We have developed modified protocols for confocal intravital monitoring of thrombus development in mouse mesenteric vessels following laser-induced injury. By including tetramethyrhodamine-labeled dextran (70,000 mw) in the blood, unlabeled cells (leukocytes and reticulocytes) form black silhouettes where the red fluorescent dye is excluded in the confocal image. In addition, platelets were labeled with rat monoclonal anti-mouse CD41/61 (Emfret Analytics, Germany) mixed with secondary goat anti-rat IgG antibody labeled with Alexa Fluor 488 and human fibrinogen was labeled with Alexa Fluor 350. Thus, high resolution confocal images of the developing thrombus included platelets in green, fibrin in blue, plasma in red and cellular components in black. Near real-time video recordings indicate the importance of flow parameters on the developing clot. In particular, turbulent flow patterns around the developing thrombus contribute to the incorporation of blood cells in the thrombus leading to the development of thrombi with a heterogeneous domain structure. Conceivably, different mechano-elastic properties of the heterogeneous domains might contribute to thrombus instability as the thrombus is subject to varying stresses and strains. In parallel with the modified experimental vascular injury model, we have begun development of a computational model of thrombus development. The modeling framework consists of a stochastic and discrete Cellular Potts Model (CPM) to describe platelet and cellular interactions and continuous submodels to describe hydrodynamic and biochemical reactions. The components of the computational model include the vessel wall, platelets (in resting and activated states), blood cells, coagulation reactions, fibrin formation, and hydrodynamic parameters. By varying input variables (such as flow rate, blood viscosity, coagulant activities) and monitoring properties of the developing thrombus, one can modify and validate the computational model. While our experimental systems enable us to determine essential components required for thrombus development, a validated model could help identify threshold levels of key components involved in thrombogenesis. This may be particularly useful in identifying hemostatic risk factors if these threshold levels are within the normal range found in the human population.
Disclosure: No relevant conflicts of interest to declare.