The saliva of blood-feeding insects contains a variety of antihemostatic agents that aid in obtaining an uninterrupted flow of blood to the mouthparts. These include vasodilators, platelet aggregation inhibitors, and anticoagulants. Most of these substances are proteinaceous, and a number of their targets have been identified. However, the molecular mechanisms for their actions and the structural features responsible for their activity are largely unknown. This proposal focuses on two types of antihemostatic agents from the saliva of the triatomine bug Rhodnius prolixus, the anticoagulant nitrophorin 2 and three proteins known as salivary antiplatelet lipocalins (SAPLs). Nitrophorins are a group of four multifunctional hemeproteins which transport nitric oxide and bind histamine. Nitrophorin 2 is the only member of the group possessing anticoagulant activity as well. SAPLs 1-3 inhibit collagen-mediated platelet aggregation and thrombin activity. Surprisingly, both the nitrophorins and SAPLs belong to the lipocalin protein family despite their completely different activities. The hypotheses tested in these studies are that the anticoagulant activity of nitrophorin 2 and the antiplatelet activity of SAPLs 1-3 are due to interaction of these molecules with specific proteinaceous targets or binding of essential ligands.
The specific aims of the study are to analyze the mechanisms and structural determinants of activity of nitrophorin 2 and SAPLs 1-3 using functional assays, X-ray crystallography, and site-directed mutagenesis.
