Adhesion molecules in hemostasis and platelet function

在这个项目中，我们将进一步研究在过去的资助期间所做的两个关键观察：1）P-选择素 诱导白细胞形成促凝血微粒（MP），和2）血小板活化 金属蛋白酶ADAM 17（TACE）导致血小板清除。 在第一个目标中，我们将研究IPS或重组P-选择素诱导MP产生的细胞生物学 分子（P-sel-lg），在体外和体内。工作假设是MP从血浆中释放出来 膜通过一个出芽过程，采用用于形成多泡的细胞机器 内体和逆转录病毒出芽。我们将研究泛素化的重要性， 细胞骨架在这个过程中。剪切力对MP生成及粘附分子的影响 将与项目3一起检查参与MP招募的血栓。通过项目2，我们将 表征过表达可溶性P-选择素的小鼠（deltaCT-P-sel小鼠）中产生的MP的细胞骨架 和表面蛋白质。将在细胞因子突变小鼠（细丝蛋白嵌合体，凝溶胶蛋白-/-）中研究MP生成。 组织因子胞浆区在组织因子靶向MP及其促凝调节中的作用 将在表达缺乏CT结构域的TF的小鼠（deltaCT-TF小鼠）中研究活性。 在第二个目标中，我们建议研究TACE在血小板生物学中的作用。我们假设信号 调节TACE的蛋白水解活性的途径对于血小板在止血中的功能 和调节血小板清除率。我们将研究细胞内加工和亚细胞 TACE在血小板活化或线粒体损伤时的定位。我们假设，在血小板中， TACE移位至质膜与其活化一致。我们将解决信号 从线粒体损伤到TACE活性的途径，重点是AMP活化蛋白激酶（AMPK）， 代谢应激敏感激酶和p38 MAP激酶。在体外和体内动脉和静脉模型中 血栓形成，我们将确定TACE的作用和激酶参与其激活血栓 阵在这些研究中，我们将使用转基因小鼠和激活激酶的抑制剂。 在第三个目标中，我们将解决与输血生物学直接相关的问题： （线粒体损伤或老化）血小板从循环中清除，以及如何提高血小板的疗效 通过将TACE抑制剂添加到血小板浓缩物和/或共输注P-sel-lg来进行输血。 TACE通过GPIb脱落和/或GPIb-V-IX聚集调节血小板清除的假设 复合物，P-选择素，通过产生促凝血MP，将加强止血， 血小板减少的小鼠。

In this project we will further investigate two key observations made in the past funding period: 1) P-selectin induces the formation of pro-coagulant microparticles (MPs) from leukocytes, and 2) activation of the platelet metalloproteinase ADAM17 (TACE) leads to platelet clearance. In the first aim, we will study the cell biology of MP production induced by IPS or a recombinant P-selectin molecule (P-sel-lg), in vitro and in vivo. The working hypothesis is that MPs are released from the plasma membrane through a budding process that employs cellular machinery used for the formation of multivesicular endosomes and for retrovirus budding. We will study the importance of ubiquitination and the cellular cytoskeleton in this process. The effect of shear stress on MP production and the adhesion molecules involved in MP recruitment to thrombi will be examined together with Project 3. With Project 2, we will characterize MPs produced in mice over-expressing soluble P-selectin (deltaCT-P-sel mice) for their cytoskeleton and surface proteins. MP generation will be studied in cytoskeleton-mutant mice (filamin chimeras, gelsolin -/-). The role of the cytoplasmic domain of tissue factor (TF) in TF targeting to MPs and in the regulation of its procoagulant activity will be studied in mice expressing TF lacking the CT domain (deltaCT-TF mice). In the second aim, we propose to study the role of TACE in platelet biology. We hypothesize that the signaling pathways that regulate the proteolytic activity of TACE are important for both platelet function in hemostasis and in the regulation of platelet clearance. We will examine the intracellular processing and the sub-cellular localization of TACE upon platelet activation or mitochondrial injury. It is our hypothesis that, in platelets, TACE translocation to the plasma membrane coincides with its activation. We will address the signaling pathways from mitochondrial injury to TACE activity with a focus on AMP-activated protein kinase (AMPK), a metabolic stress-sensing kinase, and on p38 MAP kinase. In in vitro and in vivo models of arterial and venous thrombosis, we will determine the role of TACE and the kinases involved in its activation in thrombus formation. For these studies we will use genetically modified mice and inhibitors of the activating kinases. In the third aim, we will address problems directly relevant to transfusion biology: how are damaged (mitochondrial injury or aging) platelets cleared from circulation, and how to improve the efficacy of platelet transfusions by adding TACE inhibitors to platelet concentrates and/or co-infusing P-sel-lg. We will test the hypotheses that TACE regulates platelet clearance by shedding of GPIba and/or clustering of GPIb-V-IX complexes, and that P-selectin, by producing pro-coagulant MPs, would enhance hemostasis in thrombocytopenic mice.