Svetlov S I, Siafaka-Kapadai A, Hanahan D J, Olson M S
Department of Biochemistry, University of Texas Health Science Center at San Antonio, 78284-7760, USA.
Arch Biochem Biophys. 1996 Dec 1;336(1):59-68. doi: 10.1006/abbi.1996.0532.
The profile of biochemical responses following stimulation of human platelets with 1-alkyl-2-lyso-sn-glycero-3-phosphate (ALPA), a derivative of platelet-activating factor (PAF), was investigated. In the presence of extracellular Ca2+, ALPA evoked a dose-dependent increase and sustained elevation of the intracellular free Ca2+ concentration and stimulated the formation of phosphatidic acid. Platelets released free [3H]arachidonic and [3H]oleic acid at maximal rates between 5 and 15 s following ALPA stimulation. However, in platelets labeled with myo-[3H]inositol, ALPA induced [3H]phosphoinositide breakdown and formation of [3H]inositol phosphates with slower kinetics. Intracellular Ca2+ mobilization and the release of free fatty acids and inositol phosphates were not inhibited by pretreatment of platelets with pertussis toxin (PTX) or the PAF receptor antagonist WEB 2086. Following platelet stimulation with ALPA, tyrosine phosphorylation of proteins with apparent molecular masses of 65-95, 110-130, and 145-170 kDa was increased in a time-dependent manner, while phosphorylation of 40- to 45-kDa proteins was decreased. One of the platelet proteins phosphorylated on tyrosine residues in response to ALPA was found to be PLC-gamma1. Exogenous [3H]ALPA was metabolized primarily to [1-3H]alkyl-2,3-diacylglycerol. The metabolic conversion of [3H]ALPA involved a dephosphorylation reaction, and the formation of the dephosphorylated product, [1-3H]alkyl-monoglycerol, was detected within 5 s. These data demonstrate that an ether-linked lysophosphatidic acid can activate human platelets by a PTX-insensitive mechanism which does not involve the PAF receptor. Upon stimulation of platelets, ALPA induces the activation of phospholipases A2 and C, and tyrosine phosphorylation of several cellular proteins including PLC-gamma1. These signal transduction responses in platelets are accompanied by rapid metabolism of ALPA.
研究了用血小板活化因子（PAF）的衍生物1-烷基-2-溶血-sn-甘油-3-磷酸（ALPA）刺激人血小板后的生化反应概况。在细胞外Ca2+存在的情况下，ALPA引起细胞内游离Ca2+浓度呈剂量依赖性增加并持续升高，并刺激磷脂酸的形成。在ALPA刺激后5至15秒之间，血小板以最大速率释放游离的[3H]花生四烯酸和[3H]油酸。然而，在用肌醇-[3H]肌醇标记的血小板中，ALPA诱导[3H]磷酸肌醇分解并形成[3H]肌醇磷酸，其动力学较慢。用百日咳毒素（PTX）或PAF受体拮抗剂WEB 2086预处理血小板，不会抑制细胞内Ca2+动员以及游离脂肪酸和肌醇磷酸的释放。用ALPA刺激血小板后，表观分子量为65-95 kDa、110-130 kDa和145-170 kDa的蛋白质的酪氨酸磷酸化呈时间依赖性增加，而40至45 kDa蛋白质的磷酸化则减少。发现响应ALPA在酪氨酸残基上磷酸化的一种血小板蛋白是PLC-γ1。外源性[3H]ALPA主要代谢为[1-3H]烷基-2,3-二酰基甘油。[3H]ALPA的代谢转化涉及去磷酸化反应，并且在5秒内检测到去磷酸化产物[1-3H]烷基单甘油的形成。这些数据表明，醚连接的溶血磷脂酸可以通过不涉及PAF受体的PTX不敏感机制激活人血小板。刺激血小板后，ALPA诱导磷脂酶A2和C的激活以及包括PLC-γ1在内的几种细胞蛋白的酪氨酸磷酸化。血小板中的这些信号转导反应伴随着ALPA的快速代谢。
