ML354

The detailed pharmacology and therapeutic potential of the central PAR4 receptors are poorly understood due to a lack of potent, selective, and brain-penetrant tool compounds. Despite this, robust data with biochemical and genetic tools show the therapeutic potential of PAR4 antagonists in traumatic brain injury, Alzheimer's disease, Parkinson's disease, and other neurodegenerative disorders with a neuroinflammatory component. Thus, we performed a functional HTS campaign, identified a fundamentally new PAR4 competitive inhibitor chemotype, optimized this new series (increased potency >45-fold), discovered enantiospecific activity (though opposing preference for human versus mouse PAR4), and engendered high central nervous system penetration (rat K_p's of 0.52 to 4.2 and K_p,uu's of 0.52 to 1.2).

Hemostasis is a proteolytically regulated process that requires activation of platelets and the blood coagulation cascade upon vascular injury. Activated platelets create a thrombogenic environment and amplify the coagulation process. Plant latex proteases (PLPs) have been used as therapeutic components to treat various ailments by folk healers. One of the main applications of plant latices is to stop bleeding from minor injuries and to enhance wound healing activity. Although many studies have reported the pro‐coagulant activities of PLPs, an in‐depth investigation is required to understand the mechanism of action of PLPs on platelets. Here, the effect of PLPs on platelet aggregation was studied systematically to validate the observed pharmacological effect by folk healers. Among 29 latices from the Ficus genus tested, Ficus drupacea exhibited potent pro‐coagulant and thrombin‐like activity. Drupin, a thrombin‐like cysteine protease responsible for platelet aggregation was purified from F. drupacea latex. Drupin exhibits pro‐coagulant activity and reduces the bleeding time in mice tail. It induces platelet aggregation by activating mitogen‐activated protein kinases and the nuclear factor‐κB and PI3K/Akt signalling cascade, which, in turn, phosphorylats, cytosolic phospholipase A2 leading to the release of thromboxane A2 from the granules to activate the nearby platelets to aggregate. Furthermore, we investigated the involvement of protease‐activated receptors in drupin‐induced platelet aggregation using specific protease activated receptor 1 (PAR1) and PAR4 receptor antagonists. The results confirmed that the drupin‐induced platelet aggregation was mediated by both PAR1 and PAR4, synergistically. Overall, drupin reduces the bleeding time by exerting pro‐coagulant activity and induces platelet aggregation by activating the intracellular signalling cascade.