Marshall Stuart J, Asazuma Naoki, Best Denise, Wonerow Peter, Salmon Gary, Andrews Robert K, Watson Steve P
Department of Pharmacology, University of Oxford, Mansfield Road, Oxford OX1 3QT, UK.
Biochem J. 2002 Jan 15;361(Pt 2):297-305. doi: 10.1042/0264-6021:3610297.
It has been proposed that the receptor for von Willebrand factor (vWF), glycoprotein (GP)Ib-IX-V, signals through the same pathway as the collagen receptor, GPVI, namely via Src kinases, the Fc receptor (FcR) gamma-chain and Syk, leading to tyrosine phosphorylation of phospholipase Cgamma2 (PLCgamma2). The aim of the present study was to assess the functional significance of this pathway in platelet activation by GPIb-IX-V. In washed platelets, vWF/ristocetin and vWF/botrocetin stimulate weak tyrosine phosphorylation of the FcR gamma-chain, Syk and PLCgamma2, but not the adaptor LAT (linker for activation of T-cells), which is localized to glycolipid-enriched membrane domains. Increases in tyrosine phosphorylation were blocked by the Src family kinase inhibitor, 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo-d-3,4-pyrimidine (PP1). Under the same conditions, neither stimulus induced activation of PLCgamma2 nor functional responses, such as Ca(2+) elevation, secretion or GPIIb-IIIa-dependent aggregation. In contrast, in platelet-rich plasma (PRP), threshold concentrations of ristocetin or asialo-vWF stimulated GPIb-dependent biphasic aggregation, in which the second phase was blocked by PP1. Importantly, a significant component of the initial phase and the complete second phase of aggregation was blocked by GPIIb-IIIa receptor antagonists in PRP. Higher concentrations of ristocetin stimulated GPIIb-IIIa-independent agglutination in PRP. These results demonstrate that GPIb-IX-V initiates activation of GPIIb-IIIa in PRP through an undefined pathway that is reinforced by a PP1-sensitive pathway. In contrast, activation of GPIbalpha in washed platelets does not promote functional responses.
有人提出，血管性血友病因子（vWF）的受体糖蛋白（GP）Ib-IX-V通过与胶原蛋白受体GPVI相同的途径发出信号，即通过Src激酶、Fc受体（FcR）γ链和Syk，导致磷脂酶Cγ2（PLCγ2）的酪氨酸磷酸化。本研究的目的是评估该途径在GPIb-IX-V介导的血小板激活中的功能意义。在洗涤后的血小板中，vWF/瑞斯托菌素和vWF/博托菌素刺激FcRγ链、Syk和PLCγ2发生微弱的酪氨酸磷酸化，但不刺激定位于富含糖脂膜结构域的衔接蛋白LAT（T细胞激活连接蛋白）。酪氨酸磷酸化的增加被Src家族激酶抑制剂4-氨基-5-（4-氯苯基）-7-（叔丁基）吡唑并[3,4-d]嘧啶（PP1）阻断。在相同条件下，两种刺激均未诱导PLCγ2的激活或功能性反应，如Ca(2+)升高、分泌或GPIIb-IIIa依赖性聚集。相比之下，在富血小板血浆（PRP）中，阈值浓度的瑞斯托菌素或去唾液酸vWF刺激GPIb依赖性双相聚集，其中第二相被PP1阻断。重要的是，PRP中聚集的初始阶段的一个重要组成部分和完整的第二阶段被GPIIb-IIIa受体拮抗剂阻断。更高浓度的瑞斯托菌素刺激PRP中不依赖GPIIb-IIIa的凝集。这些结果表明，GPIb-IX-V通过一条未明确的途径启动PRP中GPIIb-IIIa的激活，该途径由一条对PP1敏感的途径加强。相比之下，洗涤后血小板中GPIbalpha的激活不会促进功能性反应。
