Analysis of anti-platelet and anti-thrombotic effects of a neuron guidance molecule,semaphorin 3A

(1) Sena3A对血栓形成和动脉粥样硬化的影响为了在体内大量表达Sema3A，我们制作了Sema3A腺病毒。采用氦氖激光照射Evans蓝注射小鼠建立体内血栓形成模型。通过在视频显微镜下监测血栓形成，分析Sema3A对血栓形成的影响。(2) Sema3A抗血小板作用机制分析为了阐明Sema3A对血小板功能的抑制机制，我们研究了Sema3A对凝血酶或凝血素诱导的血小板激活信号的影响。Sema3A不影响凝血素诱导的PKC激活。相比之下，Sema3A明显抑制凝血酶诱导的P2Y_12和PI3激酶依赖性AKT活化。过量ADP的加入不能恢复Sema3A对AKT活化的抑制作用。Sema3A还抑制凝血素诱导的Rap1激活，这似乎是由PI3激酶活性调节的。Sema3A不仅抑制凝血酶，还抑制惊厥新诱导的AKT和Rapl的激活，而不影响惊厥新诱导的LAT、SLP-76、PLC□2或PKC底物的磷酸化。这些结果表明，无论激动剂的类型如何，Sema3A都能拮抗血小板中激动剂诱导的pi3k激酶途径。

(1) Effects of Sena3A on thrombosis and atherosclerosisTo express large amount of Sema3A in vivo, we made Sema3A-adenovirus. We used an in vivo thrombosis model by irradiation of helium-neon laser to Evans blue injected mice. By monitoring the production of thrombosis under video-microscope, we analyzed effects of Sema3A on thrombosis production.(2) Analysis of anti-platelet mechanism of Sema3ATo elucidate the inhibitory mechanism of platelet function by Sema3A, we investigated the impacts of Sema3A on platelet activating signaling induced by thrombin or convubxin. Sema3A did not affect thrombin-induced PKC activation. In contrast, Sema3A markedly inhibited P2Y_12 and PI3 kinase-dependent AKT activation induced by thrombin. Addition of excessive ADP could not restore the inhibition of AKT activation by Sema3A. Sema3A also inhibited thrombin-induced Rap1 activation, which appeared to be regulated by PI3 kinase activity. Sema3A inhibited not only thrombin but also convulxin-induced AKT and Rapl activation, whereas it did not affect convulxin-induced phosphorylation of LAT, SLP-76, PLC□2, or PKC substrates. These results suggested that irrespective of the type of agonists, Sema3A antagonizes PI3 kinase pathway induced by agonists in platelets.