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.

Compound heterozygous RYR1-RM mouse model reveals disease pathomechanisms and muscle adaptations to promote postnatal survival.

Pathogenic variants in the type I ryanodine receptor (RYR1) result in a wide range of muscle disorders referred to as RYR1-related myopathies (RYR1-RM). We developed the first RYR1-RM mouse model resulting from co-inheritance of two different RYR1 missense alleles (Ryr1TM/SC-ΔL mice). Ryr1TM/SC-ΔL mice exhibit a severe, early onset myopathy characterized by decreased body/muscle mass, muscle weakness, hypotrophy, reduced RYR1 expression, and unexpectedly, incomplete postnatal lethality with a plateau survival of ~50% at 12 weeks of age. Ryr1TM/SC-ΔL mice display reduced respiratory function, locomotor activity, and in vivo muscle strength. Extensor digitorum longus muscles from Ryr1TM/SC-ΔL mice exhibit decreased cross-sectional area of type IIb and type IIx fibers, as well as a reduction in number of type IIb fibers. Ex vivo functional analyses revealed reduced Ca2+ release and specific force production during electrically-evoked twitch stimulation. In spite of a ~threefold reduction in RYR1 expression in single muscle fibers from Ryr1TM/SC-ΔL mice at 4 weeks and 12 weeks of age, RYR1 Ca2+ leak was not different from that of fibers from control mice at either age. Proteomic analyses revealed alterations in protein synthesis, folding, and degradation pathways in the muscle of 4- and 12-week-old Ryr1TM/SC-ΔL mice, while proteins involved in the extracellular matrix, dystrophin-associated glycoprotein complex, and fatty acid metabolism were upregulated in Ryr1TM/SC-ΔL mice that survive to 12 weeks of age. These findings suggest that adaptations that optimize RYR1 expression/Ca2+ leak balance, sarcolemmal stability, and fatty acid biosynthesis provide Ryr1TM/SC-ΔL mice with an increased survival advantage during postnatal development.

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.

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.

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.

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.

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.

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.

Resveratrol preserves the function of human platelets stored for transfusion.

Stored platelets undergo biochemical, structural and functional changes that lead to decreased efficacy and safety of platelet transfusions. Not only do platelets acquire markers of activation during storage, but they also fail to respond normally to agonists post-storage. We hypothesized that resveratrol, a cardioprotective antioxidant, could act as a novel platelet storage additive to safely prevent unwanted platelet activation during storage, while simultaneously preserving normal haemostatic function. Human platelets treated with resveratrol and stored for 5 d released less thromboxane B2 and prostaglandin E2 compared to control platelets. Resveratrol preserved the ability of platelets to aggregate, spread and respond to thrombin, suggesting an improved ability to activate post-storage. Utilizing an in vitro model of transfusion and thromboelastography, clot strength was improved with resveratrol treatment compared to conventionally stored platelets. The mechanism of resveratrol's beneficial actions on stored platelets was partly mediated through decreased platelet apoptosis in storage, resulting in a longer half-life following transfusion. Lastly, an in vivo mouse model of transfusion demonstrated that stored platelets are prothrombotic and that resveratrol delayed vessel occlusion time to a level similar to transfusion with fresh platelets. We show resveratrol has a dual ability to reduce unwanted platelet activation during storage, while preserving critical haemostatic function.

G protein-coupled receptor kinase 5 regulates thrombin signaling in platelets.

Background: Our prior genome-wide association study of thrombin-induced platelet aggregation identified a G protein-coupled receptor kinase 5 (GRK5) noncoding variant (rs10886430-G) that is strongly associated with increased platelet reactivity to thrombin. This variant predisposes to increased risk of stroke, pulmonary embolism, and venous thromboembolism. Objectives: To determine role of platelet specific GRK5 in platelet responses to agonists and injury. Methods: Platelets from GRK5 mutant mice have been shown to have increased thrombin sensitivity, indicating that GRK5 may be a negative regulator of platelet activation. However, this has not been studied in a platelet-specific manner. We therefore used platelet-specific GRK5 mutant mice and models of thrombosis and pulmonary embolism. Results: We now demonstrate that mice lacking GRK5 specifically in platelets had a mild increase in thrombin responses in vitro and a shortened time to arterial thrombosis in vivo. In addition, platelet GRK5 mutant mice had increased thrombin but not collagen-induced thrombus burden in a mouse model of pulmonary embolism. Conclusion: These data indicate that platelet GRK5 has a significant role in limiting platelet responses to thrombin.

Long-lived lung megakaryocytes contribute to platelet recovery in thrombocytopenia models.

Lung megakaryocytes (Mks) are largely extravascular with an immune phenotype (1). Because bone marrow (BM) Mks are short-lived it has been assumed that extravascular lung Mks are constantly 'seeded' from the BM. To investigate lung Mk origins and how that impacts their functions, we developed methods to specifically label lung Mks using CFSE dye and biotin delivered oropharyngeal. Labeled lung Mks were present for up to four months, while BM Mks had a <1 week lifespan. In a parabiosis model, lung Mks were partially replaced over 1-month from a circulating source. Unlike tissue-resident macrophages, using MDS1-Cre-ERT2 TdTomato mice, we found that lung Mks arise from hematopoietic stem cells. However, studies with FlkSwitch mTmG mice showed that lung Mks are derived from a Flt3-independent lineage that does not go through a multipotent progenitor. CFSE labeling to track lung Mk-derived platelets showed that about 10% of circulating platelets are lung resident Mk-derived at steady state, but in sterile thrombocytopenia this was doubled (about 20%). Lung-derived platelets were similarly increased in a malaria infection model (Plasmodium yoelii) typified by thrombocytopenia. These studies indicate that lung Mks arise from a Flt3-negative BM source, are long-lived, and contribute more platelets during thrombocytopenia.

Platelet Ido1 expression is induced during Plasmodium yoelii infection, altering plasma tryptophan metabolites.

Platelets are immune responsive in many diseases as noted by changes in platelet mRNA in conditions such as sepsis1, atherosclerosis2, COVID-193,4, and many other inflammatory and infectious etiologies5. The malaria causing Plasmodium parasite is a persistent public health threat and significant evidence shows that platelets participate in host responses to infection. Using a mouse model of non-lethal/uncomplicated malaria, P. yoelii XNL (PyNL), infected, but not control mouse platelets expressed Ido1, a rate limiting enzyme in tryptophan metabolism that increases kynurenine at the expense of serotonin. Interferon-gamma (IFN) is a potent inducer of Ido1 and mice treated with recombinant IFN had increased platelet Ido1 and IDO1 activity. PyNL infected mice treated with anti-IFN antibody had similar platelet Ido1 and metabolic profiles to that of uninfected controls. PyNL infected mice become thrombocytopenic by day 7 post-infection and transfusion of platelets from IFN treated wild type mice, but not Ido1-/- mice, increased the plasma kynurenine to tryptophan ratio, indicating platelets are a source of post-infection IDO1 activity. We generated platelet specific Ido1 knockout mice to assess the contribution of platelet Ido1 during PyNL infection. Platelet specific Ido1-/- mice had increased death and evidence of lung thrombi which were not present in infected WT mice. Platelet Ido1 may be a significant contributor to plasma KYN in IFN driven immune processes and the loss of platelets may limit total Ido1, leading to immune and vascular dysfunction.

Thrombocytopenia Independently Leads to Monocyte Immune Dysfunction.

In addition to their well-studied hemostatic functions, platelets are immune cells. Platelets circulate at the interface between the vascular wall and leukocytes, and transient platelet-leukocyte complexes are found in both healthy and disease states, positioning platelets to provide physiologic cues of vascular health and injury. Roles for activated platelets in inducing and amplifying immune responses have received an increasing amount of research attention, but our past studies also showed that normal platelet counts are needed in healthy conditions to maintain immune homeostasis. We have now found that thrombocytopenia (a low platelet count) leads to monocyte dysfunction, independent of the cause of thrombocytopenia, in a manner that is dependent on direct platelet-monocyte CD47 interactions that regulate monocyte immunometabolism and gene expression. Compared to monocytes from mice with normal platelet counts, monocytes from thrombocytopenic mice had increased toll-like receptor (TLR) responses, including increased IL-6 production. Furthermore, ex vivo co-incubation of resting platelets with platelet naïve bone marrow monocytes, induced monocyte metabolic programming and durable changes in TLR agonist responses. Assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-Seq) on monocytes from thrombocytopenic mice showed persistently open chromatin at LPS response genes and resting platelet interactions with monocytes induced histone methylation in a CD47 dependent manner. Using mouse models of thrombocytopenia and sepsis, normal platelet numbers were needed to limit monocyte immune dysregulation and IL6 expression in monocytes from human patients with sepsis also inversely correlated with patient platelet counts. Our studies demonstrate that in healthy conditions, resting platelets maintain monocyte immune tolerance by regulating monocyte immunometabolic processes that lead to epigenetic changes in TLR-related genes. This is also the first demonstration of sterile cell interactions that regulate of innate immune-metabolism and monocyte pathogen responses.

Platelet olfactory receptor activation limits platelet reactivity and growth of aortic aneurysms.

As blood transitions from steady laminar flow (S-flow) in healthy arteries to disturbed flow (D-flow) in aneurysmal arteries, platelets are subjected to external forces. Biomechanical platelet activation is incompletely understood and is a potential mechanism behind antiplatelet medication resistance. Although it has been demonstrated that antiplatelet drugs suppress the growth of abdominal aortic aneurysms (AAA) in patients, we found that a certain degree of platelet reactivity persisted in spite of aspirin therapy, urging us to consider additional antiplatelet therapeutic targets. Transcriptomic profiling of platelets from patients with AAA revealed upregulation of a signal transduction pathway common to olfactory receptors, and this was explored as a mediator of AAA progression. Healthy platelets subjected to D-flow ex vivo, platelets from patients with AAA, and platelets in murine models of AAA demonstrated increased membrane olfactory receptor 2L13 (OR2L13) expression. A drug screen identified a molecule activating platelet OR2L13, which limited both biochemical and biomechanical platelet activation as well as AAA growth. This observation was further supported by selective deletion of the OR2L13 ortholog in a murine model of AAA that accelerated aortic aneurysm growth and rupture. These studies revealed that olfactory receptors regulate platelet activation in AAA and aneurysmal progression through platelet-derived mediators of aortic remodeling.

Lung megakaryocytes are immune modulatory cells.

Although platelets are the cellular mediators of thrombosis, they are also immune cells. Platelets interact both directly and indirectly with immune cells, impacting their activation and differentiation, as well as all phases of the immune response. Megakaryocytes (Mks) are the cell source of circulating platelets, and until recently Mks were typically only considered bone marrow-resident (BM-resident) cells. However, platelet-producing Mks also reside in the lung, and lung Mks express greater levels of immune molecules compared with BM Mks. We therefore sought to define the immune functions of lung Mks. Using single-cell RNA sequencing of BM and lung myeloid-enriched cells, we found that lung Mks, which we term MkL, had gene expression patterns that are similar to antigen-presenting cells. This was confirmed using imaging and conventional flow cytometry. The immune phenotype of Mks was plastic and driven by the tissue immune environment, as evidenced by BM Mks having an MkL-like phenotype under the influence of pathogen receptor challenge and lung-associated immune molecules, such as IL-33. Our in vitro and in vivo assays demonstrated that MkL internalized and processed both antigenic proteins and bacterial pathogens. Furthermore, MkL induced CD4+ T cell activation in an MHC II-dependent manner both in vitro and in vivo. These data indicated that MkL had key immune regulatory roles dictated in part by the tissue environment.

Novel Mechanism of Microvesicle Regulation by the Antiviral Protein Tetherin During HIV Infection.

Background Microvesicles are cell membrane-derived vesicles that have been shown to augment inflammation. Specifically, monocyte-derived microvesicles (MDMVs), which can express the coagulation protein tissue factor, contribute to thrombus formation and cardiovascular disease. People living with HIV experience higher prevalence of cardiovascular disease and also exhibit increased levels of plasma microvesicles. The process of microvesicle release has striking similarity to budding of enveloped viruses. The surface protein tetherin inhibits viral budding by physically tethering budding virus particles to cells. Hence, we investigated the role of tetherin in regulating the release of MDMVs during HIV infection. Methods and Results The plasma of aviremic HIV-infected individuals had increased levels of tissue factor + MDMVs, as measured by flow cytometry, and correlated to reduced tetherin expression on monocytes. Superresolution confocal and electron microscopy showed that tetherin localized at the site of budding MDMVs. Mechanistic studies revealed that the exposure of monocytes to HIV-encoded Tat triggered tetherin loss and subsequent rise in MDMV production. Overexpression of tetherin in monocytes led to morphologic changes in the pseudopodia directly underneath the MDMVs. Further, tetherin knockout mice demonstrated a higher number of circulating MDMVs and less time to bleeding cessation. Conclusions Our studies define a novel regulatory mechanism of MDMV release through tetherin and explore its contribution to the procoagulatory state that is frequently observed in people with HIV. Such insights could lead to improved therapies for individuals infected with HIV and also for those with cardiovascular disease.

The choline transporter Slc44a2 controls platelet activation and thrombosis by regulating mitochondrial function.

Genetic factors contribute to the risk of thrombotic diseases. Recent genome wide association studies have identified genetic loci including SLC44A2 which may regulate thrombosis. Here we show that Slc44a2 controls platelet activation and thrombosis by regulating mitochondrial energetics. We find that Slc44a2 null mice (Slc44a2(KO)) have increased bleeding times and delayed thrombosis compared to wild-type (Slc44a2(WT)) controls. Platelets from Slc44a2(KO) mice have impaired activation in response to thrombin. We discover that Slc44a2 mediates choline transport into mitochondria, where choline metabolism leads to an increase in mitochondrial oxygen consumption and ATP production. Platelets lacking Slc44a2 contain less ATP at rest, release less ATP when activated, and have an activation defect that can be rescued by exogenous ADP. Taken together, our data suggest that mitochondria require choline for maximum function, demonstrate the importance of mitochondrial metabolism to platelet activation, and reveal a mechanism by which Slc44a2 influences thrombosis.

Platelet-derived ß2m regulates age related monocyte/macrophage functions.

Platelets have central roles in both immune responses and development. Stimulated platelets express leukocyte adhesion molecules and release numerous immune modulatory factors that recruit and activate leukocytes, both at the sites of activation and distantly. Monocytes are innate immune cells with dynamic immune modulatory functions that change during the aging process, a phenomenon termed "inflammaging". We have previously shown that platelets are a major source of plasma beta-2 microglobulin (ß2M) and that ß2M induced a monocyte pro-inflammatory phenotype. Plasma ß2M increases with age and is a pro-aging factor. We hypothesized that platelet derived ß2M regulates monocyte phenotypes in the context of aging. Using wild-type (WT) and platelet specific ß2M knockout mice (Plt-ß2M-/-) mice, we found that plasma ß2M increased with age and correlated with increased circulating Ly6CHi monocytes. However, aged Plt-ß2M-/- mice had significantly fewer Ly6CHi monocytes compared to WT mice. Quantitative real-time PCR of circulating monocytes showed that WT mouse monocytes were more "pro-inflammatory" with age, while Plt-ß2M-/- derived monocytes adopted a "pro-reparative" phenotype. Older Plt-ß2M-/- mice had a significant decline in heart function compared to age matched WT mice, as well as increased cardiac fibrosis and pro-fibrotic markers. These data suggest that platelet-derived ß2M regulates age associated monocyte polarization, and a loss of platelet derived ß2M shifted monocytes and macrophages to a pro-reparative phenotype and increased pro-fibrotic cardiac responses. Platelet regulation of monocyte phenotypes via ß2M may maintain a balance between inflammatory and reparative signals that affects age related physiologic outcomes.

Platelet-derived ß2M regulates monocyte inflammatory responses.

ß-2 Microglobulin (ß2M) is a molecular chaperone for the major histocompatibility class I (MHC I) complex, hemochromatosis factor protein (HFE), and the neonatal Fc receptor (FcRn), but ß2M may also have less understood chaperone-independent functions. Elevated plasma ß2M has a direct role in neurocognitive decline and is a risk factor for adverse cardiovascular events. ß2M mRNA is present in platelets at very high levels, and ß2M is part of the activated platelet releasate. In addition to their more well-studied thrombotic functions, platelets are important immune regulatory cells that release inflammatory molecules and contribute to leukocyte trafficking, activation, and differentiation. We have now found that platelet-derived ß2M is a mediator of monocyte proinflammatory differentiation through noncanonical TGFß receptor signaling. Circulating monocytes from mice lacking ß2M only in platelets (Plt-ß2M-/-) had a more proreparative monocyte phenotype, in part dependent on increased platelet-derived TGFß signaling in the absence of ß2M. Using a mouse myocardial infarction (MI) model, Plt-ß2M-/- mice had limited post-MI proinflammatory monocyte responses and, instead, demonstrated early proreparative monocyte differentiation, profibrotic myofibroblast responses, and a rapid decline in heart function compared with WT mice. These data demonstrate a potentially novel chaperone-independent, monocyte phenotype-regulatory function for platelet ß2M and that platelet-derived 2M and TGFß have opposing roles in monocyte differentiation that may be important in tissue injury responses.

miR-451 limits CD4+ T cell proliferative responses to infection in mice.

MicroRNAs (miRNAs) are major regulators of cell responses, particularly in stressed cell states and host immune responses. Some miRNAs have a role in pathogen defense, including regulation of immune responses to Plasmodium parasite infection. Using a nonlethal mouse model of blood stage malaria infection, we have found that miR-451-/- mice infected with Plasmodium yoelii XNL cleared infection at a faster rate than did wild-type (WT) mice. MiR-451-/- mice had an increased leukocyte response to infection, with the protective phenotype primarily driven by CD4+ T cells. WT and miR-451-/- CD4+ T cells had similar activation responses, but miR-451-/- CD4+ cells had significantly increased proliferation, both in vitro and in vivo. Myc is a miR-451 target with a central role in cell cycle progression and cell proliferation. CD4+ T cells from miR-451-/- mice had increased postactivation Myc expression. RNA-Seq analysis of CD4+ cells demonstrated over 5000 differentially expressed genes in miR-451-/- mice postinfection, many of which are directly or indirectly Myc regulated. This study demonstrates that miR-451 regulates T cell proliferative responses in part via a Myc-dependent mechanism.