Kv1.3通道为靶向的新型抗血小板治疗在动脉血栓形成中的作用及机制研究

Arterial thrombosis includes a series of diseases such as myocardial infarction and stroke. Platelet activation is the central step in arterial thrombosis. The activation of platelets by stimulating factors leads to an increase in the intracellular calcium concentration ([Ca2+]i), which is essential for nearly all platelet functions like shape change, secretion and aggregation which are of particular importance for arterial thrombosis. Thus, the regulation of [Ca2+]i in platelets represents a novel strategy for preventing and treating thrombus formation. Kv1.3 is responsible for the voltage-dependent K+ conductance of platelets, which can increase Ca2+ influx and increase [Ca2+]i in platelet. In our previous work, we blocked Kv1.3 channels in platelets and found [Ca2+]i elevation and platelet aggregation were inhibited, which indicated the importance of Kv1.3 in regulating platelet functions. However, little in vivo or in vitro evidence has confirmed the mechanism and the influence on arterial thrombus formation in the case of Kv1.3 suppression. Thus, we put forward our hypothesis that Kv1.3 could modulate platelet functions through [Ca2+]i regulation and further interfere arterial thrombosis formation. In this study, we aim to use Kv1.3 blockade and gene knock-out strategy to inhibit/suppress Kv1.3 on platelets, and to explore the changes of platelet functions including aggregation, secretion and adhesion, and arterial thrombosis in mouse models, in addition to the mechanisms. This study will provide new therapeutic targets for the prevention and treatment of arterial thrombosis diseases.

调节[Ca2+]i能有效调控血小板功能，影响动脉血栓形成。Kv1.3通过改变膜电势差，影响胞外Ca2+内流，继而调控[Ca2+]i。在前期研究中，我们首次在国际上成功研发了一种新型、高特异性Kv1.3通道阻滞剂—抗hKv1.3-E314肽段单克隆抗体6E12#15（专利号：201610325161.7）。在预实验中，我们发现应用6E12#15阻滞Kv1.3通道，能抑制血小板胞内[Ca2+]i升高及其聚集功能，提示Kv1.3在调控血小板功能中起重要作用。据此我们提出假说：Kv1.3通过影响Ca2+内流，调控血小板胞内[Ca2+]i，从而影响血小板功能及动脉血栓形成。本课题拟采用血小板特异性基因敲除和通道阻滞技术，从分子、细胞及动物水平，揭示Kv1.3调控血小板功能的机制，明确Kv1.3对血小板功能的影响，探讨Kv1.3在动脉血栓形成中的作用。本研究将从新视角为动脉血栓性疾病的防治提供新思路。

研究背景及目标：血小板能在多种刺激因素下活化，常见刺激因素包括：ADP、胶原、凝血酶、肾上腺素、瑞斯托霉素、高剪切应力等。尽管各种刺激因素通过不同的受体或信号通路使血小板活化，其最终通路都能引起血小板胞内钙离子浓度([Ca2+]i)的大幅升高。胞内[Ca2+]i升高是血小板活化的中心环节，也是血小板发挥其特有功能的决定性因素。Kv1.3是一种钾离子通道，是维持静息膜电位的主要离子通道，在细胞活化时其能促使Ca2+迅速内流，提升胞内[Ca2+]i。Kv1.3通道高表达于人及小鼠血小板上，但其对血小板功能的影响不明。在本研究中，我们用Kv1.3通道单克隆抗体特异性的阻滞人及小鼠血小板Kv1.3通道，观察其对血小板功能及血栓形成的影响并探究其可能的机制。.研究结果：在研究中，基于本课题组的专利，我们生产了抗Kv1.3单克隆抗体6E12#15，它可以特异性识别人和小鼠Kv1.3通道蛋白，并充分阻断Kv1.3通道电流。我们发现6E12#15阻断Kv1.3通道后，抑制了激动剂刺激下血小板的聚集、粘附和活化。用6E12#15进行在体研究，发现其能抑制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，我们发现Kv1.3能通过调节Ca2+内流和影响胞内[Ca2+]i升高调控血小板功能，P2X1介导并参与了Kv1.3的调控作用。另外，Kv1.3−/−小鼠的血小板聚集能力受损，而体内血栓形成能力没有明显异常，这可能与Kv1.3−/−小鼠产生了过多的巨核细胞和血小板有关。.结论及科学意义：该研究显示抗Kv1.3单克隆抗体6E12#15能特异性的抑制血小板的功能及血小板介导的血栓形成，揭示了Kv1.3调控胞内[Ca2+]i及影响血小板功能的可能机制。该结果能为抗血小板治疗及动脉血栓性疾病的防治提供新的思路、靶点和潜在药物。