Changming Chen

Glucose-6-phosphate dehydrogenase (G6PD), the committed enzyme of the pentose phosphate pathway, plays a pivotal role in glucose metabolism, which is integral to platelet biology. Although G6PD deficiency is a prevalent enzyme defect worldwide, the specific role of G6PD itself in platelets remains unclear. Herein, we discover a previously unrecognized phenotype in both human patients and murine models, demonstrating that G6PD deficiency leads to elevated platelet counts through enhanced proplatelet formation (PPF). Mechanistically, we reveal a non-canonical function of G6PD in regulating actin dynamics via its interaction with an actin-binding protein, tropomodulin-3 (TMOD3). The G6PD-TMOD3 interaction, which is concentration-dependently enhanced by nicotinamide adenine dinucleotide phosphate (NADP ⁺ ), can limit actin rearrangement, thereby inhibiting PPF. Furthermore, we develop a G6PD-derived peptide, GC2P2, which potentiates the G6PD-TMOD3 interaction and significantly suppresses thrombocytopoiesis both in vitro and in vivo. In conclusion, our findings suggest that G6PD deficiency facilitates platelet biogenesis by altering TMOD3-mediated actin dynamics. The study provides new insights into thrombopoiesis and offers potential strategies for modulating platelet production.

Congenital antithrombin (AT) deficiency, primarily caused by SERPINC1 variants, is a major risk factor for venous thromboembolism (VTE). We previously reported the SERPINC1 p.M313T variant in three VTE probands with normal AT activity and antigen levels. This study aims to elucidate its pathogenetic mechanism. Thrombin generation test (TGT), thermal stability, native-urea PAGE, in vitro protein expression, and enzymatic assays were performed. Glycosylation analysis was conducted using glycosidase treatment, and structural analysis was performed through molecular dynamics simulation. AT in probands' plasma samples exhibited reduced thermostability and increased proportions of denatured and latent forms compared with normal pooled plasma. The recombinant AT-M313T protein exhibited increased inhibitory activity, consistent with findings in proband plasma based on AT activity and TGT. Despite this enhanced activity, the mutant protein demonstrated reduced thermostability and a marked tendency to transition into the latent form, potentially predisposing carriers to thrombosis under stress conditions. These characteristics may result from the introduction of abnormal O-linked glycosylation within the breach region, confirmed in both plasma-derived and recombinant AT. Molecular dynamics simulation revealed a less compact structure, with increased spacing in the shutter region and enhanced flexibility of the reactive center loop. The SERPINC1 p.M313T variant exhibits dual characteristics of high inhibitory activity and low structural stability, which together contribute to the transient AT deficiency. These findings suggest that AT deficiency may be underdiagnosed and highlight the importance of integrating techniques such as native-urea PAGE into standard diagnostic workflows to identify variants associated with structural abnormalities.

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.