Abstract 41: Discoid Platelet Aggregations Visualized by in Vivo Molecular Imaging Without Endothelium Disruption and Contribution of Inflammatory Cytokine, Ros and Integrin Signaling

Abstract

inflammation, it is vital to examine the multi-cellular kinetics in living animals. Therefore, we developed in vivo imaging technique based on single- and multi-photon microscopy, and we assessed dynamic cellular interplay in diseased conditions. The mechanism by which thrombotic vessel occlusion occurs independently of plaque development or endothelial cell (EC) disruption remains unclear, largely because of an inability to visualize thrombus formation, especially at the single-platelet level in real time. Here we demonstrate that rapidly developing thrombi composed of discoid platelets can be induced in the mesenteric vessls of living mice, enabling characterization of the kinetics of thrombosis initiation and the multicellular interrelationships during thrombus development. Platelet aggregation without EC disruption was triggered by reactive oxygen species (ROS) photochemically induced by moderate power laser irradiation. Using this technique, we elucidated that Lnk (adapter protein) regulates integrin signaling leading to stabilization of developing thrombus in vivo (Nishimura et at, 2010 JCI). In addition, we established the efficient culture system of human iPS-derived platelets, and we confirmed the functional role. These artificial platelets can circulate, and contribute to their thrombus formation in vivo, indicating the clinical usefulness considering the cell therapy for future (Takayama & Nishimmura et al, 2010 JEM). In addition, we elucidated the contribution of inflammatory cytokines, ROS, and integrin signaling to our thrombosis models (Nishimura et al, 2011 Blood). The inflammatory cytokines TNF-alpha and IL-1 could be key components of the EC response, acting through regulation of von Willebrand factor mobilization to the cell surface. Thrombus formation was then initiated by the binding of platelet GPIb-alpha to endothelial von Willebrand Factor in our model, and this effect was inhibited by the ROS scavenger N-acetylcysteine. Actin linker talin-dependent activation of alphaIIb-beta3 integrin in platelets was required for late phase thrombus stability. Our imaging can be applied to many research fields including metabolic diseases. Our imaging revealed close spatial and temporal interrelationships between angiogenesis and adipogenesis in obese adipose (Nishimura et al, 2007 Diabetes). In addition, increased inflammatory cell interactions were visualized (Nishimura et al, 2008 JCI). We also found that large numbers of CD8+ effector T cells infiltrated into obese adipose, and these cells were essential for the initiation and development of adipose inflammation (Nishimura et al, 2009 Nat Med). In sum, using our imaging system can be a powerful tools to analyze the inflammatory conditions including thrombosis. We clarified the mechanism by which thrombi are rapidly formed by discoid platelets on undisputed endothelium. The initial platelet aggregation subsequently lead to irreversible integrin- and actin-dependent thrombus development. Inflammatory cytokine signaling in ECs played pivotal role in discoid platelet aggregations in vivo.