OC 78.3 - Interferon-induced Transmembrane Protein 3 in platelets mediates granule secretion, thrombosis , and pathophysiological outcomes in sepsis

Background: Thrombosis, precipitated by dysregulated platelet responses, contributes to adverse clinical outcomes, including organ failure and death during systemic inflammation (e.g., sepsis). Previously, our lab identified interferon-induced transmembrane protein 3 (IFITM3) as a mediator of anti-viral responses in platelets and MKs during inflammation. Whether IFITM3 mediates platelet functions and subsequent pro-thrombotic responses during systemic inflammation has not been previously examined.

Aims: To define the role of platelet IFITM3 in systemic inflammation.

Methods: Cecal-ligation and Puncture (CLP) model of sepsis and Interferon alpha (IFN)-induced sterile inflammation model was performed on WT and platelet- and MK-specific IFITM3 knockout strain (IFITM3fl/fl PF4-Cre+/-). P-Selectin and CD63 expression, Platelet Factor 4 (PF4) release, platelet-leukocyte aggregates, plasma cytokines (TNF-, IL-6, CXCL1), as well as survival outcomes, platelet deposition and aggregates within organs (e.g. lung), plasma aspartate aminotransferase, lactate dehydrogenase, D-Dimer levels, were measured by flow cytometry, enzyme-linked immunosorbent assays (ELISAs), and Immunohistochemistry. In vivo collagen-epinephrine-induced pulmonary embolism model, and ex vivo thrombus formation under flow using microfluidic devices were performed.

Results: CLP sepsis or IFN-induced sterile inflammation upregulated IFITM3 in platelets which were associated with significantly enhanced secretion of P-Selectin, PF4, and CD63, and increased mortality in a collagen-epinephrine-induced murine pulmonary embolism model. Biochemical assays demonstrated IFITM3 interacts with VAMP-8, the primary v-SNARE that drives platelet secretion. Platelets lacking IFITM3 showed reduced granule secretion and reduced platelet-leukocyte aggregate formation. Sepsis-induced mortality, platelet-leukocyte aggregates, platelet deposition and aggregates within the lung, and markers of organ failure and thrombosis (e.g., aspartate aminotransferase, lactate dehydrogenase, D-Dimer), and thrombus formation under flow were all significantly reduced in the absence of IFITM3 in CLP sepsis.

Conclusion(s): Collectively, our findings suggest that IFITM3 is a novel regulator of secretion and aggregation in platelets and contributes to thrombotic events during systemic inflammation.