嵌合水蛭肽的构建与活性分析

血管成形术或动脉粥样斑块破裂等因素所致血管壁损伤而引起的血栓形成过程中 ,血小板的激活和凝血酶的形成起着关键作用 .因此 ,抗血小板和抗凝是治疗血栓的两个重要方面 .血小板膜糖蛋白GPⅡb Ⅲa受体拮抗剂 ,如含Arg Gly Asp(RGD)序列的多肽 ,在临床上已显示了良好的抗血小板     

含RGD模体的人白细胞介素18(IL-18)突变体的酵母表达及其抗血小板聚集活性

利用定点突变及DNA重组技术,在人白细胞介素18(IL18)cDNA序列中插入GGC序列,使IL18第39位精氨酸残基和第40位天冬氨酸残基之间插入一个甘氨酸残基,从而构建了RGD模体.此重组的cDNA序列构建入表达质粒pPIC9K,并转化Pichiapastoris酵母GS115,利用表达系统进行了高效表达.用SephadexG100凝胶过滤纯化表达产物,获得初步纯化的蛋白.对该蛋白进行了血小板聚集抑制实验和对GPⅡbⅢa与Fn结合的抑制实验.含RGD模体的重组IL18(IL18RGD)显示了较强的体外抑制血小板聚集活性,IC50=8.8μmolL;并具有与GPⅡbⅢa的竞争结合活性,IC50=8.0μmolL.该含有RGD模体的重组IL18仍保存对PBMC诱导产生IFNγ能力.结果表明,此IL18RGD嵌合体在具有抗炎,抗感染的同时增添了新的抑制血小板聚集功能.     

含RGD的配基与其受体相互作用研究进展

RGD配基与其受体相互作用目前已成为一个研究热点, 许多粘附蛋白是通过RGD序列与其整合素结合的, 它参与许多生物学过程. 文章主要就RGD序列肽与糖蛋白Ⅱb／Ⅲa的相互作用及其在抑制血小板聚集上进行了较为全面的综述, 并介绍了RGD序列肽在基础科学及医疗上的应用前景.     

重组白眉蝮蛇去整合素定点突变对其生物活性的影响

RGD为存在于许多糖蛋白配体中的氨基酸序列,对整合素具有识别作用.此序列也发现于许多蛇毒去整合素分子中.采用基因克隆技术从大连产白眉蝮蛇的毒腺中克隆出的去整合素adinbitor是含73个氨基酸残基的去整合素,分子中含有12个半胱氨酸和RGD模体.实验证明,adinbitor作为去整合素的新成员,具有典型的抗ADP诱导的人血小板聚集作用和抗肿瘤血管新生作用.为了将adinbitor的这2种功能分开,采用PCR基因定点突变的方法,将其cDNA序列中RGD模体改变成KGD.重组adinbitor(KGD)在E.coli BL21得到表达,并通过His•Bind亲和层析予以纯化.实验发现,adinbitor对ADP诱导的人血小板聚集具有明显抑制作用,其IC50=85 nmol/L,明显优于adinbitor(RGD) （IC50=150 nmol/L）.然而,与adinbitor(KGD)相比,adinbitor(KGD)则丧失了对血管生成的抑制作用.结果说明,adinbitor(KGD)可作为专一的抗人血小板聚集药具有潜在的开发前景.     

剪切应力诱导血小板聚集

剪切应力诱导血小板聚集（shear-induced platelet aggregation, SIPA）是指在高剪切流场诱导下血小板表面的膜糖蛋白（GPⅠb/Ⅸ/Ⅴ和GPⅡb/Ⅲa）与血浆中的von Willebrand因子（vWF）相结合,介导血小板的活化、黏附和聚集,是动脉血栓的重要成因.SIPA还需要Ca2+,ADP/ATP等生化因素的参与,因而SIPA现象是生化因素和力学因素偶合作用的结果.细胞外Ca2+是高剪切应力诱导血小板发生聚集的必需条件,Ca2+的跨膜内流引起细胞骨架结构的改变和GPⅡb/Ⅲa的活化.近来对ADP/ATP位于血小板膜上的P2受体的研究表明,P2受体与细胞内Ca2+协同作用通过多种生化途径调控血小板的活化过程在SIPA的信号传导中起着关键的作用.从力学环境与生化反应的偶合关系入手研究SIPA现象的触发机制,深入研究SIPA现象中的信号转导通路是今后的研究热点之一.     

GPⅡb/Ⅲa抗体及ACA在系统性红斑狼疮血小板减少患者中临床意义探讨

目的:探讨血小板膜糖蛋白(GP) Ⅱb/Ⅲa抗体及抗心磷脂抗体(ACA)在系统性红斑狼疮血小板减少(SLE-TP)患者中的临床意义.方法:采用酶联免疫吸附试验(ELISA)法检测SLE血小板减少组35例、非血小板减少组27例、ITP组14例及健康对照组16例血清中抗GP Ⅱb/Ⅲa抗体及ACA的水平,分析其与SLE-TP患者临床、实验室指标及SLEDAI评分的相关性;统计方法采用x2检验、Fisher确切概率法.结果:①SLE血小板减少组抗GP Ⅱb/Ⅲa抗体表达水平显著高于非血小板减少组及健康对照组(P＜0.01);②抗GP Ⅱb/Ⅲa抗体在SLE血小板减少组与ITP组间的表达差异无统计学意义;③各组间ACA阳性率差异无统计学意义;④在SLE血小板减少组,抗GP Ⅱb/Ⅲa抗体与SLEDAI评分等级有显著关联(P＜0.05),与血小板减少程度、皮疹、光过敏、脾大、关节炎、粒细胞减少、肾脏及神经系统受累等临床指标无相关性,与dsDNA、Sm、SS-A、C3、C4、Coombs实验及ACA无显著关联.结论:SLE-TP患者血清中抗GP Ⅱb/Ⅲa抗体高表达,且与疾病的活动呈正相关,可能在SLE-TP的发病过程中发挥了重要作用.     

具有抗血小板聚集功能的新型葡激酶突变体的表达、纯化及性质

[目的]为解决溶栓后再栓塞问题,构建N-端含RGD(Arg-Gly-Asp)序列的葡激酶双功能突变体.研究突变体的表达和纯化,并进行性质分析.[方法]将突变后的葡激酶突变体序列连入pBV220质粒,转化大肠杆菌BL21进行表达.阳离子交换、凝胶过滤和阴离子交换三步层析法纯化表达产物,采用溶圈法对纯化产物进行生物学活性测定,并测定纯化产物对血小板聚集的抑制效应.[结果]PAGE扫描结果显示,葡激酶突变体蛋白在大肠杆菌BL21中的表达量约占菌体蛋白总量的40%～50%;三步层析纯化后,HPLC测定其纯度可达95%.酪蛋白凝胶板溶圈法测得其比活性分别为10.8×104和11.0×104HU/mg,与野生型葡激酶活性相当;且具有明显的抗血小板聚集活性,血小板聚集仪测定其血小板聚集抑制率分别为10.72%和19.71%,明显高于野生型葡激酶血小板聚集抑制率.本实验利用pBV220载体高效表达了葡激酶突变体基因,得到了高纯度、高活性的突变体蛋白,为葡激酶生产产业化和临床应用奠定了良好的基础.     

抗凝活性肽RGD226基因构建、表达、产物纯化及活性分析

通过PCR法 ,将来源于蛇毒蛋白质eristostatin中的一段含有RGD(Arg Gly Asp)序列的十三肽(SRVARGDWNDDYS)的基因 ,以一个无胰岛素活性但保留天然免疫原性的胰岛素原突变体(PJG4 0 1)基因为模板 ,置换其B2 8和A1位之间的连接肽基因 ,构建成了能展示RGD功能序列的人源化分子 (RGD2 2 6 )的基因 .通过该基因在大肠杆菌中的表达、产物分离纯化 ,得到高纯度RGD2 2 6 .该RGD肽对由ADP诱导的体外人血小板聚集的半抑制浓度IC50 为 2 2 3μmol L .其胰岛素免疫活性是PJG4 0 1的 15 1% ,提示其在一定程度上保留了胰岛素原的免疫原性 .其受体结合活性不到PJG4 0 1的 0 1% ,说明其胰岛素的生物活性基本丧失 .动物实验证实 ,RGD2 2 6在延长小鼠出血时间上具有较明显的作用     

一种含精-甘-天冬氨酰三肽人纤溶酶原K区缺失突变体在巴斯德毕赤酵母中的表达、纯化与特性

为获得具有抗血小板聚集作用的重组人纤溶酶原 (hPLG),本研究尝试了一种含有精-甘-天冬氨酰 (RGD) 三肽的hPLG K区缺失突变体 (RGD-hPLG-?K)。首先,从pDNR-LIB-HPLG.中克隆出HPLG-?K。然后定点突变激活环内的Pro559为Asp559,形成RGD模序。构建的pPICZαA-RGD-HPLG-?K电转化巴斯德毕赤酵母GS115,甲醇诱导表达后可产生0.16 g/L培液的RGD-hPLG-?K,Ni-NTA层析后纯度可达90％以上;Western blotting证实所获RGD-hPLG-?K可与兔抗hPLG抗血清反应;其24 h尿激酶激活速率和纤溶活性与hPLG-?K无显著差别 (P=0.630,n=5);经尿激酶激活后,RGD-hPLG-?K的血小板聚集抑制率 (21.8％±1.57％) 显著高于hPLG-?K (3.8％±0.33％) (P=0.000,n=5)。表明成功构建、表达了一种具有抗血小板聚集活性的hPLG突变体,为研究新型多功能溶栓药物奠定了基础。     

血小板功能负性调控机制

在血液循环系统中,血小板在抑制因子的作用下,处于静息状态。当机体出血或外界因素刺激时,血小板活化,产生聚集、黏附和释放反应,释放出二磷酸腺苷(ADP)、花生四烯酸(AA)、血小板活化因子和5-羟色胺等物质,招募更多的血小板黏附于出血处,从而启动凝血过程,发挥止血作用。当止血反应完成后,血小板发生解聚,恢复到静息状态。然而,在病理条件下,血小板的内在解聚能力下降,形成过度活化的血小板,产生病理性血栓,导致急性缺血性心血管疾病的发生。临床使用抗血小板药物控制血小板的活化,治疗急性缺血性心血管疾病。然而,目前临床上常用的抗血小板药物发挥抗血小板活化作用的同时,影响了血小板正常的生理性止血作用,产生出血等副作用。因此,我们需要研发新型抗血小板药物,使其既能发挥抗血小板作用,又能减少出血等副作用。本文将对血小板负性调控机制进行综述,为进一步研究抗血小板药物提供思路。     

Variations of glycoprotein IIb–IIIa (αIIb/β3-integrin) content in healthy donors. The influence on platelet aggregation activity and efficacy of aspirin action

The effects of content of a fibrinogen receptor, glycoprotein (GP) IIb–IIIa (αIIb/β3-integrin), GP IIIa genetic polymorphism (substitution Leu33Pro), and fibrinogen concentration in blood plasma on platelet aggregation activity have been investigated in a group of healthy volunteers. In 35 examined donors the GP IIb–IIIa content on platelet surface varied from 40 to 71 × 10³ per platelet. Repeated measurements revealed that the GP IIb–IIIa content coefficient of variation was 9.5%, and deviations from mean levels did not exceed 20%. The level and the rate of platelet aggregation induced by ADP (1.25–20 μM) correlated with GP IIb–IIIa number (r from 0.315 to 0.591) and were higher in the group of donors with high in comparison with low GP IIb–IIIa content (>60 and (40–50) × 10^?3 per platelet, respectively). Aspirin, the inhibitor of thromboxane A₂ synthesis, partially suppressed ADP-induced platelet aggregation. The level of residual aggregation in the presence of aspirin also correlated with GP IIb–IIIa content and increased in subjects with high receptor content. Parameters of ADP-induced aggregation did not differ in donors with genotypes GP IIIa Pro33(?) (Leu33Leu33, n = 20) and Pro33(+) (Leu33Pro33, n = 13, and Pro33Pro33, n = 2) genotype. GP IIb–IIIa content was also not affected by GP IIIa polymorphism. No significant correlations were found between the level and rate of platelet aggregation and fibrinogen concentration in blood plasma. The data obtained indicate that the effects of variations of GP IIb–IIIa content on platelet aggregation are higher than GP IIIa Leu33Pro polymorphism and variations of fibrinogen concentration. High GP IIb–IIIa content is associated with increased platelet aggregation activity and decreased efficacy of aggregation inhibition by aspirin.     

From a peptide lead to an orally active peptidomimetic fibrinogen receptor antagonist

Summary Antagonists of the platelet fibrinogen receptor (GP IIb/IIIa receptor) are expected to be a new promising class of antithrombotic agents. The binding of fibrinogen to the fibrinogen receptor depends on an Arg-Gly-Asp-Ser (RGDS) tetrapeptide recognition motif. Structural modifications of the RGDS lead have led to the discovery of a non-peptide RGD mimetic GP IIb/IIIa antagonist20 (S 1197). Compound20 inhibits dose-dependently and reversibly human platelet aggregation. Modeling studies based on structure-activity data revealed the following structural features of the drug as important for receptor binding: the amidino group, the carboxylate group, hydrophobic substitutions at the carboxyl-terminus and at the side chain carrying the positive charge, the carboxyl-terminal NH group of the β-amino acid as a hydrogen bond donor and one oxygen atom of the hydantoin as a hydrogen bond acceptor. The ethyl ester prodrug of20 (S 5740) is an orally active antithrombotic agent which has the potential to be used to treat and prevent thrombotic diseases in humans.     

From a peptide lead to an orally active peptidomimetic fibrinogen receptor antagonist

Antagonists of the platelet fibrinogen receptor (GP IIb/IIIa receptor) are expected to be a new promising class of antithrombotic agents. The binding of fibrinogen to the fibrinogen receptor depends on an Arg-Gly-Asp-Ser (RGDS) tetrapeptide recognition motif. Structural modifications of the RGDS lead have led to the discovery of a non-peptide RGD mimetic GP IIb/IIIa antagonist 20 (S 1197). Compound 20 inhibits dose-dependently and reversibly human platelet aggregation. Modeling studies based on structure–activity data revealed the following structural features of the drug as important for receptor binding: the amidino group, the carboxylate group, hydrophobic substitutions at the carboxyl-terminus and at the side chain carrying the positive charge, the carboxyl-terminal NH group of the -amino acid as a hydrogen bond donor and one oxygen atom of the hydantoin as a hydrogen bond acceptor. The ethyl ester prodrug of 20 (S 5740) is an orally active antithrombotic agent which has the potential to be used to treat and prevent thrombotic diseases in humans.     

Aminopeptidase resistant Arg-Gly-Asp analogs are stable in plasma and inhibit platelet aggregation.

Tetrapeptides containing the sequence Arg-Gly-Asp (RGD) antagonize fibrinogen binding to its platelet receptor (gp IIb/IIIa, integrin alpha IIb beta 3) and inhibit platelet aggregation in vitro. The peptides RGDS and RGDY(Me)-NH2 were rapidly degraded when incubated in human, rat, and dog plasma. HPLC analysis indicated that amino acids were sequentially removed from the peptide N-terminus, and this degradation was prevented by the aminopeptidase inhibitor bestatin. Analogs of RGDY(Me)-NH2 with an acetylated or deleted alpha-amino group were prepared. Both analogs were stable when incubated in plasma, blocked 125I-fibrinogen binding to activated platelets (IC50 = 10-30 microM) and inhibited ADP induced platelet aggregation (IC50 = 10-30 microM). This study concludes that aminopeptidase rapidly degrades RGD peptides in plasma, an important issue for in vivo testing of RGD peptides and analogs. RGD analogs intrinsically stabilized against aminopeptidase are stable in plasma and are important tools for antithrombotic studies involving antagonism of gp IIb/IIIa.     

Unique pathway of thrombin-induced platelet aggregation mediated by glycoprotein Ib

Thrombin plays a central role in normal and abnormal hemostatic processes. It is assumed that alpha-thrombin activates platelets by hydrolyzing the protease-activated receptor (PAR)-1, thereby exposing a new N-terminal sequence, a tethered ligand, which initiates a cascade of molecular reactions leading to thrombus formation. This process involves cross-linking of adjacent platelets mediated by the interaction of activated glycoprotein (GP) IIb/IIIa with distinct amino acid sequences, LGGAKQAGDV and/or RGD, at each end of dimeric fibrinogen molecules. We demonstrate here the existence of a second alpha-thrombin-induced platelet-activating pathway, dependent on GP Ib, which does not require hydrolysis of a substrate receptor, utilizes polymerizing fibrin instead of fibrinogen, and can be inhibited by the Fab fragment of the monoclonal antibody LJIb-10 bound to the GP Ib thrombin-binding site or by the cobra venom metalloproteinase, mocarhagin, that hydrolyzes the extracellular portion of GP Ib. This alternative alpha-thrombin pathway is observed when PAR-1 or GP IIb/IIIa is inhibited. The recognition sites involved in the cross-linking of polymerizing fibrin and surface integrins via the GP Ib pathway are different from those associated with fibrinogen. This pathway is insensitive to RGDS and anti-GP IIb/IIIa antibodies but reactive with a mutant fibrinogen, gamma407, with a deletion of the gamma-chain sequence, AGDV. The reaction is not due to simple trapping of platelets by the fibrin clot, since ligand binding, signal transduction, and second messenger formation are required. The GP Ib pathway is accompanied by mobilization of internal calcium and the platelet release reaction. This latter aspect is not observed with ristocetin-induced GP Ib-von Willebrand factor agglutination nor with GP Ib-von Willebrand factor-polymerizing fibrin trapping of platelets. Human platelets also respond to gamma-thrombin, an autoproteolytic product of alpha-thrombin, through PAR-4. Co-activation of the GP Ib, PAR-1, and PAR-4 pathways elicit synergistic responses. The presence of the GP Ib pathway may explain why anti-alpha-thrombin/anti-platelet regimens fail to completely abrogate thrombosis/restenosis in the cardiac patient.     

Changes in the platelet membrane glycoprotein IIb.IIIa complex during platelet activation

Platelet activation is accompanied by the appearance on the platelet surface of approximately 45,000 receptor sites for fibrinogen. The binding of fibrinogen to these receptors is required for platelet aggregation. Although it is established that the fibrinogen receptor is localized to a heterodimer complex of the membrane glycoproteins, IIb and IIIa, little is known about the changes in this complex during platelet activation that result in the expression of the receptor. In the present studies, we have developed and characterized a murine monoclonal anti-platelet antibody, designated PAC-1, that binds to activated platelets, but not to unstimulated platelets. PAC-1 is a pentameric IgM that binds to agonist-stimulated platelets with an apparent Kd of 5 nM. Binding to platelets is dependent on extracellular Ca2+ (KCa = 0.4 microM) but is not dependent on platelet secretion. Platelets stimulated with ADP or epinephrine bind 10,000-15,000 125I-PAC-1 molecules/platelet while platelets stimulated with thrombin bind 20,000-25,000 molecules/platelet. Several lines of evidence indicate that PAC-1 is specific for the glycoprotein IIb.IIIa complex. First, PAC-1 binds specifically to the IIb.IIIa complex on Western blots. Second, PAC-1 does not bind to thrombasthenic platelets or to platelets preincubated with ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid at 37 degrees C, both of which lack the intact IIb.IIIa complex. Third, PAC-1 competitively inhibits the binding of 125I-A2A9, and IgG monoclonal antibody that is specific for the IIb.IIIa complex. Fourth, the antibody inhibits fibrinogen-mediated platelet aggregation. These data demonstrate that PAC-1 recognizes an epitope on the IIb.IIIa complex that is located near the platelet fibrinogen receptor. Platelet activation appears to cause a Ca2+-dependent change involving the glycoprotein IIb.IIIa complex that exposes the fibrinogen receptor and, at the same time, the epitope for PAC-1.     

Peptides as inhibitors of thrombin coagulation activity and of thrombocyte aggregation]

The analysis of literature and our own data of regulatory peptides influence on the blood coagulation system is presenting. Various natural and synthetic peptides inhibit the activity of thrombin and platelet aggregation. Direct specific inhibitors of thrombin are peptides developed on the base of D-Phe-Pro-Arg sequence. Strong specific inhibitors of the prothrombinase complex factor Xa were isolated from tissues and saliva of the blood-sucking organisms. These inhibitors decrease thrombin generation at the early stage of blood coagulation cascade Anticoagulating peptides from the tick Ornithodoros moubata tissue (TAP), the recombinant rTAP from the saliva glands of tick Ornithodoros savignyi and peptide with even greater anticoagulating activity from saliva glands of fly Glossina morsitans morsitans were isolated and characterised. For complete and reliable suppression of thrombus formation simultaneous administration of thrombin and platelet aggregation inhibitors is necessary. Main terminal stage of platelet aggregation is the interaction of receptor GP IIb/IIIa with adhesive fibrinogen sequence Arg-Pro-Asp (RGD). Peptides derived on the base of this sequence compete with fibrinogen in reaction with platelet receptors. A lot of corresponding peptidomimetics were synthesised, e.g. MK-852, RO-44 and particularly effective compound integrelin. Many direct platelet aggregation inhibitors were found in snake venoms. Recombinant peptide TAP mentioned above exerts both antithrombin and antiaggregation activity. Peptides and peptide mimetics of this type rapidly and irreversibly bound with receptor GP IIb/IIIa. They have short half life time in the blood plasma. Their preference in comparison with other drugs is particularly rapid and strong action. In our experiments it was demonstrated, that simple proline-containing peptides Pro-Gly, Trp-Pro, Pro-Gly-Pro (putative fragments of collagen and elastin) possesses significant antithrombotic and anticoagulant potential in vitro and in in vivo. Perhaps these peptides are members on intrinsic complex of haemostasis regulators.     

Triflavin, an antiplatelet Arg-Gly-Asp-containing peptide, is a specific antagonist of platelet membrane glycoprotein IIb-IIIa complex

Triflavin, an antiplatelet peptide containing Arg-Gly-Asp, purified from Trimeresurus flavoviridis venom, inhibits aggregation of human platelets stimulated by a variety of agonists. It blocks aggregation through interference with fibrinogen binding to its specific receptor on the platelet surface membrane in a competitive manner, but it has no apparent effect on intracellular events, such as thromboxane B2 formation, phosphoinositides breakdown and intracellular Ca2+ mobilization of thrombin-activated platelets. In this study, we determined the complete sequence of triflavin, which is composed of a single polypeptide chain of 70 amino acids. Its sequence is rich in cysteine and contains Arg-Gly-Asp at residues 49-51 in the carboxy-terminal domain. Triflavin shows about 68% identity of amino acid sequence with trigramin, which is a specific antagonist of the fibrinogen receptor associated with glycoprotein IIb/IIIa complex. [125I]Triflavin binds to unstimulated and ADP-stimulated platelets in a saturable manner and its Kd values are estimated to be 76 and 74 nM, respectively; the corresponding numbers of binding sites are 31,029 and 34,863 per platelet, respectively. [125I]Triflavin binding is blocked by Gly-Arg-Gly-Asp-Ser in a competitive manner. EDTA, the Arg-Gly-Asp-containing peptides (including naturally occurring polypeptides, trigramin and rhodostomin), and monoclonal antibody, 7E3, raised against GP IIb/IIIa complex, inhibit [125I]triflavin binding to unstimulated and ADP-stimulated human platelets. In conclusion, triflavin specifically binds to fibrinogen receptor associated with GP IIb/IIIa complex and its binding site is located at or near GP IIb/IIIa complex, overlapping with those of 7E3 and another Arg-Gly-Asp-containing polypeptide, rhodostomin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inhibition of platelet function by abciximab or high-dose tirofiban in patients with STEMI undergoing primary PCI: a randomised trial

BackgroundIn patients with ST-segment elevation myocardial infarction (STEMI) undergoing primary PCI, few data exist on the magnitude of platelet activation, aggregation and dosing of glycoprotein (GP) IIb/IIIa receptor inhibitors. Methods Sixty STEMI patients were randomised to abciximab, to high-dose tirofiban or to no additional GP IIb/IIIa inhibitor treatment. Platelet activation (P-selectin expression) was measured using flow cytometry and the level of inhibition of platelet aggregation was assessed using the Plateletworks assay. Additionally, the PFA-100 with the collagen/adenosine-diphosphate cartridge (CADP) was used to compare the levels of platelet inhibition. All measurements were performed at baseline (T₀), immediately after (T₁), 30 minutes (T₂), 60 minutes (T₃) and 120 minutes (T₄) after primary PCI. Results The level of platelet activation in both GP IIb/IIIa receptor inhibitor treated groups was significantly lower compared with the control group at all time points after primary PCI (p=0.04). Also the administration of the currently recommended dose of abciximab resulted in significantly lower levels of inhibition of aggregation compared with high-dose tirofiban (p<0.0001). In addition, the CADP closure times were significantly prolonged in both GP IIb/IIIa inhibitor treated groups compared with the control group at time points T1 (p=0.006) and T4 (p<0.0001). Conclusion The administration of high-dose tirofiban resulted in a significantly higher inhibition of platelet aggregation compared with the currently recommended dose of abciximab. Large clinical trials are needed to assess whether this laboratory superiority of high-dose tirofiban translates into higher clinical efficacy. (Neth Heart J 2007;15:375-81.)     

Synthesis of RGD analogs as potential vectors for targeted drug delivery

RGD analogs bind to integrin receptors with high affinity and therefore have the potential to be used as vectors for the targeted delivery of pharmaceutical agents to designated sites. Critical to this application is the ability to synthesize RGD analogs with different side chain functional groups that allow for the ready tethering of pharmaceutical agents without sacrificing their affinity for the target receptor significantly. A series of RGD analogs intended to be used as delivery vectors of pharmaceutical agents were prepared and evaluated for their ability to inhibit platelet aggregation by binding to glycoprotein IIb/IIIa. Among them, compound 11 showed the lowest IC50 against platelets activated by ADP. It was found that such RGD analogs could tolerate side chain modification fairly well with various functional groups attached such as amide, amine, ester, protected amine and poly(ethylene glycol). The fact that the compound with a side chain modification of poly(ethylene glycol) retained high affinity for glycoprotein IIb/IIIa (IC50 150 nM) suggests the feasibility of tethering fairly large pharmaceutical agents to such RGD analogs without significant sacrifice of their affinity to the intended receptor.