Gallet C, Rosa J P, Habib A, Lebret M, Lévy-Tolédano S, Maclouf J
U. INSERM 348, Hôpital Lariboisière, 8 rue Guy Patin, 75475 Paris Cedex 10, France.
J Biol Chem. 1999 Aug 13;274(33):23610-6. doi: 10.1074/jbc.274.33.23610.
Thromboxane A(2) (TxA(2)) is a potent vasoconstrictor and platelet agonist. Pharmacological studies have defined two classes of thromboxane receptors (TPs) in human platelets; sites that bind the agonist 1S-(1,2(5Z),3-(1E,3S),4)-7- 3-(3-hydroxy-4-(4'-iodophenoxy)-1-butenyl)-7-oxabicyclo-2.2. 1-heptan-2-yl-5-heptenoic acid (I-BOP) with high affinity support platelet shape change, whereas low affinity sites that bind irreversibly the antagonist GR 32191 transduce platelet aggregation. As the mechanisms of signal transduction involved in platelet aggregation begin to be elucidated, few results concern those involved in platelet shape change, which is independent of the engagement of GPIIb/IIIa. To elucidate the respective role of the two classes of pharmacological binding sites of TPs in shape change, platelets were incubated with I-BOP at low concentrations or stimulated by I-BOP at high concentrations after pretreatment with GR 32191 or activated with low concentrations of 8-epi-prostaglandin F(2)alpha. Under these three conditions, there is a rapid stimulation of protein tyrosine phosphorylation of the 80/85-kDa doublet identified as the cytoskeletal protein cortactin. Tyrosine phosphorylation of cortactin is kinetically correlated with the occurrence of shape change. These biochemical and morphological events are both inhibited by SQ 29548, a TP antagonist, indicating the specificity of the signal. Since tyrosine kinase Syk was activated early during platelet activation, we examined the possibility that cortactin is a potential substrate of Syk in TxA(2)-induced platelet shape change. p72 Syk phosphorylation and kinase activity took place during the period when platelets were changing shape upon low concentrations of I-BOP stimulation. Furthermore, cortactin was associated with Syk, and this association increases along with the level of phosphorylation. These data suggest a novel pathway for a G protein-coupled TxA(2) high affinity receptor to the protein-tyrosine kinase Syk, which is associated with cortactin in the very early steps of platelet activation.
血栓素A2（TxA2）是一种强效的血管收缩剂和血小板激动剂。药理学研究已确定人类血小板中有两类血栓素受体（TPs）；与激动剂1S-(1,2(5Z),3-(1E,3S),4)-7- 3-(3-羟基-4-(4'-碘苯氧基)-1-丁烯基)-7-氧杂双环-2.2.1-庚烷-2-基-5-庚烯酸（I-BOP）高亲和力结合的位点支持血小板形状改变，而与拮抗剂GR 32191不可逆结合的低亲和力位点则介导血小板聚集。随着血小板聚集中涉及的信号转导机制开始被阐明，很少有结果涉及与血小板形状改变相关的机制，而血小板形状改变独立于糖蛋白IIb/IIIa的参与。为了阐明TPs的两类药理学结合位点在形状改变中的各自作用，将血小板与低浓度的I-BOP孵育，或在用GR 32191预处理后用高浓度的I-BOP刺激，或用低浓度的8-表-前列腺素F2α激活。在这三种条件下，可快速刺激被鉴定为细胞骨架蛋白皮层肌动蛋白的80/85-kDa双峰的蛋白酪氨酸磷酸化。皮层肌动蛋白的酪氨酸磷酸化在动力学上与形状改变的发生相关。这些生化和形态学事件均被TP拮抗剂SQ 29548抑制，表明该信号具有特异性。由于酪氨酸激酶Syk在血小板激活早期就被激活，我们研究了皮层肌动蛋白是否是Syk在TxA2诱导的血小板形状改变中的潜在底物。在低浓度I-BOP刺激下血小板发生形状改变的期间，发生了p72 Syk磷酸化和激酶活性。此外，皮层肌动蛋白与Syk相关，并且这种相关性随着磷酸化水平的增加而增加。这些数据提示了一条从G蛋白偶联的TxA2高亲和力受体到蛋白酪氨酸激酶Syk的新途径，该途径在血小板激活的早期阶段与皮层肌动蛋白相关。
