Bannakunin: a dual-target Kunitz inhibitor bridging anticoagulation (FXa/XIIa) and anti-platelet (α2β1/P2Y12) pathways

Thrombosis is a major cause of morbidity and mortality worldwide. Consequently, there is an ongoing search for efficacious and safe anti-thrombotic drugs. Haematophagous animals have developed a large variety of salivary bioactive components to counteract host haemostatic responses. We aim to discover anti-thrombotic agents with dual anti-platelet and anticoagulant activities. A novel single Kunitz domain inhibitor (Bannakunin) precursor cloned from the salivary glands complementary DNA (cDNA) library of blood-sucking black fly Simulium bannaense was expressed in Escherichia coli. Recombinant Bannakunin was purified by Immobilised Metal Affinity Chromatography and High-Performance Liquid Chromatography. The secondary structure was determined by circular dichroism spectroscopy. The anti-thrombotic activity was evaluated through carotid artery thrombosis and tail vein thrombosis models. The inhibitory activity was evaluated using serine protease inhibition assays, SPR and molecular docking. The regulation on platelets was assessed by platelet aggregation, clot retraction and platelet spreading assays. Subsequently, its target receptors and signalling pathways were investigated through western blotting, enzyme-linked immunosorbent assay (ELISA) and flow cytometry. Recombinant Bannakunin demonstrated significant anti-thrombotic efficacy in murine FeCl3-induced carotid artery and carrageenan-induced tail vein thrombosis models and did not induce bleeding complications. Simultaneously, Bannakunin markedly prolonged prothrombin time (PT) and activated partial thromboplastin time (aPTT) in human plasma. Further investigation revealed that Bannakunin could inhibit the activity of the coagulation factors FXa and FXIIa, as well as the activities of elastase, trypsin, and plasma kallikrein, but it did not inhibit thrombin and FXIa. Surface plasmon resonance studies have shown that Bannakunin binds to the active sites of human elastase (KD: 1.95 nM) and human FXa (KD: 42.9 nM) with the highest affinity. Intriguingly, we observed that Bannakunin significantly inhibited clot retraction, as well as platelet aggregation and spreading. Mechanistically, Bannakunin inhibited collagen-induced platelet activation by downregulating the integrin α2β1-mediated Src/Syk/PLCγ2 signalling pathway and the release of Ca2+, TXB2 and ATP. Furthermore, Bannakunin could effectively inhibit ADP-induced platelet activation through blocking P2Y12 receptor, decreasing the activation of PI3K/Akt signalling pathways and upregulating the level of cAMP. These findings enrich our understanding of the anti-platelet functions of Kunitz-type inhibitors and position Bannakunin as a promising molecular template for the development of novel anti-thrombotic drugs.