Purification, cloning, expression, and mechanism of action of a novel platelet aggregation inhibitor from the salivary gland of the blood-sucking bug, Rhodnius prolixus

Ivo M.B. Francischetti, José M.C. Ribeiro, Donald Champagne, John Andersen

Research output: Contribution to journalArticlepeer-review

Abstract

Rhodnius prolixus aggregation inhibitor 1 (RPAI-1), a 19-kDa protein isolated from the salivary gland of R. prolixus, was purified by strong cation exchange and reverse-phase high performance liquid chromatographies. Based on 49 amino-terminal amino acid sequences of RPAI-1, primers were produced to generate probes to screen an R. prolixus salivary gland cDNA library. A phage containing the full-length clone of RPAI-1 codes for a mature protein of 155 amino acids. RPAI-1 shows sequence homology to triabin and pallidipin, lipocalins from Triatoma pallidipennis. The cDNA sequence was cloned in Pet17B Escherichia coli expression vector, producing an active peptide. RPAI-1 inhibits human platelet-rich plasma aggregation triggered by low concentrations of ADP, collagen, arachidonic acid, thromboxane A2 mimetics (U46619), and very low doses of thrombin and convulxin. Here we show that ADP is the target of RPAI-1 since (i) RPAI-1 inhibits ADP-dependent large aggregation formation and secretion triggered by U46619, without affecting Ca2+ increase and shape change; (ii) ADP restored the inhibition of U46619-induced platelet aggregation by RPAI-1, (iii) PGE1-induced increase of cAMP (which is antagonized by U46619 in an ADP-dependent manner) was restored by RPAI-1, (iv) RPAI-1 inhibits low concentrations of ADP- mediated responses of indomethacin-treated platelets, and (v) RPAI-1 binds to ADP, as assessed by large zone chromatography. RPAI-1 affects neither integrin α2β1- nor glycoprotein VI-mediated platelet responses. We conclude that RPAI-1 is the first lipocalin described that inhibits platelet aggregation by a novel mechanism, binding to ADP.

Original languageEnglish (US)
Pages (from-to)12639-12650
Number of pages12
JournalJournal of Biological Chemistry
Volume275
Issue number17
DOIs
StatePublished - Apr 28 2000
Externally publishedYes

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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