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中观察到的两个关键问题：1)P-选择素 诱导白细胞形成促凝血微粒(MPS)，以及2)激活血小板 金属蛋白酶ADAM17(TACE)具有清除血小板的功能。 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)，体外和体内。工作假说是MPS从血浆中释放出来 膜通过萌发过程，使用细胞机械来形成多囊泡 内体和逆转录病毒萌芽。我们将研究泛素化的重要性和细胞 细胞骨架在这一过程中的作用。剪切力对MP产量和黏附分子的影响 参与MP招募血栓的人将与项目3一起检查。在项目2中，我们将 细胞骨架高表达可溶性P-选择素的小鼠(DeltaCT-P-sel小鼠)产生的MPS的特征 和表面蛋白。MP的产生将在细胞骨架突变的小鼠(细丝嵌合体，明胶蛋白-/-)中进行研究。 组织因子胞浆结构域在Tf靶向MPS及其促凝剂调节中的作用 活性将在缺乏CT结构域的表达Tf的小鼠(deltaCT-Tf小鼠)中进行研究。 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。我们假设信号是 调节TACE蛋白分解活性的途径对两种血小板止血功能都很重要 以及对血小板清除的调节。我们将研究细胞内处理和亚细胞 血小板活化或线粒体损伤时TACE的定位。我们的假设是，在血小板中， TACE向质膜的移位与其激活相一致。我们将解决信令问题 线粒体损伤到TACE活性的途径，重点是AMP激活的蛋白激酶(AMPK)， 代谢应激敏感激酶，以及p38上的MAP激酶。动脉和静脉的体外和体内模型 血栓形成，我们将确定TACE的作用以及参与其激活的激酶在血栓中的作用 队形。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：如何受到损害 (线粒体损伤或衰老)从循环中清除的血小板，以及如何提高血小板的疗效 通过在浓缩血小板中添加TACE抑制剂和/或联合输注P-SEL-LG进行输血。我们将测试 TACE通过GPIba脱落和/或GPIb-V-IX聚集调节血小板清除的假说 P-选择素通过产生促凝血剂MPS，将增强血管内皮细胞的止血。 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.