Guanylate cyclase-cyclic GMP signaling pathway in platelet disaggregation associated with dissociation of PAF-receptor complex

PAF分子与其受体的持续结合是人类和家兔血小板长期聚集所必需的。另一方面，被PAF充分聚集的人和兔血小板缓慢分解，但被PAF受体拮抗剂WEB-2086和Y-24180快速分解。加入Y-24180后，PAF迅速与其受体分离，血小板鸟苷酸环化酶（GC）活性和细胞内cGMP水平升高，随后血小板解体，细胞表面p -选择素消失，但[Ca^<2+>]i未发生改变。此外，GC激活剂YC-1 （1 × 10^<-5> M）和cGMP磷酸二酯酶抑制剂双嘧达摩（3 × 10^<-6> M）抑制PAF （2 × 10^<-10> M）诱导的家兔血小板聚集，并通过增加细胞内cGMP水平使PAF聚集的血小板分解。CPT-cGMP （0.2 mM）而不是CPT-cAMP （0.2 mM）也能分解PAF （2 × 10^<-10> M）聚集的血小板。而NO-d依赖性更强的可溶性GC抑制剂ODQ （1 × 10^<-5> M）抑制硝普钠诱导的血小板中cGMP的产生，当Y-24180分解paf聚集的血小板时，ODQ不影响cGMP的产生。我们成功地从人巨核细胞系MEG01和CMKII-5中分离出与中性粒细胞PAF受体相同的编码PAF受体的cDNA。来自PAF受体+/+小鼠的血小板被PAF聚集并被PAF受体拮抗剂阻断，而来自PAF受体-/-小鼠的血小板不被PAF聚集。PAF拮抗剂Y24180和Rho激酶抑制剂Y27632协同分解PAF刺激的血小板。这些结果表明，血小板PAF受体与中性粒细胞的信号通路相同，但通过血小板PAF受体的信号通路具有特殊性。综上所述，这些观察结果表明，除了no依赖性sGC外，GC在血小板分解与paf受体复合物解离相关的信号转导机制中发挥重要作用。少

Continuous binding of the PAF molecule to its receptor is necessary for the long-term aggregation of human and rabbit platelets. On the other hand, human and rabbit platelets fully aggragated by PAF underwent slow disaggregation but were rapidly disaggregated by the PAF receptor antagonists WEB-2086 and Y-24180. PAF dissociated promptly from its receptor when Y-24180 was added, in parallel with elevation in platelet guanylate cyclase (GC) activity and intracellular cGMP level, followed by platelet disaggregation and disappearance of P-selectin on the cell surface, but no alteration in [Ca^<2+>]i. In addition, GC activator YC-1 (1 x 10^<-5> M) and cGMP phosphodiesterase inhibitor dipyridamole (3 x 10^<-6> M) suppressed PAF (2 x 10^<-10> M)-induced rabbit platelet aggregation, and moreover disaggregated the PAF-aggregated platelets by increasing intracellular cGMP level. CPT-cGMP (0.2 mM) but not CPT-cAMP (0.2 mM) also disaggregated PAF (2 x 10^<-10> M)-aggregated platelets. Whereas NO-d … More ependent soluble GC inhibitor ODQ (1 x 10^<-5> M) inhibited sodium nitroprusside-induced cGMP production in the platelets, ODQ did not affect the cGMP production when Y-24180 disaggregated PAF-aggregated platelets.We succeded in isolating a cDNA encoding PAF receptor from a human megakaryocytic cell line MEG01 and CMKII-5 cDNA, that is the same as neutrophile PAF receptor. Whereas platelets from PAF receptor+/+ mice were aggregated by PAF and the aggregation was blocked by PAF receptor antagonists, platelets from PAF receptor-/- mice were not aggregated by PAF. PAF antagonist Y24180 and Rho kinase inhibitor Y27632 synergistically disaggregated PAF-stimulted platelets. These results indicate that platelet PAF receptor is the same as that of neutrophile, but the signalling pathway through platelet PAF receptor is characteristic.In conclusion, these observations indicate that GC beside NO-dependent sGC plays an important role in signal transduction mechanism in platelet disaggregation associated with dissociation of PAF-receptor complex. Less