Activation of encrypted tissue factor during thrombus formation in vivo

描述（由申请人提供）：作为宿主防御机制，凝血级联反应的启动需要在时间和位置上进行严格调节。死亡的主要原因，包括中风、心脏病发作和肺栓塞，都与病理性血栓形成有关。目前的凝血教学已经从体外研究中演变而来，在体外研究中，单独研究了这个复杂系统的分离组分。我的长期目标是阐明体内启动凝血的机制，并确定如何调节这一关键步骤。最近开发的成像技术允许在真实的时间内研究活体动物中血栓形成部位的蛋白质相互作用。组织因子（TF）是启动血液凝固、凝血酶形成和纤维蛋白生成所必需的。组织因子被屏蔽在循环之外，只有在血管损伤时才暴露出来，这是一个教条。然而，TF已经在循环微粒以及白细胞和内皮细胞上被鉴定。最近的研究导致了新的建议，即TF的氧化导致二硫键的形成，作为一个分子开关，改变潜在的TF的促凝形式。我们假设在体内血栓形成和纤维蛋白生成过程中激活的循环中存在加密形式的TF。这可能是TF参与血栓形成的一种新机制。我们基于以下观察结果提出这一假设：1）循环TF在生长的血栓中积累的体内证据，2）通过抗原测量的循环TF浓度和功能-循环抗原之间的差异远远超过启动凝血所需的量，和3）组织因子中Cys 186-Cys 209二硫键的破坏降低其促凝血活性，通过形成相同的二硫键这是可逆的。基于这些观察结果，我们将使用活体显微镜和同种型特异性抗体在体内表征活性和加密的组织因子同种型。我们将表征已知抗体的同种型特异性，并已开发出新的抗体使用重组噬菌体展示技术。这些抗体将允许可视化TF亚型在激光诱导的血栓形成在活的小鼠。此外，将采用目标个体组织（包括单核细胞和内皮细胞）中TF缺陷的小鼠模型来研究这些组织在TF介导的体内血栓形成中的作用。

DESCRIPTION (provided by applicant): As a mechanism of host defense, the initiation of the blood coagulation cascade requires tight regulation both in its timing and location. Major causes of death, including stroke, heart attack and pulmonary embolism, are each linked to pathologic thrombosis. Current teaching of coagulation has evolved from in vitro studies in which isolated components of this complex system are studied individually. My long-term goal is to elucidate the mechanism initiating coagulation in vivo and to determine how this critical step is regulated. Recently developed imaging technologies allow for the study of protein interactions at the site of thrombus formation in a living animal in real time. Tissue factor (TF) is required to initiate blood coagulation, thrombin formation and fibrin generation. It has been dogma that tissue factor is shielded from the circulation and is only exposed by vascular injury. However, TF has been identified on circulating microparticles, as well as on leukocytes and endothelial cells. Recent studies have led to the novel proposal that oxidation of TF leads to disulfide bond formation, acting as a molecular switch that changes latent TF to a procoagulant form. We hypothesize that an encrypted form of TF exists within the circulation that is activated during thrombus formation and fibrin generation in vivo. This would be a novel mechanism of TF involvement in thrombosis. We base this hypothesis on the following observations: 1) in vivo evidence of circulating TF accumulating in a growing thrombus, 2) a discrepancy between circulating TF concentration as measured by antigen and function - circulating antigen far exceeds the amount required to initiate coagulation and 3) disruption of the Cys186-Cys209 disulfide bond in tissue factor decreases its procoagulant activity, and this is reversible by forming this same disulfide bond. Based on these observations, we will characterize active and encrypted tissue factor isoforms in vivo using intravital microscopy and isoform-specific antibodies. We will characterize known antibodies for isoform specificity and have developed novel antibodies using recombinant phage display technology. These antibodies will allow visualization of TF isoforms during laser-induced thrombus formation in a living mouse. Furthermore, mouse models deficient in TF in individual tissues of interest, including monocytes and endothelial cells, will be employed to investigate the role of these tissues in TF-mediated thrombus formation in vivo.