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Aberrant coagulation and thrombosis are associated with severe COVID-19 post-SARS-CoV-2 infection, yet the underlying mechanism remains obscure. Here we show that serum levels of SARS-CoV-2 envelope (E) protein are associated with coagulation disorders of COVID-19 patients, and intravenous administration of the E protein is able to potentiate thrombosis in mice. Through protein pull-down and mass spectrometry, we find that CD36, a transmembrane glycoprotein, directly binds with E protein and mediates hyperactivation of human and mouse platelets through the p38 MAPK-NF-κB signaling pathway. Conversely, the pharmacological blockade of CD36 or p38 notably attenuates human platelet activation induced by the E protein. Similarly, the genetic deficiency of CD36, as well as the pharmacological inhibition of p38 in mice, significantly diminishes E protein-induced platelet activation and thrombotic events. Together, our study reveals a critical role for the CD36-p38 axis in E protein-induced platelet hyperactivity, which could serve as an actionable target for developing therapies against aberrant thrombotic events related to the severity and mortality of COVID-19.

BACKGROUND: Anti-β2GP1 (β2-glycoprotein 1) antibodies are the primary pathogenic antibody to promote thrombosis in antiphospholipid syndrome (APS), yet the underlying mechanism remains obscure. We aimed to explore the intracellular pathway that mediated platelet activation. METHODS: Platelets were isolated from patients with APS and subjected to RNA sequencing. Platelet aggregation, the release of platelet granules, platelet spreading, and clot retraction were detected to evaluate platelet activation. We purified anti-β2GP1 antibodies from patients with APS and the total IgG from healthy donors to stimulate platelets with/without FcγRIIA (Fcγ receptor IIA) blocking antibody or Akt (protein kinase B) inhibitor. Platelet-specific Sin1 (stress-activated protein kinase–interacting protein) deficiency mice were established. The thrombus model of inferior vena cava flow restriction, ferric chloride–induced carotid injury model, and laser-induced vessel wall injury in cremaster arterioles model were constructed after administration of anti-β2GP1 antibodies. RESULTS: Combined RNA sequencing and bioinformatics analysis suggested that APS platelets exhibited increased levels of mRNA associated with platelet activation, which was in line with the hyperactivation of APS platelets in response to stimuli. Platelet activation in APS platelets was accompanied by upregulation of the mTORC2 (mammalian target of the rapamycin complex 2)/Akt pathway and increased levels of SIN1 phosphorylation at threonine 86. Anti-β2GP1 antibody derived from patients with APS enhanced platelet activation and upregulated the mTORC2/Akt pathway. Moreover, the Akt inhibitor weakened the potentiating effect of the anti-β2GP1 antibody on platelet activation. Notably, Sin1 deficiency suppresses anti-β2GP1 antibody–enhanced platelet activation in vitro and thrombosis in all 3 models. CONCLUSIONS: This study elucidated the novel mechanism involving the mTORC2/Akt pathway, which mediates the promotion of platelet activation and induction of thrombosis by the anti-β2GP1 antibody. The findings suggest that SIN1 may be a promising therapeutic target for the treatment of APS.

The short life span of platelets is a major challenge to platelet transfusion services because of the lack of effective intervention. Here, we found that the accumulation of long-chain acylcarnitines (LCACs) is responsible for mitochondrial damage and platelet storage lesion. Further studies showed that the blockade of fatty acid oxidation and the activation of AMP-activated protein kinase (AMPK)/acetyl-CoA carboxylase/carnitine palmitoyltransferase 1 (CPT1) pathways that promote fatty acid metabolism are important reasons for the accumulation of LCACs. The excessive accumulation of LCACs can cause mitochondrial damage and a short life span of stored platelets. The mechanism study elucidated that NAD+ exhaustion and the subsequent decrease in sirtuin 3 (Sirt3) activity caused an increase in the level of CPT2 K79 acetylation, which is the primary cause of the blockade of fatty acid oxidation and the accumulation of LCACs. Blocking LCAC generation with the inhibitors of AMPK or CPT1, the agonists of Sirt3, and antioxidants tremendously retarded platelet storage lesion in vitro and prolonged the survival of stored platelets in vivo posttransfusion with single or combined use. In summary, we discovered that CPT2 acetylation attenuates fatty acid oxidation and exacerbates platelet storage lesion and may serve as a new target for improving platelet storage quality.

INTRODUCTION:To assess the clinical performance and correlations of automated chemiluminescence assay (CIA) and enzyme-linked immunosorbent assay (ELISA) for detecting antiphospholipid (aPL) antibodies in the diagnosis of antiphospholipid syndrome (APS). METHODS:The study recruited 505 subjects, including 192 with APS, 193 with connective tissue diseases other than APS, and 120 healthy donors. We measured anticardiolipin (aCL) and anti-β2-glycoprotein I (anti-β2GPI) antibodies IgG, IgM, and IgA in all the samples using both CIA and ELISA. RESULTS:Total agreement between the two methods ranged from 83.50% for anti-β2GPI IgG antibodies to 92.76% for anti-β2GPI IgM antibodies in all the groups. Anti-β2GPI and aCL IgG assays showed the highest Spearman's rho coefficients (anti-β2GPI IgG = 0.742, aCL IgG = 0.715). Anti-β2GPI IgG CIA showed the highest sensitivity for diagnosis of APS at 80.21%, which was significantly higher than the sensitivity of anti-β2GPI IgG ELISA (52.08%). For diagnosis of APS, anti-β2GPI IgG CIA had the best discrimination power with the area under the curves (AUC) of 0.922, followed by aCL IgG CIA (AUC of 0.905). While the CIA AUC was slightly higher in all cases, the difference was not statistically significant. CONCLUSION:CIA measurements had a good agreement and correlation with comparative ELISA assays. The CIA anti-β2GPI IgG however was significantly more sensitive for APS diagnosis. The two assay methodologies showed comparable predictive powers and support the value of the CIA method for improved diagnosis and management of patients with APS.

IgG immune complexes contribute to the etiology and pathogenesis of numerous autoimmune disorders, including heparin-induced thrombocytopenia, systemic lupus erythematosus, rheumatoid- and collagen-induced arthritis, and chronic glomerulonephritis. Patients suffering from immune complex-related disorders are known to be susceptible to platelet-mediated thrombotic events. Though the role of the Fc receptor, FcγRIIa, in initiating platelet activation is well understood, the role of the major platelet adhesion receptor, integrin αIIbβ3, in amplifying platelet activation and mediating adhesion and aggregation downstream of encountering IgG immune complexes is poorly understood. The goal of this investigation was to gain a better understanding of the relative roles of these two receptor systems in immune complex-mediated thrombotic complications. Human platelets, and mouse platelets genetically engineered to differentially express FcγRIIa and αIIbβ3, were allowed to interact with IgG-coated surfaces under both static and flow conditions, and their ability to spread and form thrombi evaluated in the presence and absence of clinically-used fibrinogen receptor antagonists. Although binding of IgG immune complexes to FcγRIIa was sufficient for platelet adhesion and initial signal transduction events, platelet spreading and thrombus formation over IgG-coated surfaces showed an absolute requirement for αIIbβ3 and its ligands. Tyrosine kinases Lyn and Syk were found to play key roles in IgG-induced platelet activation events. Taken together, our data suggest a complex functional interplay between FcγRIIa, Lyn, and αIIbβ3 in immune complex-induced platelet activation. Future studies may be warranted to determine whether patients suffering from immune complex disorders might benefit from treatment with anti-αIIbβ3-directed therapeutics.

Murine paired immunoglobulin-like receptors B (PIRB), as the ortholog of human leukocyte immunoglobulin-like receptor B2 (LILRB2), is involved in a variety of biological functions. Here, we found that PIRB and LILRB2 were expressed in mouse and human platelets, respectively. PIRB intracellular domain deletion (PIRB-TM) mice had thrombocythemia and significantly higher proportions of megakaryocytes in bone marrow. Agonist-induced aggregation and spreading on immobilized fibrinogen were facilitated in PIRB-TM platelets. The rate of clot retraction in platelet-rich plasma containing PIRB-TM platelets was also increased. Characterization of signaling confirmed that PIRB associated with phosphatases Shp1/2 in platelets. The phosphorylation of Shp1/2 was significantly downregulated in PIRB-TM platelets stimulated with collagen-related peptide (CRP) or on spreading. The results further revealed that the phosphorylation levels of the linker for activation of T cells, SH2 domain-containing leukocyte protein of 76kDa, and phospholipase C were enhanced in PIRB-TM platelets stimulated with CRP. The phosphorylation levels of FAK Y397 and integrin β3 Y759 were also enhanced in PIRB-TM platelet spread on fibrinogen. The PIRB/LILRB2 ligand angiopoietin-like-protein 2 (ANGPTL2) was expressed and stored in platelet α-granules. ANGPTL2 inhibited agonist-induced platelet aggregation and spreading on fibrinogen. The data presented here reveal that PIRB and its ligand ANGPTL2 possess an antithrombotic function by suppressing collagen receptor glycoprotein VI and integrin αIIbβ3-mediated signaling.