Aging-related Alterations in mTOR Signaling Promote Platelet Hyperreactivity and Thrombosis.

BACKGROUND AND OBJECTIVES: Aging is an independent risk factor for the development of cardiovascular, thrombotic and other chronic diseases. However, mechanisms of platelet hyperactivation in aging remain poorly understood. Here, we examine whether and how aging alters intracellular signaling in platelets to support platelet hyperactivity and thrombosis. METHODS: Quantitative mass spectrometry with tandem mass tag (TMT) labeling systematically measured protein phosphorylation in platelets from healthy aged (>65 years) and young human (<45 years) subjects. The role of platelet mTOR in aging-induced platelet hyperreactivity was assessed using pharmacological mTOR inhibition and a platelet-specific mTOR-deficient mouse model (mTORplt-/-). RESULTS: Quantitative phosphoproteomics uncovered differential site-specific protein phosphorylation within mTOR, Rho GTPase and MAPK pathways in platelets from aged donors. Western blot confirmed constitutive activation of the mTOR pathway in platelets from both aged humans and mice, which was associated with increased aggregation compared to young controls. Inhibition of mTOR either with Torin 1 in aged humans, or genetic deletion in aged mice, reversed platelet hyperreactivity. In a collagen-epinephrine pulmonary thrombosis model, aged wild-type (mTORplt+/+) mice succumbed significantly faster compared to young controls, while time to death of aged mTORplt-/- mice was similar to young mTORplt+/+ mice. Mechanistically, we noted increased Rac1 activation and levels of mitochondrial reactive oxygen species in resting platelets from aged mice, as well as increased p38 phosphorylation upstream of thromboxane generation following agonist stimulation. CONCLUSION: Aging-related changes in mTOR phosphorylation enhance Rac1 and p38 activation, to enhance thromboxane generation, platelet hyperactivity and thrombosis.

Heterozygous Eif4nif1 Stop Gain Mice Replicate the Primary Ovarian Insufficiency Phenotype in Women.

We created the c.1286C>G stop-gain mutation found in a family with primary ovarian insufficiency (POI) at age 30 years. The Eif4enif1 C57/Bl6 transgenic mouse model contained a floxed exon 10-19 cassette with a conditional knock-in cassette containing the c.1286C>G stop-gain mutation in exon 10. The hybrid offspring of CMV- Cre mice with Eif4enif1 WT/flx mice were designated Eif4enif1 WT/ Δ for simplicity. A subset of female heterozygotes ( Eif4enif1 WT/ Δ ) had no litters. In those with litters, the final litter was earlier (5.4±2.6 vs. 10.5±0.7 months; p=0.02). Heterozygous breeding pair ( Eif4enif1 WT/ Δ x Eif4enif1 WT/ Δ ) litter size was 60% of WT litter size (3.9±2.0 vs. 6.5±3.0 pups/litter; p <0.001). The genotypes were 35% Eif4enif1 WT/flx and 65% Eif4enif1 WT/ Δ , with no homozygotes. Homozygote embryos did not develop beyond the 4-8 cell stage. The number of follicles in ovaries from Eif4enif1 WT/ Δ mice was lower starting at the primordial (499±290 vs. 1445±381) and primary follicle stage (1069±346 vs. 1450±193) on day 10 (p<0.05). The preantral follicle number was lower starting on day 21 (213±86 vs. 522±227; p<0.01). Examination of ribosome protected mRNAs (RPR) demonstrated altered mRNA expression. The Eif4enif1 stop-gain mice replicate the POI phenotype in women. The unique mouse model provides a platform to study regulation of protein translation across oocyte and embryo development in mammals.

Differences in testing for drugs of abuse amongst racial and ethnic groups at children's hospitals.

OBJECTIVES: Racial and ethnic differences in drug testing have been described among adults and newborns. Less is known regarding testing patterns among children and adolescents. We sought to describe the association between race and ethnicity and drug testing at US children's hospitals. We hypothesized that non-Hispanic White children undergo drug testing less often than children from other groups. METHODS: We conducted a retrospective cohort study of emergency department (ED)-only encounters and hospitalizations for children diagnosed with a condition for which drug testing may be indicated (abuse or neglect, burns, malnutrition, head injury, vomiting, altered mental status or syncope, psychiatric, self-harm, and seizure) at 41 children's hospitals participating in the Pediatric Health Information System during 2018 and 2021. We compared drug testing rates among (non-Hispanic) Asian, (non-Hispanic) Black, Hispanic, and (non-Hispanic) White children overall, by condition and patient cohort (ED-only vs. hospitalized) and across hospitals. RESULTS: Among 920,755 encounters, 13.6% underwent drug testing. Black children were tested at significantly higher rates overall (adjusted odds ratio [aOR]: 1.18; 1.05-1.33) than White children. Black-White testing differences were observed in the hospitalized cohort (aOR: 1.42; 1.18-1.69) but not among ED-only encounters (aOR: 1.07; 0.92-1.26). Asian, Hispanic, and White children underwent testing at similar rates. Testing varied by diagnosis and across hospitals. CONCLUSIONS: Hospitalized Black children were more likely than White children to undergo drug testing at US children's hospitals, though this varied by diagnosis and hospital. Our results support efforts to better understand and address healthcare disparities, including the contributions of implicit bias and structural racism.

Standardised Measurements for Monitoring and Comparing Multiphoton Microscope Systems.

The goal of this protocol is to enable better characterisation of multiphoton microscopy hardware across a large user base. The scope of this protocol is purposefully limited to focus on hardware, touching on software and data analysis routines only where relevant. The intended audiences are scientists using and building multiphoton microscopes in their laboratories. The goal is that any scientist, not only those with optical expertise, can test whether their multiphoton microscope is performing well and producing consistent data over the lifetime of their system.

Immunothrombosis in neurovascular disease.

A State of the Art lecture titled "Immunothrombosis in Neurovascular Diseases" was presented at the International Society on Thrombosis and Haemostasis Congress in 2023. Despite significant clinical advancements in stroke therapy, stroke remains a prominent contributor to both mortality and disability worldwide. Brain injury resulting from an ischemic stroke is a dynamic process that unfolds over time. Initially, an infarct core forms due to the abrupt and substantial blockage of blood flow. In the subsequent hours to days, the surrounding tissue undergoes gradual deterioration, primarily driven by sustained hypoperfusion, programmed cell death, and inflammation. While anti-inflammatory strategies have proven highly effective in experimental models of stroke, their successful translation to clinical use has proven challenging. To overcome this translational hurdle, a better understanding of the distinct immune response driving ischemic stroke brain injury is needed. In this review article, we give an overview of current knowledge regarding the immune response in ischemic stroke and the contribution of immunothrombosis to this process. We discuss therapeutic approaches to overcome detrimental immunothrombosis in ischemic stroke and how these can be extrapolated to other neurovascular diseases, such as Alzheimer's disease and multiple sclerosis. Finally, we summarize relevant new data on this topic presented during the 2023 International Society on Thrombosis and Haemostasis Congress.

Reciprocal stabilization of coagulation factor XIII-A and -B subunits is a determinant of plasma FXIII concentration.

ABSTRACT: Transglutaminase factor XIII (FXIII) is essential for hemostasis, wound healing, and pregnancy maintenance. Plasma FXIII is composed of A and B subunit dimers synthesized in cells of hematopoietic origin and hepatocytes, respectively. The subunits associate tightly in circulation as FXIII-A2B2. FXIII-B2 stabilizes the (pro)active site-containing FXIII-A subunits. Interestingly, people with genetic FXIII-A deficiency have decreased FXIII-B2, and therapeutic infusion of recombinant FXIII-A2 (rFXIII-A2) increases FXIII-B2, suggesting FXIII-A regulates FXIII-B secretion, production, and/or clearance. We analyzed humans and mice with genetic FXIII-A deficiency and developed a mouse model of rFXIII-A2 infusion to define mechanisms mediating plasma FXIII-B levels. Like humans with FXIII-A deficiency, mice with genetic FXIII-A deficiency had reduced circulating FXIII-B2, and infusion of FXIII-A2 increased FXIII-B2. FXIII-A-deficient mice had normal hepatic function and did not store FXIII-B in liver, indicating FXIII-A does not mediate FXIII-B secretion. Transcriptional analysis and polysome profiling indicated similar F13b levels and ribosome occupancy in FXIII-A-sufficient and -deficient mice and in FXIII-A-deficient mice infused with rFXIII-A2, indicating FXIII-A does not induce de novo FXIII-B synthesis. Unexpectedly, pharmacokinetic/pharmacodynamic modeling of FXIII-B antigen after rFXIII-A2 infusion in humans and mice suggested FXIII-A2 slows FXIII-B2 loss from plasma. Accordingly, comparison of free FXIII-B2 vs FXIII-A2-complexed FXIII-B2 (FXIII-A2B2) infused into mice revealed faster clearance of free FXIII-B2. These data show FXIII-A2 prevents FXIII-B2 loss from circulation and establish the mechanism underlying FXIII-B2 behavior in FXIII-A deficiency and during rFXIII-A2 therapy. Our findings reveal a unique, reciprocal relationship between independently synthesized subunits that mediate an essential hemostatic protein in circulation. This trial was registered at www.ClinicalTrials.com as #NCT00978380.

Mitofusin-2 Regulates Platelet Mitochondria and Function.

BACKGROUND: Single-nucleotide polymorphisms linked with the rs1474868 T allele (MFN2 [mitofusin-2] T/T) in the human mitochondrial fusion protein MFN2 gene are associated with reduced platelet MFN2 RNA expression and platelet counts. This study investigates the impact of MFN2 on megakaryocyte and platelet biology. METHODS: Mice with megakaryocyte/platelet deletion of Mfn2 (Mfn2-/- [Mfn2 conditional knockout]) were generated using Pf4-Cre crossed with floxed Mfn2 mice. Human megakaryocytes were generated from cord blood and platelets isolated from healthy subjects genotyped for rs1474868. Ex vivo approaches assessed mitochondrial morphology, function, and platelet activation responses. In vivo measurements included endogenous/transfused platelet life span, tail bleed time, transient middle cerebral artery occlusion, and pulmonary vascular permeability/hemorrhage following lipopolysaccharide-induced acute lung injury. RESULTS: Mitochondria was more fragmented in megakaryocytes derived from Mfn2-/- mice and from human cord blood with MFN2 T/T genotype compared with control megakaryocytes. Human resting platelets of MFN2 T/T genotype had reduced MFN2 protein, diminished mitochondrial membrane potential, and an increased rate of phosphatidylserine exposure during ex vivo culture. Platelet counts and platelet life span were reduced in Mfn2-/- mice accompanied by an increased rate of phosphatidylserine exposure in resting platelets, especially aged platelets, during ex vivo culture. Mfn2-/- also decreased platelet mitochondrial membrane potential (basal) and activated mitochondrial oxygen consumption rate, reactive oxygen species generation, calcium flux, platelet-neutrophil aggregate formation, and phosphatidylserine exposure following dual agonist activation. Ultimately, Mfn2-/- mice showed prolonged tail bleed times, decreased ischemic stroke infarct size after cerebral ischemia-reperfusion, and exacerbated pulmonary inflammatory hemorrhage following lipopolysaccharide-induced acute lung injury. Analysis of MFN2 SNPs in the iSPAAR study (Identification of SNPs Predisposing to Altered ALI Risk) identified a significant association between MFN2 and 28-day mortality in patients with acute respiratory distress syndrome. CONCLUSIONS: Mfn2 preserves mitochondrial phenotypes in megakaryocytes and platelets and influences platelet life span, function, and outcomes of stroke and lung injury.

Renin-Angiotensin System Components and Arachidonic Acid Metabolites as Biomarkers of COVID-19.

Through the ACE2, a main enzyme of the renin-angiotensin system (RAS), SARS-CoV-2 gains access into the cell, resulting in different complications which may extend beyond the RAS and impact the Arachidonic Acid (ArA) pathway. The contribution of the RAS through ArA pathways metabolites in the pathogenesis of COVID-19 is unknown. We investigated whether RAS components and ArA metabolites can be considered biomarkers of COVID-19. We measured the plasma levels of RAS and ArA metabolites using an LC-MS/MS. Results indicate that Ang 1-7 levels were significantly lower, whereas Ang II levels were higher in the COVID-19 patients than in healthy control individuals. The ratio of Ang 1-7/Ang II as an indicator of the RAS classical and protective arms balance was dramatically lower in COVID-19 patients. There was no significant increase in inflammatory 19-HETE and 20-HETE levels. The concentration of EETs was significantly increased in COVID-19 patients, whereas the DHETs concentration was repressed. Their plasma levels were correlated with Ang II concentration in COVID-19 patients. In conclusion, evaluating the RAS and ArA pathway biomarkers could provide helpful information for the early detection of high-risk groups, avoid delayed medical attention, facilitate resource allocation, and improve patient clinical outcomes to prevent long COVID incidence.

The predominant PAR4 variant in individuals of African ancestry worsens murine and human stroke outcomes.

Protease-activated receptor 4 (PAR4) (gene F2RL3) harbors a functional dimorphism, rs773902 A/G (encoding Thr120/Ala120, respectively) and is associated with greater platelet aggregation. The A allele frequency is more common in Black individuals, and Black individuals have a higher incidence of ischemic stroke than White individuals. However, it is not known whether the A allele is responsible for worse stroke outcomes. To directly test the in vivo effect of this variant on stroke, we generated mice in which F2rl3 was replaced by F2RL3, thereby expressing human PAR4 (hPAR4) with either Thr120 or Ala120. Compared with hPAR4 Ala120 mice, hPAR4 Thr120 mice had worse stroke outcomes, mediated in part by enhanced platelet activation and platelet-neutrophil interactions. Analyses of 7,620 Black subjects with 487 incident ischemic strokes demonstrated the AA genotype was a risk for incident ischemic stroke and worse functional outcomes. In humanized mice, ticagrelor with or without aspirin improved stroke outcomes in hPAR4 Ala120 mice, but not in hPAR4 Thr120 mice. P selectin blockade improved stroke outcomes and reduced platelet-neutrophil interactions in hPAR4 Thr120 mice. Our results may explain some of the racial disparity in stroke and support the need for studies of nonstandard antiplatelet therapies for patients expressing PAR4 Thr120.

Platelet mitochondria: the mighty few.

PURPOSE OF REVIEW: Platelet mitochondrial dysfunction is both caused by, as well as a source of oxidative stress. Oxidative stress is a key hallmark of metabolic disorders such as dyslipidemia and diabetes, which are known to have higher risks for thrombotic complications. RECENT FINDINGS: Increasing evidence supports a critical role for platelet mitochondria beyond energy production and apoptosis. Mitochondria are key regulators of reactive oxygen species and procoagulant platelets, which both contribute to pathological thrombosis. Studies targeting platelet mitochondrial pathways have reported promising results suggesting antithrombotic effects with limited impact on hemostasis in animal models. SUMMARY: Targeting platelet mitochondria holds promise for the reduction of thrombotic complications in patients with metabolic disorders. Future studies should aim at validating these preclinical findings and translate them to the clinic.

Safety and predictors of the success of lumbar punctures performed by a medicine procedure service.

BACKGROUND: Medicine procedure services (MPS) increasingly perform bedside procedures, including lumbar punctures (LPs). Success rates and factors associated with LP success performed by MPS have not been well described. OBJECTIVE: We identified patients undergoing LP by an MPS September 2015 to December 2020. We identified demographic and clinical factors, including patient position, body mass index (BMI), use of ultrasound, and trainee participation. We performed multivariable analysis to identify factors associated with LP success and complications. MAIN OUTCOME AND MEASURES: We identified 1065 LPs among 844 patients. Trainees participated in 82.2%; ultrasound guidance was used in 76.7% of LPs. The overall success rate was 81.3% with 7.8% minor and 0.1% major complications. A minority of LPs were referred to radiology (15.2%) or were traumatic (11.1%). In multivariable analysis, BMI > 30 kg/m2 (odds ratio [OR] 0.32, 95% confidence interval [CI] 0.21-0.48), prior spinal surgery (OR 0.50, 95% CI 0.26-0.87), and Black race (OR 0.62, 95% CI 0.41-0.95) were associated with decreased odds of successful LP; trainee participation (OR 2.49, 95% CI 1.51-4.12) was associated with increased odds. Ultrasound guidance (OR 0.53, 95% CI 0.31-0.89) was associated with lower odds of traumatic LP. RESULTS: In a large cohort of patients undergoing LP by an MPS, we identified high success and low complication rates. Trainee participation was associated with increased odds of success, while obesity, prior spinal surgery, and Black race were associated with decreased odds of success. Ultrasound guidance was associated with lower odds of a traumatic LP. Our data may help proceduralists in planning and assist in shared decision-making.

Corrigendum: Neonatal NET-Inhibitory Factor improves survival in the cecal ligation and puncture model of polymicrobial sepsis by inhibiting neutrophil extracellular traps.

[This corrects the article DOI: 10.3389/fimmu.2022.1046574.].

Tissue factor activity of small and large extracellular vesicles in different diseases.

Background: Circulating procoagulant extracellular vesicles (EVs) are increased in diseases, such as cancer, sepsis, and COVID-19. EV tissue factor (TF) activity is associated with disseminated intravascular coagulation in sepsis and venous thrombosis in patients with pancreatic cancer and COVID-19. EVs are commonly isolated by centrifugation at ∼20,000 g. Objectives: In this study, we analyzed the TF activity of 2 EV populations enriched for large and small EVs in patients with either sepsis, pancreatic cancer, or COVID-19. Methods: EVs were isolated from plasma by sequential centrifugation at 20,000 g (large EVs, LEVs) and then 100,000 g (small EVs, SEVs). We analyzed EVs from plasma prepared from whole blood samples from healthy individuals with or without lipopolysaccharide (LPS) stimulation as well as EVs from plasma samples from patients with either sepsis, pancreatic cancer, or COVID-19. TF-dependent (EV-TF activity) and TF-independent factor Xa (FXa) generation of the EVs was measured. Results: LPS increased EV-TF activity in LEVs but not SEVs. Similarly, in 2 patients with sepsis who had EV-TF activity above the background of the assay we observed EV-TF activity in LEVs but not SEVs. Patients with pancreatic cancer or COVID-19 had circulating EV-TF activity in both LEVs and SEVs. Conclusion: We recommend that EVs are isolated from plasma from patients by centrifugation at 100,000 g rather than 20,000 g to obtain a more accurate measure of levels of circulating EV-TF activity.

Neutrophil extracellular trap inhibition improves survival in neonatal mouse infectious peritonitis.

BACKGROUND: Treatment of neonatal peritonitis and sepsis is challenging. Following infection, neutrophils elaborate neutrophil extracellular traps (NETs)-extracellular lattices of decondensed chromatin decorated with antimicrobial proteins. NETs, however, can augment pathogenic inflammation causing collateral damage. We hypothesized that NET inhibition would improve survival in experimental neonatal infectious peritonitis. METHODS: We induced peritonitis in 7 to 10-day-old mice by intraperitoneal injection with cecal slurry. We targeted NETs by treating mice with neonatal NET-Inhibitory Factor (nNIF), an endogenous NET-inhibitor; Cl-amidine, a PAD4 inhibitor; DNase I, a NET degrading enzyme, or meropenem (an antibiotic). We determined peritoneal NET and cytokine levels and circulating platelet-neutrophil aggregates. Survival from peritonitis was followed for 6 days. RESULTS: nNIF, Cl-amidine, and DNase I decreased peritoneal NET formation and inflammatory cytokine levels at 24 h compared to controls. nNIF, Cl-amidine, and DNase I decreased circulating platelet-neutrophil aggregates, and NET-targeting treatments significantly increased survival from infectious peritonitis compared to controls. Finally, nNIF administration significantly improved survival in mice treated with sub-optimal doses of meropenem even when treatment was delayed until 2 h after peritonitis induction. CONCLUSIONS: NET inhibition improves survival in experimental neonatal infectious peritonitis, suggesting that NETs participate pathogenically in neonatal peritonitis and sepsis. IMPACT: 1. Neutrophil extracellular trap formation participates pathogenically in experimental neonatal infectious peritonitis. 2. NET-targeting strategies improve outcomes in a translational model of neonatal infectious peritonitis. 3. NET inhibition represents a potential target for drug development in neonatal sepsis and infectious peritonitis.

Investigation of Neutrophil Extracellular Traps as Potential Mediators in the Pathogenesis of Non-Acute Subdural Hematomas: A Pilot Study.

Non-acute subdural hematomas (NASHs) are a cause of significant morbidity and mortality, particularly with recurrences. Although recurrence is believed to involve a disordered neuroinflammatory cascade involving vascular endothelial growth factor (VEGF), this pathway has yet to be completely elucidated. Neutrophil extracellular traps (NETs) are key factors that promote inflammation/apoptosis and can be induced by VEGF. We investigated whether NETs are present in NASH membranes, quantified NET concentrations, and examined whether NET and VEGF levels are correlated in NASHs. Samples from patients undergoing NASH evacuation were collected during surgery and postoperatively at 24 and 48 h. Fluid samples and NASH membranes were analyzed for levels of VEGF, NETs, and platelet activation. NASH samples contained numerous neutrophils positive for NET formation. Myeloperoxidase-DNA complexes (a marker of NETs) remained elevated 48 h postoperatively (1.06 ± 0.22 day 0, 0.72 ± 0.23 day 1, and 0.83 ± 0.33 day 2). VEGF was also elevated in NASHs (7.08 ± 0.98 ng/mL day 0, 3.40 ± 0.68 ng/mL day 1, and 6.05 ± 1.8 ng/mL day 2). VEGF levels were significantly correlated with myeloperoxidase-DNA levels. These results show that NETs are increasing in NASH, a finding that was previously unknown. The strong correlation between NET and VEGF levels indicates that VEGF may be an important mediator of NET-related inflammation in NASH.