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.

BACKGROUND: Single-nucleotide polymorphisms linked with the rs1474868 T allele ( MFN2 [mitofusin-2] T/T) in the human mitochondrial fusion protein MFN2 gene are associated with reduced platelet MFN2 RNA expression and platelet counts. This study investigates the impact of MFN2 on megakaryocyte and platelet biology. METHODS: Mice with megakaryocyte/platelet deletion of Mfn2 ( Mfn2 ^−/− [ Mfn2 conditional knockout]) were generated using Pf4-Cre crossed with floxed Mfn2 mice. Human megakaryocytes were generated from cord blood and platelets isolated from healthy subjects genotyped for rs1474868. Ex vivo approaches assessed mitochondrial morphology, function, and platelet activation responses. In vivo measurements included endogenous/transfused platelet life span, tail bleed time, transient middle cerebral artery occlusion, and pulmonary vascular permeability/hemorrhage following lipopolysaccharide-induced acute lung injury. RESULTS: Mitochondria was more fragmented in megakaryocytes derived from Mfn2 ^−/− mice and from human cord blood with MFN2 T/T genotype compared with control megakaryocytes. Human resting platelets of MFN2 T/T genotype had reduced MFN2 protein, diminished mitochondrial membrane potential, and an increased rate of phosphatidylserine exposure during ex vivo culture. Platelet counts and platelet life span were reduced in Mfn2 ^−/− mice accompanied by an increased rate of phosphatidylserine exposure in resting platelets, especially aged platelets, during ex vivo culture. Mfn2 ^−/− also decreased platelet mitochondrial membrane potential (basal) and activated mitochondrial oxygen consumption rate, reactive oxygen species generation, calcium flux, platelet-neutrophil aggregate formation, and phosphatidylserine exposure following dual agonist activation. Ultimately, Mfn2 ^−/− mice showed prolonged tail bleed times, decreased ischemic stroke infarct size after cerebral ischemia-reperfusion, and exacerbated pulmonary inflammatory hemorrhage following lipopolysaccharide-induced acute lung injury. Analysis of MFN2 SNPs in the iSPAAR study (Identification of SNPs Predisposing to Altered ALI Risk) identified a significant association between MFN2 and 28-day mortality in patients with acute respiratory distress syndrome. CONCLUSIONS: Mfn2 preserves mitochondrial phenotypes in megakaryocytes and platelets and influences platelet life span, function, and outcomes of stroke and lung injury.

Platelet-derived extracellular vesicles (PEVs) play important roles in hemostasis and thrombosis. There are three major types of PEVs described based on their size and characteristics, but newer types may continue to emerge owing to the ongoing improvement in the methodologies and terms used to define various types of EVs. As the literature on EVs is growing, there are continuing attempts to standardize protocols for EV isolation and reach consensus in the field. This review provides information on mechanisms of PEV production, characteristics, cellular interaction, and their pathological role, especially in autoimmune and infectious diseases. We also highlight the mechanisms through which PEVs can activate parent cells in a feedback loop.

Background: Reactive oxygen species (ROS) contribute to platelet hyperactivation during aging. Several oxidative pathways and antioxidant enzymes have been implicated; however, their mechanistic contributions during aging remain elusive. We hypothesized that mitochondria are an important source of platelet ROS and that mitochondrial SOD2 (superoxide dismutase) protects against mitochondrial ROS-driven platelet activation and thrombosis during aging. Methods: We studied littermates of platelet-specific SOD2-knockout (SOD2 ^fl/fl Pf4Cre, pSOD2-KO) and control (SOD2 ^fl/fl ) mice at young (4–5 months) or old (18–20 months) ages. We examined agonist-induced platelet activation, platelet-dependent thrombin generation potential, and susceptibility to in vivo thrombosis. Results: Platelet α _{II b} β3 activation, aggregation, and adhesion were increased to similar extents in aged mice of both genotypes compared with young mice. In contrast, the age-dependent increases in mitochondrial and total cellular ROS, calcium elevation, and phosphatidylserine exposure were augmented in platelets from pSOD2-KO mice compared with control mice. Aged pSOD2-KO mice showed increased platelet-dependent thrombin generation compared with aged control mice. In vivo, aged pSOD2-KO mice exhibited enhanced susceptibility to carotid artery and pulmonary thrombosis compared to aged control mice. Adoptive transfer of platelets from aged pSOD2-KO but not aged control mice increased thrombotic susceptibility in aged host mice, suggesting a prothrombotic effect of platelet pSOD2 deficiency. Treatment with avasopasem manganese (GC4419), a SOD mimetic, decreased platelet mitochondrial pro-oxidants, cellular ROS levels, and inhibited procoagulant platelet formation and arterial thrombosis in aged mice. Conclusions: Platelet mitochondrial ROS contributes to age-related thrombosis and endogenous SOD2 protects from platelet-dependent thrombin generation and thrombosis during aging.

Background: COVID-19-associated coagulopathy increases mortality in patients with COVID-19 infection. The International Society of Thrombosis and Hemostasis (ISTH) has published a guidance document recommending thromboprophylaxis with low-molecular weight heparin (LMWH) for all hospitalized patients with COVID-19. Despite standard dose LMWH thromboprophylaxis, arterial and venous thrombotic events occur in up to 30-40% of COVID-19 patients admitted to the intensive care unit (ICU). It is not known if patients with coagulopathy from COVID-19 benefit from higher levels of prophylactic anticoagulation therapy. No data exist regarding the comparative safety or efficacy of intermediate dose versus standard prophylactic dose LMWH. Study Design and Methods: This is a multi-center, randomized, open-label study (NCT04360824) comparing standard prophylactic dose enoxaparin (40 mg SC daily if BMI < 30 kg/m2; 30 mg SC twice daily or 40 mg SC twice daily if BMI ≥ 30 kg/m2) versus intermediate-weight adjusted dose enoxaparin (1 mg/kg SC daily if BMI < 30 kg/m2 or 0.5 mg/kg SC twice daily if BMI ≥ 30 kg/m2) in hospitalized patients with laboratory-confirmed SARS-CoV-2 infection who are either admitted to an intensive care unit or have a modified ISTH Overt DIC score ≥ 3. Objectives: The primary endpoint is to compare all-cause 30-day mortality of patients treated with standard prophylactic dose versus intermediate-dose enoxaparin. The secondary endpoints are to determine whether the use of standard prophylactic dose versus intermediate dose enoxaparin impacts major bleeding, arterial thrombosis, venous thrombosis, time to intubation or transfusion of blood products. The exploratory endpoints are to determine the effects of SARS-CoV-2 infection on laboratory coagulation parameters to better understand the mechanism of COVID-19 coagulopathy compared to healthy control subjects; parameters include citrullinated histone H3 (H3Cit), a component of neutrophil extracellular traps. The null hypothesis is that intermediate dose enoxaparin (intervention arm) will have a mortality rate that is not less than standard prophylactic dose enoxaparin (standard of care arm). Assuming a true margin of 20%, a sample size of 82 patients in each arm of the study is required to have 80% power when testing for a difference. Baseline Data: Three centers are currently enrolling patients. As of August 1, 2020, we have enrolled 52 patients (28 males and 24 females) with a median age of 61 years (range 24-80). 51% were Caucasian, 36% Hispanic, and 16% other ethnicities. Median BMI was 31 kg/m2 (range 20-52). Baseline D-Dimer was elevated in 95% of participants with a median of 2.23 mg/mL (range 0.39-40.32). The majority of participants had normal baseline PT, PTT, and platelet levels. All patients had fibrinogen levels over 100 mg/dL with a median of 607 mg/dL. Plasma H3Cit was elevated significantly in patients with COVID-19 (n = 21) vs healthy control subjects (n = 10) (6.48 vs 1.09 ng/mL, P = 0.001). Conclusion: Baseline data for the first 52 patients enrolled confirm the presence of multiple risk factors for thrombosis such as elevated BMI, D dimer, and plasma markers of neutrophil extracellular traps. In contrast to initial reports from China, the prevalence of thrombocytopenia and hypofibrinogenemia was very low in our cohort. Hispanics represented 36% of our study population, compared with 19% of overall COVID-19 infections in the region, suggesting a higher severity of illness in this subpopulation. The enrollment of this trial is ongoing with the addition of sites that have larger African American populations. As safety and efficacy data mature, this study will help clarify the optimum dose of prophylactic enoxaparin to prevent COVID-19 associated coagulopathy. Disclosures No relevant conflicts of interest to declare.