H₂N（CH₂）₇-CO-Gly-Asp-Trp的设计、合成及抗血小板作用的研究

【作者】 王晓霞；

【导师】 章汴生； 牛勃；

【作者基本信息】 山西医科大学 ， 生物化学与分子生物学， 2002， 硕士

【摘要】 目的 1．设计高活性、高稳定性、高特异性的RGD类似物：H₂N（CH₂）₇-CO-Gly-Asp-Trp。 2．固相法合成H₂N（CH₂）₇-CO-Gly-Asp-Trp，并纯化、鉴定。 3．测定H₂N（CH₂）₇-CO-Gly-Asp-Trp的抗血小板活性、稳定性及其对血浆凝血功能的影响。 4．H₂N（CH₂）₇-CO-Gly-Asp-Trp的作用机理研究。 5．H₂N（CH₂）₇-CO-Gly-Asp-Trp对血小板GPⅡbⅢa受体的特异性研究。 方法 1．以天然产物或内源性物质RGD为先导化合物，按照活性物质依赖何种机制发挥生物效应探索构效关系的本质，对RGD先导化合物进行结构修饰、取代、改造，最终进一步提高其活性，降低其副作用。 2．采用固相法合成H₂N（CH₂）₇-CO-Gly-Asp-Trp，并经RP-HPLC纯化，以及元素分析、质谱鉴定结构。 3．采用比浊法测定H₂N（CH₂）₇-CO-Gly-Asp-Trp抑制血小板聚集的活性以及在血浆中的稳定性。利用血凝仪测定其对血浆凝血功能的影响。 4．通过流式细胞技术验证H₂N（CH₂）₇-CO-Gly-Asp-Trp的作用机理以及对血小板活化的影响。 5．利用体外HUVEC脱粘附实验及MTT法，定量研究H₂N（CH₂）₇-CO-Gly-Asp-Trp使HUVEC产生脱粘附的IC₅₀并与抑制血小板聚集所需IC₅₀相比，确定它的特异性大小。 结果 1．羧基端以疏水性最强的色氨酸取代，氨基端以非蛋白氨基酸：ω—氨基辛酸取代精氨酸，我们设计出了序列：H₂N（CH₂）₇-CO-Gly-Asp-Trp。 2．固相法合成粗肽后经RP-HPLC纯化，其纯度在98％以上，元素分析以 山西医科人学 生物化学与分子生物学专业 硕士学位论文 一 及质谱确证了其结构与理论值相符。 3．HZN（CHZ），－CO－GlyASp－TFp抑制血小板聚集的IC；0为4．71土2．52uM， 与阴性对照组、则性对照组相比，均为P＜O刀1。不仅对＊OP诱导的血．小 板聚集具有抑制作川，而且对其它诱聚剂如胶原诱导的血小板聚集也有很《强的抑制作川。比外＊N)，－＊＊－0卜＊s卜np＆血浆中孵育3h仍保持100％ 的活性，而RGDS的活性儿乎完全丧人。另外，血浆凝血．功能各项指标TT、 PT、APTT均在上常范围内。 4．随着AoGDW浓度的增加，CD41抗体与活化血小板GPll b／llla受体结合 率卜现厂降趋势，相对下阴性对照组，P＜0．01，但n41抗体与静十血．小 板Gpllb八＊a受体结合率与阴性对照组相比P＞0．05。另外给药后，m62p 抗体与静十血小板P选择素的结合率与阴性对照组比，PMO．05。 5定性研究：随着作川时间延长，Curled－up现象增多，随之出现cell一 free area，脱落细胞数量相应增多。定量研究：MTT染色后，随着AoGDW 浓度的增加，OD值逐渐减少，粘附抑制率逐渐增加，与阴性对照组相比， P＜0 of。HUVEC产生脱粘附的 IC。；．为 16．5士0．68mM，这一浓度人约是抑 制 50％血小板聚集所需浓度的 3500倍。 结论 通过本课题设计、合成RGD类似物，解决了一些多肽类治疗剂存在的 稳定性及特异性差等缺点，得到了高活性、高稳定性、高特异性的 H。N（CH。），－CO－G！yAsP－TrP。针对纤维蛋白原与血小板Gpllb／llla受体的 偶联过程而设计的RGD类似物，以其高效、完全、特异优于目前常用的抗 血小板药，因而必将对抑制血小板聚集以及抑制血栓形成产生明确的治疗 作用。更多还原

【Abstract】 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.更多还原