Intravital imaging of the spatiotemporal dynamics of fibrinolysis in a murine model of vascular injury Free

Fibrinolysis plays a key role in maintaining hemostatic balance by removing unnecessary or excessive thrombi, thereby maintaining vessel patency. Plasmin, the central fibrinolytic protease, degrades fibrin within thrombi and is generated from plasminogen primarily by tissue-type plasminogen activator (tPA). As plasmin degrades fibrin, it exposes C-terminal lysine residues on the fibrin surface. These residues enhance the binding of plasminogen and tPA, spatiotemporally promoting plasmin generation and amplifying fibrinolysis through a positive feedback loop. Whereas hyperfibrinolysis leads to excessive bleeding, hypofibrinolysis promotes thrombosis and has been implicated in the pathophysiology of myocardial infarction, stroke, and deep vein thrombosis (DVT). Given the critical role of fibrinolysis in hemostasis, reliable models to investigate its physiological and pathological mechanisms are essential. Importantly, there is a lack of animal models that enable spatiotemporal assessment of fibrinolytic potential in vivo.To address this gap, we developed a novel intravital imaging model using state-of-the-art multi-photon-excitation (MPE) microscopy to enable real-time visualization of fibrinolytic activity in the mesenteric venules of live mice. We aimed to assess both fibrin degradation and plasminogen accumulation following administration of exogenous tPA in a ferric-chloride (FeCl₃) - induced vascular injury model. C57BL/6 (WT) mice were administered with Alexa Fluor-568-conjugated Glu-plasminogen (Glu-plg), Evans blue to visualize vasculature, and Alexa Fluor-488-conjugated anti-fibrin mAb to visualize fibrin. Following mouse anesthesia, mesenteric venules were exposed and subjected to 4% FeCl₃ for two minutes. Fibrin formation was confirmed 30 minutes post-injury. Real-time image sequences were recorded immediately after tPA administration for 30 min. To evaluate the involvement of fibrin C-terminal lysine residues in the accumulation of Glu-plg within the thrombus, mice were pre-treated with carboxypeptidase B (CPB), which removes C-terminal lysine residues from the fibrin surface.Ferric chloride induced fibrin-rich, non-occlusive thrombus formation in approximately 90% of mice. tPA administration induced fibrinolysis in a dose-dependent manner. Notably, fibrin degradation closely correlated with Glu-plg accumulation, presumably at sites of, exposed on the fibrin surface, C-terminal lysine residues. Pretreatment with CPB, significantly reduced Glu-plasminogen binding and subsequently impaired subsequent fibrin degradation.We developed a novel intravital imaging model that enables real-time visualization of fibrinolysis in live mice. This system offers a powerful tool to study the biology of the fibrinolytic system under physiological and pathological conditions and offers a platform evaluate potential pharmacological strategies regulating fibrinolysis.