Role of Kv1.3 Channels in Platelet Functions and Thrombus Formation

Objective: Platelet activation by stimulatory factors leads to an increase in intracellular calcium concentration ([Ca2+](i)), which is essential for almost all platelet functions. Modulation of Ca(2+)influx and [Ca2+](i)in platelets has been emerging as a possible strategy for preventing and treating platelet-dependent thrombosis. Voltage-gated potassium 1.3 channels (Kv1.3) are highly expressed in platelets and able to regulate agonist-evoked [Ca2+](i)increase. However, the role of Kv1.3 channels in regulating platelet functions and thrombosis has not yet been elucidated. In addition, it is difficult to obtain a specific blocker for this channel, since Kv1.3 shares identical drug-binding sites with other K(+)channels. Here, we investigate whether specific blockade of Kv1.3 channels by monoclonal antibodies affects platelet functions and thrombosis. Approach and Results: In this study, we produced the anti-Kv1.3 monoclonal antibody 6E12#15, which could specifically recognize both human and mouse Kv1.3 proteins and sufficiently block Kv1.3 channel currents. We found Kv1.3 blockade by 6E12#15 inhibited platelet aggregation, adhesion, and activation upon agonist stimulation. In vivo treatment with 6E12#15 alleviated thrombus formation in a mesenteric arteriole thrombosis mouse model and protected mice from collagen/epinephrine-induced pulmonary thromboembolism. Furthermore, we observed Kv1.3 regulated platelet functions by modulating Ca(2+)influx and [Ca2+](i)elevation, and that this is mediated in part by P2X(1). Interestingly,Kv1.3(-/-)mice showed impaired platelet aggregation while displayed no abnormalities in in vivo thrombus formation. This phenomenon was related to more megakaryocytes and platelets produced inKv1.3(-/-)mice compared with wild-type mice. Conclusions: We showed specific inhibition of Kv1.3 by the novel monoclonal antibody 6E12#15 suppressed platelet functions and platelet-dependent thrombosis through modulating platelet [Ca2+](i)elevation. These results indicate that Kv1.3 could act as a promising therapeutic target for antiplatelet therapies.

目的：刺激因子激活血小板会导致细胞内钙浓度（[Ca2+](i)）升高，这对几乎所有血小板功能都至关重要。调节血小板中Ca(2+)内流和[Ca2+](i)已逐渐成为预防和治疗血小板依赖性血栓形成的一种可能策略。电压门控钾离子1.3通道（Kv1.3）在血小板中高度表达，并且能够调节激动剂诱发的[Ca2+](i)升高。然而，Kv1.3通道在调节血小板功能和血栓形成中的作用尚未阐明。此外，由于Kv1.3与其他K(+)通道具有相同的药物结合位点，因此很难获得针对该通道的特异性阻断剂。在此，我们研究单克隆抗体对Kv1.3通道的特异性阻断是否会影响血小板功能和血栓形成。 方法和结果：在本研究中，我们制备了抗Kv1.3单克隆抗体6E12#15，它能够特异性识别人和小鼠的Kv1.3蛋白，并充分阻断Kv1.3通道电流。我们发现6E12#15对Kv1.3的阻断抑制了激动剂刺激下的血小板聚集、黏附和激活。在体内用6E12#15治疗可减轻肠系膜小动脉血栓形成小鼠模型中的血栓形成，并保护小鼠免受胶原蛋白/肾上腺素诱导的肺血栓栓塞。此外，我们观察到Kv1.3通过调节Ca(2+)内流和[Ca2+](i)升高来调节血小板功能，并且这部分是由P2X(1)介导的。有趣的是，Kv1.3(-/-)小鼠表现出血小板聚集受损，但在体内血栓形成方面未显示异常。这种现象与Kv1.3(-/-)小鼠相比野生型小鼠产生更多的巨核细胞和血小板有关。 结论：我们表明新型单克隆抗体6E12#15对Kv1.3的特异性抑制通过调节血小板[Ca2+](i)升高抑制了血小板功能和血小板依赖性血栓形成。这些结果表明Kv1.3可作为抗血小板治疗的一个有前景的治疗靶点。