We propose to study a series of six insect salivary proteins that facilitate the taking of a blood meal. These proteins all interfere with hemostasis - the body's means of preventing blood loss. They accomplish this through a variety of mechanisms, including the delivery of nitric oxide to induce vasodilation, the sequestering of histamine to interfere with inflammation, and the direct inhibition of the blood coagulation cascade. Remarkably, five of the proteins, all from the insect Rhodnius prolixus, display at least five distinct antihemostatic activities that are all apparently built into the same protein fold. Our initial interest was to uncover the mechanism by which four of the proteins use heme to reversibly bind and transport nitric oxide (NO) - a compound that is normally toxic to hemoproteins such as hemoglobin, and yet is a second messenger in most (possible all) animal cells. We are now in a position to answer this question, based on results from X-ray crystallograpic, spectroscopic, kinetic, and thermodynamic measurements. During our initial experiments, it became clear that the greater family of salivary proteins provided the opportunity to address several additional questions of biological interest, including those pertaining to protein evolution, biochemical signaling, molecular dynamics, inflammation during blood-feeding by insects, and physiological aspects of blood coagulation. The proposed studies are to (1) determine the crystal structures of wild-type and mutant forms of the proteins; (2) measure the kinetics and thermodynamics of NO transport by the proteins; (3) determine the mechanistic role of a unique NO- induced conformational change in the proteins; (4) uncover those features of the proteins that give rise to anticoagulation; and (5) to begin study of related NO signaling proteins. Although beyond the scope of the present proposal, it also possible that our studies will lead to practical therapies such as for the reduced spread of Chagas' disease, the trypanosome for which is delivered by the Rhodnius insect, or development of anticoagulants, vasodilators, anti-inflammatory agents, or blood substitutes.