Inhibiting thromboinflammation via interleukin-4 binding to platelet glycoprotein VI and suppression of thrombosis

Inflammation and coagulation are intricately linked. Although some studies have documented pro-inflammatory drivers of coagulation, the role of anti-inflammatory cytokines and the underlying mechanisms in the modulation of coagulation, platelet function, and thrombosis remain poorly characterized. In this study, we demonstrated that interleukin-4 (IL-4), an anti-inflammatory cytokine, plays a crucial role in regulating platelet function and thrombus formation. IL-4 inhibited collagen-induced intracellular calcium mobilization, P-selectin expression, ATP release, integrin αIIbβ3 activation, platelet spreading, and platelet aggregation. IL-4 binds to residues 38-46 within GPVI's extracellular collagen-binding domain and suppresses downstream signaling by inhibiting phosphorylation of phospholipase Cγ2 and protein kinase C. A GPVI-derived interfering peptide (RR9) binding to IL-4 disrupted the IL-4-GPVI complex and reversed the inhibitory effects of IL-4 on platelets. In vivo, IL-4 overexpression or intravenous administration inhibited platelet intracellular calcium mobilization, ATP release, and aggregation, prolonged bleeding time, and attenuated both arterial and venous thrombosis. Conversely, IL-4 knockout, anti-IL-4 antibody treatment, or RR9 treatment enhanced platelet reactivity, promoted hemostasis, and aggravated thrombosis. Our findings reveal that IL-4 negatively regulates platelet function through direct interaction with GPVI, thereby facilitating crosstalk among inflammation, hemostasis, and thrombosis. Therefore, modulating IL-4 levels provides a novel strategy for treatment of thrombotic disorders.