A murine stable thrombosis model enabling therapeutic evaluation of antithrombotic drugs

Over recent decades, numerous experimental models have been developed to investigate arterial thrombosis and evaluate the efficacy of antithrombotic therapies. A major limitation of these models lies in the biphasic dynamics of a thrombus, which prevents assessment of its destabilization/disaggregation/thrombolysis after pharmacological treatment. In this study, we developed a stable thrombosis model based on the external application of 2 pieces of 7.5% FeCl₃-saturated Whatman papers to the carotid artery. Histological analysis and transmission electron microscopy revealed no exposure of the subendothelial matrix. Intravital microscopy identified platelets as the major cell type adhering to the injured vessel wall, with plateau-type kinetics of thrombus formation throughout the experiment. Doppler probe measurements detected the arrest of blood flow at 14 ± 3 minutes, when the thrombus reached its maximum area. Intravital and histological studies further showed that thrombus composition changed over time after injury. At 45 minutes, thrombi were enriched in red blood cells, with a shift in the platelet-fibrin composition toward fibrin predominance compared with thrombi at 15 minutes. To evaluate the efficacy of known antithrombotic agents in this model, inhibitors were injected 15 minutes after initiation of thrombosis. Video microscopy revealed that 0.4 mg/mL cangrelor (P2Y₁₂ inhibitor) or 4 mg/mL GR 144053 trihydrochloride (αIIbβ3 blocker) induced complete disaggregation, whereas 50 IU/mL heparin or 4 mg/mL recombinant tissue plasminogen activator induced destabilization of the thrombus. In contrast, 3.2 mg/mL aspirin, 40 IU/mL unfractionated heparin, or 4 mg/mL hirudin had no effect on a preformed thrombus. Altogether, this model constitutes a valuable platform for assessing therapeutic strategies aimed at stabilizing or dissolving established thrombi.