The Identification And Molecular Mechanism Of Antiplatelet Peptides Derived From Oat, Highland Barley, And Buckwheat

Cardiovascular disease caused by thrombosis has become one of the major threats to human health. Platelet aggregation is the key target in the thrombosis. The study on platelet aggregation inhibitors has great significance for the prevention and treatment of thrombosis caused by platelet aggregation. In this paper, we report the identification and molecular mechanism of anti-platelet peptides released from oat, highland barley, and buckwheat proteins, the main results are as follows.(1) The albumin, globulin, and glutelin fractions were extracted sequentially from oat, highland barley, and buckwheat. In vitro gastrointestinal digestion, tryptic digestion, and alcalase digestion were used to get peptides. Anti-platelet activities of these peptides were tested. The results show that all peptides showed high antiplatelet activity, with IC50 values of 0.282 mg/ml (oat flour gastrointestinal peptides,6 h) to 2.496 mg/ml (highland barley glutelin tryptic peptides,14 h) in a dose-dependent manner.(2) The oat globulin tryptic peptides were ultra-filtered into three fractions of small (MW<3kDa), middle (3kDa-10kDa), and big (MW>10kDa). Anti-platelet activities of the three fractions were tested. The potent peptides with anti-platelet activity were identified using nano-LC-ESI-MS/MS (nano-liquid chromatography-electrospray ionization tandem mass spectroscopy). These results suggest that the three fractions strongly inhibit platelet aggregation, and that the small fractionshave greater antiplatelet activities than do the big fractions, which is 73.11 ± 0.95%. And thirty-eight peptides with more than seven residues were identified in the oat globulin tryptic peptides (MW<3kDa). Most of them come from oat 11S globulin and oat 12S globulin 2.(3) The inhibitory activity of the oat anti-platelet peptides (OAPP) on arachidonic acid (AA) pathway was assessed using the cyclooxygenase-1 (COX1) and thromboxane-B2 (TXB2) ELISA kit. Interactions between OAPP and COX1 were assessed using computational docking analysis. OAPP exerted strong antiplatelet effect by inhibiting COX1, which caused a subsequent reduction in TXA2 production. Results of computational modeling revealed that nine peptides, including ALPIDVLANAYR, EFLLAGNNKR, GEEFGAFTPK, QLAQIPR, LQAFEPLR, ALPVDVLANAYR, GEEFDAFTPK, QKEFLLAGNNK, and TNPNSMVSHIAGK bound the COX1 active centers with low binding energy (-6.5 to -7.5 kcal/mol).This is the first report to identify antiplatelet peptides from grain hydrolysates and the binding modes at the molecular level, leading to their possible use as functional food ingredients to prevent thrombosis.