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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-hour 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=0.05), and by the presence of an inhibitor (OR 4.29, p=0.04), increased BMI (OR 4.49 and 6.09 for overweight and obese, respectively, compared to normal BMI, each p

Content validity is often lacking in tools for the assessment of competency in medical specialty and subspecialty trainees. The primary aim of this study was to develop a protocol for early test assessment in pediatric hematology using iterative rounds of expert evaluation in the context of explicit knowledge goals. We used the pediatric hematology content of the American Board of Pediatrics as a test case for assessment protocol development. We found that the use of expert input and three rounds of test development increased test content validity. The instrument developed here has the potential to improve assessment of competency in pediatric hematology. This approach also may be useful in the development of competency assessment tools for other medical subspecialties.

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. Methods: We conducted a multi-center, open-label, randomised 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. Findings: A total of 176 patients (99 males and 77 females) underwent randomisation. 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 (P=0.31 by chi-square test). Unadjusted Cox proportional hazards modeling demonstrated similar mortality with intermediate and standard dose enoxaparin (hazard ratio, 0.67; 95% confidence interval, 0.33 to 1.37; P=0.28. 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 only 2% of patients in each arm. Interpretation: 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. Trial Registration: The trial was registered with ClinicalTrials.gov (NCT04360824). Funding Statement: National Institutes of Health Clinical and Translational Science Award. Declaration of Interests: SRL has served as a paid consultant for Novo Nordisk, UniQure, and Argenx, all outside the scope of the submitted work. All other authors declare no competing interests. SRB has served as an editor or committee member for Wiley, Abbott, and Boston Scientific Corporation. All other authors declare no competing interests. Ethics Approval Statement: The trial protocol was approved by the local institutional review boards of the participating sites. Written informed consent was obtained from all patients or their legally authorized representatives.

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.

Objective: The role of glycolytic enzyme PKM2 (pyruvate kinase muscle 2) in smooth muscle cell (SMC) phenotype switching and neointimal hyperplasia is poorly understood. We determined the role of PKM2 in SMC phenotype switching and neointimal hyperplasia. Approach and Results: We show that PKM2 is expressed in the SMC-rich neointima of the murine carotid artery and peri-strut areas in bare-metal stented human coronary arteries. PDGF-BB (platelet-derived growth factor-BB) stimulation upregulates the expression of PKM2 in cultured murine and human coronary SMC. To provide conclusive evidence for PKM2 in SMC function, we generated SMC-specific PKM2 −/− mutant strain. We report that PKM2 deletion in SMC reduces injury-induced neointimal hyperplasia by inhibiting SMC proliferation and migration, suppressing synthetic phenotype, and reducing aerobic glycolysis associated with decreased ERK (extracellular signal-regulated kinase), mTOR (mammalian target of rapamycin), and STAT3 (signal transducer and activator of transcription 3) signaling. Furthermore, we show that nuclear PKM2 interacts with STAT3 and β-catenin and regulates transcription of MEK5 (mitogen/extracellular signal-regulated kinase kinase-5), cyclin D1, GLUT1 (glucose transporter 1), and LDHA (lactate dehydrogenase A). Treatment of human coronary SMC with ML265, an activator that induces PKM2 tetramerization and blocks its nuclear translocation, inhibited proliferation, migration, and phenotypic switching. Perivascular application of PKM2 activator reduced neointimal hyperplasia in mice. Conclusions: These findings reveal that PKM2 is a key regulator of SMC function in vascular remodeling and implicates PKM2 as a potential target to reduce neointimal hyperplasia.

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.

Rationale: The glycolytic enzyme pyruvate kinase muscle 2 (PKM2) 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. Objective: We determined whether myeloid cell-specific PKM2 regulates efferocytosis and atherosclerosis. Methods and Results: We generated novel myeloid cell-specific PKM2 -/- mice on Ldlr-deficient background (PKM2 mye-KO Ldlr -/- ). Controls were littermate PKM2 WT Ldlr -/- mice. To rule out sex-based differences, male and female mice were placed on a high-fat "Western" diet for 14 weeks, starting at eight weeks. PKM2 was upregulated in macrophages of Ldlr -/- mice fed the Western diet compared with a control 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 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 pro-inflammatory genes, including MCP-1, IL-1β, and IL-12. Myeloid cell-specific PKM2 -/- mice exhibited reduced apoptosis concomitant with enhanced macrophage efferocytosis and upregulation of LRP1 in macrophages in vitro and atherosclerotic lesions in vivo . Silencing LRP1 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. Conclusion: Genetic deletion or limiting PKM2 nuclear translocation in myeloid cells reduces atherosclerosis by suppressing inflammation and enhancing efferocytosis.

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.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.Of 65 patients with acute VTE, 51 % (33/65) had catheter-related thrombosis (CRT), including 71 % (19/27) of patientslt;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 lt; 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).Low serum albumin levels are highly prevalent among pediatric patients with VTE, especially in those patients with CRT.

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 α9β1 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 α9β1 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 ( α9 fl/fl LysMCre +/− ), hyperlipidemic ( α9 fl/fl LysMCre +/− 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)-1β levels. α9 −/− mice were less susceptible to FeCl 3 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 α9β1 improves short- and long-term functional outcomes in stroke models with preexisting comorbidities by limiting cerebral thrombosis and inflammation.

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 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.

Background: Severe COVID-19 leads to inflammation and coagulopathy with progression to multiple organ failure. Several prothrombotic mechanisms have been proposed, including NETosis with release of citrullinated histones, platelet hyperactivation, and generation of procoagulant microvesicles. Aims: We hypothesized that histones derived from NETosis promote platelet activation and release of microvesicles that mechanistically contribute to thrombin generation and thrombosis in COVID-19. Methods: Platelet poor plasma (PPP) was prepared from 136 COVID-19 patients enrolled in a multicenter randomized clinical trial comparing standard prophylactic dose to intermediate dose enoxaparin in hospitalized patients with COVID-19 (NCT04360824). We also prepared PPP and washed platelets from 53 healthy subjects. We measured citrullinated histone H3 (H3Cit) by ELISA, microvesicles by flow cytometry, and thrombin generation using the Calibrated Automated Thrombogram. Ex vivo platelet adhesion and thrombus growth on collagen was measured in a microfluidic chamber. Results: Compared to healthy subjects, COVID-19 patients had elevated plasma levels of H3Cit and platelet-derived microvesicles. When PPP was triggered with exogenous tissue factor and phospholipids, higher endogenous thrombin potential, peak thrombin, and velocity index were observed in COVID-19 patients compared to healthy subjects. Increased thrombin generation with COVID-19 PPP was evident even when triggered without added phospholipids, suggesting the presence of procoagulant microvesicles in COVID-19 plasma. Enhanced thrombin generation also was observed after transfer of microvesicles isolated from COVID-19 patient plasma to control PPP. Incubation of control platelets with PPP from COVID-19 patients but not healthy subjects caused increased platelet-dependent thrombin generation and larger platelet-thrombi under venous shear stress. These effects of COVID-19 plasma on platelet-dependent thrombin generation and platelet-thrombi formation were inhibited by a histone aptamer. Conclusions: We conclude that histone-mediated platelet activation promotes thrombosis in COVID-19 in part via increased thrombin generation driven by microvesicles.

Introduction: The underlying cause of coronary artery disease (CAD) is atherosclerosis, which is a pathological response to chronic inflammation and hyperlipidemia. The onset of atherogenesis is characterized by infiltration of myeloid cells, including monocytes followed by fatty streak formation and progressive accumulation of smooth muscle cells (SMCs). These microenvironmental changes dictate the balance between inflammatory and anti-inflammatory macrophages. Pyruvate kinase M2 (PKM2), a glycolytic enzyme, is highly expressed in activated proinflammatory macrophages. The mechanistic role of PKM2 in atherosclerosis remains unknown. Hypothesis: We hypothesize that PKM2 promotes macrophage migration in response to MCP-1 and mediates atherosclerosis by increasing inflammation. Methods and Results: PKM2 was upregulated in macrophages of Ldlr -/- mice fed a high-fat "Western" diet compared with a control chow diet. We generated the novel myeloid cell-specific PKM2 fl/fl LysMCre +/- on a Ldlr-deficient background (PKM2 fl/fl LysMCre +/- Ldlr -/- ) and evaluated atherosclerosis after 14 weeks high-fat “Western” diet feeding. Controls were littermate PKM2 fl/fl LysMCre -/- Ldlr -/- mice. 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. (P-/- mice compared with control mice that was associated with decreased plasma levels of pro-inflammatory cytokines, including MCP-1, and reduced transmigration of macrophages in response to MCP-1. Macrophages isolated from myeloid-specific PKM2 -/- mice fed a high-fat "Western" diet exhibited reduced expression of pro-inflammatory genes, including MCP-1, IL-1β, IL-12, and increased expression of the anti-inflammatory genes Arg1 and IL-10. Inhibiting PKM2 nuclear translocation in bone marrow-derived macrophages led to a significant reduction in MCP-1 and IL-1β levels and reduced transmigration of macrophages. Conclusion: Genetic deletion of PKM2 in myeloid cells reduces atherosclerosis by limiting inflammation.

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.

Essentials Long-term recurrence risk after a first unprovoked VTE with negative d-dimer levels is uncertain. Anticoagulation was stopped if d-dimer was negative, and was continued if d-dimer was positive. Five years after stopping anticoagulants, recurrent VTE was 30% in men and 17% in women. Negative d-dimers do not justify stopping anticoagulants in most men but appear to in most women. BACKGROUND The long-term risk of recurrence in patients with a first unprovoked venous thromboembolism (VTE) who have negative d-dimer results is uncertain. OBJECTIVES To determine this risk, including in subgroups based on sex. PATIENTS AND METHODS ln a prospective interventional cohort study of 410 patients with a first unprovoked VTE, anticoagulants were stopped if d-dimer was negative on therapy and 1 month after stopping therapy. Other patients remained on anticoagulant therapy. We previously reported findings after a mean of 2.2 years. The current report includes 3 years of additional follow-up in 293 of these patients. RESULTS During a median follow-up of 5.0 years, recurrent VTE after stopping therapy in response to negative d-dimer testing was 5.1% (95% confidence interval [CI], 3.6-6.5) per patient-year overall, 7.5% (95% CI, 5.5-10.0) in men, 3.8% (95% CI, 2.0-6.6) in women with VTE not associated with estrogens, and 0.4% (95% CI, 0.0-2.3) in women with VTE associated with estrogens (P

Background N8‐GP is an extended half‐life recombinant factor VIII developed for prophylaxis and treatment of bleeds in patients with hemophilia A. Objective To assess pharmacokinetic (PK) characteristics of N8‐GP in previously treated patients with severe hemophilia A, model the time spent at hemophilia thresholds of ≥1 and ≤5 IU/dL (moderate) or >5 IU/dL (mild) FVIII levels during N8‐GP prophylaxis, and investigate the relationship between N8‐GP half‐life and von Willebrand factor (vWF). Methods PK assessments were obtained from patients with severe hemophilia A (FVIII < 1 IU/dL) participating in 4 clinical trials: pathfinder 1 (20‐60 years); pathfinder 2 (12‐17 and ≥18 years); pathfinder 5 (0‐11 years), and pathfinder 7 (25‐71 years). All PK profiles were assessed after washout and considered single‐dose PK profiles. Pre‐ and postdose FVIII activity at steady state was measured at all visits. Results From 69 patients, 108 PK profiles of N8‐GP 50 IU/kg were assessed. Adults/adolescents received 50 IU/kg every 4 days, achieving mean trough levels of 3.0 IU/dL (95% confidence interval, 2.6‐3.5, adults) and 2.7 IU/dL (1.8‐4.0, adolescents). Children received 60 IU/kg twice weekly, leading to mean trough levels of 1.2 IU/dL (0.8‐1.6, 0‐ to 5‐year‐olds) and 2.0 IU/dL (1.5‐2.7, 6‐ to 11‐year‐olds). PK modeling predicted children dosed every 3 days and adults/adolescents dosed every 3 to 4 days would maintain FVIII levels >5 and >1 IU/dL for >80% and 100% of the time, respectively. N8‐GP half‐life correlated linearly with von Willebrand factor levels in adults/adolescents, less in children. Conclusions Prophylaxis with fixed intervals (Q4D/twice weekly) and fixed weight‐based dosing (50/60 IU/kg) ensured >1 IU/dL FVIII trough levels in both adults and children.

A series of recent epidemiological studies have implicated the endogenous nonproteinogenic amino acid l -homoarginine as a novel candidate cardiovascular risk factor. The association between homoarginine levels and the risk of adverse cardiovascular outcomes is inverse (ie, high cardiovascular risk is predicted by low rather than high homoarginine levels), which makes it plausible to normalize systemic homoarginine levels via oral supplementation. The emergence of homoarginine as a potentially treatable protective cardiovascular risk factor has generated a wave of hope in the field of cardiovascular prevention. Herein, we review the biochemistry, physiology, and metabolism of homoarginine, summarize the strengths and weaknesses of the epidemiological evidence linking homoarginine to cardiovascular disease and its potential protective cardiovascular effects, and identify priorities for future research needed to define the clinical utility of homoarginine as a prognostic factor and therapeutic target in cardiovascular disease.

Deficiency of the Nox2 (gp91phox) catalytic subunit of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase is a genetic cause of X-linked chronic granulomatous disease, a condition in which patients are prone to infection resulting from the loss of oxidant production by neutrophils. Some studies have suggested a role for superoxide derived from Nox2 NADPH oxidase in platelet activation and thrombosis, but data are conflicting. Using a rigorous and comprehensive approach, we tested the hypothesis that genetic deficiency of Nox2 attenuates platelet activation and arterial thrombosis. Our study was designed to test the genotype differences within male and female mice. Using chloromethyl-dichlorodihydrofluorescein diacetate, a fluorescent dye, as well as high-performance liquid chromatography analysis with dihydroethidium as a probe to detect intracellular reactive oxygen species (ROS), we observed no genotype differences in ROS levels in platelets. Similarly, there were no genotype-dependent differences in levels of mitochondrial ROS. In addition, we did not observe any genotype-associated differences in platelet activation, adhesion, secretion, or aggregation in male or female mice. Platelets from chronic granulomatous disease patients exhibited similar adhesion and aggregation responses as platelets from healthy subjects. Susceptibility to carotid artery thrombosis in a photochemical injury model was similar in wild-type and Nox2-deficient male or female mice. Our findings indicate that Nox2 NADPH oxidase is not an essential source of platelet ROS or a mediator of platelet activation or arterial thrombosis in large vessels, such as the carotid artery.

Introduction Prophylaxis with replacement factor IX (FIX) reduces bleeding frequency and improves quality of life in haemophilia B patients. With prophylaxis, the likelihood of bleeding is lowered with increasing trough levels. New products with extended half-life (EHL) can maintain high factor activity levels over prolonged periods, compared with standard FIX products. Aim To evaluate the safety, efficacy and pharmacokinetics of the new recombinant FIX EHL product, nonacog beta pegol (N9-GP), using pooled data, with a focus on-but not limited to-prophylaxis at 40 IU/kg. Methods N9-GP has been investigated in males with congenital haemophilia B and FIX activity ≤2% in the paradigm™ clinical trial programme. This analysis includes pooled data from five completed paradigm™ trials conducted in previously treated adults, adolescents and children, focusing on results of prophylaxis with 40 IU/kg once-weekly intravenous dosing. Results In total, 115 previously treated patients were exposed to N9-GP. Of 54 patients (47%) treated with N9-GP 40 IU/kg once-weekly prophylaxis, 72% experienced no spontaneous bleeds over 1 year. In all patients receiving 40 IU/kg once-weekly, median overall annualized bleeding rate (ABR) was 1.03 (interquartile range 0.00; 2.89); median spontaneous ABR was 0.00 (0.00; 0.80). No patients developed inhibitors. Estimated mean steady-state trough levels with N9-GP 40 IU/kg once-weekly were ≥15% overall; 27.3% in adolescents and adults. Conclusion N9-GP 40 IU/kg once-weekly was well tolerated and effective in preventing bleeding, maintaining mean FIX activity levels ≥15% across all age groups. N9-GP may provide a new treatment option for preventing bleeding in haemophilia B patients.

Essentials Signaling by Gas6 through Tyro3/Axl/Mer receptors is essential for stable platelet aggregation. UNC2025 is a small molecule inhibitor of the Mer tyrosine kinase. UNC2025 decreases platelet activation in vitro and thrombus formation in vivo. UNC2025's anti-platelet effect is synergistic with inhibition of the ADP receptor, P2Y12. SummaryBackground Growth arrest-specific protein 6 signals through the TAM (TYRO-3–AXL–MERTK) receptor family, mediating platelet activation and thrombus formation via activation of the aggregate-stabilizing αIIbβ3 integrin. Objective To describe the antithrombotic effects mediated by UNC2025, a small-molecule MERTK tyrosine kinase inhibitor. Methods MERTK phosphorylation and downstream signaling were assessed by immunoblotting. Light transmission aggregometry, flow cytometry and microfluidic analysis were used to evaluate the impact of MERTK inhibition on platelet activation and stability of aggregates in vitro. The effects of MERTK inhibition on arterial and venous thrombosis, platelet accumulation at microvascular injury sites and tail bleeding times were determined with murine models. The effects of combined treatment with ADP–P2Y1&12 pathway antagonists and UNC2025 were also evaluated. Results and Conclusions Treatment with UNC2025 inhibited MERTK phosphorylation and downstream activation of AKT and SRC, decreased platelet activation, and protected animals from pulmonary embolism and arterial thrombosis without increasing bleeding times. The antiplatelet effect of UNC2025 was enhanced in combination with ADP–P2Y1&12 pathway antagonists, and a greater than additive effect was observed when these two agents with different mechanisms of inhibition were coadministered. TAM kinase signaling represents a potential therapeutic target, as inhibition of this axis, especially in combination with ADP–P2Y pathway antagonism, mediates decreased platelet activation, aggregate stability, and thrombus formation, with less hemorrhagic potential than current treatment strategies. The data presented here also demonstrate antithrombotic activity mediated by UNC2025, a novel translational agent, and support the development of TAM kinase inhibitors for clinical applications.

Introduction: N8-GP is an extended half-life recombinant factor VIII product indicated for use in patients with hemophilia A. The pathfinder trials evaluated routine prophylaxis and bleed control in previously treated patients aged ≥12 years (pathfinder2) and patients aged Methods: In the pathfinder2 trial, patients aged ≥12 years with severe hemophilia A were administered N8-GP 50 IU/kg every 4 days (Q4D) as routine prophylaxis or 20 to 70 IU/kg as on-demand treatment. Patients aged Results: See Table for efficacy in prophylaxis (median annualized bleeding rates [ABRs]) and bleed treatment (success rate). Median ABRs tended to decrease from 0.84 (global) and 0.86 (US) in pathfinder2 to 0.00 (global) and 0.00 (US) in pathfinder8 in patients aged ≥12 years on the prophylaxis regimen. In addition, in patients aged Overall, there were 59 treatment-related adverse events (AEs) in patients on the prophylactic regimen in pathfinder2, 13 of which occurred in US patients, and 11 treatment-related AEs in patients treated on-demand, 3 of which occurred in US patients. In pathfinder5, there were 16 treatment-related AEs, 7 of which occurred in US patients. In pathfinder8, there were 6 treatment-related AEs in patients aged ≥12 years, 3 of which were in US patients. In addition, there was 1 treatment-related AE in a non-US patient aged 5 Bethesda units. The patient returned to their previous FVIII product, and their inhibitor status was negative at follow-up. Conclusions: Efficacy of N8-GP was sustained during the main and extension pathfinder trials in both global and US patients. In addition, a favorable safety profile was maintained throughout the course of the program. Figure 1 Figure 1. Disclosures Wheeler: Novo Nordisk A/S: Consultancy; Bayer: Consultancy; BioMarin: Consultancy; HEMA Biologics: Consultancy; Spark: Consultancy; Takeda: Consultancy; UniQure: Consultancy. Kearney: Grifols: Research Funding; Daiichi Sankyo: Research Funding; CVS Pharmacy: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novo Nordisk Inc: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Bioverativ/Sanofi: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Staber: Bayer, CSL Behring, Sanofi, Takeda: Membership on an entity's Board of Directors or advisory committees. Wufsus: Novo Nordisk Inc: Current Employment. Ostrow: Novo Nordisk Inc: Current Employment. Lentz: Novo Nordisk Inc: Consultancy, Honoraria, Research Funding.

Background: COVID-19-associated coagulopathy increases mortality in patients with COVID-19 infection. The International Society of Thrombosis and Hemostasis (ISTH) has published a guidance document recommending thromboprophylaxis with low-molecular weight heparin (LMWH) for all hospitalized patients with COVID-19. Despite standard dose LMWH thromboprophylaxis, arterial and venous thrombotic events occur in up to 30-40% of COVID-19 patients admitted to the intensive care unit (ICU). It is not known if patients with coagulopathy from COVID-19 benefit from higher levels of prophylactic anticoagulation therapy. No data exist regarding the comparative safety or efficacy of intermediate dose versus standard prophylactic dose LMWH. Study Design and Methods: This is a multi-center, randomized, open-label study (NCT04360824) comparing standard prophylactic dose enoxaparin (40 mg SC daily if BMI < 30 kg/m2; 30 mg SC twice daily or 40 mg SC twice daily if BMI ≥ 30 kg/m2) versus intermediate-weight adjusted dose enoxaparin (1 mg/kg SC daily if BMI < 30 kg/m2 or 0.5 mg/kg SC twice daily if BMI ≥ 30 kg/m2) in hospitalized patients with laboratory-confirmed SARS-CoV-2 infection who are either admitted to an intensive care unit or have a modified ISTH Overt DIC score ≥ 3. Objectives: The primary endpoint is to compare all-cause 30-day mortality of patients treated with standard prophylactic dose versus intermediate-dose enoxaparin. The secondary endpoints are to determine whether the use of standard prophylactic dose versus intermediate dose enoxaparin impacts major bleeding, arterial thrombosis, venous thrombosis, time to intubation or transfusion of blood products. The exploratory endpoints are to determine the effects of SARS-CoV-2 infection on laboratory coagulation parameters to better understand the mechanism of COVID-19 coagulopathy compared to healthy control subjects; parameters include citrullinated histone H3 (H3Cit), a component of neutrophil extracellular traps. The null hypothesis is that intermediate dose enoxaparin (intervention arm) will have a mortality rate that is not less than standard prophylactic dose enoxaparin (standard of care arm). Assuming a true margin of 20%, a sample size of 82 patients in each arm of the study is required to have 80% power when testing for a difference. Baseline Data: Three centers are currently enrolling patients. As of August 1, 2020, we have enrolled 52 patients (28 males and 24 females) with a median age of 61 years (range 24-80). 51% were Caucasian, 36% Hispanic, and 16% other ethnicities. Median BMI was 31 kg/m2 (range 20-52). Baseline D-Dimer was elevated in 95% of participants with a median of 2.23 mg/mL (range 0.39-40.32). The majority of participants had normal baseline PT, PTT, and platelet levels. All patients had fibrinogen levels over 100 mg/dL with a median of 607 mg/dL. Plasma H3Cit was elevated significantly in patients with COVID-19 (n = 21) vs healthy control subjects (n = 10) (6.48 vs 1.09 ng/mL, P = 0.001). Conclusion: Baseline data for the first 52 patients enrolled confirm the presence of multiple risk factors for thrombosis such as elevated BMI, D dimer, and plasma markers of neutrophil extracellular traps. In contrast to initial reports from China, the prevalence of thrombocytopenia and hypofibrinogenemia was very low in our cohort. Hispanics represented 36% of our study population, compared with 19% of overall COVID-19 infections in the region, suggesting a higher severity of illness in this subpopulation. The enrollment of this trial is ongoing with the addition of sites that have larger African American populations. As safety and efficacy data mature, this study will help clarify the optimum dose of prophylactic enoxaparin to prevent COVID-19 associated coagulopathy. Disclosures No relevant conflicts of interest to declare.

The hemostatic response to vascular injury culminates in a fibrin clot network that forms an initial barrier to blood loss and also contributes to microbial host defense. Fibrinogen is cleaved by thrombin into fibrin monomers that spontaneously polymerize into protofibrils and form the extensive fiber networks characteristic of blood clots. In this issue of the JCI, Macrae and colleagues characterize an alternative fibrin structure in which fibrinogen and fibrin assemble into a continuous 2D film at the exterior face of the fibrin clot network. Fibrin films connect to the underlying fiber network through tethering fibers and provide a protective barrier to microbial infiltration. These findings shed new light on a previously overlooked mechanism of fibrin assembly at the clot surface and provide a link between hemostasis and innate immunity.

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.

Clinicians write a billion free text notes per year. These notes are typically replete with errors of all types. No established automated method can extract data from this treasure trove. The practice of medicine therefore remains haphazard and chaotic, resulting in vast economic waste. The lexeme hypotheses are based on our analysis of how records are created. They enable a computer system to predict what issue a clinician will need to address next, based on the environment in which the clinician is working, and what responses the clinician has selected to date. The system uses a lexicon storing the issues (queries) and a range of responses to the issues. When the clinician selects a response, a text fragment is added to the output file. In the first phase of this work, the notes of 69 returning hemophilia patients were scrutinized, and the lexicon was expanded to 847 lexeme queries and 7995 responses to enable the construction of completed notes. The quality of lexeme-generated notes from 20 consecutive subjects was then compared to the clinicians’ conventional clinic notes. The system generated grammatically correct notes. In comparison to the traditional clinic note, the lexeme-generated notes were more complete (88 % compared with 62 %), and had less typographical and grammatical errors (0.8 versus 3.5 errors per note). The system notes and traditional notes averaged about 800 words, but the traditional notes had a much wider distribution of lengths. The note-creation rate from marshalling the data to completion using the system averaged 80 wpm, twice as fast as the typical clinician can type. The lexeme method generates more complete, grammatical and organized notes faster than traditional methods. The notes are completely computerized at inception, and they incorporate prompts for clinicians to address otherwise overlooked items. This pilot justifies further exploration of this methodology.

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

Curry, Nicola/0000-0002-3849-0688; Escobar, Miguel/0000-0002-2944-0240 WOS: 000470929100022 PubMed: 30817066 Introduction Turoctocog alfa pegol (N8-GP) is a site-specific, 40 kDa glycoPEGylated recombinant factor VIII (FVIII) product with an extended half-life. The comprehensive main phase of the pivotal pathfinder 2 trial showed N8-GP dosed every 4 days (Q4D) provided favourable safety and efficacy for preventing bleeds in 175 patients with haemophilia A. Aim and methods We investigated the safety and efficacy of N8-GP prophylaxis when administered weekly (Q7D) for 24 weeks to patients with low bleeding rates in the pathfinder 2 extension trial. Patients (>= 12 years) with 50% of patients experienced no bleeds. Median ABR for overall, joint, spontaneous, traumatic and muscle was 0.00 for both cohorts. Overall estimated success rate for treating bleeding episodes was 87.5%; 94.7% of bleeds were controlled with

Background— The fibronectin-splicing variant containing extra domain A (Fn-EDA) is present in negligible amounts in the plasma of healthy humans but markedly elevated in patients with comorbid conditions, including diabetes mellitus and hypercholesterolemia, which are risk factors for stroke. It remains unknown, however, whether Fn-EDA worsens stroke outcomes in such conditions. We determined the role of Fn-EDA in stroke outcome in a model of hypercholesterolemia, the apolipoprotein E–deficient (Apoe −/− ) mouse. Methods and Results— In a transient cerebral ischemia/reperfusion injury model, Apoe −/− mice expressing fibronectin deficient in EDA (Fn-EDA −/− Apoe −/− mice) exhibited smaller infarcts and improved neurological outcomes at days 1 and 8 ( P −/− mice). Concomitantly, intracerebral thrombosis [assessed by fibrin(ogen) deposition] and postischemic inflammation (phospho–nuclear factor-κB p65, phospho–IκB kinase α/β, interleukin 1β, and tumor necrosis factor-α) within lesions of Fn-EDA −/− Apoe −/− mice were markedly decreased ( P −/− mice). In an FeCl 3 injury–induced carotid artery thrombosis model, thrombus growth rate and the time to occlusion were prolonged in Fn-EDA −/− Apoe −/− mice ( P −/− mice). Genetic ablation of TLR4 improved stroke outcome in Apoe −/− mice ( P −/− Apoe −/− mice. Bone marrow transplantation experiments revealed that nonhematopoietic cell–derived Fn-EDA exacerbates stroke through Toll-like receptor-4 expressed on hematopoietic cells. Infusion of a specific inhibitor of Fn-EDA into Apoe −/− mouse 15 minutes after reperfusion significantly improved stroke outcome. Conclusions— Hypercholesterolemic mice deficient in Fn-EDA exhibit reduced cerebral thrombosis and less inflammatory response after ischemia/reperfusion injury. These findings suggest that targeting Fn-EDA could be an effective therapeutic strategy in stroke associated with hypercholesterolemia.

Essentials Factor VIIa binds activated platelets to promote hemostasis in hemophilia patients with inhibitors. The interactions and sites responsible for platelet-FVIIa binding are not fully understood. Endothelial cell protein C receptor (EPCR) is expressed on activated human platelets. EPCR binding enhances the efficacy of a FVIIa variant and could impact design of new therapeutics.Background High-dose factor VIIa (FVIIa) is routinely used as an effective bypassing agent to treat hemophilia patients with inhibitory antibodies that compromise factor replacement. However, the mechanism by which FVIIa binds activated platelets to promote hemostasis is not fully understood. FVIIa-DVQ is an analog of FVIIa with enhanced tissue factor (TF)-independent activity and hemostatic efficacy relative to FVIIa. Our previous studies have shown that FVIIa-DVQ exhibits greater platelet binding, thereby suggesting that features in addition to lipid composition contribute to platelet-FVIIa interactions. Objectives Endothelial cell protein C receptor (EPCR) also functions as a receptor for FVIIa on endothelial cells. We therefore hypothesized that an interaction with EPCR might play a role in platelet-FVIIa binding. Methods/results In the present study, we used flow cytometric analyses to show that platelet binding of both FVIIa and FVIIa-DVQ is partially inhibited in the presence of excess protein C or an anti-EPCR antibody. This decreased binding results in a corresponding decrease in the activity of both molecules in FXa and thrombin generation assays. Enhanced binding to EPCR was sufficient to account for the increased platelet binding of FVIIa-DVQ compared with wild-type FVIIa. As EPCR protein expression has not previously been shown in platelets, we confirmed the presence of EPCR in platelets using immunofluorescence, flow cytometry, immunoprecipitation, and mass spectrometry. Conclusions This work represents the first demonstration that human platelets express EPCR and suggests that modulation of EPCR binding could be utilized to enhance the hemostatic efficacy of rationally designed FVIIa analogs.

Activated neutrophils release neutrophil extracellular traps (NETs) via a pathway dependent on reactive oxygen species (ROS). NETs are enriched in extracellular DNA and histones, and contribute to venous thrombosis. Nox2-containing NADPH oxidase is a major upstream mediator of ROS-dependent release of NETs. Neutrophils from mice deficient in Nox2 or patients with X-linked chronic granulomatous disease due to Nox2 deficiency do not produce NETs, suggesting a role for Nox2 in venous thrombosis. We hypothesized that mice genetically deficient in Nox2 are protected from venous thrombosis. We studied male and female mice deficient in Nox2 (Nox2-KO) and their wild type (WT) littermates at 10-14 weeks of age (n=7-13/group). Using electron spin resonance and H2C-DCFH-DA staining, we confirmed that neutrophils from Nox2-KO mice do not produce ROS at baseline or upon PMA-activation. Neutrophils from WT mice displayed a dose-dependent release of NETs upon PMA-stimulation (SYTOX green staining using fluorescence microscopy), whereas Nox2-KO mice did not produce NETs. We next examined susceptibility to venous thrombosis in a stasis model of inferior vena cava (IVC) ligation. After 48 hours of ligation, both male and female Nox2-KO mice developed significantly smaller thrombi compared with WT mice (P

Activated neutrophils release neutrophil extracellular traps (NETs) via a pathway dependent on reactive oxygen species (ROS). NETs are enriched in extracellular DNA and histones, and contribute to venous thrombosis. Nox2-containing NADPH oxidase is a major upstream mediator of ROS-dependent release of NETs. Neutrophils from mice deficient in Nox2 or patients with X-linked chronic granulomatous disease due to Nox2 deficiency do not produce NETs, suggesting a role for Nox2 in venous thrombosis. We hypothesized that mice genetically deficient in Nox2 are protected from venous thrombosis. We studied male and female mice deficient in Nox2 (Nox2-KO) and their wild type (WT) littermates at 10-14 weeks of age (n=7-13/group). Using electron spin resonance and H2C-DCFH-DA staining, we confirmed that neutrophils from Nox2-KO mice do not produce ROS at baseline or upon PMA-activation. Neutrophils from WT mice displayed a dose-dependent release of NETs upon PMA-stimulation (SYTOX green staining using fluorescence microscopy), whereas Nox2-KO mice did not produce NETs. We next examined susceptibility to venous thrombosis in a stasis model of inferior vena cava (IVC) ligation. After 48 hours of ligation, both male and female Nox2-KO mice developed significantly smaller thrombi compared with WT mice (P

Introduction Rural and critical access hospitals rely on the “drip and ship” practice using helicopter emergency medical services (HEMS). But those helicopter flights are an unusual environment with physical factors such as vibration and accelerations that could potentially affect the stability, and pharmacological properties of IV rtPA, an issue that has not been previously addressed. Materials and Methods This was a prospective cohort study of consecutive acute ischemic stroke patients receiving IV rtPA through a Comprehensive Stroke Center from November 2015 to February 2017 to measure the effects of HEMS on the integrity and activity of rtPA by collecting residual medication left in the vial. Clinical Trial Registration http://www.clinicaltrials.gov . NCT02752256 Results A total of 33 patients and rtPA samples were included; 18 patients who presented directly to the Comprehensive Stroke Center emergency department and 15 patients who received rtPA during air ambulance transfer. The median rtPA antigen concentration in the residual medication vial was 3.04 mg/mL (IQR: 1.24-3.87) in the HEMS group and 1.91 mg/mL (IQR: 1.33-2.60) in the controls (P = .168). There were no significant differences in rtPA activity or specific activity between the HEMS and control groups and there was no association between total HEMS flight time on overall rtPA specific activity. Conclusions In summary, this study provides supportive evidence of the lack of a detrimental effect of the HEMS physical environment on the integrity of rtPA, therefore endorsing current drip and ship practices without infusion adjustments.

We read with great interest the recent article by Liu et al1 on the role of phosphoinositide 3-kinase (PI3K) in platelet activation and thrombosis. Liu et al demonstrated that mice lacking platelet-specific VPS34, a PI3K isoform, exhibit diminished platelet activation and aggregation and are protected from accelerated thrombosis. This phenotype was associated with loss of translocation of the p40phox cytoplasmic subunit of NADPH (nicotinamide adenine dinucleotide phosphate) oxidase to the plasma membrane, suggesting that VPS34 may regulate platelet reactive oxygen species (ROS) generation and subsequent platelet activation by mediating assembly of a functional NADPH oxidase. The authors did not directly measure …

Hyperhomocysteinemia is a risk factor for stroke; however the mechanisms by which elevated homocysteine leads to stroke are poorly defined. In a murine model of cystathionine β synthase (CBS) deficiency, we tested the hypothesis that mild to severe elevation in plasma total homocysteine (tHcy) increases susceptibility to cerebral infarction through N-methyl-D-aspartate (NMDA) receptor activation and is associated with altered permeability of the blood brain barrier. We studied male Cbs -/- mice conditionally expressing a zinc-inducible mutated human CBS (I278T) transgene along with Cbs+/- and Cbs+/+ littermates at 10-14 weeks of age. The human transgene was allowed to express only until weaning to overcome the early mortality of Cbs-/- mice. A mild to severe increase in plasma total homocysteine levels (tHcy) were observed in Cbs +/- and Cbs -/- mice (6.1±0.3 and 309±18 μM respectively) compared with Cbs+/+ littermates (3.1±0.6 μM, PCbs +/- and Cbs -/- mice exhibited significant increase in cerebral infarct size following ischemia-reperfusion (12.2±3% in Cbs+/+ mice, vs 35.1±7.7% in Cbs+/- and 27.7±7.8% in Cbs-/- mice, PCbs+/- and Cbs -/- mice (3.8±0.8 and 4.8±0.2 EB/g respectively) compared to Cbs+/+ mice (1.7±0.4 EB/g, PCbs+/- mice from cerebral ischemia reperfusion injury and reduced blood brain barrier permeability in Cbs+/- mice. These data provide evidence that both NMDA receptor activation and blood brain barrier disruption contribute to the mechanism of stroke in hyperhomocysteinemia and that modulation of the NMDA pathway may represent a novel therapeutic target.

In this issue of Blood, Nazir et al demonstrate that activated protein C (aPC) protects from ischemia-reperfusion injury by suppressing the activation of NLRP3 inflammasomes, revealing a novel target of the anti-inflammatory pathway triggered by biased agonism of proteinase-activated receptor 1 (PAR-1).1

SummaryBackground Vatreptacog alfa, a recombinant human factor VIIa (rFVIIa) analog developed to improve the treatment of bleeds in hemophilia patients with inhibitors, differs from native FVIIa by three amino acid substitutions. In a randomized, double-blind, crossover, confirmatory phase III trial (adept™2), 8/72 (11%) hemophilia A or B patients with inhibitors treated for acute bleeds developed anti-drug antibodies (ADAs) to vatreptacog alfa. Objectives To characterize the formation of anti-vatreptacog alfa ADAs in hemophilia patients with inhibitors. Methods/patients This was a post hoc analysis of adept™2. Immunoglobulin isotype determination, specificity analysis of rFVIIa cross-reactive antibodies, epitope mapping of rFVIIa single mutant analogs and pharmacokinetic (PK) profiling were performed to characterize the ADAs. Results Immunoglobulin isotyping indicated that the ADAs were of the immunoglobulin G subtype. In epitope mapping, none of the rFVIIa single mutant analogs (V158D, E296V or M298Q) contained the complete antibody epitope, confirming that the antibodies were specific for vatreptacog alfa. In two patients, for whom PK profiling was performed both before and after the development of ADAs, vatreptacog alfa showed a prolonged elimination phase following ADA development. During the follow-up evaluation, the rFVIIa cross-reactivity disappeared after the last vatreptacog alfa exposure, despite continued exposure to rFVIIa as part of standard care. Conclusions Results from the vatreptacog alfa phase III trial demonstrate that the specific changes made, albeit relatively small, to the FVIIa molecule alter its clinical immunogenicity.

Objective— Cellular fibronectin containing extra domain A (EDA + -FN) is abundant in the arteries of patients with atherosclerosis. Several in vitro studies suggest that EDA + -FN interacts with Toll-like receptor 4 (TLR4). We tested the hypothesis that EDA + -FN exacerbates atherosclerosis through TLR4 in a clinically relevant model of atherosclerosis, the apolipoprotein E–deficient ( Apoe −/− ) mouse. Approach and Results— The extent of atherosclerosis was evaluated in whole aortae and cross sections of the aortic sinus in male and female EDA −/− Apoe −/− mice (which lack EDA + -FN), EDA fl/fl Apoe −/− mice (which constitutively express EDA + -FN), and control Apoe −/− mice fed a high-fat Western diet for 14 weeks. Irrespective of sex, EDA fl/fl Apoe −/− mice exhibited a 2-fold increase in atherosclerotic lesions (aorta and aortic sinus) and macrophage content within plaques, whereas EDA −/− Apoe −/− mice exhibited reduced atherosclerotic lesions ( P Apoe −/− , n=10–12 mice/group), although cholesterol and triglyceride levels and circulating leukocytes were similar. Genetic ablation of TLR4 partially reversed atherosclerosis exacerbation in EDA fl/fl Apoe −/− mice ( P EDA −/− Apoe −/− mice. Purified cellular FN, which contains EDA, potentiated dose-dependent NFκB-mediated inflammation (increased phospho-NFκB p65/NFκB p65, tumor necrosis factor-α, and interleukin-1β) in bone marrow–derived macrophages from EDA −/− Apoe −/− mice but not from EDA −/− TLR4 −/− Apoe −/− mice. Finally, using immunohistochemistry, we provide evidence for the first time that EDA + -FN colocalizes with macrophage TLR4 in murine aortic lesions and human coronary artery atherosclerotic plaques. Conclusions— Our findings reveal that TLR4 signaling contributes to EDA + -FN–mediated exacerbation of atherosclerosis. We suggest that EDA + -FN could be a therapeutic target in atherosclerosis.

Objective— Clinical evidence suggests an association between oxidative stress and vascular disease, and in vitro studies have demonstrated that reactive oxygen species can have prothrombotic effects on vascular and blood cells. It remains unclear, however, whether elevated levels of reactive oxygen species accelerate susceptibility to experimental thrombosis in vivo. Approach and Results— Using a murine model with genetic deficiency in superoxide dismutase-1 (SOD1), we measured susceptibility to carotid artery thrombosis in response to photochemical injury. We found that SOD1-deficient ( Sod1 −/− ) mice formed stable arterial occlusions significantly faster than wild-type ( Sod1 +/+ ) mice ( P Sod1 −/− mice also developed significantly larger venous thrombi than Sod1 +/+ mice after inferior vena cava ligation ( P Sod1 −/− mice ( P Sod1 +/+ mice), and generation of activated protein C in response to infusion of thrombin in vivo was decreased in Sod1 −/− mice ( P Sod1 +/+ mice). SOD1 deficiency had no effect on the expression of thrombomodulin, endothelial protein C receptor, or tissue factor in lung or levels of protein C in plasma. Exposure of human thrombomodulin to superoxide in vitro caused oxidation of multiple methionine residues, including critical methionine 388, and a 40% decrease in thrombomodulin-dependent activation of protein C ( P P Conclusions— SOD prevents thrombomodulin methionine oxidation, promotes protein C activation, and protects against arterial and venous thrombosis in mice.