The contribution of platelet-activating factor (PAF) to platelet deposition and oedema formation induced by exogenous soluble mediators, zymosan particles and associated with a reversed passive Arthus (RPA) reaction in rabbit skin was investigated by use of a novel long-acting PAF receptor antagonist, UK-74,505. 2. Oedema formation and platelet accumulation were simultaneously measured by i.v. injection of [125I]-albumin and 111In-labelled rabbit platelets. UK-74,505 was either administered i.v. or used to pretreat radiolabelled platelets in vitro before their injection into recipient animals. Platelets pretreated with UK-74,505 were also labelled with the fluorescent calcium indicator, Fura-2, to assess their ex vivo reactivity to PAF at the end of the in vivo experiment. 3. UK-74,505 (0.5 mg kg-1), administered i.v., inhibited PAF-induced oedema formation, but did not affect oedema induced by zymosan particles, bradykinin (BK), histamine, formyl-methionyl-leucylphenylalanine (FMLP), zymosan-activated plasma (ZAP, as a source of C5a des Arg), leukotriene B4 (LTB4) or interleukin-8 (IL-8). 4. UK-74,505, administered i.v. also suppressed the small platelet accumulation induced by exogenous PAF, but had no effect on accumulation induced by IL-8 or ZAP. Although oedema induced by zymosan was not affected by i.v. UK-74,505, zymosan-induced platelet accumulation was significantly attenuated by the antagonist. 5. The RPA reaction in rabbit skin was associated with marked oedema formation and platelet accumulation which were both inhibited by i.v. UK-74,505. 6. In vitro, UK-74,505 inhibited aggregation and the increase in intracellular calcium concentration induced by PAF in rabbit washed platelets in a concentration-dependent manner (IC50 = 1.6 x 10-8 M and 1.1 x 10-8 M, respectively). Platelets pretreated with 10-6 M UK-74,505, and maintained at 37 degrees C,were unresponsive to PAF, whilst responding normally to thrombin, for up to 4 h.7. In a second series of in vivo experiments, platelets were labelled with 111In and loaded with Fura-2.The platelets were then pretreated with 10-6 M UK-74,505, washed, and injected into recipient rabbits.These platelets, prepared from blood samples taken at the end of the in vivo experiments, exhibited an 80% reduction in their response to PAF as measured ex vivo with Fura-2. However, in contrast to the effects of i.v. UK-74,505, platelets pretreated with the antagonist did accumulate effectively in the RPA reaction, a significant reduction only being observed in responses at the lowest antibody dose. In addition, pretreatment of platelets had no effect on the small platelet accumulation induced by PAF.8. These results suggest that PAF is an important mediator of oedema formation and platelet accumulation in the RPA reaction in rabbit skin. However, they question the role of PAF receptors on platelets in this model. The results also indicate that PAF may be involved in platelet accumulation induced by zymosan in rabbit skin.
摘要
采用新型长效血小板活化因子（PAF）受体拮抗剂UK-74,505，研究了PAF在兔皮肤中外源性可溶性介质、酵母聚糖颗粒诱导的血小板沉积和水肿形成以及与反向被动Arthus（RPA）反应中的作用。2. 通过静脉注射[125I] -白蛋白和111In标记的兔血小板同时测定水肿形成和血小板聚集。UK-74,505可静脉注射给药，或在将放射性标记的血小板注射入受体动物之前用于体外预处理。用UK-74,505预处理的血小板还用荧光钙指示剂Fura-2标记，以在体内实验结束时评估其对PAF的离体反应性。3. 静脉注射UK-74,505（0.5 mg kg-1）可抑制PAF诱导的水肿形成，但不影响酵母聚糖颗粒、缓激肽（BK）、组胺、甲酰甲硫氨酰亮氨酰苯丙氨酸（FMLP）、酵母聚糖活化血浆（ZAP，作为C5a des Arg的来源）、白三烯B4（LTB4）或白细胞介素-8（IL-8）诱导的水肿。4. 静脉注射UK-7,505也可抑制外源性PAF诱导的少量血小板聚集，但对IL-8或ZAP诱导的聚集无影响。虽然酵母聚糖诱导的水肿不受静脉注射UK-74,505的影响，但拮抗剂可显著减轻酵母聚糖诱导的血小板聚集。5. 兔皮肤中的RPA反应与明显的水肿形成和血小板聚集有关，两者均受静脉注射UK-74,505的抑制。6. 在体外，UK-74,505以浓度依赖性方式抑制兔洗涤血小板中PAF诱导的聚集和细胞内钙浓度升高（IC50分别为1.6×10-8 M和1.1×10-8 M）。用10-6 M UK-74,505预处理并维持在37℃的血小板对PAF无反应，而对凝血酶正常反应，长达4小时。7. 在第二系列体内实验中，血小板用111In标记并加载Fura-2。然后用10-6 M UK-74,505预处理血小板，洗涤后注射入受体兔。从体内实验结束时采集的血样制备的这些血小板，用Fura-2离体测量显示其对PAF的反应降低了80%。然而，与静脉注射UK-74,505的效果相反，用拮抗剂预处理的血小板在RPA反应中确实有效聚集，仅在最低抗体剂量的反应中观察到显著降低。此外，血小板预处理对PAF诱导的少量血小板聚集无影响。8. 这些结果表明，PAF是兔皮肤RPA反应中水肿形成和血小板聚集的重要介质。然而，它们对该模型中血小板上PAF受体的作用提出了质疑。结果还表明，PAF可能参与兔皮肤中酵母聚糖诱导的血小板聚集。
