Pharmacological inhibition of Septins with Forchlorfenuron attenuates thrombus formation in experimental thrombotic mice models with modulating multiple signaling pathways in platelets.

INTRODUCTION: The Septin family of cytoskeletal proteins is abundant in platelets. When these proteins are functionally blocked using the compound forchlorfenuron (FCF), it hampers the normal activation processes of purified human platelets. OBJECTIVES: To evaluate the in vivo effects of FCF on physiological haemostasis and pathological thrombosis in mice and to investigate possible molecular mechanisms. METHODS: The impact of FCF on haemorrhage risk in the brain, liver, and tail of mice was investigated. Using several experimental models, thrombus development in the lung, mesenteric arteries, and postcava was studied. Functional assays were performed on mice and human platelets, both with and without FCF pretreatment. These tests included aggregation, granule release, ROS production, integrin αIIbß3 activation, cytoskeletal remodeling imaging, and clot retraction. RESULTS: Neither oral nor intravenous administration of FCF showed any apparent impairment of haemostasis in the tissues studied, but only later administration resulted in a significant reduction in thrombus formation in different mice vessel types. FCF generally inhibited agonist-induced platelet aggregation, degranulation, ROS burst, morphological expansion on the fibrinogen matrix with completely disordered dynamic organizations of the cytoskeleton for septin, tubulin and actin. In addition, FCF was found to antagonise agonist-induced dephosphorylation of VASP (Ser239) and PI3K/AKT and ERK1/2 phosphorylation. CONCLUSION: FCF showed preferences in attenuating pathological thrombus formation, apart from physiological haemostasis, with possible mechanisms to prevent cytoskeletal remodelling and signal transduction of AKT, ERK1/2 and VASP signalling pathways, suggesting that Septin may serve as a promising target for the prevention and treatment of thrombotic diseases.

Repurposing of rilpivirine for preventing platelet ß3 integrin-dependent thrombosis by targeting c-Src active autophosphorylation.

BACKGROUND: HIV-infected individuals are known to be at higher risk for thrombotic cardiovascular disease (CVD), which may also be differentially affected by components of anti-HIV drugs. To identify the effects of a series of FDA-approved anti-HIV drugs on platelet aggregation in humans, focusing on the novel pharmacological effects of rilpivirine (RPV), a reverse transcriptase inhibitor, on platelet function both in vitro and in vivo and the mechanisms involved. METHODS AND RESULTS: In vitro studies showed that RPV was the only anti-HIV reagent that consistently and efficiently inhibited aggregation elicited by different agonists, exocytosis, morphological extension on fibrinogen, and clot retraction. Treatment of mice with RPV significantly prevented thrombus formation in FeCl3-injured mesenteric vessels, postcava with stenosis surgery, and ADP -induced pulmonary embolism models without defects in platelet viability, tail bleeding, and coagulation activities. RPV also improved cardiac performance in mice with post-ischemic reperfusion. A mechanistic study revealed that RPV preferentially attenuated fibrinogen-stimulated Tyr773 phosphorylation of ß3-integrin by inhibiting Tyr419 autophosphorylation of c-Src. Molecular docking and surface plasmon resonance analyses showed that RPV can bind directly to c-Src. Further mutational analysis showed that the Phe427 residue of c-Src is critical for RPV interaction, suggesting a novel interaction site for targeting c-Src to block ß3-integrin outside-in signaling. CONCLUSION: These results demonstrated that RPV was able to prevent the progression of thrombotic CVDs by interrupting ß3-integrin-mediated outside-in signaling via inhibiting c-Src activation without hemorrhagic side effects, highlighting RPV as a promising reagent for the prevention and therapy of thrombotic CVDs.