Sibakazin: a novel black fly derived non-classical kazal protease inhibitor, targeting antiplatelet (P2Y12/GPVI) pathways without perturbing coagulation.

While Kazal-type inhibitors from hematophagous animals are well-known anticoagulants, their roles in platelet regulation remain largely unexplored. Here, we report the characterization of Sibakazin, a single-domain Kazal-type inhibitor from the salivary glands of the blood-sucking black fly Simulium bannaense. Structurally, Sibakazin possesses a non-canonical acidic N-terminus and methionine at the P1 site. Consistent with this distinctive structure, Sibakazin exhibited no activity against key serine proteases of the coagulation cascade (e.g., thrombin, FXa) and did not affect clotting times (aPTT, PT), distinguishing it from classical anticoagulants. Intriguingly, Sibakazin demonstrated potent antithrombotic efficacy in vivo, suppressing both FeCl3-induced arterial and carrageenan-induced venous thrombosis in mice without increasing bleeding risk. Mechanistically, Sibakazin exerts its effects through dual inhibition of platelet activation. It directly targets glycoprotein VI (GPVI), suppressing the downstream Src/Syk/PLCγ2/Akt signaling cascade to block thrombus initiation, while simultaneously inhibiting the ADP receptor P2Y12, disrupting the PI3K/Akt pathway to attenuate secondary amplification signals. This dual blockade effectively inhibits platelet aggregation, spreading, and clot retraction. Our findings identify Sibakazin as a novel dual-targeting antiplatelet agent with potent safe antithrombotic properties, expanding the functional landscape of Kazal-type inhibitors and providing a promising contribution for next generation therapeutic development.