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

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 A 2 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 Ca 2؉ increase and shape change; (ii) ADP restored the inhibition of U46619-induced platelet aggregation by RPAI-1, (iii) PGE 1 -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 indomethacintreated platelets, and (v) RPAI-1 binds to ADP, as assessed by large zone chromatography. RPAI-1 affects neither integrin ␣ 2 ␤ 1 -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.Normal hemostasis is initiated when platelets are exposed to subendothelial matrix, where they adhere to collagen via specific cell-surface receptors. This adhesion step is followed by platelet activation that is accompanied by synthesis and release of pro-aggregatory molecules such as TXA 2 1 and ADP, which amplify platelet responses to collagen and recruit additional platelets to the site of injury, respectively (1). The concerted action of collagen, ADP, and TXA 2 activates specific signaling pathways, generating a number of second messengers and leading to the functional expression of integrin ␣ IIb ␤ 3 , the fibrinogen receptor on platelets (1). Binding of fibrinogen to platelets mediates platelet aggregation, the final common response of activated platelets to most agonists. In addition to platelet aggregation, normal hemostasis involves activation of the blood coagulation cascade and vasoconstriction, making it a very redundant system (2). Redundancy is also observed in terms of the receptors involved in platelet responses for each specific agonist. It has now become clear that physiologic platelet agonists stimulate platelets by more than one receptor, and specific platelet responses involve distinct receptors. Accordingly, it has been shown that integrin ␣ 2 ␤ 1 receptor mediates platelet adhesion to collagen, whereas GPVI induces most of the signal involved in plat...

Exploring the sialome of the blood-sucking bug Rhodnius prolixus

“…1-3) had sequences matching three of the six novel NP, as indicated in Table 2. Four novel cDNA sequences match proteins previously described as Rhodnius platelet aggregation inhibitor proteins (RPAI) (Francischetti et al, 2000). These four sequences have a clear signal peptide indicative of secretion.…”

“…The remaining clusters similar to RPAI produce various degrees of sequence similarities to RPAI-1 or RPAI-2 and may represent novel salivary proteins in Rhodnius. RPAI-1 and RPAI-2 inhibit platelet aggregation by strongly binding to adenosine nucleotides (Francischetti et al, 2000). ADP at the submicromolar concentrations normally found in the plasma decreases the threshold for collagen-induced platelet aggregation.…”

“…These four sequences have a clear signal peptide indicative of secretion. RPAI-1 has been shown (Francischetti et al, 2000 to bind adenosine derivatives and inhibit platelet aggregation. Except for RPAI-6, which has five cysteines, the remaining sequences have six conserved cysteines indicative of three disulfide bonds (Fig.…”

“…When probing in their victim's skin for blood, Rhodnius inject a salivary cocktail that contains apyrase, an enzyme that hydrolyses ADP, a nucleotide released by injured cells and a potent inducer of platelet aggregation (Sarkis et al, 1986). Additionally, a lipocalin with very high affinity for ADP mops up any residual nucleotide (Francischetti et al, 2000). Serotonin and thromboxane A 2 , which are potent vasoconstrictors released by platelets, are also neutralized (Ribeiro, 1982;Ribeiro and Sarkis, 1982).…”

Patrones electroforéticos de hemoproteínas salivares (nitroforinas) de Rhodnius colombiensis y Rhodnius prolixus (Hemiptera, Reduviidae, Triatominae)

“…En los triatominos se han encontrado varias sustancias relacionadas con estas funciones. En el caso del género Rhodnius, hasta el momento se han reportado ocho proteínas salivares: tres factores que contrarrestan la agregación plaquetaria, llamados inhibidores de agregación de R. prolixus (RPAI 1-3) (proteínas lipocalinas) (4,5); una apirasa que se encarga de hidrolizar el ADP bloqueando la agregación plaquetaria (4,6), y, por último, las nitroforinas (NPs), un grupo de cuatro hemoproteínas denominadas NP1 a NP4 según su relativa abundancia en las glándulas salivares (7). Las nitroforinas se pueden agrupar en dos conjuntos con base en las relaciones de sus secuencias de aminoácidos; es así como NP1 y NP4 son idénticas en un 90%, y NP2 y NP3 son idénticas en un 80% (4,7,8).…”