Pretreatment with a dual antiplatelet and anticoagulant (APAC) reduces ischemia-reperfusion injury in a mouse model of temporary middle cerebral artery occlusion-implications for neurovascular procedures.

BACKGROUND: Several neurovascular procedures require temporary occlusion of cerebral arteries, leading to ischemia of unpredictable length, occasionally causing brain infarction. Experimental models of cerebral ischemia-reperfusion injury have established that platelet adhesion and coagulation play detrimental roles in reperfusion injury following transient cerebral ischemia. Therefore, in a model of cerebral ischemia-reperfusion injury (IRI), we investigated the therapeutic potential of a dual antiplatelet and anticoagulant (APAC) heparin proteoglycan mimetic which is able to bind to vascular injury sites. METHODS: Brain ischemia was induced in mice by transient occlusion of the right middle cerebral artery for 60 min. APAC, unfractionated heparin (UFH) (both at heparin equivalent doses of 0.5 mg/kg), or vehicle was intravenously administered 10 min before or 60 min after the start of ischemia. At 24 h later, mice were scored for their neurological and motor behavior, and brain damage was quantified. RESULTS: Both APAC and UFH administered before the onset of ischemia reduced brain injury. APAC and UFH pretreated mice had better neurological and motor functions (p < 0.05 and p < 0.01, respectively) and had significantly reduced cerebral infarct sizes (p < 0.01 and p < 0.001, respectively) at 24 h after transient occlusion compared with vehicle-treated mice. Importantly, no macroscopic bleeding complications were observed in either APAC- or UFH-treated animals. However, when APAC or UFH was administered 60 min after the start of ischemia, the therapeutic effect was lost, but without hemorrhaging either. CONCLUSIONS: Pretreatment with APAC or UFH was safe and effective in reducing brain injury in a model of cerebral ischemia induced by transient middle cerebral artery occlusion. Further studies on the use of APAC to limit ischemic injury during temporary occlusion in neurovascular procedures are indicated.

Involvement of peptidylarginine deiminase 4 in eosinophil extracellular trap formation and contribution to citrullinated histone signal in thrombi.

BACKGROUND: Extracellular traps formed by neutrophils (NETs) and eosinophils (EETs) have been described in coronary thrombi, contributing to thrombus stability. A key mechanism during NET formation is histone modification by the enzyme PAD4. Citrullinated histones, the product of PAD4 activity, are often attributed to neutrophils. Eosinophils also express high levels of PAD4. OBJECTIVES: We aimed to explore the contribution of PAD4 to EET formation. METHODS: We performed immunohistological analyses on thrombi, including a large, intact, and eosinophil-containing thrombus retrieved from the right coronary artery using an aspiration catheter and stroke thrombi from thrombectomy retrieval. We studied eosinophils for their capability to form PAD4-dependent EETs in response to strong ET-inducing agonists as well as activated platelets and bacteria. RESULTS: Histopathology and immunofluorescence microscopy identified a coronary thrombus rich in platelets and neutrophils, with distinct areas containing von Willebrand factor and citrullinated histone H3 (H3Cit). Eosinophils were also identified in leukocyte-rich areas. The majority of the H3Cit+ signal colocalized with myeloperoxidase, but some colocalized with eosinophil peroxidase, indicating EETs. Eosinophils isolated from healthy volunteers produced H3Cit+ EETs, indicating an involvement of PAD4 activity. The selective PAD4 inhibitor GSK484 blocked this process, supporting PAD4 dependence of H3Cit+ EET release. Citrullinated histones were also present in EETs produced in response to live Staphylococci. However, limited evidence for EETs was found in mouse models of venous thrombosis or infective endocarditis. CONCLUSION: As in NETosis, PAD4 can catalyze the formation of EETs. Inhibition of PAD4 decreases EET formation, supporting the future utility of PAD4 inhibitors as possible antithrombotic agents.

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.

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.

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.

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.

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.].

Endovascular mechanical thrombectomy in a child with COVID-19: Clot analysis reveals a novel pathway in the neuroinflammatory cascade resulting in large-vessel occlusion.

Large-vessel occlusion is rare in children, but its results can be devastating and may lead to recurrent strokes, persistent neurological deficits, and decreased quality of life. Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has yielded extrapulmonary effects and multiorgan diseases, many of which are neurological manifestations. There is a paucity of literature in pediatric patients about large-vessel occlusion in the setting of COVID-19 infection. We discuss a nine-year-old child who presented with a left middle cerebral artery occlusion and underwent revascularization with a Thrombolysis in Cerebral Infarction grade 3 reperfusion approximately three weeks after COVID-19 diagnosis. The patient harbored concerning signs and symptoms of multisystem inflammatory syndrome in children. This case emphasizes the importance of recognizing SARS-CoV-2 and the propensity for thrombosis in a delayed fashion, which can lead to severe stroke in young people.

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.

IFITM3 regulates fibrinogen endocytosis and platelet reactivity in nonviral sepsis.

Platelets and megakaryocytes are critical players in immune responses. Recent reports suggest infection and inflammation alter the megakaryocyte and platelet transcriptome to induce altered platelet reactivity. We determined whether nonviral sepsis induces differential platelet gene expression and reactivity. Nonviral sepsis upregulated IFN-induced transmembrane protein 3 (IFITM3), an IFN-responsive gene that restricts viral replication. As IFITM3 has been linked to clathrin-mediated endocytosis, we determined whether IFITM3 promoted endocytosis of α-granule proteins. IFN stimulation enhanced fibrinogen endocytosis in megakaryocytes and platelets from Ifitm+/+ mice, but not Ifitm-/- mice. IFITM3 overexpression or deletion in megakaryocytes demonstrated IFITM3 was necessary and sufficient to regulate fibrinogen endocytosis. Mechanistically, IFITM3 interacted with clathrin and αIIb and altered their plasma membrane localization into lipid rafts. In vivo IFN administration increased fibrinogen endocytosis, platelet reactivity, and thrombosis in an IFITM-dependent manner. In contrast, Ifitm-/- mice were completely rescued from IFN-induced platelet hyperreactivity and thrombosis. During murine sepsis, platelets from Ifitm+/+ mice demonstrated increased fibrinogen content and platelet reactivity, which was dependent on IFN-α and IFITMs. Platelets from patients with nonviral sepsis had increases in platelet IFITM3 expression, fibrinogen content, and hyperreactivity. These data identify IFITM3 as a regulator of platelet endocytosis, hyperreactivity, and thrombosis during inflammatory stress.

MAPK-interacting kinase 1 regulates platelet production, activation, and thrombosis.

The MAPK-interacting kinase (Mnk) family includes Mnk1 and Mnk2, which are phosphorylated and activated in response to extracellular stimuli. Mnk1 contributes to cellular responses by regulating messenger RNA (mRNA) translation, and mRNA translation influences platelet production and function. However, the role of Mnk1 in megakaryocytes and platelets has not previously been studied. The present study investigated Mnk1 in megakaryocytes and platelets using both pharmacological and genetic approaches. We demonstrate that Mnk1, but not Mnk2, is expressed and active in human and murine megakaryocytes and platelets. Stimulating human and murine megakaryocytes and platelets induced Mnk1 activation and phosphorylation of eIF4E, a downstream target of activated Mnk1 that triggers mRNA translation. Mnk1 inhibition or deletion significantly diminished protein synthesis in megakaryocytes as measured by polysome profiling and [35S]-methionine incorporation assays. Depletion of Mnk1 also reduced megakaryocyte ploidy and proplatelet forming megakaryocytes in vitro and resulted in thrombocytopenia. However, Mnk1 deletion did not affect the half-life of circulating platelets. Platelets from Mnk1 knockout mice exhibited reduced platelet aggregation, α granule secretion, and integrin αIIbß3 activation. Ribosomal footprint sequencing indicated that Mnk1 regulates the translation of Pla2g4a mRNA (which encodes cPLA2) in megakaryocytes. Consistent with this, Mnk1 ablation reduced cPLA2 activity and thromboxane generation in platelets and megakaryocytes. In vivo, Mnk1 ablation protected against platelet-dependent thromboembolism. These results provide previously unrecognized evidence that Mnk1 regulates mRNA translation and cellular activation in platelets and megakaryocytes, endomitosis and thrombopoiesis, and thrombosis.

Procoagulant platelets: novel players in thromboinflammation.

Platelets play a key role in maintaining hemostasis. However, dysregulated platelet activation can lead to pathological thrombosis or bleeding. Once a platelet gets activated, it will either become an aggregatory platelet or eventually a procoagulant platelet with both types playing distinct roles in thrombosis and hemostasis. Although aggregatory platelets have been extensively studied, procoagulant platelets have only recently come into the spotlight. Procoagulant platelets are a subpopulation of highly activated platelets that express phosphatidylserine and P-selectin on their surface, allowing for coagulation factors to bind and thrombin to be generated. In recent years, novel roles for procoagulant platelets have been identified and they have increasingly been implicated in thromboinflammatory diseases. Here, we provide an up-to-date review on the mechanisms resulting in the formation of procoagulant platelets and how they contribute to hemostasis, thrombosis, and thromboinflammation.

Neutrophil extracellular traps regulate ischemic stroke brain injury.

Ischemic stroke prompts a strong inflammatory response, which is associated with exacerbated outcomes. In this study, we investigated mechanistic regulators of neutrophil extracellular trap (NET) formation in stroke and whether they contribute to stroke outcomes. NET-forming neutrophils were found throughout brain tissue of ischemic stroke patients, and elevated plasma NET biomarkers correlated with worse stroke outcomes. Additionally, we observed increased plasma and platelet surface-expressed high-mobility group box 1 (HMGB1) in stroke patients. Mechanistically, platelets were identified as the critical source of HMGB1 that caused NETs in the acute phase of stroke. Depletion of platelets or platelet-specific knockout of HMGB1 significantly reduced plasma HMGB1 and NET levels after stroke, and greatly improved stroke outcomes. We subsequently investigated the therapeutic potential of neonatal NET-inhibitory factor (nNIF) in stroke. Mice treated with nNIF had smaller brain infarcts, improved long-term neurological and motor function, and enhanced survival after stroke. nNIF specifically blocked NET formation without affecting neutrophil recruitment after stroke. Importantly, nNIF also improved stroke outcomes in diabetic and aged mice and was still effective when given 1 hour after stroke onset. These results support a pathological role for NETs in ischemic stroke and warrant further investigation of nNIF for stroke therapy.

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

Introduction: Neutrophil extracellular traps (NETs) clear pathogens but may contribute Q8 pathogenically to host inflammatory tissue damage during sepsis. Innovative therapeutic agents targeting NET formation and their potentially harmful collateral effects remain understudied. Methods: We investigated a novel therapeutic agent, neonatal NET-Inhibitory Factor (nNIF), in a mouse model of experimental sepsis - cecal ligation and puncture (CLP). We administered 2 doses of nNIF (1 mg/ kg) or its scrambled peptide control intravenously 4 and 10 hours after CLP treatment and assessed survival, peritoneal fluid and plasma NET formation using the MPO-DNA ELISA, aerobic bacterial colony forming units (CFU) using serial dilution and culture, peritoneal fluid and stool microbiomes using 16S rRNA gene sequencing, and inflammatory cytokine levels using a multiplexed cytokine array. Meropenem (25 mg/kg) treatment served as a clinically relevant treatment for infection. Results: We observed increased 6-day survival rates in nNIF (73%) and meropenem (80%) treated mice compared to controls (0%). nNIF decreased NET formation compared to controls, while meropenem did not impact NET formation. nNIF treatment led to increased peritoneal fluid and plasma bacterial CFUs consistent with loss of NET-mediated extracellular microbial killing, while nNIF treatment alone did not alter the peritoneal fluid and stool microbiomes compared to vehicle-treated CLP mice. nNIF treatment also decreased peritoneal TNF-a inflammatory cytokine levels compared to scrambled peptide control. Furthermore, adjunctive nNIF increased survival in a model of sub-optimal meropenem treatment (90% v 40%) in CLP-treated mice. Discussion: Thus, our data demonstrate that nNIF inhibits NET formation in a translationally relevant mouse model of sepsis, improves survival when given as monotherapy or as an adjuvant with antibiotics, and may play an important protective role in sepsis.

Neutrophil cathepsin G proteolysis of protease-activated receptor 4 generates a novel, functional tethered ligand.

Platelet-neutrophil interactions regulate ischemic vascular injury. Platelets are activated by serine proteases that cleave protease-activated receptor (PAR) amino termini, resulting in an activating tethered ligand. Neutrophils release cathepsin G (CatG) at sites of injury and inflammation, which activates PAR4 but not PAR1, although the molecular mechanism of CatG-induced PAR4 activation is unknown. We show that blockade of the canonical PAR4 thrombin cleavage site did not alter CatG-induced platelet aggregation, suggesting CatG cleaves a different site than thrombin. Mass spectrometry analysis using PAR4 N-terminus peptides revealed CatG cleavage at Ser67-Arg68. A synthetic peptide, RALLLGWVPTR, representing the tethered ligand resulting from CatG proteolyzed PAR4, induced PAR4-dependent calcium flux and greater platelet aggregation than the thrombin-generated GYPGQV peptide. Mutating PAR4 Ser67or Arg68 reduced CatG-induced calcium flux without affecting thrombin-induced calcium flux. Dog platelets, which contain a conserved CatG PAR4 Ser-Arg cleavage site, aggregated in response to human CatG and RALLLGWVPTR, while mouse (Ser-Gln) and rat (Ser-Glu) platelets were unresponsive. Thus, CatG amputates the PAR4 thrombin cleavage site by cleavage at Ser67-Arg68 and activates PAR4 by generating a new functional tethered ligand. These findings support PAR4 as an important CatG signaling receptor and suggest a novel therapeutic approach for blocking platelet-neutrophil-mediated pathophysiologies.