Chondroitin Sulfate as a New Profibrinolytic-Like Agent: A Preclinical Proof of Concept in a Model of Thromboembolic Stroke in Mice

BACKGROUND: Ischemic stroke requires effective reperfusion therapies to limit brain injury, yet rtPA (recombinant tissue-type plasminogen activator) efficacy is limited, particularly in platelet-rich thrombi. Neutrophil extracellular traps (NETs) and their components, especially histones and DNA, contribute to thrombolysis resistance. Chondroitin sulfate (CS), a glycosaminoglycan with high affinity for extracellular histones, may neutralize their prothrombotic effects and improve outcomes. This study aimed to evaluate the effects of CS in preclinical ischemic stroke models and its impact on components of neutrophil extracellular traps. METHODS: Two mouse models of middle cerebral artery occlusion were used: a fibrin-rich thromboembolic stroke model (rtPA-sensitive) and a platelet-rich aluminum chloride model (rtPA-resistant). Mice received intravenous CS (30-120 mg/kg), rtPA (10 mg/kg), or a combination of both. Lesion volume, tissue recanalization/reperfusion, hemorrhagic transformation, and functional connectivity were assessed via 7T magnetic resonance imaging and ultrafast Doppler imaging. In vitro coagulation-fibrinolysis assays examined the effects of neutrophil extracellular trap components on fibrin polymerization and fibrinolysis, and their modulation by CS±rtPA. RESULTS: In the fibrin-rich model, CS alone reduced lesion volume by 36% and improved recanalization, comparable to rtPA (43%), without increasing hemorrhagic transformation. CS enhanced functional connectivity recovery at 24 hours, whereas combined CS+rtPA lost these benefits. In the platelet-rich model, CS did not affect lesion size, recanalization, or hemorrhage. In vitro, histones promoted clot stabilization and altered fibrinolysis, effects fully neutralized by equimolar CS in the absence of rtPA. With rtPA, CS's neutralizing capacity was reduced, and histone-driven profibrinolysis was accentuated at higher CS doses. DNA produced opposite effects to histones, and combined DNA+histones masked histone activity, resisting inhibition by CS+DNase. CONCLUSIONS: CS mitigates histone-mediated prothrombotic effects, improves reperfusion and network recovery in fibrin-rich stroke, but loses efficacy in platelet-rich thrombi and when combined with rtPA. These findings support CS as a potential adjunct or alternative therapy, particularly for patients with contraindications to rtPA.