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.
View: Text | PDF
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

×

Figure 8

Increased IFITM3 expression in nonviral sepsis patients is associated with increased fibrinogen content and platelet hyperreactivity.

Options: View larger image (or click on image) Download as PowerPoint
Increased IFITM3 expression in nonviral sepsis patients is associated wi...
(A) Platelets from healthy donors and nonviral sepsis patients were stained for IFITM3 (yellow), fibrinogen (magenta), and actin (blue). White arrows demonstrate representative platelets positive for IFITM3 and fibrinogen (n = 3). Pink arrows indicate IFITM3-positive, fibrinogen-positive platelets. Scale bars: 1 μm. (B and C). Immunoblot of platelets isolated from healthy donors and nonviral sepsis patients and probed for fibrinogen, αIIb, and p-FAK in the presence or absence of activation by collagen (2 μg/mL, final) (n = 3). Unpaired t test. (D–G) Washed platelet aggregation using platelets isolated from healthy donors and nonviral sepsis patients in response to collagen (2 μg/mL, final) and 2MesADP (10 nM, final) (n = 5 per group). Unpaired t test. (H and I) Platelets isolated from healthy donors and nonviral sepsis patients were fractionated for lipid rafts using a sucrose density gradient and probed for IFITM3, αIIb, clathrin, LAT, and β-actin. Representative blots are shown in H and quantified in I. Exposures for each protein pair were the same. Fraction 11 contains all the nonsoluble material (n = 3 independent experiments). (J) Platelet mRNA was isolated from healthy donors (n = 5) and nonviral sepsis patients (n = 14) at day 0 (initial hospitalization) and from the same sepsis patients at day 90 (days after discharge). IFITM3 expression was measured by quantitative reverse-transcriptase PCR (RT-qPCR) normalized to GAPDH and compared with expression levels in healthy donors. Brown-Forsythe and Welch’s ANOVA tests with Dunnett’s T3 multiple comparisons test. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001.