Determinants of Thrombus Structure and Stability

描述（由申请人提供）：静脉血栓形成/血栓栓塞每年影响超过100万美国人。血栓负荷（大小）预测长期预后。随着新药的开发，减少静脉血栓负担的努力可能对美国人口产生相当大的影响。静脉“红色”血栓的主要特征是其高红细胞（RBC）含量，传统上认为这是由于静态纤维蛋白凝块中的红细胞被被动捕获所致。然而，我们新的初步数据表明，红细胞通过一种需要纤维蛋白交联酶、因子XIII （FXIII）的活性机制保留在血块中。本应用的目的是确定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，需要FXIII活性来保留静脉血栓中的红细胞，阻断或降低FXIII活性将减小静脉血栓的大小。这一假设将在三个具体目标中得到解决：1)绘制介导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；

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.