H <sub> 2 </ Sub> N (ch <sub> 2 </ Sub>) <sub> 7 </ Sub>-co - Gly-asp-trp Design, Synthesis And Antiplatelet Effects Research

RGD is tripeptide that contains arginine-glycine-aspartic acid and involves in ligand binding to platelet GPIIb/IIIa,so it plays a essential role in thrombosis formation.The studies have revealed that RGD analogs were effective inhibition of fibrinogen binding to platelet GPIlb/IIIa.So antagonists of platelet GPIIb/IIIa reprsent a new class of antiplatelet agents.Objectivel.To design highly active ,highly steady.highly specificy RGD analog.2.To synthesize ,purify,verify H2N(CH2)7-CO-Gly-Asp-Trp (AoGDW) .3.To testify activity,stability of AoGDW and influence of plasma cruor byAoGDW.4.To confirm the mechanism of action of AoGDW.5.To study HUVEC detachment caused by AoGDW and specificity ofAoGDW.Methods and resultsl.In order to confer potency,stability,and specificity to synthetic peptide based upon RGD,RGD mimics as inhibitors of platelet aggregation was designed by using of conformational constraints.Modiflcation of RGD was replacement of Arg with w -aminooctanoic acid and Asp was followed by Trp.Then RGD sequence is H2N(CH2)7-CO-Gly-Asp-Trp.2.Peptide was synthesized by the solid-phase method,purified by reversed-phase HPLC on C-18 columns.The purified peptide was at least 98% pure.Peptide structure was verified through ESI-MS and amino acid analysis.Results were consistent with theory.3.Platelet aggregation was studied using a 4-channel aggregometer(Helena) and expressed as percentage change in light transmittance compared with a just maximally aggregated sample after addition of ADP,collagen.IC50 value of inhibitors of platelet aggregation is 4.71 ±2. 52uM,compared to NS or RGDS,p <0.01.Then PRP was preincubated with AoGDW and RGDS for 3h at 37℃,with stirring at 900rpm in the aggregometer and testified rate of platelet aggregation.Result of AoGDW was achieved with improved plasma stability(100% activity after 3h),but activity of RGDS was suffered total loss of in vitro activity.We also testified TT,PT,APTT that were normal after plasma preincubated with AoGDW.4.The mechanism of action of peptide was confirmed by FCM(Flow Cytometric Messurements).The effect of AoGDW on CD41 binding to activated platelet was concentration-dependent,P < 0.01 versus negative group,but could not bind to the platelet GPIIb/IIIa on nonactivated platelet. Expression of P-selection on platelet was not affected by AoGDW. 5.HUVEC were seeded in a FCS-coated 24-well plate in 1mL of complete HUVEC media per well and grown to confluence for 2 days.The cells were then washed with serum-free medium RPMI 1640,AoGDW was added in serum-free medium RPMI 1640.At the end of 4h of incubation at 37℃,the cells were rinsed with serum-free medium RPMI 1640 to removed any nonadherent cells.Then MTT method was used to evalute percentage of HUVEC detachment.The results revealed the cells began to curled -up within 30min at concentration 22mM of AoGDW,cell-free area subsequently appeared in the monolayer as the cells retracted into ridges and the cells partly detached from culture plates,forming floating aggregates. AoGDW caused 50% HUVEC detachment at concentrations 3500-fold greater than one required to Inhibit platelet aggregation.ConclusionWe designed and synthesized H2N(CH2)7-CO-Gly-Asp-Trp as highly active .highly steady,highly specificy RGD analog.Futher studies will have to evaiute the clinical relevance of II b/IIIa inhibitors and define consequences for the furture drug development of this potent antiplatelet agents.