title: Biochemical and functional characterization of recombinant Rhodnius prolixus platelet aggregation inhibitor 1 as a novel lipocalin with high affinity for adenosine diphosphate and other adenine nucleotides.
abstract: The Rhodnius prolixus aggregation inhibitor 1 (RPAI-1) is a novel blood-sucking salivary molecule that binds to ADP and attenuates platelet aggregation. In this report, we determine the binding constants and specificity of RPAI-1 for adenine nucleotides and its functional significance. By the Hummel-Dreyer method of equilibrium gel filtration, we show that RPAI-1 binds ADP with a K(0.5) of 48.6 plus minus 12.2 nM. RPAI-1 also displays high-affinity binding to ATP, AMP, Ado, AP4A, and alpha,beta Met ADP; however, RPAI-1 does not bind to inosine, guanosine, uridine, or cytidine. Binding is not modified by EDTA, indicating that Ado structure but not phosphate groups or Ca(2+) is necessary for binding. By computer simulation, we show that RPAI-1 is more effective in scavenging low but not high concentrations of ADP, in contrast to R. prolixus apyrase. RPAI-1 inhibits in vitro the ADP-dependent platelet-rich plasma aggregation by collagen (COLL), TRAP, PAF, and A23187 but did not block platelet aggregation by ristocetin or phorbol myristate acetate (PMA) and only slightly attenuated that by convulxin. RPAI-1 prolongs the closure time as assessed with PFA-100, when COLL-Epi but not COLL-ADP cartridges are employed. RPAI-1 also affects platelet-mediated hemostasis time and COLL-induced thrombus formation at high shear as assessed with the Clot Signature Analyzer. We conclude that RPAI-1 exerts an antiplatelet effect due to scavenging of low concentrations of ADP in vitro and in vivo. RPAI-1 is the first lipocalin described so far with unique specificity for adenine nucleotides.
authors.affiliation: Laboratory of Parasitic Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of health, 4 Center Drive, Room 4/126, MSC-0425, National Institutes of Health, Bethesda, Maryland 20892-0425, USA.
authors.forename: Ivo M B
authors.initials: IM
authors.lastname: Francischetti
authors.forename: John F
authors.initials: JF
authors.lastname: Andersen
authors.forename: José M C
authors.initials: JM
authors.lastname: Ribeiro
mesh_terms: D000227:Adenine Nucleotides / Q000378:metabolism*; D002352:Carrier Proteins / Q000378:metabolism*; D003198:Computer Simulation; D006801:Humans; D019476:Insect Proteins / Q000378:metabolism*; D007700:Kinetics; D010974:Platelet Aggregation; D011485:Protein Binding; D011994:Recombinant Proteins / Q000378:metabolism; D012471:Salivary Proteins and Peptides / Q000378:metabolism*
chemical_list: D000227:Adenine Nucleotides; D002352:Carrier Proteins; D019476:Insect Proteins; C408466:RPAI-1 protein, Rhodnius prolixus; D011994:Recombinant Proteins; D012471:Salivary Proteins and Peptides