PREVENTION OF COLD INDUCED PLATELET STORAGE LESION

预防寒冷引起的血小板储存损伤

• 批准号: 6353071
• 负责人: John H Hartwig
• 金额: $ 26.95万
• 依托单位: IMMUNE DISEASE INSTITUTE, INC.
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-09-01 至 2001-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6353071
• 关键词: actins; baboons; calcium flux; chelating agents; cryopreservation; cytochalasins; gelsolin; human subject; laboratory mouse; laboratory rabbit; phosphatidylinositols; platelet activation; platelet preservation

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.

将盘式静止血小板冷却至<15度C的温度 当细胞为时，形成扭曲和改变的粘合特性 重新加热。 这种现象排除了制冷或冻结 采购用于输血的血小板。 血小板的冷却增加了 胞质的无钙导致盘状静息细胞成为球形 并组装肌动蛋白丝。 当肌动蛋白丝中的肌动蛋白丝中发生 静止的细胞被切断。 肌动蛋白组装随后发生 细丝的刺末端变成未盖。 我们为了解 冷介导的血小板形状变化的机制表明，首先 产生的药理学方法以防止感冒诱发形状 更改。 特定目标1将在体外优化该方案，然后在 评估这些存储的生存和功能的动物模型 血小板的目的是大大扩展人类的架子 血小板通过冷藏。 如果在4度C下存储2至4周 获得了，我们将通过确定这些工作是否保存来扩展这项工作 细胞可以冷冻并解冻，从而可以长期存储。 目标2 将测试冷磷酸肌醇（PPI）在冷的参与 通过将肽添加到专门与PPI结合的血小板中添加激活 并阻止凝血酶刺激的血小板肌动蛋白组件。 如果这些 磷脂是寒冷中解开的介体，这些肽 应防止细丝解蛋白和肌动蛋白组装。 如果PPI结合 肽抑制细丝解蛋白，我们将确定冷却结果是否 在综合或聚类的聚磷酸肌醇或抑制其 水解。 AIM 3将确定从中释放了哪些封盖蛋白 血小板肌动蛋白丝冷却。 在AIM 4中，我们将确定 凝胶素在冷却伴随的球体形成中的作用。 我们 将确定凝胶素介导的肌动蛋白丝碎片是否也是 正常的GPIB受体调节和细胞粘附和细胞粘附所必需的 冷却对这些过程的影响。 使用遗传学的血小板 缺少凝胶蛋白的工程老鼠，我们已经证明了凝胶林是主要的 血小板的钙依赖性切断蛋白是必不可少的 球体形成。 我们将检查形态，肌动蛋白重塑和 野生型和凝胶素无效血小板的受体调制特性 对冷却和其他激活刺激的反应。