Dhar A, Shukla S D
Department of Pharmacology, University of Missouri School of Medicine, Columbia 65212.
J Biol Chem. 1994 Mar 25;269(12):9123-7.
Antibodies to pp60v-arc and phosphotyrosine were introduced into rabbit platelets using an electropermeabilization technique. The presence of these antibodies inside platelets was detected by flow cytometry. Platelet-activating factor (PAF)-stimulated phospholipase C activity (inositol phosphate production) and aggregation were dramatically inhibited in platelets transjected with either of these antibodies. Incubation of these antibodies with intact cells (i.e. nonpermeabilized) or electrotransjection of several nonspecific antibodies/agents (e.g. goat anti-mouse IgG, mouse serum, human platelet glycoprotein Ib monoclonal antibody, and fetal calf serum) into platelets had no effect on the PAF responses. trpE (another isotype-matched control antibody of pp60v-src) and pp56lck polyclonal antibody (another src-related kinase not present in platelets) also had no effect on PAF-induced aggregation and inositol phosphate production in permeabilized platelets. This indicates that the effect of internalized pp60v-src antibody is direct and specific in platelets. Stimulation of platelets by PAF increased the association and phosphorylation of both pp60c-src (60 kDa) and phospholipase C gamma 1 (145 kDa). This study provides the first evidence in platelets for a direct and specific involvement of pp60c-src in PAF-mediated phospholipase C activation and aggregation response.
采用电通透技术将抗pp60v-arc和抗磷酸酪氨酸抗体导入兔血小板。通过流式细胞术检测血小板内这些抗体的存在情况。血小板激活因子（PAF）刺激的磷脂酶C活性（肌醇磷酸生成）和聚集在注射了这两种抗体之一的血小板中受到显著抑制。将这些抗体与完整细胞（即未通透化的细胞）孵育，或将几种非特异性抗体/试剂（如山羊抗小鼠IgG、小鼠血清、人血小板糖蛋白Ib单克隆抗体和胎牛血清）电导入血小板，对PAF反应均无影响。trpE（pp60v-src的另一种同型匹配对照抗体）和pp56lck多克隆抗体（血小板中不存在的另一种src相关激酶）对通透化血小板中PAF诱导的聚集和肌醇磷酸生成也无影响。这表明内化的pp60v-src抗体在血小板中的作用是直接且特异的。PAF刺激血小板增加了pp60c-src（60 kDa）和磷脂酶Cγ1（145 kDa）的结合及磷酸化。本研究首次在血小板中提供了证据，证明pp60c-src直接且特异性地参与PAF介导的磷脂酶C激活和聚集反应。
