Activation of encrypted tissue factor during thrombus formation in vivo

描述（申请人提供）：凝血级联作为宿主防御的一种机制，其启动时间和位置都需要严格调控。主要死亡原因，包括中风、心脏病发作和肺栓塞，都与病理性血栓形成有关。目前的凝血教学是从体外研究发展而来的，在体外研究中，这个复杂系统的分离成分被单独研究。我的长期目标是阐明体内启动凝血的机制，并确定这一关键步骤是如何调节的。最近开发的成像技术允许实时研究活体动物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。组织因子在血液循环中是被屏蔽的，只有在血管损伤时才暴露出来。然而，已在循环微粒以及白细胞和内皮细胞上发现TF。最近的研究提出了新的建议，即氧化TF导致二硫键的形成，作为分子开关，将潜在的TF转变为促凝剂形式。我们假设一种加密形式的TF存在于循环中，在体内血栓形成和纤维蛋白生成过程中被激活。这可能是TF参与血栓形成的一种新的机制。我们的假设基于以下观察结果：1)体内证据表明循环TF在不断增长的血栓中积累；2)抗原和功能测量的循环TF浓度之间的差异-循环抗原远远超过启动凝血所需的量；3)组织因子中Cys186-Cys209二硫键的破坏降低了其促凝活性，这是通过形成相同的二硫键来逆转的。基于这些观察结果，我们将使用活体显微镜和同种异构体特异性抗体在体内表征活性和加密的组织因子同种异构体。我们将对已知抗体的异构体特异性进行表征，并利用重组噬菌体展示技术开发出新的抗体。这些抗体将允许可视化的TF异构体在激光诱导血栓形成在一个活的老鼠。此外，在单个组织中缺乏TF的小鼠模型，包括单核细胞和内皮细胞，将被用来研究这些组织在TF介导的血栓形成中的作用。

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.