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Generation of a liposome based platelet substitute : insertion of GPIb-IX into liposomes Brockmann, Nadine

Abstract

The provision of platelets for transfusion is a major function of a blood transfusion service. Since platelets have a short 5-day shelf life, may carry known and theoretical risks of donor derived transfusion products, and the demand exceeds supply, it is necessary to look to the creation of alternative products. A liposome based platelet substitute is one of such alternatives. The initial response in the formation of a platelet aggregate during a thrombotic event is the adhesion of the platelets to the damaged endothelium. This occurs via the plasma protein von Willebrand factor (vWF) that becomes activated in the blood in response to turbulent blood flow. Collagen-immobilized vWF binds to the platelet glycoprotein Ib-IX-V (GPIb-IX-V) complex, which is the initial platelet receptor involved in adhesion. Intracellular signaling linked to GPIb-IX-V binding leads to platelet activation, shape change and granule release. During this study, GPIb-IX was purified from outdated platelet concentrates and was inserted into 100 - 200 nm unilamellar liposome vesicles containing 25 mole% cholesterol, 10 mole% l,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) and 65 mole% l,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC). From 25 platelet concentrates, 0.925 mg GPIb-IX was purified which represented 18% of the protein in that sample. Outdated platelet concentrates were lysed with Triton X-100. The lysate was first subjected to the affinity chromatography on a wheat germ agglutinin agarose (WGA-agarose) column, and in some cases, was followed by an anion exchange mono- Q-Sepharose column. The final purification after WGA-agarose was 180,000 fold from platelet concentrates. GPIb-IX was incorporated into the liposomes using detergent exchange dialysis. Flow cytometry revealed that GPIb as well as GPIX was present on the external liposome surface. This result was confirmed by fluorescence microscopy. To assess the function of liposomal GPIb-IX, the liposomes were exposed to vWF, ristocetin and collagen type III. Results showed that the presence of GPIb-IX in the liposomes significantly enhanced vWF binding to the liposomes; therefore, the liposome product generated may act as a platelet aggregation enhancer.

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