Department of Pharmaceutical Sciences, School of Pharmacy, The University of Texas El Paso, El Paso, TX 79902, USA.
Department of Pharmacy Practice, Irma Lerma Rangel College of Pharmacy, Texas A&M University, Kingsville, TX 78363, USA.
Int J Mol Sci. 2022 Aug 8;23(15):8794. doi: 10.3390/ijms23158794.
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.
血清素（5-羟色胺或 5-HT）已知是一种弱血小板激动剂，参与血栓形成。虽然 5-HT 本身不能诱导血小板聚集，但从α颗粒中分泌出来时，它会与 G 蛋白偶联受体（GPCR；即 5HTR）结合，从而放大血小板功能反应（例如聚集）。因此，5HTR 介导的反应更多地参与了正在生长的血栓中血小板聚集的二次放大。因此，尽管 5-HT 可以被视为血小板激活的弱诱导剂，但它是 ADP、胶原和肾上腺素等激动剂触发聚集的重要放大器，从而增强血栓形成。5HTR/5-HT 信号通路是科学界和医学界感兴趣的，因为它与几种心血管疾病的发生有关。然而，由于这些试剂具有有害的副作用，并且/或者缺乏选择性等原因，迄今为止，开发 5HTR 拮抗剂作为心血管疾病的治疗剂的努力都失败了。鉴于研究努力确定 5HTR 配体结合域位于第二细胞外环（EL2；氨基酸 P-N）中，我们开发了一种针对 EL2 区域的抗体，即 5HTRAb，并在血小板中对其药理活性进行了表征。因此，我们利用来自健康人类供体以及 C57BL/6J 小鼠（10-12 周龄）的血小板来分析 5HTRAb 在体外、离体和体内对血小板激活、血栓形成以及 5HTR 配体结合的抑制作用。我们的结果表明，5HTRAb 抑制 5-HT 增强的血小板体外和离体激活，但对激动剂诱导的激活没有明显影响。与 P2Y12 拮抗剂氯吡格雷和 5HTR 拮抗剂酮色林不同，5HTRAb 还发现可延长血栓闭塞时间，且不会调节尾部出血时间。此外，发现 5HTRAb 通过直接拮抗血小板 5HTR 来实现这一点。我们的研究结果表明，定制的 5HTRAb 具有血小板功能阻断活性，并可防止血栓形成，而不会损害正常止血。
