CREATION OF A PLATELET SUBSTITUTE USING RECOMBINANT PLATELET MEMBRANE GLYCOPROTEINS

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、同种异体免疫反应和昂贵的费用。为了克服这些问题，现在应该考虑临床应用血小板替代品。理想情况下，人工血小板应该在体内起到血小板的作用。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的开发更困难。血小板血栓只能在血管损伤部位形成，在正常循环中不能激活或聚集血小板。只有数量有限的物质被研究用来替代血小板。据报道，其中一些化合物在体外与黏附配体或正常血小板发生反应，或在体内有效地增强血小板减少或血小板病态动物的止血功能。目前还没有关于血小板替代品对…有效的报道更多的止血在大型临床研究中。血小板糖蛋白(GP)Ib/IX复合体是von Willebrand因子(VWF)的受体，在原发性止血中发挥关键作用。我们已经在CHO细胞中表达了Gplba的一个结构域(成熟GPIbalpha的1-302个残基)，它保留了vWF结合功能(Murata Metals等人)。生物化学：15474,1991年)。为了创造一个候选的血小板替代品，我们将这个重组片段(GammaGP1balpha)掺入到由卵磷脂、胆固醇和磷脂酰甘油(摩尔比为10：5：2)组成的脂质体中，并在体外和体内评价了它们的功能。在一些实验中，用罗丹明标记脂类。使用FITC标记的抗GPIba单抗，通过流式细胞仪检测脂质体表面的GammaGPIbalpha。用聚集仪PA 100(日本小和)监测γ-GPIbalpha脂质体的凝集作用，该仪器测量光散射的变化。加入vWF和ristocelin(R)可引起γ-GPIbalpha脂质体的特异性凝集，这种凝集可被抗vWF的单抗NMC-4完全消除。荧光显微镜下也证实了罗丹明标记脂质体的凝集作用。接下来，我们检查是否会发生异源聚集，即脂质体与血小板的结合。先将富含血小板血浆(PRP)与罗丹明标记的γ-GPα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物中呈强阳性。当以较低的血小板浓度(20-80×10~(-1)/ml)与PRP混合时，γ-GPα-脂质体呈剂量依赖性地增强PA-100测定的R诱导的血小板凝集。综上所述，GPIbalpha脂质体被引入到血小板聚集体中，并增强了血小板的凝集性。γ-GPIbalpha脂质体可以结合vWF并在体内积累血栓，作为血栓传感器、药物输送系统或支持血小板减少者止血的血小板替代品。利用人工建立动-静脉分流的大鼠血栓模型进行的体内研究表明，输注的GammaGPIbalpha脂质体特异性地聚集在血栓形成部位，提示它们可能被结合到血栓中，并可能支持体内止血。较少

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