PREVENTION OF COLD INDUCED PLATELET STORAGE LESION

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Cooling of discoid resting platelets to temperatures <15 degrees C causes shape distortions and altered adhesive properties when the cells are rewarmed. This phenomenon precludes refrigeration or freezing of platelets procured for transfusion. Cooling of platelets increases their cytosolic free calcium causing discoid resting cells to become spherical and assemble actin filaments. Sphering occurs when actin filaments in the resting cell become severed. Actin assembly occurs subsequently when the barbed ends of filaments become uncapped. Our efforts to understand the mechanism of cold-mediated platelet shape change have suggested a first- generation pharmacological approach to prevent the cold induced shape changes. SPECIFIC AIM 1 will optimize this regimen in vitro and then test it in animal models to evaluate the survival and function of these stored platelets with the goal of extending greatly the shelf-like of the human platelet through cold-storage. If storage at 4 degrees C for 2 to 4 weeks is obtained, we will extend this work by determining if these preserved cells can be frozen and thawed thereby allowing long-term storage. AIM 2 will test the involvement of polyphosphoinositides (ppI's) in cold activation by adding peptides to platelets that bind specifically to ppIs and block thrombin-stimulated platelet actin assembly. If these phospholipids are the mediators of uncapping in the cold, these peptides should prevent filament uncapping and actin assembly. If the ppI-binding peptides inhibit filament uncapping, we will determine if cooling results in synthesis or clustering of the polyphosphoinositides, or inhibits their hydrolysis. AIM 3 will determine which capping proteins are released from the platelet actin filaments by cooling. In AIM 4, we will establish the role of gelsolin in the sphere formation that accompanies cooling. We will determine if gelsolin-mediated actin filament fragmentation is also necessary for normal GpIb receptor modulation and cell adhesion and the effect of cooling on these processes. Using platelets from genetically engineered mice lacking gelsolin, we have shown that gelsolin, the major calcium-dependent severing protein of the platelet, is essential for this sphere formation. We will examine the morphology, actin remodeling, and receptor modulation properties of wild type and gelsolin null platelets in response to cooling and other activating stimuli.