SNARE proteins in platelet alpha-granule secretion

描述(申请人提供)：富含血小板的动脉血栓可导致中风、外周血管疾病和心肌梗死中的组织梗塞。在血栓形成过程中，血小板分泌其颗粒内容物。最丰富的血小板颗粒，即α颗粒，含有促进血栓传播的黏附分子、凝血因子和血管活性因子。我们使用了一个通透性的血小板分泌模型来确定SNARE蛋白在导致血小板α颗粒分泌的膜融合事件中的作用。然而，激动剂诱导的血小板刺激导致SNARE蛋白介导膜融合的机制仍然很不清楚。我们发现α-颗粒的分泌需要磷脂酰肌醇(4，5)-二磷酸的合成。这些研究证实了II型磷脂酰肌醇5-磷酸-4-激酶在α颗粒分泌中的作用。然而，合成α-颗粒分泌所需的磷脂酰肌醇(4，5)-二磷酸的合成途径尚不清楚。本提案特定目的1中描述的实验将确定I型磷脂酰肌醇4-磷酸5-激酶和II型磷脂酰肌醇5-磷酸4-激酶在血小板α-颗粒分泌中的相对贡献。这些研究还将确定I型磷脂酰肌醇5-磷酸4-激酶是否在血小板α颗粒分泌过程中作为蛋白激酶C的下游效应因子。磷脂酰肌醇(4，5)-二磷酸调节血小板肌动蛋白细胞骨架的重塑和α颗粒的分泌。特定目的2中描述的实验将确定肌动蛋白细胞骨架是否介导磷脂酰肌醇(4，5)-二磷酸刺激血小板α颗粒分泌的作用。具体目标3中描述的实验将确定肌动蛋白细胞骨架在α颗粒分泌过程中指导SNARE蛋白复合体形成的作用。这些研究将确定α颗粒分泌所需的关键激活途径，并揭示肌动蛋白细胞骨架和血小板分泌机制之间的相互作用，这对血小板α颗粒分泌是重要的。

DESCRIPTION (provided by applicant): Platelet-rich, arterial thrombi mediate tissue infarction in stroke, peripheral vascular disease, and myocardial infarction. During thrombus formation, platelets secrete their granule contents. The most abundant platelet granule, the alpha-granule, contains adhesion molecules, coagulation factors, and vasoactive factors that contribute to thrombus propagation. We have used a permeabilized platelet secretory model to define a role for SNARE proteins in membrane fusion events leading to platelet alpha-granule secretion. The mechanisms by which agonist-induced stimulation of the platelet results in SNARE protein-mediated membrane fusion, however, remain largely unknown. We have found that phosphatidylinositol (4,5)-bisphosphate synthesis is required for alpha-granule secretion. These studies demonstrated a role for type II phosphatidylinositol 5-phosphate 4-kinase in alpha-granule secretion. The synthetic pathway responsible for the synthesis of phosphatidylinositol (4,5)-bisphosphate required for alpha-granule secretion, however, is poorly characterized. Experiments described in Specific Aim 1 of this proposal will determine the relative contributions of type I phosphatidylinositol 4-phosphate 5-kinase and type II phosphatidylinositol 5-phosphate 4-kinase in platelet alpha-granule secretion. These studies will also determine whether type I phosphatidylinositol 5-phosphate 4-kinase serves as a downstream effector of protein kinase C during platelet alpha-granule secretion. Phosphatidylinositol (4,5)-bisphosphate mediates both remodeling of the platelet actin cytoskeleton and secretion of alpha-granules. Experiments described in Specific Aim 2 will determine whether the actin cytoskeleton mediates the effects of phosphatidylinositol (4,5)-bisphosphate in stimulating platelet alpha-granule secretion. Experiments described in Specific Aim 3 will define the role of the actin cytoskeleton in directing SNARE protein complex formation during alpha-granule secretion. These studies will define a critical activation pathway required for alpha-granule secretion and reveal interactions between the actin cytoskeleton and platelet secretory machinery that are important for platelet alpha-granule secretion.