Aging and chronic inflammation are independent risk factors for the development of atherothrombosis and cardiovascular disease. We hypothesized that aging-associated inflammation promotes the development of platelet hyperreactivity and increases thrombotic risk during aging. Functional platelet studies in aged-frail adults and old mice demonstrated that their platelets are hyperreactive and form larger thrombi. We identified tumor necrosis factor α (TNF-α) as the key aging-associated proinflammatory cytokine responsible for platelet hyperreactivity. We further showed that platelet hyperreactivity is neutralized by abrogating signaling through TNF-α receptors in vivo in a mouse model of aging. Analysis of the bone marrow compartments showed significant platelet-biased hematopoiesis in old mice reflected by increased megakaryocyte-committed progenitor cells, megakaryocyte ploidy status, and thrombocytosis. Single-cell RNA-sequencing analysis of native mouse megakaryocytes showed significant reprogramming of inflammatory, metabolic, and mitochondrial gene pathways in old mice that appeared to play a significant role in determining platelet hyperreactivity. Platelets from old mice (where TNF-α was endogenously increased) and from young mice exposed to exogenous TNF-α exhibited significant mitochondrial changes characterized by elevated mitochondrial mass and increased oxygen consumption during activation. These mitochondrial changes were mitigated upon TNF-α blockade. Similar increases in platelet mitochondrial mass were seen in platelets from patients with myeloproliferative neoplasms, where TNF-α levels are also increased. Furthermore, metabolomics studies of platelets from young and old mice demonstrated age-dependent metabolic profiles that may differentially poise platelets for activation. Altogether, we present previously unrecognized evidence that TNF-α critically regulates megakaryocytes resident in the bone marrow niche and aging-associated platelet hyperreactivity and thrombosis.

Hematopoietic stem cells (HSCs) are capable of entering the cell cycle to replenish the blood system in response to inflammatory cues; however, excessive proliferation in response to chronic inflammation can lead to either HSC attrition or expansion. The mechanism(s) that limit HSC proliferation and expansion triggered by inflammatory signals are poorly defined. Here, we show that long-term HSCs (HSCLT) rapidly repress protein synthesis and cell cycle genes following treatment with the proinflammatory cytokine interleukin (IL)-1. This gene program is associated with activation of the transcription factor PU.1 and direct PU.1 binding at repressed target genes. Notably, PU.1 is required to repress cell cycle and protein synthesis genes, and IL-1 exposure triggers aberrant protein synthesis and cell cycle activity in PU.1-deficient HSCs. These features are associated with expansion of phenotypic PU.1-deficient HSCs. Thus, we identify a PU.1-dependent mechanism triggered by innate immune stimulation that limits HSC proliferation and pool size. These findings provide insight into how HSCs maintain homeostasis during inflammatory stress.

Anucleate platelets, long viewed as merely cell fragments with a limited repertoire of rapid-acting hemostatic functions, are now recognized to have a complex and dynamic transcriptome mirroring that of many nucleated cells. The field of megakaryocyte and platelet transcriptomics has been rapidly growing, particularly with the advent of newer technologies such as next-generation RNA-sequencing. Studies interrogating the megakaryocyte and platelet transcriptome have led to a number of key insights into human health and disease. In this brief focused review, we will discuss some of the recent discoveries made through transcriptome analysis of megakaryocytes and platelets. We will also highlight the utility of integrating ribosome footprint analysis to augment discoveries. Both bulk and single-cell sequencing approaches will be reviewed, along with comparative studies between human and murine platelets under basal healthy settings and during acute systemic inflammatory diseases.

Background Growth arrest-specific protein 6 signals through the TAM (TYRO-3-AXL-MERTK) receptor family, mediating platelet activation and thrombus formation via activation of the aggregate-stabilizing α β integrin. Objective To describe the antithrombotic effects mediated by UNC2025, a small-molecule MERTK tyrosine kinase inhibitor. Methods MERTK phosphorylation and downstream signaling were assessed by immunoblotting. Light transmission aggregometry, flow cytometry and microfluidic analysis were used to evaluate the impact of MERTK inhibition on platelet activation and stability of aggregates in vitro. The effects of MERTK inhibition on arterial and venous thrombosis, platelet accumulation at microvascular injury sites and tail bleeding times were determined with murine models. The effects of combined treatment with ADP-P2Y pathway antagonists and UNC2025 were also evaluated. Results and Conclusions Treatment with UNC2025 inhibited MERTK phosphorylation and downstream activation of AKT and SRC, decreased platelet activation, and protected animals from pulmonary embolism and arterial thrombosis without increasing bleeding times. The antiplatelet effect of UNC2025 was enhanced in combination with ADP-P2Y pathway antagonists, and a greater than additive effect was observed when these two agents with different mechanisms of inhibition were coadministered. TAM kinase signaling represents a potential therapeutic target, as inhibition of this axis, especially in combination with ADP-P2Y pathway antagonism, mediates decreased platelet activation, aggregate stability, and thrombus formation, with less hemorrhagic potential than current treatment strategies. The data presented here also demonstrate antithrombotic activity mediated by UNC2025, a novel translational agent, and support the development of TAM kinase inhibitors for clinical applications.

Inflammation is central to the pathogenesis of pulmonary vascular remodeling and pulmonary hypertension (PH). Inflammation precedes remodeling in preclinical models, thus supporting the concept that changes in immunity drive remodeling in PH. Platelets are recognized as mediators of inflammation, but whether platelets contribute to hypoxia-driven inflammation has not been studied. We utilized a murine hypoxia model to test the hypothesis that platelets drive hypoxia-induced inflammation. We evaluated male and female 9-week-old normoxic and hypoxic mice and in selected experiments included hypoxic thrombocytopenic mice. Thrombocytopenic mice were generated with an anti-GP1ba rat IgG antibody. We also performed immunostaining of lung sections from failed donor controls and patients with idiopathic pulmonary arterial hypertension. We found that platelets are increased in the lungs of hypoxic mice and hypoxia induces platelet activation. Platelet depletion prevents hypoxia-driven increases in the pro-inflammatory chemokines CXCL4 and CCL5 and attenuates hypoxia-induced increase in plasma CSF-2. Pulmonary interstitial macrophages are increased in the lungs of hypoxic mice; this increase is prevented in thrombocytopenic mice. To determine the potential relevance to human disease lung sections from donors and patients with advanced iPAH were immunostained for the platelet specific protein CD41. We observed iPAH lungs had a two-fold increase in CD41, compared to controls. Our data provide evidence that platelets are increased in the lungs and activated in mice with hypoxia-induced inflammation and provides rationale for the further study of the potential contribution of platelets to inflammatory mediated vascular remodeling and PH.