R. prolixus saliva contains bioactive anti-hemostatic lipocalin, nitrophorin (NP). NPs are multifunctional proteins of 21 KDa. During blood feeding, NO bound to NP is released at the site of feeding. Upon release of NO, NP binds histamine released by the hosts’ mast cell causing vasodilation, antiinflammation and anticoagulation. In R. prolixus, several NPs (NP1-7) have been described. Among them, NP2 inhibits formation of factor X activating complex by binding to Factor IXa in the intrinsic blood coagulation cascade. NP2 is a specific inhibitor of Xase with a Ki of 6.2 nm while NP3 is less active. NP1 and NP4 show no activity. Also, differences in sequence exist between NP2 and other NPs. Crystal structure suggests that NP2 and NP4 present highly different surfaces to factor Xase complex. NP2 has 4 fewer residues at the C-terminal than NP4 resulting in the exposure of beta barrel face to solvent. Our goal is to identify the structure-function relationships of NP2. To achieve this, charged amino acid residues that are likely to be involved in FXase interaction were mutated by site directed mutagenesis. The mutants were cloned into PET 17b, expressed in E. coli. The recombinant NP2 was purified and assayed for FX inhibition. Results from kinetic assays and surface plasmon resonance will be presented.