Pula Giordano, Schuh Kai, Nakayama Keiko, Nakayama Keiichi I, Walter Ulrich, Poole Alastair W
Department of Pharmacology, School of Medical Sciences, University of Bristol, United Kingdom.
Blood. 2006 Dec 15;108(13):4035-44. doi: 10.1182/blood-2006-05-023739. Epub 2006 Aug 29.
Protein kinase Cdelta (PKCdelta) has been shown by pharmacologic approaches to negatively regulate collagen-induced platelet aggregation. Here we addressed the molecular and cellular mechanisms underlying this negative regulation. Using PKCdelta-/- platelets, we show that the mechanism did not involve altered inside-out signaling to integrin alpha(IIb)beta3 and did not affect early signaling events downstream of GPVI, because there was no difference in tyrosine phosphorylation of PLCgamma2 between wild-type and PKCdelta-/- platelets. There was also no increase in secretion of dense granule content, in contrast to studies using rottlerin where secretion was enhanced. Importantly, however, there was marked enhancement of filopodia generation in PKCdelta-/- platelets upon adhesion to collagen compared with wild-type platelets. Filopodia play an essential role regulating adhesive events leading to platelet aggregation by increasing platelet-platelet contact. We show that the critical effector for PKCdelta is vasodilator-stimulated phosphoprotein (VASP), a major regulator of actin cytoskeleton dynamics. PKCdelta physically interacts with VASP constitutively and regulates its phosphorylation on Ser157. In VASP-/- platelets, the enhancement of filopodia generation, actin polymerization, and platelet aggregation by rottlerin is ablated. PKCdelta is therefore a critical negative regulator of filopodia, and hence platelet aggregation, through a functional interaction with the actin organizer VASP.
药理学方法已表明蛋白激酶Cδ（PKCδ）对胶原诱导的血小板聚集具有负调控作用。在此，我们探讨了这种负调控背后的分子和细胞机制。使用PKCδ基因敲除小鼠的血小板，我们发现该机制不涉及整合素α（IIb）β3外向内信号转导的改变，也不影响GPVI下游的早期信号事件，因为野生型和PKCδ基因敲除小鼠的血小板中PLCγ2的酪氨酸磷酸化没有差异。与使用rottlerin（分泌增强）的研究不同，致密颗粒内容物的分泌也没有增加。然而，重要的是，与野生型血小板相比，PKCδ基因敲除小鼠的血小板在黏附于胶原时丝状伪足生成显著增强。丝状伪足通过增加血小板-血小板接触在调节导致血小板聚集的黏附事件中起重要作用。我们发现PKCδ的关键效应分子是血管舒张刺激磷蛋白（VASP），它是肌动蛋白细胞骨架动力学的主要调节因子。PKCδ与VASP持续发生物理相互作用并调节其Ser157位点的磷酸化。在VASP基因敲除小鼠的血小板中，rottlerin对丝状伪足生成、肌动蛋白聚合和血小板聚集的增强作用被消除。因此，PKCδ通过与肌动蛋白组织者VASP的功能相互作用，是丝状伪足以及血小板聚集的关键负调控因子。
