A Novel Peptide Motif for Platelet Fibrinogen Receptor Recognition

Abstract

To develop a specific antagonist of platelet ␣IIb␤3 using small linear peptides, we synthesized a series of hexapeptides that did not have an Arg-Gly-Asp (RGD) sequence and examined their anti-platelet activity and their specificity for ␣IIb␤3. We found a novel motif sequence, Pro-X1-X2-X3-Asp-X4, where X1 to X4 were all L-form ␣-amino acids, which specifically inhibited aggregation of human platelets at submicromolar concentrations. The Pro residue at the N terminus was essential to the anti-platelet activity, and the acetylation of the imino group of this residue also resulted in the complete loss of the activity. The results of the binding assay using purified human platelet ␣IIb␤3 and placental ␣v␤3 and those of the cell adhesion assay suggest that this motif peptide is highly specific for platelet ␣IIb␤3 among other integrins. Flow cytometric studies using an fluorescein isothiocyanate-labeled RGD peptide showed that this motif peptide inhibited the binding of an RGD peptide to activated platelets, suggesting that it has the same inhibitory mode as RGD peptides. Conformational analysis of this motif peptide and an RGD-containing peptide suggests that the imino group of the Pro residue may substitute for the role of the guanidino group of the Arg residue of the RGD sequence.Integrins are heterodimeric cell surface receptor molecules and are thought to be particularly important mediators of cell adhesion, cell migration, and adhesion-dependent intracellular signaling. Until now, more than 20 integrin receptors have been identified, and many of them recognize the Arg-Gly-Asp (RGD) 1 sequence as a common recognition motif within their putative ligands upon ligand-receptor interaction (1-4). The ligand binding specificity has been studied by many investigators, and it has been revealed that some integrins show apparent redundancy in integrin-ligand interactions. For example, ␣v␤3, a so-called vitronectin receptor, can bind several apparently dissimilar ligands such as vitronectin, fibrinogen, fibronectin, von Willebrand factor, osteopontin, and bone sialoprotein, all of which have the RGD sequence (reviewed in Refs. 5 and 6). In addition, the same RGD site within a ligand can be recognized by several different integrins. These apparent redundancies in integrin-ligand interactions are very interesting aspects of integrin receptors and may be beneficial to the cell adhesion of many types of cells to extracellular matrix proteins.We are interested in developing small peptides that are specific for each integrin as a tool for investigating ligandintegrin interactions and as an agent for therapeutic uses. Recently, many small peptides containing the RGD sequence have been synthesized, and their specificity toward integrins has been examined (7,8). Most of the linear RGD peptides have shown very low specificity among many types of integrin receptors including platelet ␣IIb␤3 (fibrinogen receptor), ␣v␤3 (vitronectin receptor), and ␣5␤1 (fibronectin receptor). In the case of ␣IIb␤3 and ␣v␤3, both integrins have the same...

Ayadualin, a novel RGD peptide with dual antihemostatic activities from the sand fly Lutzomyia ayacuchensis, a vector of Andean-type cutaneous leishmaniasis

“…[38]. Consistent with previous studies using reducing agents-treated disintegrins [39,40] and synthetic cyclic and linear RGD peptides [41][42][43], a disulfide bond structure composed of two cysteine residues flanking the RGD sequence was essential for the antiplatelet action of ayadualin since substitution of the cysteines to serine residues abrogated the activity.…”

“…longipalpis saliva [18,19], strongly suggesting that the peptide has no antiplatelet activity. Another motif for platelet fibrinogen receptor, the PXXXDX sequence [42], was found in ayadualin at amino acid 46-51 of the immature protein (Fig. 1A).…”

Conserved Amblyomma americanum tick Serpin19, an inhibitor of blood clotting factors Xa and XIa, trypsin and plasmin, has anti-haemostatic functions

“…The observation that pre-incubating rAAS19 with platelets for 15 min prior to addition of the agonist had minimal effect suggests that the observed rAAS19 anti-platelet aggregation effects were mediated through inhibition of thrombin activity. However, it is interesting to note that the “RGD” motif, which is known to interfere with platelet aggregation binding the integrin GPIIb-IIIa on activated platelets (Varon et al, 1993; Katada et al, 1997) is conserved in AAS19 and its homologues. If this motif was functional, pre-incubation with platelets should interfere with platelet aggregation function.…”

The use of microcalorimetry to characterize tetanus neurotoxin, pertussis toxin and filamentous haemagglutinin

“…The 105 kDa PT is a typical A-B type multicomponent toxin composed of a catalytic subunit (A-subunit or subunit S1) and a cell-binding pentamer (B-oligomer composed of subunits S2 and S3, two copies of S4, and S5) [8]. PT exerts its toxic activity by the S1-catalysed transfer of ADP-ribose from NAD + to the α-subunit of regulatory G-proteins (G i α) [9] which prevents G i α from inhibiting adenylate cyclase.…”