CREATION OF A PLATELET SUBSTITUTE USING RECOMBINANT PLATELET MEMBRANE GLYCOPROTEINS

血小板输注的缺点和局限性包括有限的供应、传染病的传播、发热反应、同种免疫的发展和高成本。临床应用血小板替代品，现在应该考虑克服这些问题。理想情况下，人工血小板应在体内充当血小板。然而，由于血小板功能的复杂性，人工血小板的开发可能比红细胞替代品更困难。血小板血栓应仅在血管损伤部位形成，在正常循环中不应有血小板活化或聚集。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。没有血小板替代品被报道对 关于我们 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，其在原发性止血中起关键作用。我们已经在CHO细胞中表达了保留vWF结合功能的Gplba结构域（成熟GPI β的残基1-302）（Murata Metal等，J Biol Chem 266：15474，1991）。为了创建一个候选的血小板替代品，我们已经将这个重组片段（gammaGP 1balpha）到脂质体组成的卵磷脂，胆固醇，磷脂酰甘油（10：5：2，摩尔比），并评估其功能在体外和体内。对于一些实验，脂质用罗丹明标记。使用FITC标记的抗GPIba单克隆抗体通过流式细胞术可检测脂质体表面上的γ GPIba。通过测量光散射变化的聚集计PA 100（Kowa，Japan）监测γ GPIb-脂质体的凝集。加入vWF和瑞斯托菌素（R）引起γ GPI α-脂质体的特异性凝集，其被抗vWF单克隆抗体NMC-4完全消除。罗丹明标记的脂质体的凝集也证明了荧光显微镜。我们接下来检查了异源聚集，即，将发生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γ-GPIb-脂质体混合，并加入R以诱导血小板凝集。罗丹明荧光在血小板聚集体中呈强阳性。当γ-GPIb-脂质体与PRP在低血小板浓度（20- 80 × 10 - 6/ml）下混合时，如通过PA 100评估的，γ-GPIb-脂质体剂量依赖性地增强R-诱导的血小板凝集。总之，GPIa-脂质体被掺入血小板聚集体中并增强血小板凝集。γ GPI β-脂质体可结合vWF并在体内生长的血栓上积聚，用作血栓的传感器、药物递送系统或血小板替代物，支持血栓形成中的止血individuals.in使用具有人工产生的动静脉分流的大鼠血栓形成模型的体内研究表明，输注的γ GPI β-脂质体特异性地积聚到血栓形成部位，这表明它们可掺入血栓中并可支持体内止血。少

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