Cold-induced activation of human platelets is associated with the passage of the platelets through their thermotropic phase transition. Anti-freeze glycoproteins (AFGPs) from the blood of certain Arctic and Antarctic fishes are capable of stabilizing human platelets during chilling and storage below the phase transition (4 C) for up to 21 days in vitro. Recent studies suggest that these AFGPs stabilize platelets in part, by preventing lateral phase separation of platelet membrane phospholipids. Such storage conditions would most certainly increase the ease of blood banking this valuable resource, however, prior to human clinical trials, it is essential to determine whether AFGPs can stabilize platelets from several potential animal models. The specific goals of the current project are to: (1) determine if passage the thermotropic phase transition correlates with cold-activation in a variety of animal model systems and to elucidate underlying events during storage, (2) to provide evidence concerning the mechanism by which AFGPs inhibit these events, including possible inhibition of microvesiculation and lateral phase separation, (3) determine the AFGP- platelet recovery and survival times in our animal model systems in vivo. Several techniques will be employed to accomplish these goals. Fourier-transform infrared spectroscopy will be used to monitor the membrane phospholipid phase transitions and lateral phase separations. Fluorescence spectroscopy will be employed for measuring vesiculation and lateral phase separation. Fluorescence activated cell scanning will measure the expression of glycoproteins Ib, IIb-IIIa and P-selectin on the plasma membrane, as well as to determine platelet recovery and survival times in vivo. In addition we have developed a unique shapemeter to quantify the level of shape change, activation and agonist responsiveness of these platelets during and after cold-storage.
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