Rhodnius prolixus aggregation inhibitor (RPAI-1) is a 19-kDa. lipocalin isolated from the salivary gland of the Chagas' Disease vector Rhodnius prolixus. RPAI-1 was cloned in a PET17b expression vector and expressed in E. coli as an active molecule. RPAI-1 inhibits platelet aggregation by low concentrations of collagen, arachidonic acid and U46619 and very low doses of thrombin and convulxin. The effects of RPAI-1 on platelets were studied using U46619 as an agonist. RPAI-1 dose-depently inhibits 1146619-induced platelet aggregation with a IC50 ~ 200-400 uM. However, RPAI-1 does not inhibit shape change or calcium increase triggered by U46619, but it attenuates U46619-mediated granule secretion and characteristicaly, large platelet aggregates formation. Control experiments indicate that RPAI-1 does not bind to U46619 or arachidonic acid, nor it affects [3H]U46619 binding to platelets. In addition, RPAI-1 is less effective as an inhibitor in platelets that have been desensitized to U466I9, or platelets whose secretion has been impaired, or bovine platelets that are insensitive to TXA2 and ADP. Taken together, our results suggest that RPAI-1 affects a distal point of platelet aggregation triggered by U46619, most likely by attenuation of the pro-aggregatory effects of released ADP. Indeed, we show that RPAI-1 inhibits platelet aggregation triggered by ADP at concentrations lower than 1 uM, and it blocks ADP-dependent platelet responses, including ADP-mediated U46619-induced cAMP-decrease stimulated by PGE1. In addition, the effects of RPAI-1 on U46619-induced platelet aggregation are overcame by sub-aggregating concentrations of ADP. By means of Hummel-Dreyer methods to study ligand-ligand interations.we show that RPAI-1 binds ADP and adenosine nucleotides with nanomolar affinity, as follows: ADP (48.9 nM), ATP (14.8 nM), AMP (31.8 nM), adenosine (60.0 nM), diadenosine tetraphosphates (18.9 nM), and alpha-beta-methylene ADP (26.9 nM). RPAI-1 does not interact with inosine, guanosine, uridine, and cytidine. These data indicate that RPAI-1 is a high-affinity ADP binding lipocalin that affects platelet function by scavenging low, although physiological important concentrations of ADP that are not readly destroyed by apyrases. Since ADP mediates platelet aggregation by low concentrations of most agonists, we suggest that, in vivo, RPAI-1 function by increasing the threshold of platelet activation to most agonists, by removing free ADP from the medium.
|Original language||English (US)|
|Issue number||11 PART I|
|State||Published - 2000|
ASJC Scopus subject areas
- Cell Biology