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.

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.

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.

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.

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.

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.

Identification of activators of ERK5 transcriptional activity by high-throughput screening and the role of endothelial ERK5 in vasoprotective effects induced by statins and antimalarial agents.

Because ERK5 inhibits endothelial inflammation and dysfunction, activating ERK5 might be a novel approach to protecting vascular endothelial cells (ECs) against various pathological conditions of the blood vessel. We have identified small molecules that protect ECs via ERK5 activation and determined their contribution to preventing cardiac allograft rejection. Using high-throughput screening, we identified certain statins and antimalarial agents including chloroquine, hydroxychloroquine, and quinacrine as strong ERK5 activators. Pitavastatin enhanced ERK5 transcriptional activity and Kruppel-like factor-2 expression in cultured human and bovine ECs, but these effects were abolished by the depletion of ERK5. Chloroquine and hydroxychloroquine upregulated ERK5 kinase activity and inhibited VCAM-1 expression in an ERK5-dependent but MAPK/ERK kinase 5- and Kruppel-like factor 2/4-independent manner. Leukocyte rolling and vascular reactivity were used to evaluate endothelial function in vivo, and we found that EC-specific ERK5 knockout (ERK5-EKO) mice exhibited increased leukocyte rolling and impaired vascular reactivity, which could not be corrected by pitavastatin. The role of endothelial ERK5 in acute cardiac allograft rejection was also examined by heterotopic grafting of the heart obtained from either wild-type or ERK5-EKO mice into allomismatched recipient mice. A robust increase in both inflammatory gene expression and CD45-positive cell infiltration into the graft was observed. These tissue rejection responses were inhibited by pitavastatin in wild-type but not ERK5-EKO hearts. Our study has identified statins and antimalarial drugs as strong ERK5 activators and shown that ERK5 activation is preventive of endothelial inflammation and dysfunction and acute allograft rejection.

Platelet factor 4 mediates vascular smooth muscle cell injury responses.

Activated platelets release many inflammatory molecules with important roles in accelerating vascular inflammation. Much is known about platelet and platelet-derived mediator interactions with endothelial cells and leukocytes, but few studies have examined the effects of platelets on components of the vascular wall. Vascular smooth muscle cells (VSMCs) undergo phenotypic changes in response to injury including the production of inflammatory molecules, cell proliferation, cell migration, and a decline in the expression of differentiation markers. In this study, we demonstrate that the platelet-derived chemokine platelet factor 4 (PF4/CXCL4) stimulates VSMC injury responses both in vitro and in vivo in a mouse carotid ligation model. PF4 drives a VSMC inflammatory phenotype including a decline in differentiation markers, increased cytokine production, and cell proliferation. We also demonstrate that PF4 effects are mediated, in part, through increased expression of the transcription factor Krüppel-like factor 4. Our data indicate an important mechanistic role for platelets and PF4 in VSMC injury responses both in vitro and in vivo.

Platelet induction of the acute-phase response is protective in murine experimental cerebral malaria.

Platelets are most recognized as the cellular mediator of thrombosis, but they are increasingly appreciated for their immunomodulatory roles, including responses to Plasmodium infection. Platelet interactions with endothelial cells and leukocytes contribute significantly to the pathogenesis of experimental cerebral malaria (ECM). Recently, it has been suggested that platelets not only have an adverse role in cerebral malaria, but platelets may also be protective in animal models of uncomplicated malaria. We now demonstrate that these diverse and seemingly contradictory roles for platelets extend to cerebral malaria models and are dependent on the timing of platelet activation during infection. Our data show that platelets are activated very early in ECM and have a central role in initiation of the acute-phase response to blood-stage infection. Unlike platelet depletion or inhibition postinfection, preinfection platelet depletion or treatment with a platelet inhibitor is not protective. Additionally, we show that platelet-driven acute-phase responses have a major role in protecting mice from ECM by limiting parasite growth. Our data now suggest that platelets have a complex role in ECM pathogenesis: platelets help limit parasite growth early postinfection, but with continued platelet activation as the disease progresses, platelets contribute to ECM-associated inflammation.

Platelets present antigen in the context of MHC class I.

Platelets are most recognized for their vital role as the cellular mediator of thrombosis, but platelets also have important immune functions. Platelets initiate and sustain vascular inflammation in many disease conditions, including arthritis, atherosclerosis, transplant rejection, and severe malaria. We now demonstrate that platelets express T cell costimulatory molecules, process and present Ag in MHC class I, and directly activate naive T cells in a platelet MHC class I-dependent manner. Using an experimental cerebral malaria mouse model, we also demonstrate that platelets present pathogen-derived Ag to promote T cell responses in vivo, and that platelets can be used in a cell-based vaccine model to induce protective immune responses. Our study demonstrates a novel Ag presentation role for platelets.

Monocyte-derived Dll4 is a novel contributor to persistent systemic inflammation in HIV patients.

Background: In people living with HIV (PLWH) on combination antiretroviral therapy (cART), persistent systemic inflammation is a driving force for the progression of comorbidities, such as cardiovascular and cerebrovascular diseases. In this context, monocyte- and macrophage-related inflammation rather than T cell activation is a major cause of chronic inflammation. However, the underlying mechanism of how monocytes cause persistent systemic inflammation in PLWH is elusive. Methods and Results: In vitro, we demonstrated that lipopolysaccharides (LPS) or tumor necrosis factor alpha (TNFα), induced a robust increase of Delta-like ligand 4 (Dll4) mRNA and protein expression in human monocytes and Dll4 secretion (extracellular Dll4, exDll4) from monocytes. Enhanced membrane-bound Dll4 (mDll4) expression in monocytes triggered Notch1 activation to promote pro-inflammatory factors expression. Dll4 silencing and inhibition of Nocth1 activation diminished the LPS or TNFα -induced inflammation. exDll4 releases in response to cytokines occurred in monocytes but not endothelial cells or T cells. In clinical specimens, we found that PLWH, both male and female, on cART, showed a significant increase in mDll4 expression, activation of Dll4-Notch1 signaling, and inflammatory markers in monocytes. Although there was no sex effect on mDII4 in PLWH, plasma exDll4 was significantly elevated in males but not females compared to HIV uninfected individuals. Furthermore, exDll4 plasma levels paralleled with monocytes mDll4 in male PLWH. Circulating exDll4 was also positively associated with pro-inflammatory monocytes phenotype and negatively associated with classic monocytes phenotype in male PLWH. Conclusion: Pro-inflammatory stimuli increase Dll4 expression and Dll4-Notch1 signaling activation in monocytes and enhance monocyte proinflammatory phenotype, contributing to persistent systemic inflammation in male and female PLWH. Therefore, monocyte mDll4 could be a potential biomarker and therapeutic target of systemic inflammation. Plasma exDll4 may also play an additional role in systemic inflammation but primarily in men.

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.