Bannaensin, a Two-Kunitz-Domain Black Fly Salivary Protein Suppresses Host Hemostasis by Targeting FXa and Platelet Signaling

While single-domain Kunitz-type inhibitors in black fly salivary glands have been well characterized, limited information is available regarding the roles and mechanisms of inhibitors containing multiple Kunitz domains during the blood-feeding process. Here, a two-Kunitz-domain inhibitor (Bannaensin) precursor cloned from the salivary glands cDNA library of blood-sucking black fly Simulium bannaense, was expressed in Escherichia coli. Recombinant Bannaensin prolonged plasma recalcification time, activated partial thromboplastin time (aPTT), and prothrombin time (PT), and exhibited high-affinity binding to FXa (Kd = 21.91 nM) and trypsin (Kd = 6.168 nM), indicating potent anticoagulant activity. Intriguingly, in addition to direct serine protease inhibitory activity, Bannaensin significantly suppressed clot retraction, platelet aggregation and spreading. Mechanistically, it suppressed collagen-induced platelet aggregation by blocking integrin α2β1 receptor and the activation of Src/Syk/PLCγ2 signaling pathways, which reduced intracellular calcium and Thromboxane A2 (TXA2) release. Additionally, Bannaensin inhibited ADP-induced platelet aggregation by functioning as a P2Y12 receptor antagonist and suppressing its downstream PI3K/Akt signaling pathway. In mouse thrombosis models, Bannaensin effectively prevented FeCl3-induced carotid artery thrombosis and carrageenan-induced tail vein thrombosis without causing significant bleeding. Our data enrich the understanding of salivary functional diversity in hematophagous insects, providing new insights into the molecular adaptations underlying hematophagy. Specifically, we reveal a previously unrecognized evolutionary strategy where a two-Kunitz-domain architecture simultaneously modulates host platelet function and coagulation pathways. This dual-targeting mechanism not only highlights the sophisticated anti-hemostatic armory of black flies but also emphasizes the potential of Bannaensin as a promising lead molecule for antithrombotic therapy.