SSGE and DEE, new peptides isolated from a soy protein hydrolysate that inhibit platelet aggregation

Abstract

A soy protein hydrolysate was found to inhibit rat platelet aggregation induced by ADP, an aggregating agent. To find out its principal antiplatelet peptide(s), the soy protein hydrolysate was separated successively by gel filtration chromatography, reverse-phase HPLC, and cation exchange HPLC. During the course of separation, we observed that most fractions had antiplatelet effects, which suggests that most peptides have some degree of antiplatelet effect. Following the inhibitory fractions, we purified and identified two new peptides, SSGE and DEE, by LC–electrospray ionization MS and peptide sequencing. Both peptides were highly hydrophilic. The concentrations to obtain 50% inhibition (IC₅₀) of the aggregation intensity were approximately 480 and 460 μM, respectively, for SSGE and DEE.

Introduction

It is generally accepted that platelet adhesion and aggregation plays an important role in the pathogenesis in thrombosis, particularly arteriothrombosis (Gorden, 1981; Knapp, Reilly, Alessandrini, & Fitzgerald, 1986). Peptides have been shown to inhibit platelet aggregation and thrombosis, as reviewed by Rutherfurd and Gill (2000) and Fiat et al. (1993). C-terminal sequence of human fibrinogen γ-chain (HHLGGAKQAGDV) and tetrapeptide sequences [RGD(S or F)], residues 572–575 or 95–98 of the fibrinogen α-chain (Andriex et al., 1989), a undecapeptide (MAIPPKKNQDK), glycomacropeptide (GMP) (Jollès et al., 1986) and a pentapeptide (KNQDK) (Jollès & Caen, 1991), corresponding, respectively, to residues 106–116 and 112–116 of cow κ-casein, and KRDS, a tetrapeptide with a sequence corresponding to residues 39–42 of human lactotransferrin (Mazoyer et al., 1990) inhibited platelet aggregation and fibrinogen binding to ADP-activated platelets. Peptide fractions from soybean paste inhibited ADP-induced platelet aggregation (Shon et al., 1996). Whole κ-casein inhibited thrombin-induced platelet aggregation and GMP inhibited both thrombin and ADP-induced platelet aggregation (Drouet et al., 1990). In vivo (in an experimental model of arteriolar thrombosis), intravenous injection of RGDS and KRDS (Mazoyer et al., 1990), and GMP (Drouet et al., 1990) inhibited thrombogenesis.

In the course of development of biologically active soy protein hydrolysates, a soy protein hydrolysate was found to inhibit rat platelet aggregation induced by ADP, an aggregating agent. To find out its principal antiplatelet peptide(s), the soy protein hydrolysate was successively separated by gel filtration chromatography, reverse-phase HPLC, and cation exchange HPLC. During the course of separation, we observed that most fractions had antiplatelet effects, which suggests that most peptides have some degree of antiplatelet effect. Following the inhibitory fractions, we have purified and identified two new peptides, DEE and SSGE, and determined their inhibitory effects on platelet aggregation in rat in vitro.