A Potent Fibrinolytic Enzyme Alp1 from <i>Cordyceps militaris</i> : From Sequence-Function Characterization to in Vivo Antithrombotic Application

Alp1, a potent fibrinolytic enzyme from <i>Cordyceps militaris</i> CM03, was characterized and identified as an S8 family alkaline serine protease. Molecular simulations revealed that the pro-peptide enhances mature peptide flexibility, which is crucial for functional folding and catalytic accessibility. Furthermore, heterologous expression confirmed its role as an essential intramolecular chaperone. Alp1 exhibits optimal activity at 60 °C and pH 7-11, yielding an activity of 1779 U/mL. This activity is enhanced by Ca²⁺ and Mn²⁺, but inhibited by specific metal ions (Fe³⁺, Fe²⁺, Cu²⁺) and protease inhibitors (PMSF, TPCK). Alp1 directly degrades fibrin and all three fibrinogen chains (α, β, γ). <i>In vitro</i> and <i>in vivo</i> (rat thrombosis model) assays demonstrated its potent thrombolytic and anticoagulant properties, outperforming urokinase and matching streptokinase efficacy. Thus, Alp1 warrants further investigation as a potential candidate for next-generation thrombolytic applications and functional food development.