The Platelet Napoleon Complex-Small Cells, but Big Immune Regulatory Functions.

Platelets have dual physiologic roles as both cellular mediators of thrombosis and immune modulatory cells. Historically, the thrombotic function of platelets has received significant research and clinical attention, but emerging research indicates that the immune regulatory roles of platelets may be just as important. We now know that in addition to their role in the acute thrombotic event at the time of myocardial infarction, platelets initiate and accelerate inflammatory processes that are part of the pathogenesis of atherosclerosis and myocardial infarction expansion. Furthermore, it is increasingly apparent from recent studies that platelets impact the pathogenesis of many vascular inflammatory processes such as autoimmune diseases, sepsis, viral infections, and growth and metastasis of many types of tumors. Therefore, we must consider platelets as immune cells that affect all phases of immune responses.

Thrombocytopenia Independently Leads to Changes in Monocyte Immune Function.

BACKGROUND: While platelets have well-studied hemostatic functions, platelets are immune cells that circulate at the interface between the vascular wall and white blood cells. The physiological implications of these constant transient interactions are poorly understood. Activated platelets induce and amplify immune responses, but platelets may also maintain immune homeostasis in healthy conditions, including maintaining vascular integrity and T helper cell differentiation, meaning that platelets are central to both immune responses and immune quiescence. Clinical data have shown an association between low platelet counts (thrombocytopenia) and immune dysfunction in patients with sepsis and extracorporeal membrane oxygenation, further implicating platelets as more holistic immune regulators, but studies of platelet immune functions in nondisease contexts have had limited study. METHODS: We used in vivo models of thrombocytopenia and in vitro models of platelet and monocyte interactions, as well as RNA-seq and ATAC-seq (assay for transposase-accessible chromatin with sequencing), to mechanistically determine how resting platelet and monocyte interactions immune program monocytes. RESULTS: Circulating platelets and monocytes interact in a CD47-dependent manner to regulate monocyte metabolism, histone methylation, and gene expression. Resting platelet-monocyte interactions limit TLR (toll-like receptor) signaling responses in healthy conditions in an innate immune training-like manner. In both human patients with sepsis and mouse sepsis models, thrombocytopenia exacerbated monocyte immune dysfunction, including increased cytokine production. CONCLUSIONS: Thrombocytopenia immune programs monocytes in a manner that may lead to immune dysfunction in the context of sepsis. This is the first demonstration that sterile, endogenous cell interactions between resting platelets and monocytes regulate monocyte metabolism and pathogen responses, demonstrating platelets to be immune rheostats in both health and disease.

Markers of platelet activation foR identification of late onset sEpsis in infaNTs: PARENT study protocol.

BACKGROUND: Newborns are at high risk of sepsis. At present there is no definitive "rule in" blood test for sepsis at the point of clinical concern. A positive blood culture remains the gold standard test for neonatal sepsis, however laboratory markers that correlate prospectively with culture positive sepsis could aid clinicians in making decisions regarding administration of empiric antibiotic therapies. METHODS: This multi-site, prospective observational study will take place in two neonatal intensive care units (National Maternity Hospital and Rotunda Hospital, Dublin). Neonates born at less than 34 weeks will be enroled and informed consent obtained prior to late onset sepsis work up. If at any point subsequently during their neonatal intensive care stay they develop signs and symptoms of possible sepsis requiring blood culture, an additional sodium citrate sample will be obtained. Infants will be categorised into three groups as follows: (i) culture positive sepsis, (ii) culture negative sepsis where an infant receives 5 days of antibiotics (iii) non sepsis. Our primary outcome is to establish if differential platelet/endothelial activation can prospectively identify neonatal culture positive late onset sepsis. TRIAL REGISTRATION NUMBER: NCT05530330 IMPACT: Preterm infants are a high risk group for the development of sepsis which is a major cause of mortality in this population. Platelets have been associated with host response to invasive bacterial infections both in animal models and translational work. A positive blood culture is the gold standard test for neonatal sepsis but can be unreliable due to limited blood sampling in the very low birth weight population. This study hopes to establish if platelet/endothelial associated plasma proteins can prospectively identify late onset neonatal sepsis.

Platelet and Megakaryocyte Roles in Innate and Adaptive Immunity.

Classically, platelets have been described as the cellular blood component that mediates hemostasis and thrombosis. This important platelet function has received significant research attention for >150 years. The immune cell functions of platelets are much less appreciated. Platelets interact with and activate cells of all branches of immunity in response to pathogen exposures and infection, as well as in response to sterile tissue injury. In this review, we focus on innate immune mechanisms of platelet activation, platelet interactions with innate immune cells, as well as the intersection of platelets and adaptive immunity. The immune potential of platelets is dependent in part on their megakaryocyte precursor providing them with the molecular composition to be first responders and immune sentinels in initiating and orchestrating coordinated pathogen immune responses. There is emerging evidence that extramedullary megakaryocytes may be immune differentiated compared with bone marrow megakaryocytes, but the physiological relevance of immunophenotypic differences are just beginning to be explored. These concepts are also discussed in this review. The immune functions of the megakaryocyte/platelet lineage have likely evolved to coordinate the need to repair a vascular breach with the simultaneous need to induce an immune response that may limit pathogen invasion once the blood is exposed to an external environment.

Transfusion of Adult, but Not Neonatal, Platelets Promotes Monocyte Trafficking in Neonatal Mice.

BACKGROUND: Thrombocytopenia is common in preterm neonates. Platelet transfusions are sometimes given to thrombocytopenic neonates with the hope of reducing the bleeding risk, however, there are little clinical data to support this practice, and platelet transfusions may increase the bleeding risk or lead to adverse complications. Our group previously reported that fetal platelets expressed lower levels of immune-related mRNA compared with adult platelets. In this study, we focused on the effects of adult versus neonatal platelets on monocyte immune functions that may have an impact on neonatal immune function and transfusion complications. METHODS: Using RNA sequencing of postnatal day 7 and adult platelets, we determined age-dependent platelet gene expression. Platelets and naive bone marrow-isolated monocytes were cocultured and monocyte phenotypes determined by RNA sequencing and flow cytometry. An in vivo model of platelet transfusion in neonatal thrombocytopenic mice was used in which platelet-deficient TPOR (thrombopoietin receptor) mutant mice were transfused with adult or postnatal day 7 platelets and monocyte phenotypes and trafficking were determined. RESULTS: Adult and neonatal platelets had differential immune molecule expression, including Selp. Monocytes incubated with adult or neonatal mouse platelets had similar inflammatory (Ly6Chi) but different trafficking phenotypes, as defined by CCR2 and CCR5 mRNA and surface expression. Blocking P-sel (P-selectin) interactions with its PSGL-1 (P-sel glycoprotein ligand-1) receptor on monocytes limited the adult platelet-induced monocyte trafficking phenotype, as well as adult platelet-induced monocyte migration in vitro. Similar results were seen in vivo, when thrombocytopenic neonatal mice were transfused with adult or postnatal day 7 platelets; adult platelets increased monocyte CCR2 and CCR5, as well as monocyte chemokine migration, whereas postnatal day 7 platelets did not. CONCLUSIONS: These data provide comparative insights into adult and neonatal platelet transfusion-regulated monocyte functions. The transfusion of adult platelets to neonatal mice was associated with an acute inflammatory and trafficking monocyte phenotype that was platelet P-sel dependent and may have an impact on complications associated with neonatal platelet transfusions.

Platelet MHC class I mediates CD8+ T-cell suppression during sepsis.

Circulating platelets interact with leukocytes to modulate host immune and thrombotic responses. In sepsis, platelet-leukocyte interactions are increased and have been associated with adverse clinical events, including increased platelet-T-cell interactions. Sepsis is associated with reduced CD8+ T-cell numbers and functional responses, but whether platelets regulate CD8+ T-cell responses during sepsis remains unknown. In our current study, we systemically evaluated platelet antigen internalization and presentation through major histocompatibility complex class I (MHC-I) and their effects on antigen-specific CD8+ T cells in sepsis in vivo and ex vivo. We discovered that both human and murine platelets internalize and proteolyze exogenous antigens, generating peptides that are loaded onto MHC-I. The expression of platelet MHC-I, but not platelet MHC-II, is significantly increased in human and murine platelets during sepsis and in human megakaryocytes stimulated with agonists generated systemically during sepsis (eg, interferon-γ and lipopolysaccharide). Upregulation of platelet MHC-I during sepsis increases antigen cross-presentation and interactions with CD8+ T cells in an antigen-specific manner. Using a platelet lineage-specific MHC-I-deficient mouse strain (B2Mf/f-Pf4Cre), we demonstrate that platelet MHC-I regulates antigen-specific CD8+ T-cell proliferation in vitro, as well as the number and functional responses of CD8+ T cells in vivo, during sepsis. Loss of platelet MHC-I reduces sepsis-associated mortality in mice in an antigen-specific setting. These data identify a new mechanism by which platelets, through MHC-I, process and cross-present antigens, engage antigen-specific CD8+ T cells, and regulate CD8+ T-cell numbers, functional responses, and outcomes during sepsis.

ß2M Signals Monocytes Through Non-Canonical TGFß Receptor Signal Transduction.

RATIONALE: Circulating monocytes can have proinflammatory or proreparative phenotypes. The endogenous signaling molecules and pathways that regulate monocyte polarization in vivo are poorly understood. We have shown that platelet-derived ß2M (ß-2 microglobulin) and TGF-ß (transforming growth factor ß) have opposing effects on monocytes by inducing inflammatory and reparative phenotypes, respectively, but each bind and signal through the same receptor. We now define the signaling pathways involved. OBJECTIVE: To determine the molecular mechanisms and signal transduction pathways by which ß2M and TGF-ß regulate monocyte responses both in vitro and in vivo. METHODS AND RESULTS: Wild-type- (WT) and platelet-specific ß2M knockout mice were treated intravenously with either ß2M or TGF-ß to increase plasma concentrations to those in cardiovascular diseases. Elevated plasma ß2M increased proinflammatory monocytes, while increased plasma TGFß increased proreparative monocytes. TGF-ßR (TGF-ß receptor) inhibition blunted monocyte responses to both ß2M and TGF-ß in vivo. Using imaging flow cytometry, we found that ß2M decreased monocyte SMAD2/3 nuclear localization, while TGF-ß promoted SMAD nuclear translocation but decreased noncanonical/inflammatory (JNK [jun kinase] and NF-κB [nuclear factor-κB] nuclear localization). This was confirmed in vitro using both imaging flow cytometry and immunoblots. ß2M, but not TGF-ß, promoted ubiquitination of SMAD3 and SMAD4, that inhibited their nuclear trafficking. Inhibition of ubiquitin ligase activity blocked noncanonical SMAD-independent monocyte signaling and skewed monocytes towards a proreparative monocyte response. CONCLUSIONS: Our findings indicate that elevated plasma ß2M and TGF-ß dichotomously polarize monocytes. Furthermore, these immune molecules share a common receptor but induce SMAD-dependent canonical signaling (TGF-ß) versus noncanonical SMAD-independent signaling (ß2M) in a ubiquitin ligase dependent manner. This work has broad implications as ß2M is increased in several inflammatory conditions, while TGF-ß is increased in fibrotic diseases. Graphic Abstract: A graphic abstract is available for this article.

Effect of Single Housing on Innate Immune Activation in Immunodeficiency Virus-Infected Pigtail Macaques ( Macaca nemestrina ) as a Model of Psychosocial Stress in Acute HIV Infection.

OBJECTIVE: Simian immunodeficiency virus (SIV) infection of macaques recapitulates many aspects of HIV pathogenesis and is similarly affected by both genetic and environmental factors. Psychosocial stress is associated with immune system dysregulation and worse clinical outcomes in people with HIV. This study assessed the impact of single housing, as a model of psychosocial stress, on innate immune responses of pigtailed macaques ( Macaca nemestrina ) during acute SIV infection. METHODS: A retrospective analysis of acute SIV infection of 2- to si6-year-old male pigtailed macaques was performed to compare the innate immune responses of socially ( n = 41) and singly ( n = 35) housed animals. Measures included absolute monocyte count and subsets, and in a subset ( n ≤ 18) platelet counts and activation data. RESULTS: SIV infection resulted in the expected innate immune parameter changes with a modulating effect from housing condition. Monocyte number increased after infection for both groups, driven by classical monocytes (CD14 + CD16 - ), with a greater increase in socially housed animals (227%, p < .001, by day 14 compared with preinoculation time points). Platelet numbers recovered more quickly in the socially housed animals. Platelet activation (P-selectin) increased by 65% ( p = .004) and major histocompatibility complex class I surface expression by 40% ( p = .009) from preinoculation only in socially housed animals, whereas no change in these measures occurred in singly housed animals. CONCLUSIONS: Chronic psychosocial stress produced by single housing may play an immunomodulatory role in the innate immune response to acute retroviral infection. Dysregulated innate immunity could be one of the pathways by which psychosocial stress contributes to immune suppression and increased disease severity in people with HIV.

Sex-Specific Platelet Activation Through Protease-Activated Receptors Reverses in Myocardial Infarction.

OBJECTIVE: The platelet phenotype in certain patients and clinical contexts may differ from healthy conditions. We evaluated platelet activation through specific receptors in healthy men and women, comparing this to patients presenting with ST-segment-elevation myocardial infarction and non-ST-segment-elevation myocardial infarction. Approach and Results: We identified independent predictors of platelet activation through certain receptors and a murine MI model further explored these findings. Platelets from healthy women and female mice are more reactive through PARs (protease-activated receptors) compared with platelets from men and male mice. Multivariate regression analyses revealed male sex and non-ST-segment-elevation myocardial infarction as independent predictors of enhanced PAR1 activation in human platelets. Platelet PAR1 signaling decreased in women and increased in men during MI which was the opposite of what was observed during healthy conditions. Similarly, in mice, thrombin-mediated platelet activation was greater in healthy females compared with males, and lesser in females compared with males at the time of MI. CONCLUSIONS: Sex-specific signaling in platelets seems to be a cross-species phenomenon. The divergent platelet phenotype in males and females at the time of MI suggests a sex-specific antiplatelet drug regimen should be prospectively evaluated.

Estrogen activates endothelial exocytosis.

Estrogen therapy is used to treat patients with post-menopausal symptoms, such as hot flashes and dyspareunia. Estrogen therapy also decreases the risk of fractures from osteoporosis in post-menopausal women. However, estrogen increases the risk of venous thromboembolic events, such as pulmonary embolism, but the pathways through which estrogen increase the risk of thromboembolism is unknown. Here, we show that estrogen elicits endothelial exocytosis, the key step in vascular thrombosis and inflammation. Exogenous 17ß-estradiol (E2) stimulated endothelial exocytosis of Weibel-Palade bodies (WPBs), releasing von Willebrand factor (vWF) and interleukin-8 (IL-8). Conversely, the estrogen antagonist ICI-182,780 interfered with E2-induced endothelial exocytosis. The ERα agonist propyl pyrazole triol (PPT) but not the ERß agonist diarylpropionitrile (DPN) induced vWF release, while ERα silencing counteracted vWF release by E2, suggesting that ERα mediates this effect. Exocytosis triggered by E2 occurred rapidly within 15 min and was not inhibited by either actinomycin D or cycloheximide. On the contrary, it was inhibited by the pre-treatment of U0126 or SB203580, an ERK or a p38 inhibitor, respectively, suggesting that E2-induced endothelial exocytosis is non-genomically mediated by the MAP kinase pathway. Finally, E2 treatment enhanced platelet adhesion to endothelial cells ex vivo, which was interfered with the pre-treatment of ICI-182,780 or U0126. Taken together, our data show that estrogen activates endothelial exocytosis non-genomically through the ERα-MAP kinase pathway. Our data suggest that adverse cardiovascular effects such as vascular inflammation and thrombosis should be considered in patients before menopausal hormone treatment.

The Protective Role of Natriuretic Peptide Receptor 2 against High Salt Injury in the Renal Papilla.

Mutations in natriuretic peptide receptor 2 (Npr2) gene cause a rare form of short-limbed dwarfism, but its physiological effects have not been well studied. Human and mouse genetic data suggest that Npr2 in the kidney plays a role in salt homeostasis. Herein, we described anatomic changes within renal papilla of Npr2 knockout (Npr2-/-) mice. Dramatic reduction was found in diuresis, and albuminuria was evident after administration of 1% NaCl in drinking water in Npr2-/- and heterozygous (Npr2+/-) mice compared with their wild-type (Npr2+/+) littermates. There was indication of renal epithelial damage accompanied by high numbers of red blood cells and inflammatory cells (macrophage surface glycoproteins binding to galectin-3) and an increase of renal epithelial damage marker (T-cell Ig and mucin domain 1) in Npr2-/- mice. Addition of 1% NaCl tended to increase apoptotic cells (cleaved caspase 3) in the renal papilla of Npr2-/- mice. In vitro, genetic silencing of the Npr2 abolished protective effects of C-type natriuretic peptide, a ligand for Npr2, against death of M-1 kidney epithelial cells exposed to 360 mmol/L NaCl. Finally, significantly lower levels of expression of the NPR2 protein were detected in renal samples of hypertensive compared with normotensive human subjects. Taken together, these findings suggest that Npr2 is essential to protect renal epithelial cells from high concentrations of salt and prevent kidney injury.

Evaluation of the procoagulant properties of a newly developed platelet modified lysate product.

BACKGROUND: Platelet transfusion is associated with logistical problems with the national storage guidelines of platelets. This results in decreased function in vivo as a result of the platelet storage lesion, and complications such as allergic or hemolytic reactions and thrombosis. We evaluated a new, freshly prepared platelet modified lysate (PML) product designed to be more procoagulant than fresh and stored platelets. METHODS: Fresh platelets were concentrated, sonicated, and centrifuged to produce PML. Samples of both washed and unwashed PML were evaluated for particle size, concentration, and activity, and then tested for clot kinetics and thrombin generation. PML samples were also stored at various temperatures for durations up to 6 months and evaluated for clot kinetics and thrombin generation throughout. RESULTS: PML showed significantly higher concentration of platelet microparticles, increased procoagulant properties, and increased thrombin generation as compared to fresh and stored platelets. In addition, PML maintained its clot kinetics over a 6-month storage period with variable storage conditions. CONCLUSIONS: The newly proposed PML product is more procoagulant, stable, and has additional potential applications than currently available platelet products. Further studies will be performed to assess its functions in vivo and to assess thrombotic potential.

Acetylcholine Inhibits Platelet Activation.

Platelets are key mediators of thrombosis. Many agonists of platelet activation are known, but fewer endogenous inhibitors of platelets, such as prostacyclin and nitric oxide (NO), have been identified. Acetylcholinesterase inhibitors, such as donepezil, can cause bleeding in patients, but the underlying mechanisms are not well understood. We hypothesized that acetylcholine is an endogenous inhibitor of platelets. We measured the effect of acetylcholine or analogs of acetylcholine on human platelet activation ex vivo. Acetylcholine and analogs of acetylcholine inhibited platelet activation, as measured by P-selectin translocation and glycoprotein IIb IIIa conformational changes. Conversely, we found that antagonists of the acetylcholine receptor, such as pancuronium, enhance platelet activation. Furthermore, drugs inhibiting acetylcholinesterase, such as donepezil, also inhibit platelet activation, suggesting that platelets release acetylcholine. We found that NO mediates acetylcholine inhibition of platelets. Our data suggest that acetylcholine is an endogenous inhibitor of platelet activation. The cholinergic system may be a novel target for antithrombotic therapies.

Platelet CD36 promotes thrombosis by activating redox sensor ERK5 in hyperlipidemic conditions.

Atherothrombosis is a process mediated by dysregulated platelet activation that can cause life-threatening complications and is the leading cause of death by cardiovascular disease. Platelet reactivity in hyperlipidemic conditions is enhanced when platelet scavenger receptor CD36 recognizes oxidized lipids in oxidized low-density lipoprotein (oxLDL) particles, a process that induces an overt prothrombotic phenotype. The mechanisms by which CD36 promotes platelet activation and thrombosis remain incompletely defined. In this study, we identify a mechanism for CD36 to promote thrombosis by increasing activation of MAPK extracellular signal-regulated kinase 5 (ERK5), a protein kinase known to be exquisitely sensitive to redox stress, through a signaling pathway requiring Src kinases, NADPH oxidase, superoxide radical anion, and hydrogen peroxide. Pharmacologic inhibitors of ERK5 blunted platelet activation and aggregation in response to oxLDL and targeted genetic deletion of ERK5 in murine platelets prevented oxLDL-induced platelet deposition on immobilized collagen in response to arterial shear. Importantly, in vivo thrombosis experiments after bone marrow transplantation from platelet-specific ERK5 null mice into hyperlipidemic apolipoprotein E null mice showed decreased platelet accumulation and increased thrombosis times compared with mice transplanted with ERK5 expressing control bone marrows. These findings suggest that atherogenic conditions critically regulate platelet CD36 signaling by increasing superoxide radical anion and hydrogen peroxide through a mechanism that promotes activation of MAPK ERK5.

Hypoxia and Ischemia Promote a Maladaptive Platelet Phenotype.

OBJECTIVE: Reduced blood flow and tissue oxygen tension conditions result from thrombotic and vascular diseases such as myocardial infarction, stroke, and peripheral vascular disease. It is largely assumed that while platelet activation is increased by an acute vascular event, chronic vascular inflammation, and ischemia, the platelet activation pathways and responses are not themselves changed by the disease process. We, therefore, sought to determine whether the platelet phenotype is altered by hypoxic and ischemic conditions. APPROACH AND RESULTS: In a cohort of patients with metabolic and peripheral artery disease, platelet activity was enhanced, and inhibition with oral antiplatelet agents was impaired compared with platelets from control subjects, suggesting a difference in platelet phenotype caused by the disease. Isolated murine and human platelets exposed to reduced oxygen (hypoxia chamber, 5% O2) had increased expression of some proteins that augment platelet activation compared with platelets in normoxic conditions (21% O2). Using a murine model of critical limb ischemia, platelet activity was increased even 2 weeks postsurgery compared with sham surgery mice. This effect was partly inhibited in platelet-specific ERK5 (extracellular regulated protein kinase 5) knockout mice. CONCLUSIONS: These findings suggest that ischemic disease changes the platelet phenotype and alters platelet agonist responses because of changes in the expression of signal transduction pathway proteins. Platelet phenotype and function should, therefore, be better characterized in ischemic and hypoxic diseases to understand the benefits and limitations of antiplatelet therapy.

Novel Thrombotic Function of a Human SNP in STXBP5 Revealed by CRISPR/Cas9 Gene Editing in Mice.

OBJECTIVE: To identify and characterize the effect of a SNP (single-nucleotide polymorphism) in the STXBP5 locus that is associated with altered thrombosis in humans. GWAS (genome-wide association studies) have identified numerous SNPs associated with human thrombotic phenotypes, but determining the functional significance of an individual candidate SNP can be challenging, particularly when in vivo modeling is required. Recent GWAS led to the discovery of STXBP5 as a regulator of platelet secretion in humans. Further clinical studies have identified genetic variants of STXBP5 that are linked to altered plasma von Willebrand factor levels and thrombosis in humans, but the functional significance of these variants in STXBP5 is not understood. APPROACH AND RESULTS: We used CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated 9) techniques to produce a precise mouse model carrying a human coding SNP rs1039084 (encoding human p. N436S) in the STXBP5 locus associated with decreased thrombosis. Mice carrying the orthologous human mutation (encoding p. N437S in mouse STXBP5) have lower plasma von Willebrand factor levels, decreased thrombosis, and decreased platelet secretion compared with wild-type mice. This thrombosis phenotype recapitulates the phenotype of humans carrying the minor allele of rs1039084. Decreased plasma von Willebrand factor and platelet activation may partially explain the decreased thrombotic phenotype in mutant mice. CONCLUSIONS: Using precise mammalian genome editing, we have identified a human nonsynonymous SNP rs1039084 in the STXBP5 locus as a causal variant for a decreased thrombotic phenotype. CRISPR/Cas9 genetic editing facilitates the rapid and efficient generation of animals to study the function of human genetic variation in vascular diseases.

Role of Axl in T-Lymphocyte Survival in Salt-Dependent Hypertension.

OBJECTIVE: Survival of immune and nonimmune cells relies on Axl, a receptor tyrosine kinase, which is implicated in hypertension. Activated T lymphocytes are involved in regulation of high blood pressure. The goal of the study was to investigate the role of Axl in T-lymphocyte functions and its contribution to salt-dependent hypertension. APPROACH AND RESULTS: We report increased apoptosis in peripheral blood from Axl(-/-) mice because of lower numbers of white blood cells mostly lymphocytes. In vitro studies showed modest reduction in interferon gamma production in Axl(-/-) type 1 T helper cells. Axl did not affect basic proliferation capacity or production of interleukin 4 in Axl(-/-) type 2 T helper cells. However, competitive repopulation of Axl(-/-) bone marrow or adoptive transfer of Axl(-/-) CD4(+) T cells to Rag1(-/-) mice showed robust effect of Axl on T lymphocyte expansion in vivo. Adoptive transfer of Axl(-/-) CD4(+) T cells was protective in a later phase of deoxycorticosterone-acetate and salt hypertension. Reduced numbers of CD4(+) T cells in circulation and in perivascular adventitia decreased vascular remodeling and increased vascular apoptosis in the late phase of hypertension. CONCLUSIONS: These findings suggest that Axl is critical for survival of T lymphocytes, especially during vascular remodeling in hypertension.

Associations between ambient wood smoke and other particulate pollutants and biomarkers of systemic inflammation, coagulation and thrombosis in cardiac patients.

BACKGROUND: Increased particulate air pollution has been associated with both an increased risk of myocardial infarction (MI) and adverse changes in cardiac biomarkers. Up to 30% of ambient wintertime fine particles (PM2.5) in Rochester, NY are from wood burning. Our study examined associations between ambient levels of a marker of wood smoke (Delta-C) and other particulate air pollutants and biomarkers of inflammation, coagulation and thrombosis. METHODS: We measured blood concentrations of C-reactive protein (CRP), D-dimer, fibrinogen, P-selectin, platelet factor 4 (PF-4), von Willebrand factor (vWF), and myeloperoxidase (MPO) of 135 patients undergoing cardiac catheterization during the winters of 2011-2013. We coupled these data with hourly ambient concentrations of Delta-C, black carbon (BC; marker of traffic pollution), and ultrafine (10-100nm; UFP), accumulation mode (100-500nm; AMP), and fine particles (<2.5µm; PM2.5). Using linear regression models, we estimated the change in each biomarker associated with increased pollutant concentrations at intervals between 1 and 96h preceding blood collection. RESULTS: Each 0.13µg/m3 increase in Delta-C concentration in the prior 12h was associated with a 0.91% increase in fibrinogen levels (95% CI=0.23%, 1.59%), but unexpectedly in the prior 48h, each 0.17µg/m3 increase in Delta-C concentration was associated with a 2.75% decrease in MPO levels (95% CI=-5.13%,-0.37%). We did not see associations between Delta-C concentrations and any other biomarkers. Interquartile range (IQR) increases in PM2.5, BC, UFP, and AMP concentrations were generally associated with increased CRP and fibrinogen, but not PF4, D-dimer, vWF, or P-selectin. CONCLUSIONS: In a population of cardiac patients, we noted adverse changes in fibrinogen associated with increased concentrations of a marker of wood smoke. Increases in PM2.5, BC, AMP, and UFP concentrations in the previous 96h were also associated with adverse changes in markers of systemic inflammation and coagulation, but not with markers of endothelial cell dysfunction or platelet activation.

Platelet Extracellular Regulated Protein Kinase 5 Is a Redox Switch and Triggers Maladaptive Platelet Responses and Myocardial Infarct Expansion.

BACKGROUND: Platelets have a pathophysiologic role in the ischemic microvascular environment of acute coronary syndromes. In comparison with platelet activation in normal healthy conditions, less attention is given to mechanisms of platelet activation in diseased states. Platelet function and mechanisms of activation in ischemic and reactive oxygen species-rich environments may not be the same as in normal healthy conditions. Extracellular regulated protein kinase 5 (ERK5) is a mitogen-activated protein kinase family member activated in hypoxic, reactive oxygen species-rich environments and in response to receptor-signaling mechanisms. Prior studies suggest a protective effect of ERK5 in endothelial and myocardial cells after ischemia. We present evidence that platelets express ERK5 and that platelet ERK5 has an adverse effect on platelet activation via selective receptor-dependent and receptor-independent reactive oxygen species-mediated mechanisms in ischemic myocardium. METHODS AND RESULTS: Using isolated human platelets and a mouse model of myocardial infarction (MI), we found that platelet ERK5 is activated post-MI and that platelet-specific ERK5(-/-) mice have less platelet activation, reduced MI size, and improved post-MI heart function. Furthermore, the expression of downstream ERK5-regulated proteins is reduced in ERK5(-/-) platelets post-MI. CONCLUSIONS: ERK5 functions as a platelet activator in ischemic conditions, and platelet ERK5 maintains the expression of some platelet proteins after MI, leading to infarct expansion. This demonstrates that platelet function in normal healthy conditions is different from platelet function in chronic ischemic and inflammatory conditions. Platelet ERK5 may be a target for acute therapeutic intervention in the thrombotic and inflammatory post-MI environment.