Sibanin, a novel black fly-derived Kunitz protease inhibitor, prevents thrombus formation in mice by anticoagulation-antiplatelet duality

Abstract

Most Kunitz inhibitors exhibit serine protease inhibitory activity, but limited information is available on the regulation of platelet function. Herein, we report the purification and characterization of a novel single Kunitz domain inhibitor (Sibanin) from the salivary glands of the black fly Simulium bannaense. Recombinant Sibanin prolonged activated partial thromboplastin time and prothrombin time, and exhibited high-affinity binding to FXa and elastase with a KD of 5.0 nM and 1.67 nM, respectively. Moreover, Sibanin also shows strong anti-inflammatory and analgesic functions, which would facilitate blood-feeding. Of note, Sibanin markedly suppressed platelet spreading and aggregation, as well as clot retraction. Further studies showed that Sibanin dose-dependently inhibited ADP-induced platelet aggregation by acting on the P2Y12 receptor and blocking its downstream PI3K/AKT/ERK signal pathway. Furthermore, Sibanin also suppressed collagen-induced platelet aggregation by blocking the glycoprotein VI (GPVI) receptor and attenuating the activation of RAP1 signaling pathways. In addition, Sibanin prevented FeCl₃-induced arterial thrombosis and carrageenan-induced tail vein thrombosis in mice without inducing a bleeding tendency. Our findings provide new insights into the molecular and functional of Kunitz inhibitors, and will contribute to the understanding of the molecular mechanisms that mediate hematophagous lifestyle of Simulium bannaense.