMC-derived Fn-EDA in SMC phenotypic switching or its implication in neointimal hyperplasia remains unclear. Herein, using human coronary artery sections with a bare metal stent, we demonstrate the expression of Fn-EDA in the vicinity of SMC-rich neointima and peri-strut areas. In mice, Fn-EDA colocalizes with SMCs in the neointima of injured carotid arteries and promotes neointima formation in the comorbid condition of hyperlipidemia by potentiating SMC proliferation and migration. No sex-based differences were observed. Mechanistic studies suggested that Fn-EDA mediates integrin- and TLR4-dependent proliferation and migration through activation of FAK/Src and Akt1/mTOR signaling, respectively. Specific deletion of Fn-EDA in SMCs, but not in endothelial cells, reduced intimal hyperplasia and suppressed the SMC synthetic phenotype concomitant with decreased Akt1/mTOR signaling. Targeting Fn-EDA in human aortic SMCs suppressed the synthetic phenotype and downregulated Akt1/mTOR signaling. These results reveal that SMC-derived Fn-EDA potentiates phenotypic switching in human and mouse aortic SMCs and neointimal hyperplasia in the mouse. We suggest that targeting Fn-EDA could be explored as a potential therapeutic strategy to reduce neointimal hyperplasia.

Pharmacokinetics, immunogenicity, safety, and preliminary efficacy of subcutaneous turoctocog alfa pegol in previously treated patients with severe hemophilia A (alleviate 1).

BACKGROUND: The current standard of care for patients with hemophilia A is regular prophylaxis with factor VIII (FVIII) administered intravenously. Interest in subcutaneous (s.c.) administration, to potentially increase convenience, reduce the treatment burden and improve compliance, is increasing. OBJECTIVES: Evaluate the pharmacokinetics (PK), immunogenicity, safety, and preliminary efficacy of s.c. administration of turoctocog alfa pegol (s.c. N8-GP) in adult or adolescent previously treated patients (PTPs) with severe hemophilia A (alleviate 1; NCT02994407). PATIENTS/METHODS: In part A, 24 PTPs received a single dose of s.c. N8-GP (12.5, 25, 50, or 100 IU/kg) with 6 patients per cohort. PK modelling of data from part A supported a suitable dose for part B. Part B comprised a multiple dose trial in 26 PTPs; patients <60 kg received 2000 IU and patients ≥60 kg received 4000 IU s.c. N8-GP daily for 3 months. RESULTS: Single-dose s.c. N8-GP supported dose linearity. Daily prophylaxis with s.c. N8-GP appeared well tolerated and efficacious, achieving a mean trough FVIII activity close to 10% at steady state. Five patients developed anti-N8-GP binding antibodies after 42 to 91 exposure days, one of whom developed an inhibitor to FVIII. Anti-N8-GP antibody appearance was associated with a decline in FVIII plasma activity in four of the five patients. Five patients reported a total of nine treatment-requiring bleeding episodes during prophylaxis. CONCLUSIONS: Subcutaneous administration of N8-GP is associated with a high incidence of antibodies in PTPs with severe hemophilia A. Further clinical development of s.c. N8-GP has been suspended.