Determinants of Thrombus Structure and Stability

描述(申请人提供)：静脉血栓形成/血栓栓塞症每年影响100多万美国人。血栓负荷(大小)预测长期结果。随着新药的开发，减轻静脉血栓负担的努力可能会对美国人口产生相当大的影响。静脉“红色”血栓的显著特征是其高红细胞(RBC)含量，传统上认为这是由于红细胞被动地滞留在静态纤维蛋白凝块中所致。然而，我们的新的初步数据表明，红细胞通过一种需要纤维蛋白交联酶的活性机制，即因子XIII(FXIII)保留在血栓中。这项应用的目标是确定FXIII、纤维蛋白(原)、FXIII介导的纤维蛋白交联和红细胞促进静脉血栓形成的分子机制。我们的总体假设是，FXIII活性是保留静脉血栓中的红细胞所必需的，阻断或减少FXIII活性将减少静脉血栓的大小。这一假说将在三个具体目标中得到解决：1)定位介导FXIII结合的纤维蛋白原残基，并确定FXIII的激活和活性对纤维蛋白网络结构和稳定性的影响，2) 确定全血环境中血液成分(FXIII、红细胞和纤维蛋白原)对凝块大小的贡献，以及3)确定FXIII抑制剂对静脉血栓形成和稳定性的影响。我们将在体外和体内使用生化、遗传学和药理学工具来确定FXIII-纤维蛋白原轴，并确定FXIII活性和红细胞滞留的作用 静脉血栓形成。纤维蛋白(原)-FXIII的相互作用将使用溶液相结合分析、显微镜、创新的纤维蛋白分析方法和血栓弹性成像进行检测。凝块的形成将在重建的全血模型中使用来自健康人和有静脉血栓病史的患者的血浆来检查。血栓的形成和稳定性将使用静脉血栓形成和溶栓模型、免疫组织化学、超声成像和活体显微镜进行检查。这些研究将阐明FXIII活性在静脉血栓形成中的生物学作用，并确定纤维蛋白(原)和红细胞在静脉血栓形成和稳定性中的新作用。这项研究具有很高的创新性，因为它挑战了当前的范式，即红细胞被动地被困在静态的富含纤维蛋白的静脉血栓中。这项拟议的研究具有临床意义，因为它可能揭示减少美国人群静脉血栓形成的新策略。

DESCRIPTION (provided by applicant): Venous thrombosis/thromboembolism affects over 1 million Americans per year. Thrombus burden (size) predicts long-term outcome. Efforts to reduce venous thrombus burden with the development of new drugs could have a considerable impact on the US population. The defining characteristic of venous "red" thrombi is their high red blood cell (RBC) content, which was traditionally thought to result from passive trapping of RBCs in the static fibrin clot. However, our new preliminary data indicate that RBCs are retained in clots via an active mechanism that requires the fibrin cross-linking enzyme, factor XIII (FXIII) The goal of this application is to determine the molecular mechanisms by which FXIII, fibrin(ogen), FXIII-mediated fibrin cross-linking, and RBCs contribute to venous thrombosis. Our overall hypotheses are that FXIII activity is required to retain RBCs in venous thrombi, and that blocking or reducing FXIII activity will reduce venous thrombus size. This hypothesis will be addressed in three Specific Aims: 1) Map the fibrinogen residues that mediate FXIII binding and determine the impact of FXIII activation and activity on fibrin network structure and stability, 2) Determine the contributions of blood components (FXIII, RBCs, and fibrinogen) to clot size in a whole blood milieu, and 3) Determine the effect of FXIII inhibitors on the formation and stability of venous thrombi. We will employ biochemical, genetic and pharmacologic tools in vitro and in vivo to define the FXIII-fibrinogen axis and determine the role of FXIII activity and RBC retention in venous thrombosis. Fibrin(ogen)-FXIII interactions will be examined using solution phase binding assays, microscopy, innovative fibrin analytical methods, and thromboelastography. Clot formation will be examined in reconstituted whole blood models using plasmas from healthy individuals and patients with a history of venous thrombosis. Thrombus formation and stability will be examined using venous thrombosis and thrombolysis models, immunohistochemistry, ultrasound imaging, and intravital microscopy. These studies will elucidate the biological role of FXIII activity in venous thrombosis, and define novel roles for fibrin(ogen) and RBCs in venous thrombus formation and stability. The study is highly innovative because it challenges the current paradigm that RBCs are passively trapped in static, fibrin- rich venous thrombi. The proposed research is clinically significant because it may reveal new strategies to reduce venous thrombosis in the US population.