Salivary glands from blood feeding arthropods secrete several molecules that inhibit mammalian hemostasis and facilitate blood feeding and pathogen transmission. The salivary functions from Simulium guianense, the main vector of River Blindness in South America remain largely understudied. Here we have characterized a salivary protease inhibitor (Guianensin) from the blackfly Simulium guianense. Guianensin was identified in the sialotranscriptome of adult S. guianense flies and belongs to the Kunitz-domain of protease inhibitors. It targets various serine proteases involved in hemostasis and inflammation. Binding to these enzymes is highly specific to the catalytic site and is not detectable for their zymogens or the catalytic site blocked human coagulation factor Xa (FXa) and thrombin. Accordingly, Guianensin significantly increased both PT and aPTT in human plasma and consequently increased blood clotting time ex vivo. Guianensin also inhibited FXa generation and prothrombinase activity on endothelial cells. We also show that Guianensin act as a potent anti-inflammatory on FXa-induced paw edema formation in mice. The information generated by this work highlights the biological functionality of Guianensin as an antithrombotic and anti-inflammatory protein that may play significant roles in blood feeding and pathogen transmission.
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