IFITM3 regulates fibrinogen endocytosis and platelet reactivity in nonviral sepsis
Robert A. Campbell, … , Anandi Krishnan, Matthew T. Rondina
Robert A. Campbell, … , Anandi Krishnan, Matthew T. Rondina
Published October 4, 2022
Citation Information: J Clin Invest. 2022;132(23):e153014. https://doi.org/10.1172/JCI153014.
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Research Article Hematology Inflammation

IFITM3 regulates fibrinogen endocytosis and platelet reactivity in nonviral sepsis

Abstract

Platelets and megakaryocytes are critical players in immune responses. Recent reports suggest infection and inflammation alter the megakaryocyte and platelet transcriptome to induce altered platelet reactivity. We determined whether nonviral sepsis induces differential platelet gene expression and reactivity. Nonviral sepsis upregulated IFN-induced transmembrane protein 3 (IFITM3), an IFN-responsive gene that restricts viral replication. As IFITM3 has been linked to clathrin-mediated endocytosis, we determined whether IFITM3 promoted endocytosis of α-granule proteins. IFN stimulation enhanced fibrinogen endocytosis in megakaryocytes and platelets from Ifitm+/+ mice, but not Ifitm–/– mice. IFITM3 overexpression or deletion in megakaryocytes demonstrated IFITM3 was necessary and sufficient to regulate fibrinogen endocytosis. Mechanistically, IFITM3 interacted with clathrin and αIIb and altered their plasma membrane localization into lipid rafts. In vivo IFN administration increased fibrinogen endocytosis, platelet reactivity, and thrombosis in an IFITM-dependent manner. In contrast, Ifitm–/– mice were completely rescued from IFN-induced platelet hyperreactivity and thrombosis. During murine sepsis, platelets from Ifitm+/+ mice demonstrated increased fibrinogen content and platelet reactivity, which was dependent on IFN-α and IFITMs. Platelets from patients with nonviral sepsis had increases in platelet IFITM3 expression, fibrinogen content, and hyperreactivity. These data identify IFITM3 as a regulator of platelet endocytosis, hyperreactivity, and thrombosis during inflammatory stress.

Authors

Robert A. Campbell, Bhanu Kanth Manne, Meenakshi Banerjee, Elizabeth A. Middleton, Abigail Ajanel, Hansjorg Schwertz, Frederik Denorme, Chris Stubben, Emilie Montenont, Samantha Saperstein, Lauren Page, Neal D. Tolley, Diana L. Lim, Samuel M. Brown, Colin K. Grissom, Douglas W. Sborov, Anandi Krishnan, Matthew T. Rondina

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Figure 9

IFITM deficiency reduces platelet reactivity in murine model of sepsis.

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IFITM deficiency reduces platelet reactivity in murine model of sepsis. ...
(A and B) Sepsis was induced in Ifitm+/+ mice by CLP, and Ifitm3 expression in flow-sorted CD41+ bone marrow megakaryocytes and CD45-depleted platelets was examined at the indicated times by RT-PCR. Ifitm3 expression was normalized to Gapdh and compared with that control mice. n = 6–12 (A); n = 5–27 (B). *P ≤ 0.05; ****P ≤ 0.0001, 1-way ANOVA with Dunnett’s post hoc test compared with control. (C) Platelet Ifitm3 protein expression in sham or CLP Ifitm+/+ mice was examined by immunoblot at day 3 of CLP. n = 3. (D–G) Sepsis was induced in Ifitm+/+ and Ifitm–/– mice by CLP, and platelets were isolated at day 3 after CLP for wash platelet aggregation in response to 2MesADP (10 nM, final D and E) and collagen (2 μg/mL, final F and G). Platelet aggregation was compared with that of sham-operated control in either Ifitm +/+ or Ifitm –/– mice (n = 3 per group). *P ≤ 0.05, 1-way ANOVA with Šidák’s multiple comparisons test. (H–J) Sepsis was induced in Ifitm+/+ mice by CLP. One hour before and 6 hours after CLP, mice were treated with 1 mg (total, i.p.) of either an anti–IFN-αR1 antibody or control IgG. Washed platelets were isolated at day 3 after CLP and aggregation assessed in response to 2MesADP. (H and I). Platelet Ifitm3 expression was also measured by immunoblot (Supplemental Figure 23). (J). One-way Kruskal Wallis test with Dunn’s multiple comparisons test or 1-way ANOVA with Tukey’s multiple comparisons test (J). **P ≤ 0.01; ****P ≤ 0.0001. n = 6–8 (H and I); n = 3–4 (J).