A Novel Antibody Targeting the Second Extracellular Loop of the Serotonin 5-HT2A Receptor Inhibits Platelet Function

Serotonin (5-hydroxytriptamine or 5-HT) is known to be a weak platelet agonist, and is involved in thrombus formation. While 5-HT cannot induce platelet aggregation on its own, when secreted from the alpha granules, it binds to its G-protein Coupled Receptor (GPCR; i.e., 5HTR), thereby acting to amplify platelet functional responses (e.g., aggregation). Thus, 5HTR-mediated responses are more involved in the secondary amplification of platelet aggregation in the growing thrombus. Therefore, even though 5-HT can be seen as a weak inducer of platelet activation, it is an important amplifier of aggregation triggered by agonists such as ADP, collagen, and epinephrine, thereby enhancing thrombogenesis. The 5HTR/5HT signaling pathway is of clinical interest to the scientific and medical communities as it has been implicated in the genesis of several forms of cardiovascular disorders. However, efforts to develop antagonists for 5HTR as therapeutic agents in cardiovascular diseases have thus far failed due to these reagents having deleterious side-effects, and/or to lack of selectivity, amongst other reasons. In light of research efforts that identified that the 5HTR ligand binding domain resides in the second extracellular loop (EL2; amino acids P-N), we developed an antibody, i.e., referred to as 5HTRAb, against the EL2 region, and characterized its pharmacological activity in the context of platelets. Thus, we utilized platelets from healthy human donors, as well as C57BL/6J mice (10-12 weeks old) to analyze the inhibitory effects of the 5HTRAb on platelet activation in vitro, ex vivo, and on thrombogenesis in vivo as well as on 5HTR ligand binding. Our results indicate that the 5HTRAb inhibits 5-HT-enhanced platelet activation in vitro and ex vivo, but has no apparent effects on that which is agonist-induced. The 5HTRAb was also found to prolong the thrombus occlusion time, and it did so without modulating the tail bleeding time, in mice unlike the P2Y12 antagonist clopidogrel and the 5HTR antagonist ketanserin. Moreover, it was found that the 5HTRAb does so by directly antagonizing the platelet 5HTR. Our findings document that the custom-made 5HTRAb exhibits platelet function blocking activity and protects against thrombogenesis without impairing normal hemostasis.