Membrane Phospholipids: The Key Regulators of Tissue Factor Encryption/Decryption

在血管损伤时，血浆凝血因子VII(FVII)和激活的FVII(FVIIa)一起进入 与辅因子组织因子(Tf)接触，Tf在管壁内的细胞中组成性表达。 FVIIa与Tf形成络合物使FVIIa和Tf的催化活性显著增强 触发转铁蛋白介导的血液凝固。某些疾病条件可诱导循环血中转铁蛋白的表达 细胞和血管内皮细胞，从而允许循环血液和转铁蛋白之间直接接触，从而导致 血栓形成。虽然转铁蛋白介导的血液凝固是维持止血的关键，但异常的 激活组织因子介导的凝血系统导致血栓形成，这是急性心肌梗死的诱发事件 脑梗塞、缺血性中风和败血症。因此，对TF表达和活性的适当调节是 这不仅对维持止血平衡至关重要，而且对一般人的健康也至关重要。通常，大多数 在细胞中表达的Tf保持加密，几乎没有促凝活性，足以实现 止血，但不会引起血管内凝血。细胞损伤增强组织因子促凝血活性 大大不改变转铁蛋白抗原水平，即将隐匿性转铁蛋白转化为血栓前转铁蛋白。转铁蛋白促凝剂 细胞内的活动受各种翻译后机制的动态控制。我们最近的研究 揭示了神经鞘磷脂(SM)在细胞膜外叶中负责维持 Tf处于加密状态，且SM水解会激活Tf并释放Tf微泡(MVS)。SM 新陈代谢在许多疾病中都会改变，包括动脉粥样硬化、糖尿病、败血症和癌症， 同样的疾病环境会导致TF的异常激活。目前的提议是以上述小说为基础的 研究发现并建议探讨SM代谢在调节Tf- 中介止血、血栓形成和炎症。目标1侧重于阐明SM通过 代谢调节TF促凝血活性，而Aim 2研究SM代谢是否 影响止血和血栓形成。目标3中提出的实验将检验这一假设 炎症诱导的SM代谢改变在转铁蛋白激活和转铁蛋白介导中起关键作用 凝血障碍。目标4侧重于研究SM代谢改变是否通过 转铁蛋白活性的调节。在拟议的研究中，我们将操纵巨噬细胞中的SM水平， 内皮细胞，以及其他类型的细胞，通过过度表达或下调涉及的各种酶 在SM代谢中或使用这些酶的特定药理抑制剂。我们将雇佣不同的 SM代谢异常基因敲除小鼠及其止血血栓模型的研究 新发现的机制的病理生理学相关性。我们建议的研究将导致 我们对TF介导的凝血如何在各种疾病环境中被激活的理解发生了范式转变。 它们还可能导致开发新的、有针对性的干预措施来预防血栓形成。

Upon vascular injury, plasma clotting factor VII (FVII) along with traces of activated FVII (FVIIa) come into contact with the cofactor tissue factor (TF), which is expressed constitutively in cells within the vessel wall. Complex formation of FVIIa with TF results in a marked enhancement of the catalytic activity of FVIIa and triggers TF-mediated blood coagulation. Certain disease conditions induce TF expression in circulating blood cells and vascular endothelial cells and thus allow direct contact between circulating blood and TF that leads to thrombosis. While TF-mediated blood coagulation is essential to maintain hemostasis, the aberrant activation of TF-mediated blood coagulation leads to thrombosis, the precipitating event in acute myocardial infarction, ischemic stroke, and sepsis. Therefore, the proper regulation of TF expression and the activity is critical for not only to the maintenance of the hemostatic balance but also for health in general. Typically, most of the TF expressed in cells stays encrypted with very little procoagulant activity that is sufficient to achieve hemostasis but not to cause intravascular coagulation. Cellular injury enhances TF procoagulant activity greatly without altering TF antigen levels, i.e., transforming cryptic TF to prothrombotic TF. TF procoagulant activity in cells is controlled dynamically by a variety of post-translational mechanisms. Our recent studies revealed that sphingomyelin (SM) in the outer leaflet of the plasma membrane is responsible for maintaining TF in an encrypted state and that hydrolysis of SM activates TF and releases TF+ microvesicles (MVs). SM metabolism is altered in many disease settings, including atherosclerosis, diabetes, sepsis, and cancer, the same disease settings that induce aberrant activation of TF. The current proposal is built on the above novel findings and proposes to investigate the pathophysiologic relevance of SM metabolism in regulation of TF- mediated hemostasis, thrombosis, and inflammation. Aim 1 focuses on elucidating mechanisms by which SM metabolism regulates TF procoagulant activity, whereas Aim 2 investigates whether SM metabolism influences hemostasis and thrombosis. Experiments proposed in Aim 3 will test the hypothesis that acute inflammation-induced alterations in SM metabolism play a key role in TF activation and TF-mediated coagulopathy. Aim 4 focuses on investigating whether altered SM metabolism contributes to inflammation via the regulation of TF activity. In the proposed studies, we will manipulate SM levels in macrophages, endothelial cells, and other cell types by the overexpression or down regulation of various enzymes involved in the SM metabolism or using specific pharmacological inhibitors of these enzymes. We will employ various knock-out mice with altered SM metabolism and murine models of hemostasis and thrombosis to investigate the pathophysiologic relevance of the newly identified mechanism. Our proposed studies will lead to a paradigm shift in our understanding of how TF-mediated coagulation is activated in various disease settings. They may also lead to the development of novel, targeted interventions to prevent thrombosis.