Pertinaxin, a salivary Kunitz-type protease inhibitor from Simulium pertinax, inhibits factor Xa and impairs plasma coagulation

Abstract Salivary protease inhibitors are key components of the anti-hemostatic repertoire that enables hematophagous arthropods to maintain blood flow during feeding. Here, we characterized Pertinaxin, a previously unstudied Kunitz-type protease inhibitor identified in the salivary gland transcriptome of Simulium pertinax . Pertinaxin displays the conserved structural features of the Kunitz family and shares substantial sequence similarity with other black fly salivary inhibitors. Recombinant Pertinaxin delayed human plasma coagulation in complementary assays and inhibited selected serine proteases, with factor Xa (FXa) emerging as a prominent target. FXa inhibition occurred in the nanomolar range, whereas thrombin was not significantly inhibited and elastase activity was only minimally affected. Kinetic analyses further supported a concentration-dependent inhibitory effect of Pertinaxin on FXa activity. Together, these characterize Pertinaxin as a functionally active salivary Kunitz-type protease inhibitor with anticoagulant activity and expand our understanding of the antihemostatic repertoire of S. pertinax and the molecular strategies employed by simuliids during blood feeding.