Efficient Mining for Structurally Diverse Fusicoccane-Type Diterpenoids as Novel Antithrombotic Agents and Their Divergent Mechanisms on Platelet Inhibition.

Through genome mining, we discovered a 5/8/5 fusicoccane-type diterpenoid gene cluster (named Thm) from the biocontrol fungus Trichoderma harzianum, representing the first report of such a cluster in the genus Trichoderma. Heterologous expression of Thm in Aspergillus oryzae NSAR1 led to the isolation of 33 fusicoccane-type diterpenoids (including 30 new compounds 1-30). Structurally, compounds 1-14 were tetracyclic diterpenoids, with 1 and 2 being a class of rare fusicoccane-alkaloid hybrids. Mechanistic profiling revealed that compound 6 acts as a selective GPVI pathway antagonist, potently inhibiting Syk and PLCγ2 phosphorylation and subsequent platelet activation. In contrast, compound 31 exerts its antiplatelet effect via a distinct ROCK1-dependent pathway, suppressing cytoskeletal reorganization by reducing myosin light chain (MLC) phosphorylation and key regulator expression. Critically, both compounds demonstrate a groundbreaking therapeutic dissociation, providing robust protection against arterial and venous thrombosis without impairing normal hemostasis, thereby presenting a promising strategy for safe antithrombotic therapy.