Renesto P, Ferrer-Lopez P, Chignard M
Unité de Pharmacologie cellulaire, Unité associée IP/INSERM No. 285, Institut Pasteur, Paris, France.
Lab Invest. 1990 Apr;62(4):409-16.
Eglin C is an inhibitor of two serine proteinase-derived polymorphonuclear leucocytes (PMN) i.e., elastase and cathepsin G. Since the latter has recently been shown to be involved in the activation of platelets by stimulated PMN, the effects of recombinant eglin C in the PMN-platelet cooperation model were studied. First, the inhibitory capacity of eglin C against purified cathepsin G was measured spectrophotometrically by following hydrolysis of a specific synthetic substrate, N-succinyl-ala-ala-pro-phe-para-nitroanilide. The inhibition of the enzymatic activity of 180 nM (5 micrograms/ml) cathepsin G was directly proportional to eglin C concentration and reached 100% with 2 micrograms/ml (240 nM). Platelet activation generated by a submaximal concentration of cathepsin G (200 nM) was also totally suppressed by 2 micrograms/ml of eglin C. Inhibition was specific (a 100 times higher concentration of eglin C did not alter platelet activation induced by thrombin), and surmountable (an increase of cathepsin G concentration reduced the eglin C effect). Thus, the mechanism of inhibition by eglin C of cathepsin G-induced platelet activation could be explained by a stoichiometric relation between eglin C and cathepsin G as previously described. Investigations were then performed with the PMN-platelet cooperation model, using two distinct stimuli, N-formylmethionylleucylphenylalanine (FMLP) or recombinant human C5a, at submaximal concentrations, 2.10(-7) M and 10(-7) M, respectively. A concentration-dependent inhibition of platelet activation aggregation and serotonin release-lambda was observed. Eglin C used at 10 micrograms/ml and 25 micrograms/ml totally blocked the platelet responses induced by recombinant human C5a and FMLP, respectively. Leucotriene B4, but also thromboxane B2 production measured by radioimmunoassays, were observed under FMLP activation. In the presence of eglin C, thromboxane B2 formation was totally suppressed, whereas leucotriene B4 synthesis was still effective. In fact, the mechanism of inhibition of eglin C is located neither on PMN (leucotriene B4 formation by FMLP-activated PMN was not affected), nor on platelets (response to thrombin was unchanged). The target is most probably cathepsin G since eglin C suppressed thromboxane B2 formation by platelets challenged by this serine proteinase. These results constitute an argument in favor of the implication of cathepsin G in the PMN-mediated platelet activation. Moreover, they reinforce the hypothesis that this mechanism could be operating under in vivo pathologic conditions, since eglin C is capable of preventing or ameliorating some experimental pulmonary diseases.
埃格林C是两种丝氨酸蛋白酶衍生的多形核白细胞（PMN）即弹性蛋白酶和组织蛋白酶G的抑制剂。由于最近发现后者参与了受刺激的PMN对血小板的激活作用，因此研究了重组埃格林C在PMN - 血小板相互作用模型中的作用。首先，通过跟踪特定合成底物N - 琥珀酰 - 丙氨酸 - 丙氨酸 - 脯氨酸 - 苯丙氨酸 - 对硝基苯胺的水解，用分光光度法测定埃格林C对纯化的组织蛋白酶G的抑制能力。180 nM（5微克/毫升）组织蛋白酶G的酶活性抑制与埃格林C浓度成正比，在2微克/毫升（240 nM）时达到100%。2微克/毫升的埃格林C也完全抑制了亚最大浓度（200 nM）的组织蛋白酶G所产生的血小板激活。抑制作用具有特异性（埃格林C浓度高出100倍时不会改变凝血酶诱导的血小板激活），且是可克服的（组织蛋白酶G浓度增加会降低埃格林C的作用）。因此，埃格林C抑制组织蛋白酶G诱导的血小板激活的机制可以用先前描述的埃格林C与组织蛋白酶G之间的化学计量关系来解释。然后使用PMN - 血小板相互作用模型进行研究，分别使用两种不同的刺激物，亚最大浓度的N - 甲酰甲硫氨酰亮氨酰苯丙氨酸（FMLP）或重组人C5a，浓度分别为2×10⁻⁷ M和10⁻⁷ M。观察到血小板激活聚集和5 - 羟色胺释放 - λ呈浓度依赖性抑制。10微克/毫升和25微克/毫升的埃格林C分别完全阻断了重组人C5a和FMLP诱导产生的血小板反应。在FMLP激活下观察到了白三烯B4以及通过放射免疫测定法测定的血栓素B2的产生。在存在埃格林C的情况下，血栓素B2的形成被完全抑制，而白三烯B4的合成仍然有效。实际上，埃格林C的抑制机制既不位于PMN上（FMLP激活的PMN形成白三烯B4不受影响），也不位于血小板上（对凝血酶的反应未改变）。最有可能的靶点是组织蛋白酶G，因为埃格林C抑制了受这种丝氨酸蛋白酶刺激的血小板中血栓素B2的形成。这些结果支持了组织蛋白酶G参与PMN介导的血小板激活的观点。此外，它们强化了这样一种假设，即这种机制可能在体内病理条件下起作用，因为埃格林C能够预防或改善一些实验性肺部疾病。
