Chignard M, Selak M A, Smith J B
Proc Natl Acad Sci U S A. 1986 Nov;83(22):8609-13. doi: 10.1073/pnas.83.22.8609.
Human neutrophils and platelets were loaded with the intracellular calcium indicator fura-2. The chemotactic peptide N-formyl-Met-Leu-Phe (fMet-Leu-Phe) induced a rapid elevation of cytosolic free calcium in cytochalasin B-treated neutrophils but failed to increase the cytosolic calcium in platelets. On the other hand, when unloaded neutrophils were incubated together with autologous fura-2-loaded platelets, fMet-Leu-Phe stimulated a 6-fold increase in platelet cytosolic calcium subsequent to a brief lag. Parallel experiments demonstrated that the addition of fMet-Leu-Phe to neutrophil/platelet incubates also elicited platelet aggregation and serotonin release. Platelet activation showed a positive correlation with the concentration of fMet-Leu-Phe added to the mixed cell population. Cell-free supernatants prepared from fMet-Leu-Phe-stimulated neutrophils were capable of inducing platelet calcium mobilization, aggregation, and secretion. The amount of platelet-activating material present in the supernatant was proportional to the number of activated neutrophils. Preincubation of platelets with BN 52021, acetylsalicylic acid, SQ-29,548, or hirudin did not modify the aggregation response induced by the supernatant collected from fMet-Leu-Phe-activated neutrophils, suggesting that the material was not 1-alkyl-2-acetyl-sn-glycero-3-phosphocholine (paf-acether), arachidonic acid, thromboxane A2, or thrombin. Pretreatment of the neutrophil supernatant with an ADP (creatine phosphate/creatine phosphokinase) or a superoxide/peroxide (superoxide dismutase/catalase) scavenging system also had no effect on aggregation or secretion, indicating that these substances did not participate in platelet activation. The biological activity present in the neutrophil supernatant was destroyed by heat and inactivated by treatment with phenylmethylsulfonyl fluoride, indicating that it is a protein and most probably an enzyme with serine protease activity. These data provide the direct observation of secondary signal transmission to platelets following primary activation of neutrophils. We propose the name neutrophilin for the neutrophil-derived mediator.
将人中性粒细胞和血小板用细胞内钙指示剂fura-2进行负载。趋化肽N-甲酰基-甲硫氨酰-亮氨酰-苯丙氨酸（fMet-Leu-Phe）可使经细胞松弛素B处理的中性粒细胞胞质游离钙迅速升高，但未能使血小板胞质钙增加。另一方面，当未负载的中性粒细胞与自体fura-2负载的血小板一起孵育时，fMet-Leu-Phe在短暂延迟后刺激血小板胞质钙增加了6倍。平行实验表明，向中性粒细胞/血小板孵育物中添加fMet-Leu-Phe也会引发血小板聚集和5-羟色胺释放。血小板活化与添加到混合细胞群体中的fMet-Leu-Phe浓度呈正相关。由fMet-Leu-Phe刺激的中性粒细胞制备的无细胞上清液能够诱导血小板钙动员、聚集和分泌。上清液中存在的血小板活化物质的量与活化中性粒细胞的数量成正比。血小板与BN 52021、乙酰水杨酸、SQ-29548或水蛭素预孵育不会改变从fMet-Leu-Phe活化的中性粒细胞收集的上清液诱导的聚集反应，这表明该物质不是1-烷基-2-乙酰基-sn-甘油-3-磷酸胆碱（血小板活化因子）、花生四烯酸、血栓素A2或凝血酶。用ADP（磷酸肌酸/磷酸肌酸激酶）或超氧化物/过氧化物（超氧化物歧化酶/过氧化氢酶）清除系统预处理中性粒细胞上清液对聚集或分泌也没有影响，表明这些物质不参与血小板活化。中性粒细胞上清液中的生物活性被加热破坏，并用苯甲基磺酰氟处理使其失活，表明它是一种蛋白质，很可能是一种具有丝氨酸蛋白酶活性的酶。这些数据提供了对中性粒细胞初次活化后向血小板的二次信号传递的直接观察。我们提议将中性粒细胞衍生的介质命名为嗜中性粒细胞素。
