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.

将盘状静息血小板冷却至 <15 摄氏度会导致 当细胞被 重新加热。 这种现象妨碍了冷藏或冷冻 采购血小板用于输血。 血小板冷却可增加其 胞质游离钙导致盘状静息细胞变成球形 并组装肌动蛋白丝。 当肌动蛋白丝位于 休眠细胞被切断。 随后发生肌动蛋白组装 细丝的带刺末端变得无盖。 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- 产生预防感冒致形的药理学方法 变化。 具体目标 1 将在体外优化该方案，然后在体内进行测试 动物模型来评估这些储存的存活和功能 血小板的目的是大大延长人类的架子 血小板通过冷藏。 如果在 4°C 下储存 2 至 4 周 获得后，我们将通过确定这些是否保留来扩展这项工作 细胞可以冷冻和解冻，从而可以长期保存。 目标2 将测试多磷酸肌醇 (ppI) 在寒冷中的参与 通过向血小板中添加与 ppIs 特异性结合的肽来激活 并阻断凝血酶刺激的血小板肌动蛋白组装。 如果这些 磷脂是在寒冷条件下开盖的介质，这些肽 应防止丝体脱帽和肌动蛋白组装。 如果 ppI 结合 肽抑制细丝脱帽，我们将确定冷却结果是否有效 参与多磷酸肌醇的合成或聚集，或抑制其 水解。 AIM 3 将确定哪些加帽蛋白从 通过冷却血小板肌动蛋白丝。 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。 使用遗传血小板 缺乏凝溶胶蛋白的工程小鼠，我们已经证明凝溶胶蛋白是主要的 血小板的钙依赖性切断蛋白对此至关重要 球体形成。 我们将检查形态、肌动蛋白重塑和 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。