| Budget Amount *help |
¥2,600,000 (Direct Cost: ¥2,600,000)
Fiscal Year 1998: ¥400,000 (Direct Cost: ¥400,000)
Fiscal Year 1997: ¥900,000 (Direct Cost: ¥900,000)
Fiscal Year 1996: ¥1,300,000 (Direct Cost: ¥1,300,000)
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| Research Abstract |
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
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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
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