PREVENTION OF COLD INDUCED PLATELET STORAGE LESION

将盘状静息血小板冷却至<15 ℃的温度， 形状变形和改变的粘合性能， 重新加热 这种现象妨碍了冷藏或冷冻。 为输血准备的血小板 血小板的冷却增加了它们的 胞浆游离钙导致盘状静止细胞变成球形 并组装肌动蛋白丝 当细胞中的肌动蛋白丝 休眠细胞被切断。 肌动蛋白装配随后发生， 细丝的倒钩末端变得无帽。 我们努力了解 冷介导的血小板形状变化的机制提出了第一个- 代药理学方法，以防止寒冷诱导的形状 变化 SPECIFIC AIM 1将在体外优化该方案，然后在 动物模型来评估这些储存的细胞的存活和功能， 目的是大大延长人类的货架状 血小板通过冷藏。 如果在4摄氏度下储存2至4周， 获得，我们将通过确定这些保留的 细胞可以冷冻和解冻，从而允许长期储存。 目的2 将测试多磷酸肌醇（ppI）在感冒中的作用， 通过向血小板中添加肽来活化，所述肽特异性地结合到PPI 并阻断凝血酶刺激的血小板肌动蛋白组装。 如果这些 磷脂是在寒冷中脱帽的介质，这些肽 应防止细丝脱帽和肌动蛋白组装。 如果ppI结合 肽抑制丝脱帽，我们将确定是否冷却结果 在聚磷酸肌醇的合成或聚簇中，或抑制其 水解 AIM 3将决定哪些加帽蛋白从 血小板肌动蛋白丝。 在AIM 4中，我们将建立 凝溶胶蛋白在伴随冷却的球体形成中的作用。 我们 将确定凝溶胶蛋白介导的肌动蛋白丝断裂是否也 正常GpIb受体调节和细胞粘附所必需的， 冷却对这些过程的影响。 利用血小板从基因上 缺乏凝溶胶蛋白的工程小鼠，我们已经证明，凝溶胶蛋白，主要的 血小板的钙依赖性切割蛋白，是必不可少的， 球体形成。 我们将研究形态学，肌动蛋白重塑， 野生型和凝溶胶蛋白无效血小板的受体调节特性 对冷却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.