Abstract 9389: Thrombus Formation Processes are Dependent on Endothelial Injuries: Examined by in vivo Two-photon Molecular Imaging

Abstract

Aim: The cellular mechanisms of thrombotic diseases associated with cardiovascular events remains unclear, largely because of an inability to visualize thrombus formation. Thus, we developed in vivo imaging technique based on single- and multi-photon microscopy and laser injuries technique to revealed the multicellular processes during thrombus development. Methods: We visualized the cell dynamics including single platelet behavior, and assessed dynamic cellular interplay in two thrombosis models using two photon microscopy to CAG-eGFP mice (Figure a, b). Results: First, we visualized that rapidly developing thrombi composed of discoid platelets without EC disruption was triggered by ROS induced by laser irradiation (Figure c). In this model, thrombus consisted by discoid platelet aggregations without leukocyte recruitment and not affected by Gr-1 antibody. The second model is, thrombus with EC disruption. High power laser induced EC erosion and extravasations of circulating leukocytes with thrombus development. Inflammatory cytokine, adhesion molecules dynamically control these two processes, and Gr-1 antibody significantly suppressed these steps. (Figure d) Leukocyte was immediately recruited into the subendothelial layers with bleeding and hemostatic reactions, and TLR4 signaling contributed to these steps. Pretreatmet of LPS or ischemic procedures markedly enhanced these steps. Thrombus included calcium activated cores and deformed platelets. Immigrated leukocyte also showed the increase of intracellular calcium. Summary: These results indicated that endothelial function, especially inflammatory status, determined the thrombotic reaction. Leukocyte also contributed with TLR4 signaling. In sum, using our imaging system can be a powerful tool to analyze thrombus formation and evaluate the therapeutic strategies.