Slupsky J R, Cawley J C, Kaplan C, Zuzel M
University Department of Haematology, Royal Liverpool Hospital, U.K.
Br J Haematol. 1997 Feb;96(2):275-86. doi: 10.1046/j.1365-2141.1997.d01-2011.x.
The use of agonist monoclonal antibodies (mAbs) to probe the signalling function of platelet membrane proteins is severely limited by the dependence of the mAb effect on Fc-FcgammaRII interaction. Furthermore, in addition to its anchoring role, the FcgammaRII receptor itself generates a stimulation signal resulting in granule secretion. Platelet stimulation by the released granule contents can then further obscure the original activation signal. Here we demonstrate that these problems are largely overcome by the use of platelets which had been degranulated with thrombin prior to stimulation with mAbs. We found that, like intact cells, degranulated platelets could also be activated and induced to aggregate by mAbs against a 67 kD membrane protein (known as PTA1) and CD9, and by crosslinked CD32 (FcgammaRII). However, the signal generated by crosslinked FcgammaRII was weak compared with that induced by the other monoclonal antibodies. Thus, by diminishing the FcgammaRII signal contribution, we have succeeded for the first time to clearly dissect the target antigen signal from that generated by FcgammaRII. In addition to differences in the degree of aggregation, analysis of the signals generated by each mAb showed differences in Ca2+ fluxes and protein phosphorylation. Moreover, the signals generated by CD9 and PTA1 antigens differed significantly in their sensitivity to PKC inhibition or ADP-ribosylation of the small GTP-binding protein rhoA. Despite these differences, the signals initiated by all three antigens converged to a common signalling pathway which included activation of tyrosine kinase(s). The pattern of protein phosphorylation strongly resembled that induced by gpIIb/IIIa-mediated platelet interaction with macromolecular ligands and by mutual cell contact. The multiple intercellular links formed by mAb would have a similar effect since the Fc-receptor anchorage required for antigen stimulation is already known to be provided by adjacent cells. The present findings suggest that the function of both CD9 and PTA1 antigens is closely associated with gpIIb/IIIa activation.
利用激动剂单克隆抗体（mAb）来探究血小板膜蛋白的信号传导功能，因mAb效应依赖于Fc-FcγRII相互作用而受到严重限制。此外，除了其锚定作用外，FcγRII受体本身还会产生刺激信号，导致颗粒分泌。释放的颗粒内容物对血小板的刺激随后会进一步掩盖原始的激活信号。在此，我们证明，通过使用在用mAb刺激之前已用凝血酶脱颗粒的血小板，这些问题在很大程度上得以克服。我们发现，与完整细胞一样，脱颗粒的血小板也可被针对67 kD膜蛋白（称为PTA1）和CD9的mAb以及交联的CD32（FcγRII）激活并诱导聚集。然而，与其他单克隆抗体诱导的信号相比，交联的FcγRII产生的信号较弱。因此，通过减少FcγRII信号贡献，我们首次成功地将靶抗原信号与FcγRII产生的信号清晰地分开。除了聚集程度的差异外，对每种mAb产生的信号分析显示，Ca2+通量和蛋白质磷酸化存在差异。此外，CD9和PTA1抗原产生的信号在对PKC抑制或小GTP结合蛋白rhoA的ADP核糖基化的敏感性方面存在显著差异。尽管存在这些差异，但由所有三种抗原引发的信号都汇聚到一条共同的信号通路，其中包括酪氨酸激酶的激活。蛋白质磷酸化模式与gpIIb/IIIa介导的血小板与大分子配体相互作用以及细胞间相互接触诱导的模式非常相似。mAb形成的多个细胞间连接可能具有类似的作用，因为已知抗原刺激所需的Fc受体锚定是由相邻细胞提供的。目前的研究结果表明，CD9和PTA1抗原的功能都与gpIIb/IIIa激活密切相关。
