Abstract

The thrombotic cellular mechanisms associated with cardiovascular events remains unclear, largely because of an inability to visualize thrombus formation. In addition, the contribution of endothelial cell (EC) injuries to thrombus formation processes are unclear, and we developed in vivo imaging technique based on single- and multi-photon microscopy to revealed the multicellular processes during thrombus development (Figure a,b). We visualized the cell dynamics including single platelet behavior, and assessed dynamic cellular interplay in two thrombosis models. First, we visualized that rapidly developing thrombi composed of discoid platelets without EC disruption was triggered by ROS photochemically induced by moderate power laser irradiation (Figure c). In this model, thrombus consisted by discoid platelet aggregations without leukocyte recruitment. 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. (Figure d)

Using this technique, we elucidated that Lnk (adapter protein) regulates integrin signaling leading to stabilization of developing thrombus without EC disruption. Specifically, adhesion molecules dynamically control these processes. Thrombus formation was initiated by the binding of platelet GPIb-alpha to endothelial von Willebrand Factor in this model, and actin linker talin-dependent activation of alphaIIb-beta3 integrin in platelets was required for late phase thrombus stability.

As for the thrombus formation with EC disruption, chemokine expressions in endothelium and leukocyte (especially neutrophils) recruitment played a significant role in these processes. TLR4 signaling also contributed to these steps.

In sum, using our imaging system can be a powerful tool to analyze thrombus formation and evaluate the therapeutic strategies.

Disclosures:

No relevant conflicts of interest to declare.

Author notes

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