Purification, Cloning, Expression, and Mechanism of Action of a Novel Platelet Aggregation Inhibitor from the Salivary Gland of the Blood-sucking Bug, Rhodnius prolixus

doi:10.1074/jbc.275.17.12639

Purification, Cloning, Expression, and Mechanism of Action of a Novel Platelet Aggregation Inhibitor from the Salivary Gland of the Blood-sucking Bug, *Rhodnius prolixus**

From the ^‡Laboratory of Parasitic Diseases, NIAID, National Institutes of Health, Bethesda, Maryland 20892-0425, the ^¶Department of Entomology, University of Georgia, Athens, Georgia 30602, and the ^‖Department of Biochemistry, University of Arizona, Tucson, Arizona 85721

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 Pet17BEscherichia 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.

Footnotes

↵* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵§ To whom correspondence should be addressed: Bldg. 4, Rm. 126, 4 Center Dr., MSC 0425, Bethesda, MD 20892-0425. Tel.: 301-496-3066; Fax: 301-402-4941; E-mail: jribeiro@nih.gov.
Abbreviations:

TXA₂

thromboxane A₂

BSA

bovine serum albumin

NO

nitric oxide

PCR

polymerase chain reaction

PG

prostaglandin

RPAI-1

R. prolixus aggregation inhibitor 1

ELISA

enzyme-linked immunosorbent assay

PRP

platelet-rich plasma

PAGE

polyacrylamide gel electrophoresis

DTT

dithiothreitol

HPLC

high performance liquid chromatography

GPVI

glycoprotein VI
- Received December 14, 1999.
- Revision received February 3, 2000.
The American Society for Biochemistry and Molecular Biology, Inc.