Lipid phase separation correlates with activation in platelets during chilling

When human platelets are chilled below 22 degreesC, they spontaneously acti vate, a phenomenon that severely limits their storage life. It has previous ly been proposed that there is a correlation between cold-induced platelet activation and passage of the membranes through a liquid-crystalline to gel phase transition. Because animal models are essential for developing metho ds for cold storage of platelets, it is necessary to investigate such a cor relation in animal platelets. In this work, horse platelets were used as a model, and it was found that cold-induced morphological activation is relat ed to the lipid phase transition. Using fluorescence microscopy with the li pophilic fluorescent dye 1,1'-dioctadecyl-3,3,3',3'-tetramethyl-indo cyanin e perchlorate (Dil-C-18), and Fourier transform infrared spectroscopy (FTIR ), it was found that lipid phase separation occurs during cooling and low t emperature storage. Furthermore, removal of cholesterol from the plasma mem brane also induced a phase separation, possibly between specific phospholip id classes. Steady-state fluorescence anisotropy of 1,6-diphenyl-1,3,5-hexa triene (DPH) and trimethylammonium-DPH (TMA-DPH) were compared in cells and multilamellar vesicles (MLV) composed of platelet lipids. Cholesterol depl etion led to a decrease in the fluorescence anisotropy of the two probes, w hich can be explained by changes in the order of the phospholipid molecules . In addition, the lipid composition and fatty acid profile of the cellular phospholipids were determined. Based of the similarities between horse and human platelets, it is suggested that horse platelets may be used as a mod el for studying cold-stored platelets. The results are discussed in relatio n to the possible role of phase separation during cell signalling.