CYTOSKELETON AND PLATELET CLEARANCE

用于输血的血小板的“保质期”相对较短，为5天。随着储存量的增加，血小板 发生结构和功能变化，称为“血小板储存病变”。这些变化不仅导致 对激动剂的止血反应降低，但也加速了体内循环的清除。 已经提出了几种机制，包括血小板糖蛋白Ib复合物在血小板膜上的聚集。 血小板表面，聚糖改变导致肝Ashwell-Morell受体识别和脱落 血小板糖蛋白Ibα。磷脂酰丝氨酸（PS）暴露于储存的血小板表面是一个众所周知的标记 从流通中清除。在静息血小板中，PS位于膜双层的内叶 并且在储存期间和血小板聚集期间存在从内小叶到外小叶的跨双层运动 activation. PS暴露的衰老（或凋亡）诱导和激活依赖途径 已经描述了衰老血小板中PS的暴露通过线粒体Bcl-2家族发生， 蛋白质和（与激活诱导的PS暴露相反），它不需要升高胞质Ca+， 涉及细胞色素C释放和随后的半胱天冬酶3的活化。PS暴露同时发生 随着微粒的产生，储存的血小板制剂含有富含PS的微粒。的 维持静息血小板中磷脂不对称性的分子机制，如大多数哺乳动物 细胞是未知的。我们提出了一种新的机制，在存储过程中磷脂不对称的损失。 我们认为皮质肌动蛋白网络将PS限制在内叶，并假设在储存过程中， 细胞骨架的重组释放了对质膜双层的正常约束，导致细胞骨架的丧失。 阴离子磷脂的不对称性和跨双层运动。细胞骨架蛋白，紧密结合在 阴离子磷脂，如肌动蛋白，也可能与一个假定的 扰码相关机制。赠款的总体目标是阐明 维持新鲜血小板中的磷脂不对称性及其在储存期间的损失。本提案的具体目标 该提案的具体目标是：（1）确定皮质肌动蛋白网络的作用， 维持磷脂不对称性，（2）确定cofilin-1诱导的调节机制 PS暴露在储存的血小板中和（3）确定cofilin-1抑制对血小板存活的影响。

Platelets, harvested for transfusion, have a relatively short “shelf life” of 5 days. With increasing storage, platelets develop structural and functional changes called “platelet storage lesions”. These changes result not only in reduced hemostatic response to agonists but also in an accelerated clearance from the circulation in vivo. Several mechanisms have been proposed including clustering of the platelet glycoprotein Ib complex on the surface of platelets, alterations in glycans leading to recognition by hepatic Ashwell-Morell receptor and shedding of platelet glycoprotein Ibα. Exposure of phosphatidylserine (PS) on stored platelet surface is a well known tag for clearance from the circulation. In resting platelets, PS is located in the inner leaflet of the membrane bilayer and there is transbilayer movement from the inner to the outer leaflet during storage and during platelet activation. Both a senescence (or apoptosis)- induced and an activation-dependent pathways of PS exposure have been described. Exposure of PS in senescent platelets occurs through the mitochondrial Bcl-2 family of proteins and (in contrast to activation-induced PS exposure) it does not require elevation of cytosolic Ca+ and it involves cytochrome c release and subsequent activation of caspase 3. PS exposure occurs simultaneously with the generation of microparticles and stored platelet preparation contain PS-rich microparticles. The molecular mechanisms that maintain the phospholipid asymmetry in resting platelets, as in most mammalian cells, are not known. We propose a novel mechanism for the loss of phospholipid asymmetry during storage. We posit that the cortical actin network confines PS to the inner leaflet and hypothesize that during storage the reorganization of cytoskeleton releases normal constraints on plasma membrane bilayer leading to the loss of asymmetry and transbilayer movement of anionic phospholipids. The cytoskeletal proteins, tightly bound to anionic phospholipids such as actin, are also exteriorized to the cytoplasmic surface possibly with a putative scramblase-dependent mechanism. The overall objective of the grant is to delineate the mechanisms that maintain phospholipid asymmetry in fresh platelets and its loss during storage. Specific aims of this proposal are: The specific aims of the proposal are (1) to determine the role of the cortical actin network in maintaining the phospholipid asymmetry, (2) to define the mechanism of regulation of cofilin-1-induced PS exposure in stored platelets and (3) to determine the effect of cofilin-1 inhibition on platelet survival.