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

(1)为了在体内大量表达Sema 3A，我们制备了Sema 3A-腺病毒。我们采用氦氖激光照射伊文思蓝注射小鼠的体内血栓形成模型。通过在视频显微镜下监测血栓形成，分析Sema 3A对血栓形成的影响。(2)Sema 3A抗血小板作用机制分析为了阐明Sema 3A抑制血小板功能的机制，我们研究了Sema 3A对凝血酶或convubxin诱导的血小板活化信号的影响。Sema 3A不影响凝血酶诱导的PKC激活。而Sema 3A明显抑制凝血酶诱导的P2 Y12和PI 3激酶依赖的AKT激活。加入过量的ADP不能恢复Sema 3A对AKT激活的抑制作用。Sema 3A还抑制凝血酶诱导的Rap 1激活，这似乎是由PI 3激酶活性调节。Sema 3A不仅抑制凝血酶，而且抑制惊厥素诱导的AKT和Rapl活化，但不影响惊厥素诱导的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，Sema 3A拮抗血小板中激动剂诱导的PI 3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.