PREVENTION OF COLD INDUCED PLATELET STORAGE LESION

盘状静息血小板冷却至摄氏15度引起 当细胞被放置时，形状扭曲和粘连性能改变 又热了。这种现象排除了冷藏或冷冻的可能性 获取用于输血的血小板。血小板的降温增加了它们的 胞内游离钙导致盘状静息细胞变球形 并组装肌动蛋白细丝。当肌动蛋白细丝在 静息细胞被切断。肌动蛋白组装随后发生时， 细丝的带刺末端变得没有盖子。我们努力理解 冷介导的血小板形状改变的机制提出了第一个- 预防寒诱形的世代药理学研究进展 改变。 特定的AIM 1将在体外优化这种方案，然后在 用动物模型评估这些细胞的存活率和功能 以极大地扩展人类货架状为目标的血小板 通过冷藏获得的血小板。如果在4摄氏度下储存2至4周 ，我们将通过确定这些保存的 细胞可以被冷冻和解冻，从而允许长期储存。目标2 将测试多聚磷酸肌醇(PPI)在寒冷中的参与程度 通过向血小板中添加与PPI特异结合的多肽来激活 并阻断凝血酶刺激的血小板肌动蛋白组装。如果这些 磷脂是寒冷中解帽的媒介，这些多肽 应防止细丝脱帽和肌动蛋白组装。如果PPI绑定 多肽抑制细丝脱帽，我们将确定是否降温结果 在多磷肌醇的合成或聚集中，或抑制其 水解液。AIM 3将确定哪些封顶蛋白从 冷却后的血小板肌动蛋白细丝。在目标4中，我们将建立 明胶在伴随冷却而形成的球体中的作用。我们 将确定明胶蛋白介导的肌动蛋白细丝断裂是否也 正常的GPIB受体调节和细胞黏附所必需的 冷却对这些过程的影响。使用来自基因的血小板 缺乏明胶的转基因小鼠，我们已经证明了明胶，主要的 血小板的钙依赖切断蛋白对此是必不可少的 球体队形。我们将检查形态，肌动蛋白重塑，以及 野生型和明胶蛋白缺失型血小板的受体调节特性 对降温和其他激活刺激的反应。

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.