Activation of encrypted tissue factor during thrombus formation in vivo

描述(申请人提供)：作为一种宿主防御机制，凝血级联的启动在时间和位置上都需要严格的调控。主要的死亡原因，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，这个复杂系统的独立成分被单独研究。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。最近发展起来的成像技术可以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)是启动凝血、凝血酶形成和纤维蛋白生成所必需的。人们一直认为，组织因子被屏蔽在循环之外，只有在血管损伤时才会暴露。然而，已经在循环微粒以及白细胞和内皮细胞上发现了转铁蛋白。最近的研究导致了一种新的观点，即Tf的氧化导致二硫键的形成，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一种加密形式的转铁蛋白。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说：1)体内有证据表明循环中的TF积聚在不断生长的血栓中，2)抗原测量的循环中的TF浓度与功能循环抗原之间的差异远远超过启动凝血所需的量，3)组织因子中的Cys186-Cys209二硫键的破坏降低了其促凝血活性，这是通过形成相同的二硫键而可逆的。在这些观察的基础上，我们将使用活体显微镜和异构体特异性抗体来表征体内活跃的和加密的组织因子亚型。我们将鉴定已知抗体的异构体特异性，并利用重组噬菌体展示技术开发新的抗体。这些抗体将使激光诱导的活体小鼠血栓形成过程中的转铁蛋白亚型可视化。此外，将利用单个感兴趣的组织(包括单核细胞和内皮细胞)中缺乏转铁蛋白的小鼠模型来研究这些组织在体内转铁蛋白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.