6-(αα-二苯基乙酰哌嗪基苯基)-4,5-二氢-5-甲基-3(2H)-哒嗪酮对兔血小板聚集及TXB_2,cAMP的影响

6-(αα-二苯基乙酰哌嗪基苯基)-4,5-二氢-5-甲基-3(2H)-哒嗪酮(简称DMDP)是我院新合成的哒嗪酮的衍生物。DMDP可以显著抑制由花生四烯酸(AA(?),ADP和血小板活化因子(PAF)诱导的免血小板聚集,其IC_(50)分别为1.12±0.1.4.19±0.5和2.97±0.1μmol/L。实验还表明DMDP在1～500 μmol/L浓度范围内呈剂量依赖性地抑制兔血小板内血栓素B_2含量,但升高兔血小板内环腺苷酸水平,这可能是其抑制血小板聚集的作用机理之一。     

6-(αα-二苯基乙酰哌嗪基苯基)-4,5-二氢-5-甲基-3(2H)-哒嗪酮对兔血小板聚集及TXB2,cAMP的影响

6-(αα-二苯基乙酰哌嗪基苯基)-4,5-二氢-5-甲基-3(2H)-哒嗪酮(简称DMDP)是我院新合成的哒嗪酮的衍生物。DMDP可以显著抑制由花生四烯酸(AA(?),ADP和血小板活化因子(PAF)诱导的免血小板聚集,其IC₅₀分别为1.12±0.1.4.19±0.5和2.97±0.1μmol/L。实验还表明DMDP在1～500 μmol/L浓度范围内呈剂量依赖性地抑制兔血小板内血栓素B₂含量,但升高兔血小板内环腺苷酸水平,这可能是其抑制血小板聚集的作用机理之一。     

新型血小板GPⅡb/Ⅲa受体拮抗剂Z4A5抗血小板作用的研究

目的研究血小板GPⅡb/Ⅲa受体拮抗剂Z4A5抑制血小板聚集的活性、稳定性以及对已聚集血小板的解聚作用。方法采用比浊法测定Z4A5对体外二磷酸腺苷(aden-osine diphosphate,ADP)诱导血小板聚集的抑制作用和稳定性及对ADP诱导聚集血小板的解聚作用。结果 Z4A5浓度依赖性的抑制ADP诱导的血小板聚集,半数抑制浓度(50%concentration of inhibition,IC50)为(0.46±0.05)μmol.L-1(n=10,P<0.05);Z4A5在血浆中孵育3 h仍保持85.55%(P<0.05)的活性;10μmol.L-1 Z4A5在加入ADP诱导聚集的血小板3 min及7 min后的解聚率为分别为22.66%(P<0.05)及30.93%(P<0.01)。结论 Z4A5在体外有较强的、稳定的抑制血小板聚集作用,而且对聚集血小板有一定的解聚作用。     

氯吡格雷的药理学,临床疗效和耐受性

氯吡格雷(clopidogrel)是一种具有不可逆抑制血小板聚集的噻吩并吡啶化合物,化学结构与噻氯匹定(ticlopidine)类似.与阿司匹林等药物不同,它产生抗血小板聚集作用不是通过花生四烯酸代谢途径,而是通过抑制ADP对血小板的诱导作用.1 药效学特征1.1 作用机制 氯吡格雷通过阻断ADP受体,抑制纤维蛋白原与它的血小板受体(即糖蛋白GP Ⅱ b-Ⅲa)结合,但不是直接干扰GP Ⅱb-Ⅲ a复合物的形成,可能是间接的减少纤维蛋白耗的结合.氯吡格雷抑制健康志愿者ADP和凝血酶诱导的血小板聚集,它选择性地减少可抑制活比腺苷酸环化酶的功能性ADP受体的数目.大鼠血小板体外实验表明,氯吡格雷与血小板表面腺苷酸环化酶偶联的ADP结合是直接的、竞争性的和不可逆的.     

用转移电泳方法鉴定单克隆抗体识别的血小板抗原

用改良的转移电泳-酶联免疫方法分析研究了8株IgG抗人血小板单克隆抗体所识别的抗原。其中有3株得到阳性结果。单克隆抗体SZ-2与分子量为17万的蛋白(GPⅠ_b)相结合,SZ-21与分子量为9.2万的蛋白(GPⅢ_a)起反应,SZ-22与分子量相当于GPⅡ_b(14万)的蛋白相结合。这些结果与免疫沉淀及亲和层析研究所得的结果相符合。     

氯吡格雷致便秘 2例

氯吡格雷是一种新型的噻酚吡啶类衍生物,能选择性地阻断血小板ADP受体,抑制纤维蛋白原与其血小板受体糖蛋白GPⅡb/Ⅲa结合而发挥对血小板聚集的抑制作用。此外,氯吡格雷可降低血小板选择蛋白(P2 selectin,CD62,一种血小板脱颗粒的标志物)的表达,抑制血小板的活化。临床常用于防治血小板高聚集状态相关的心脑血管疾病和     

Nα-乙酰-精氨酰-甘氨酰-天冬氨酰-对甲氧基苯乙胺对血小板聚集的抑制

N^α-乙酰-精氨酰-甘氨酰-天冬氨酰-对甲氧基苯乙胺(W2002)是精-甘-天冬氨酸(Arg-Gly-Asp)肽类血小板聚集抑制剂. 为探讨其抗血小板聚集的活性, 分别用比浊法和血小板计数的方法测定二磷酸腺苷(ADP)及高剪切速率诱导的血小板聚集,并用放射性配体分析法测定血小板表面结合［¹²⁵I］纤维蛋白原(FGN)的含量, 以了解W2002竞争性抑制［¹²⁵I］FGN与血小板糖蛋白(GP)Ⅱb/Ⅱa结合的生物活性. 结果显示 : W2002有明显的抑制ADP诱导血小板聚集的活性, 除最低终浓度(9 μmol·L^-1)外, 其余各浓度点(270, 135, 45 μmol·L^-1)与生理盐水对照组比较差异均非常显著; 其对抗高剪切速率诱导的血小板聚集也有明确的量-效关系; 抑制［¹²⁵I］FGN与血小板结合的IC₅₀值为 (41.5±2.9)μmol·L^-1, 在老年人和青年人群中比较无明显差异. 研究提示W2002通过抑制FGN与血小板GPⅡb/Ⅲa的结合而发挥作用.     

体外循环对血小板聚集及膜蛋白的影响

目的 :探讨体外循环下心内直视手术对血小板聚集及血小板膜蛋白 GP b、GP a的影响。方法 :选择体外循环下心内直视手术患者 30例 ,分别以体外循环转机前及停机即刻为采血点 ,用流式细胞仪测定血小板膜蛋白 GP b、GP a的阳性百分率。结果 :血小板对 ADP、ADR的最大聚集率分别由转机前的 (70 .35± 10 .12 ) % ,(74 .2 7±9.4 7) %降至 (4 7.2 7± 9.2 6 ) %及 (5 0 .0 2± 8.74 ) %。转机前后相比有显著性意义 (P<0 .0 5 )。GP b、GP a 分别由转机前的 (92 .88± 4 .70 ) % ,(90 .5 4± 7.0 2 ) %降至 (70 .4 7± 7.0 2 ) %及 (70 .0 2± 5 .0 7) % ,转机前后相比亦有显著性意义 (P<0 .0 5 )。结论 :体外循环造成的血小板膜蛋白受损及血小板聚集率的下降可能引起血小板功能损害 ,从而造成术后出血     

含RGD多肽以及衍生物的舒血管作用

已经证实纤维蛋白分子内两个区域内的氨基酸序列可以介导纤维蛋白与GPIIb/Ⅲa受体的结合。除单克隆抗体我，用合成的含RGD和APLRV的小分子多肽可以封闭GPIIb/Ⅲa的这种结合功能，我们的初步研究发现，RGDS除具有抑制血小板聚集和抗血栓形成的作用外，还显示舒血管作用，为了证实RGDS相关多肽的舒血管作用，研究了RGDF，APLRV，APLRVRGDS，APLRVRGDF，在体外用NE预处理大鼠的动脉肌条后观察了上述合成多肽的舒血管作用，检测了三种剂量（10^-5mol/L,10^-6mol/L,10^-7mol/L)下收缩的肌条舒张的程度。     

甲基莲心碱对正常与高脂血人及动物血小板聚集功能的影响

用比浊法测定血小板聚集率,观察甲基莲心碱(Nef)体外给药对正常与高脂血的人、大鼠及家兔血小板聚集功能的影响。结果表明,Nef在体外抑制ADP、胶原、Adr诱导的人血小板聚集,呈明显剂量依赖性。健康人组,对ADP、Adr诱聚1min时的IC_(50)分别为59和36μmol/L;对ADP、Adr、胶原诱聚5min时的IC_(50)分别为42,89和110μmol/L。高脂血症患者组,Nef对ADP、Adr诱聚1min时的IC_(50)分别为45,38μmol/L;对ADP、Adr、胶原诱聚5min时的IC_(50)分别为36,27和16μmol/L。Nef 50、500μmol/L也能明显抑制ADP诱导的以正常或高脂饮食喂养大鼠的血小板聚集。Nef(0.3～300μmol/L)抑制ADP、胶原诱导的兔血小板聚集呈剂量依赖性。     

Vasodilation Effects of RGD Containing Peptides and De rivatives

已经证实纤维蛋白分子内两个区域内的氨基酸序列可以介导纤维蛋白与ＧＰＩｂ／Ｉ┐Ｉａ受体的结合。除单克隆抗体外，用合成的含ＲＧＤ和ＡＰＬＲＶ的小分子多肽可以封闭ＧＰＩｂ／Ｉ┐Ｉａ的这种结合功能，我们的初步研究发现，ＲＧＤＳ除具有抑制血小板聚集和抗血栓形成的作用外，还显示舒血管作用。为了证实ＲＧＤＳ相关多肽的舒血管作用，研究了ＲＧＤＦ，ＡＰＬＲＶ，ＡＰＬＲＶＲＧＤＳ，ＡＰＬＲＶＲＧＤＦ，在体外用ＮＥ预处理大鼠的动脉肌条后观察了上述合成多肽的舒血管作用，检测了三种剂量（１０┐５ｍｏｌ／Ｌ，１０┐６ｍｏｌ／Ｌ，１０┐７ｍｏｌ／Ｌ）下收缩的肌条舒张的程度。     

Antiplatelet Aggregation Effects of YIGSK and RGD Containing Peptides

观察了ＹＩＧＳＫ，ＲＧＤＳ，ＲＧＤＶ，ＲＧＤＦ，ＹＩＧＳＫＲＧＤＳ，ＹＩＧＳＫＲＧＤＶ和ＹＩＧＳＫＲＧＤＦ的抗血小板聚集作用，通过比较它们的活性，发现将ＹＩＧＳＫ和含ＲＧＤ多肽偶联可增强某些化合物的抗血小板聚集活性     

Pharmacological properties of YM-57029, a novel platelet glycoprotein IIb/IIIa antagonist

The pharmacological properties of YM-57029 [4-[4-(4-carbamimidoylphenyl)-3-oxopiperazin-1-yl]piperidino]acetic acid monohydrochloride trihydrate), a novel glycoprotein IIb/IIIa antagonist were examined in this study. YM-57029 inhibited fibrinogen binding to purified glycoprotein IIb/IIIa, 1000-fold more potently than the tetrapeptide arginine-glycine-aspartic acid-serine (RGDS). YM-57029 concentration-dependently inhibited ADP-, collagen- and high shear stress-induced platelet aggregation, strongly inhibited ATP release from platelets activated by ADP, and enhanced deaggregation of ADP-induced platelet aggregates. In a pro-aggregatory activity study, RGDS or SC-54701A ((S)-3-[3-[(4-amidinophenyl)carbamoyl]propionamido]-4-pentynoic acid monohydrochloride) caused prominent small aggregate formation. At a higher concentration, RGDS induced medium and large size aggregates, and SC-54701A induced medium aggregates. In contrast, YM-57029 produced only a few small and no larger size aggregates. Ex vivo ADP-induced platelet aggregation and platelet retention to collagen-coated plastic beads were dose-dependently inhibited by YM-57029 after intravenous bolus injection in guinea pigs. YM-57029 produced dose-dependent antithrombotic effects in carotid artery thrombosis and arterio-venous shunt thrombosis models in guinea pigs at 10 and 30 microg/kg, respectively. At these doses, YM-57029 prolonged template bleeding time. These results suggest that YM-57029 is a potent glycoprotein IIb/IIIa antagonist which has less pro-aggregatory effect. It may be a promising antiplatelet agent for thromboembolic diseases, and a good prototype for developing an orally active compound.     

A tetravalent RGD ligand for integrin-mediated cell adhesion

Monovalent RGD (arginine-glycine-aspartic acid) peptides or polymers furnished with RGD in random distributions are employed as cell-scaffolds and gene delivery vehicles. However, integrin binding to RGD is dependent on the spatial distribution (clustering) of the ligand and intrinsic integrin affinity via conformational changes (avidity). Here we have designed and expressed a polypeptide consisting of a tetrameric coiled coil and spacer facilitating polyvalent (clustered) display of integrin ligands; the RGD motif was used as proof of principle. Size-exclusion chromatography and circular dichroism showed that the polypeptide self assembled as a tetramer in solution with a defined secondary structure. Cell adhesion to surfaces coated with the polypeptide was up to 3-fold greater than that for (monovalent) RGDS peptide at equivalent concentrations. Moreover, the polypeptide in solution at concentrations >or= 1 microM inhibited cell adhesion to fibronectin-coated surfaces, while RGDS peptide in solution at concentrations up to 500 muM did not. These cell data demonstrate that the polypeptide bound integrin receptors in a polyvalent manner. The polypeptide will therefore be of use in the engineering of tissue-culture scaffolds with increased cell adhesion activity, or to targeted gene delivery vehicles, and could incorporate protein ligands in place of the RGD motif.     

含 RGD 四肽的合成与功能

在血小板依赖的血栓形成中,纤维蛋白与血小板表面GP II_b/III_a受体结合是重要步骤。在受体识别中,RGD(Arg-Gly-Asp,精·甘·天冬)是关键序列。含RGD多肽的竞争抑制作用,表现为抗血栓作用。本文合成了RGDS,RGDV和RGDF3种四肽;测定了它们的舒血管活性和抗血栓活性;讨论了溶液中的构象对生物活性的影响。在大鼠血栓模型上,用5.0μmol·kg^-1剂量,RGDS和RGDV无明显的抗血栓作用。用25μmol·kg^-1剂量,RGDF的抗血栓作用有明显的统计意义。该结果与它们在溶液中的构象相关。3种四肽对主动脉条的舒血管作用体现另一种趋势,RGDS和RGDV显示明确的舒血管活性。小剂量时RGDF的舒血管作用与对照组相比,无统计学意义。     

Antiplatelet mechanisms of TA-993 and its metabolite MB3 in ADP-induced platelet aggregation

We investigated the antiplatelet mechanisms of TA-993 [(-)-cis-3-acetoxy-5-(2-(dimethylamino)ethyl)-2, 3-dihydro-8-methyl-2-(4-methylphenyl)-1,5-benzothiazepin-4(5H)-one maleate] and its metabolite MB3 (deacetyl and N-monomethyl TA-993) in human platelets stimulated by ADP in vitro. TA-993 and MB3 concentration-dependently inhibited fibrinogen binding to the ADP-stimulated platelets as well as inhibiting platelet aggregation. The antiplatelet effect of MB3 was about 300 times more potent than those of TA-993 and a glycoprotein IIb/IIIa receptor antagonist, Arg-Gly-Asp-Ser (RGDS). Aggregation of ADP-treated fixed platelets caused by the addition of fibrinogen was inhibited by RGDS but not by TA-993 and MB3. TA-993 and MB3 inhibited ADP-induced polymerization of actin filaments. Neither TA-993 nor MB3 affected cyclic AMP and cyclic GMP levels in resting platelets, and nor suppressed the increase in intracellular Ca(2+) concentration induced by ADP. These results suggest that the antiplatelet mechanisms of TA-993 and MB3 may involve inactivation of glycoprotein IIb/IIIa receptors via inhibition of the polymerization of actin.     

Discovery of an orally active non-peptide fibrinogen receptor antagonist based on the hydantoin scaffold

Antagonists of the platelet fibrinogen receptor (GP IIb/IIIa receptor) are expected to be a promising new 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 44 (S 1197). Compound 44 inhibited, in a dose dependent and reversible manner, human and dog platelet aggregation as well as 125I-fibrinogen binding to ADP-activated human gel filtered platelets and isolated GP IIb/IIIa with K(i) values of 9 nM and 0.17 nM, respectively. A pharmacophore mapping procedure with QXP and a 3D-QSAR analysis applying the GRID/GOLPE methodology yielded a stable, rather predictive model and revealed structural features which are important for binding. Hydrophobic substitutions both at the hydantoin nucleus and at the C-terminus increase the affinity toward the fibrinogen receptor. The crystalline ethyl ester prodrug 48 (HMR 1794) is an orally active antithrombotic agent which is a promising drug candidate for the treatment of thrombotic diseases in humans.     

Effect of protein properties on display efficiency using the M13 phage display system

The M13 phage display system is a powerful technology for engineering proteins such as functional mutant proteins and peptides. In this system, it is necessary that the protein is displayed on the phage surface. Therefore, its application is often limited when a protein is poorly displayed. In this study, we attempted to understand the relationship between a protein's properties and its display efficiency using the well-known pIII and pVIII type phage display system. The display of positively charged SV40 NLS and HIV-1 Tat peptides on pill was less efficient than that of the neutrally charged RGDS peptide. When different molecular weight proteins (1.5-58 kDa) were displayed on pIII and pVIII, their display efficiencies were directly influenced by their molecular weights. These results indicate the usefulness in predicting a desired protein's compatibility with protein and peptide engineering using the phage display system.     

Halysin, an antiplatelet Arg-Gly-Asp-containing snake venom peptide, as fibrinogen receptor antagonist

By means of Sephadex G-75 and CM-Sephadex C-50 column chromatography and reverse-phase HPLC, a low molecular weight (Mr = 7500), cysteine-rich peptide, halysin, was purified from Agkistrodon halys (mamushi) snake venom. Halysin is a potent platelet aggregation inhibitor that concentration-dependently inhibited human platelet aggregation stimulated by ADP, thrombin and collagen (IC50 = 0.16 to 0.36 microM) without affecting platelet secretion. It was active in inhibiting platelet aggregation of platelet-rich plasma and whole blood. Halysin had no effect on thromboxane B2 formation of platelets or intracellular Ca2+ mobilization of Quin 2-AM loaded platelets stimulated by thrombin. It inhibited the fibrinogen-induced aggregation of elastase-treated platelets. Halysin concentration-dependently inhibited the 125I-fibrinogen binding to ADP-stimulated platelets in a competitive manner (IC50 = 0.16 microM). 125I-Halysin bound to resting platelets (Kd = 1.6 x 10(-7) M) and to ADP-stimulated platelets (Kd = 3.4 x 10(-8) M) in a saturable manner. EDTA, the Arg-Gly-Asp (RGD)-containing snake venom peptides trigamin and rhodostomin, Arg-Gly-Asp-Ser (RGDS), and Gly-Gln-Gln-His-His-Leu-Gly-Gly-Ala-Lys-Gln-Ala-Gly-Asp-Val blocked both 125I-fibrinogen binding and 125I-halysin binding to ADP-stimulated platelets. The monoclonal antibody, 7E3, raised against glycoprotein IIb-IIIa complex blocked both 125I-fibrinogen and 125I-halysin binding, whereas 10E5 had no significant effect on halysin binding to ADP-stimulated platelets, indicating that 7E3 and halysin bind to an epitope which is different from that of 10E5. RGDS concentration-dependently inhibited 125I-halysin binding in a competitive manner. We determined the primary structure of halysin which is a single peptide chain of 71 amino acid residues. An RGD sequence appeared in the carboxy-terminal domain of halysin. Halysin showed about an 85% identical sequence with trigamin which is a specific antagonist of fibrinogen receptor associated with glycoprotein IIb-IIIa complex. In conclusion, halysin inhibited platelet aggregation by interfering with fibrinogen binding to the fibrinogen receptor of the activated platelets. The RGD sequence of halysin plays an important role in the expression of its biological activity.     

AoGDW肽体外抗血小板作用的研究

目的　为寻找活性更高、作用更稳定的具有抗栓作用的RGD肽衍生物 ,我们设计合成了AoGDW肽 ,并研究其体外抗血小板聚集的活性、稳定性以及它对血浆凝血功能的影响。方法　①采用比浊法测定AoGDW肽抑制人血小板聚集的活性以及在血浆中的稳定性。②利用血凝仪测定其对血浆凝血功能的影响。结果　AoGDW肽抑制血小板聚集的 5 0 %的抑制剂浓度 (IC50 )为 (4 7± 2 5 ) μmol/L ,与阴性对照组、阳性对照组相比 ,均为P <0 0 1。在血浆中孵育 3h仍保持 10 0 %的活性。另外血浆凝血功能各项指标凝血活酶时间 (TT)、凝血酶原时间 (PT)、活化部分凝血活酶时间 (APTT)均在正常范围内。结论　AoGDW肽在体外有较强的、稳定的抑制血小板聚集的作用 ,并且不影响血浆正常的凝血功能