The Antidepressant 5-HT2A Receptor Antagonists Pizotifen and Cyproheptadine Inhibit Serotonin-Enhanced Platelet Function

Abstract

There is considerable interest in defining new agents or targets for antithrombotic purposes. The 5-HT2A receptor is a G-protein coupled receptor (GPCR) expressed on many cell types, and a known therapeutic target for many disease states. This serotonin receptor is also known to regulate platelet function. Thus, in our FDA-approved drug repurposing efforts, we investigated the antiplatelet activity of cyproheptadine and pizotifen, two antidepressant 5-HT2A Receptor antagonists. Our results revealed that cyproheptadine and pizotifen reversed serotonin-enhanced ADP-induced platelet aggregation in vitro and ex vivo. And the inhibitory effects of these two agents were found to be similar to that of EMD 281014, a 5-HT2A Receptor antagonist under development. In separate experiments, our studies revealed that these 5-HT2A receptor antagonists have the capacity to reduce serotonin-enhanced ADP-induced elevation in intracellular calcium levels and tyrosine phosphorylation. Using flow cytometry, we also observed that cyproheptadine, pizotifen, and EMD 281014 inhibited serotonin-enhanced ADP-induced phosphatidylserine (PS) exposure, P-selectin expression, and glycoprotein IIb-IIIa activation. Furthermore, using a carotid artery thrombosis model, these agents prolonged the time for thrombotic occlusion in mice in vivo. Finally, the tail-bleeding time was investigated to assess the effect of cyproheptadine and pizotifen on hemostasis. Our findings indicated prolonged bleeding time in both cyproheptadine- and pizotifen-treated mice. Notably, the increases in occlusion and bleeding times associated with these two agents were comparable to that of EMD 281014, and to clopidogrel, a commonly used antiplatelet drug, again, in a fashion comparable to clopidogrel and EMD 281014. Collectively, our data indicate that the antidepressant 5-HT2A antagonists, cyproheptadine and pizotifen do exert antiplatelet and thromboprotective effects, but similar to clopidogrel and EMD 281014, their use may interfere with normal hemostasis.

Investigation of a Thromboxane A 2 Receptor–Based Vaccine for Managing Thrombogenesis

“…Mouse platelets were prepared, as discussed previously 42, 43. Briefly, mouse blood was collected from a ventricle, and the citrated (0.38%) blood was mixed with PBS, pH 7.4, and was incubated with prostaglandin I 2 (10 ng/mL; 5 minutes), followed by centrifugation at 237 g for 10 minutes at room temperature.…”

“…Platelets (with counts adjusted, as previously described42, 43, 44) were stimulated with 1 μmol/L U46619, 5 μmol/L ADP, or 80 μmol/L TRAP4, and aggregation was measured. Each experiment was repeated at least 3 times, with blood pooled from at least 3 separate groups of 6 to 8 immunized mice.…”

“…These experiments were performed, as we previously described 42, 43, 44. Platelets from vaccinated mice (C‐EL2, C‐EL2r, and KLH) were prepared, as previously described (250 μL; 2.5×10 8 /mL), before being placed into siliconized cuvettes and stirred for 5 minutes at 37°C at 237 × g .…”

“…Flow cytometric analysis was performed on platelets from vaccinated mice (C‐EL2, C‐EL2r, and KLH), as previously discussed 42, 43, 44. Briefly, platelets (2×10 8 ) were fixed with 2% formaldehyde for 30 minutes at room temperature, before incubation with anti‐mouse glycoprotein IIb‐IIIa, anti–glycoprotein Ib, or anti–glycoprotein VI antibodies at room temperature for 30 minutes in the dark.…”

“…Flow cytometric analysis was performed on platelets from vaccinated mice (C‐EL2, C‐EL2r, and KLH), as previously discussed 42, 43, 44. Briefly, platelets (2×10 8 ) were stimulated with 1 μmol/L U46619, 5 μmol/L ADP, or 80 μmol/L TRAP4 for 3 minutes.…”