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CREATION OF A PLATELET SUBSTITUTE USING RECOMBINANT PLATELET MEMBRANE GLYCOPROTEINS

使用重组血小板膜糖蛋白创建血小板替代品

基本信息

批准号：
08671258
负责人：
MURATA Mitsuru
金额：
$ 1.66万
依托单位：
KEIO UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1996
资助国家：
日本
起止时间：
1996 至 1998
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-08671258/
关键词：
PLATELETS PLATELET SUBSTITUTES VON WILLEBRAND FACTOR TRANSFUSION RECOMBINANTS LIPOSOMES ARTIFICIAL ORGANS リポソーム

项目摘要

Drawbacks and limitations in platelet transfusion include limited supply, transmission of infectious diseases, febrile reaction, development of alloimmunization and high cost. Clinical application of platelet substitutes should now be considered to overcome these problems. Ideally, artificial platelets should act as platelets in vivo. However, there might be even more difficulties in the development of artificial platelets than that of red cell substitutes, because of the complexity of platelet functions. Platelet thrombi should be formed only at the site of vascular injury and no platelet should be activated or aggregated in normal circulation. Only a limited number of substances have been studied for platelet substitutes. Some of these were reportedly reactive with adhesive ligands or with normal platelets in vitro, or effective in enhancing the hemostatic functions in thrombocytopenic or thrombocytopathic animals in vivo. No platelet substitute has been reported to be effective for … More hemostasis in large clinical studies.Platelet glycoprotein (GP) Ib/IX complex is a receptor for von Willebrand factor (vWF), which plays a crucial role in primary hemostasis. We have expressed in CHO cells a domain of Gplba (residues 1-302 of mature GPIbalpha) that retained a vWF-binding function (Murata Metal et al. J Biol Chem 266 : 15474, 1991). To create a candidate platelet substitute, we have incorporated this recombinant fragment (gammaGP1balpha) into liposomes composed of egg lecithin, cholesterol, and phosphatidylglycerol (10 : 5 : 2, by molar ratio) and evaluated their functions in vitro and in vivo. For some experiments, lipids were labeled with rhodamine. gammaGPIbalpha on the liposome surface was detectable by flow cytometry using a FITC-labeled anti-GPIba monoclonal antibody. Agglutination of gammaGPIbalpha-liposomes was monitored by an aggregometer PA 100 (Kowa, Japan), that measures changes in light scattering. Addition of vWF and ristocelin (R) caused specific agglutination of gammaGPIbalpha-liposomes, that was completely abolished by an anti-vWF monoclonal antibody NMC-4. Agglutination of rhodamine-labeled liposomes was also demonstrated by fluorescent microscopy. We next examined whether heterologous aggregation, i.e., attachment of liposomes to platelets, would occur. Platelet-rich plasma (PRP) was first mixed with rhodamine-labeled gammaGPIbalpha-liposomes, and R was added to induce platelet agglutination. Rhodamine-fluorescence was strongly positive in platelet aggregates. When gammaGPIbalpha-liposomes were mixed with PRP at low platelet concentration (20-80x l0/ml), gammaGPIbalpha-liposomes dose-dependently enhanced R-induced platelet agglutination as assessed by PA 100. In summary, GPIbalpha-liposomes were incorporated into platelet aggregates and enhanced platelet agglutination. gammaGPIbalpha-liposomes may bind vWF and accumulate on growing thrombi in vivo, serving as a sensor of thrombi, a drug delivery system or a platelet substitute supporting hemostasis in thrombocytopenic individuals.in vivo study using rat thrombosis model that has artificially-created arterio-venous shunt showed that infusedgammaGPIbalpha-liposomes specifically accumulated to the thrombosis site suggesting that they may be incorporated into thrombus and may support hemostasis in vivo. Less

血小板输注的缺点和局限性包括供应有限、传染病传播、发热反应、异体免疫发展和费用高。现在应该考虑血小板替代品的临床应用，以克服这些问题。理想情况下，人造血小板应在体内发挥血小板的作用。然而，由于血小板功能的复杂性，人造血小板的发展可能比红细胞替代品的发展更加困难。血小板血栓只应在血管损伤部位形成，正常循环中不应激活或聚集血小板。只有有限数量的物质被研究用于血小板替代品。据报道，其中一些在体外与粘附配体或正常血小板反应，或在体内有效增强血小板减少或血小板病变动物的止血功能。在大型临床研究中，没有血小板替代品被报道对止血有效。血小板糖蛋白（GP） Ib/IX复合物是血管性血友病因子（vWF）的受体，在原发性止血中起着至关重要的作用。我们已经在CHO细胞中表达了一个保留vwf结合功能的Gplba结构域（成熟GPIbalpha残基1-302）（Murata Metal等）。中国生物医学工程学报（英文版），2004(5):444 - 444。为了创造一种候选的血小板替代品，我们将重组片段（gammaGP1balpha）整合到由卵磷脂、胆固醇和磷脂酰甘油（摩尔比为10:5:2）组成的脂质体中，并在体外和体内评估了它们的功能。在一些实验中，脂质被罗丹明标记。使用fitc标记的抗gpiba单克隆抗体，流式细胞术检测脂质体表面的gammaGPIbalpha。gammagpibalpha -脂质体的凝集用聚集体仪pa100（日本Kowa）监测，该聚集体仪测量光散射的变化。添加vWF和里斯托素(R)引起gammagpibalpha -脂质体的特异性凝集，抗vWF单克隆抗体NMC-4完全消除了这种凝集。荧光显微镜也显示罗丹明标记的脂质体具有凝集作用。我们接下来检查了是否会发生异源聚集，即脂质体与血小板的附着。首先将富血小板血浆（PRP）与罗丹明标记的gammagpibalpha -脂质体混合，加入R诱导血小板凝集。罗丹明荧光在血小板聚集体中呈强阳性。当gammagpibalpha -脂质体与低血小板浓度（20-80 × 10 /ml）的PRP混合时，经pa100评估，gammagpibalpha -脂质体剂量依赖性地增强r诱导的血小板凝集。综上所述，gpibalpha脂质体被纳入血小板聚集体并增强血小板凝集。gammagpibalpha -脂质体可以结合vWF并在体内积累在生长的血栓上，作为血栓的传感器、药物输送系统或血小板替代品，支持血小板减少个体的止血。利用人工造动-静脉分流的大鼠血栓形成模型进行体内研究，结果表明，输注的gammagpibalpha脂质体特异性地积聚到血栓形成部位，表明它们可能被纳入血栓，并可能在体内支持止血。少