Marie Lordkipanidzé

Mutations in the gene coding G6b-B, Mpig6b in mice, and MPIG6B in humans lead to myelofibrosis, progressive splenomegaly and osteosclerosis. Aberrant megakaryocyte function in bone marrow and spleen may affect bone cells through cell-cell contact or soluble factors. We aimed to assess the role of circulating factors produced by splenic cells in mediating osteosclerosis in Mpig6b ^−/− mice using splenectomy, a procedure commonly used in clinics. Splenectomy or sham surgery was performed on 32-week-old female Mpig6b ^−/− and littermate wildtype (WT) mice, when splenomegaly and long bone osteosclerosis were established. Twenty weeks later, the hematopoietic parameters and bone structure of the femur and lumbar vertebra were analyzed. Splenectomy significantly improved platelet count, but the larger platelet size persisted in Mpig6b ^−/− mice. The Mpig6b ^−/− femurs with established osteosclerosis were not affected by splenectomy. In contrast, splenectomy prevented the development of osteosclerosis in vertebrae of Mpig6b ^−/− mice. Significant vertebral bone loss and increased circulating TRACP 5b were demonstrated in splenectomized Mpig6b ^−/− mice compared to sham-operated and WT mice. Using quantitative plasma proteomics, we identified intercellular adhesion molecule 2 (ICAM2) as significantly increased in Mpig6b ^−/− mice compared to WT and reversed to normal levels after splenectomy. The interaction of membrane-bound ICAM2 and MAC1 was reported to be critical for osteoclast fusion. Our in vitro study revealed that soluble recombinant ICAM2 dose-dependently inhibited osteoclast fusion, suggesting that competitive binding of MAC1 with soluble ICAM2 prevented its interaction with membrane-bound ICAM2. Thus, we identified the soluble ICAM2 as a novel inhibitor of osteoclast fusion and mediator of osteosclerosis in Mpig6b ^−/− mice.

Brain-Derived Neurotrophic Factor (BDNF), highly enriched in platelets, contributes to vascular integrity and thrombotic responses. The rs11030119 variant, located in a BDNF intronic enhancer, has been linked to stroke recovery, but its influence on peripheral BDNF dynamics and hemostasis remains unexplored. In this study, we aimed to understand the impact of rs11030119 on circulating levels of BDNF, and its subsequent effects on thrombus formation. Twenty-six healthy individuals homozygous for either the GG or AA genotype of rs11030119 were matched for age and sex. BDNF and proBDNF levels were quantified in serum, plasma, and platelets. Platelet aggregation, ATP secretion, thrombin generation, and clot firmness were assessed. Washed platelets were also tested with recombinant BDNF. AA carriers displayed significantly lower plasma and serum BDNF levels and diminished BDNF release upon activation, despite comparable platelet BDNF content. The expression of its receptor, TrkB, on the platelet surface was also reduced in AA carriers. However, platelet reactivity, thrombin generation, and viscoelastic clot properties were preserved across genotypes. In conclusion, rs11030119 modulates circulating and platelet-releasable BDNF without impairing hemostatic function, suggesting that BDNF bioavailability may not directly correlate with thrombotic potential. These findings uncover a novel genetic mechanism controlling platelet-derived neurotrophin output.

Neutrophil extracellular traps can contribute to thrombosis via stabilization of fibrin network, which is normally conducted by plasma transglutaminase, Factor XIII-A as part of coagulation cascade. The possible presence and activity of FXIII-A in neutrophils or during NETosis are unknown. Here, we investigated potential presence of FXIII-A in neutrophils and participation in NET-fibrin(ogen) interaction in vitro. Data mining of human and mouse F13A1/F13a1 mRNA expression in whole-body scRNA sequence atlases was conducted. F13a1 mRNA and protein expression was assessed in isolated mouse bone marrow neutrophils. NETosis was induced using 12-phorbol 13-myristate acetate (PMA), and the transglutaminase activity was assessed with 5-(biotinamido)pentylamine incorporation to plasma fibronectin and a fluorescence-fibrin(ogen)-based activity assay using ATTO488-Cadaverine. Externalization of FXIII-A and its interaction with neutrophil extracellular trap (NET) markers, namely, decondensed DNA, CitH3, and MPO, were examined with immunofluorescence microscopy. NET–fibrin(ogen) interaction was investigated with and without serum and/or transglutaminase inhibitor, NC9. Effect of soluble fibrinogen and fibrin(ogen) network on NETosis was also assessed. Data mining of RNAseq atlases showed F13A1/F13a1 expression in adipose tissue, blood, and bone marrow neutrophils. mRNA expression and protein production were confirmed in isolated neutrophils where expression was comparable to that of macrophages and monocytes. FXIII-A was externalized and active as a transglutaminase and colocalized with NET markers during NETosis. FXIII-A transglutaminase activity promoted NET–fibrin(ogen) interaction and entrapment of neutrophils within fibrin(ogen) matrix. Soluble fibrinogen or fibrin(ogen) network did not induce NETosis. This study identifies neutrophils as a source of FXIII-A and suggests its role in stabilizing NET–fibrin(ogen) matrix structures.

Platelets display unexpected roles in immune and coagulation responses. Emerging evidence suggests that STING is implicated in hypercoagulation. STING is an adaptor protein downstream of the DNA sensor cyclic GMP-AMP synthase (cGAS) that is activated by cytosolic microbial and self-DNA during infections, and in the context of loss of cellular integrity, to instigate the production of type-I IFN and pro-inflammatory cytokines. To date, whether the cGAS-STING pathway is present in platelets and contributes to platelet functions is not defined. Using a combination of pharmacological and genetic approaches, we demonstrate here that megakaryocytes and platelets possess a functional cGAS-STING pathway. Our results suggest that in megakaryocytes, STING stimulation activates a type-I IFN response, and during thrombopoiesis, cGAS and STING are transferred to proplatelets. Finally, we show that both murine and human platelets contain cGAS and STING proteins, and the cGAS-STING pathway contributes to potentiation of platelet activation and aggregation. Taken together, these observations establish for the first time a novel role of the cGAS-STING DNA sensing axis in the megakaryocyte and platelet lineage.