Selak M A, Chignard M, Smith J B
Department of Pharmacology, Temple University Medical School, Philadelphia, PA 19140.
Biochem J. 1988 Apr 1;251(1):293-9. doi: 10.1042/bj2510293.
The present studies were undertaken to characterize a serine protease released by N-formyl-L-Met-L-Leu-L-Phe (fMet-Leu-Phe)-stimulated neutrophils that rapidly induces platelet calcium mobilization, secretion and aggregation. The biological activity associated with this protease was unaffected by leupeptin, was only weakly diminished by N-p-tosyl-L-Lys-chloromethane, but was strongly inhibited by alpha 1-antitrypsin, soyabean trypsin inhibitor, N-tosyl-L-Phe-chloromethane and benzoyloxycarbonyl-Gly-Leu-Phe-chloromethane (Z-Gly-Leu-PheCH2Cl). These observations indicated that the biological activity of neutrophil supernatants could be attributed to a chymotrypsin-like enzyme such as cathepsin G. Furthermore, platelet aggregation and 5-hydroxytryptamine release induced by cell-free supernatants from fMet-Leu-Phe-stimulated neutrophils were found to be blocked by antiserum to cathepsin G in a concentration-dependent manner but were unaffected by antiserum to elastase. The biological activity present in neutrophil supernatants co-purified with enzymic activity for cathepsin G during sequential Aprotinin-Sepharose affinity chromatography and carboxymethyl-Sephadex chromatography. SDS/polyacrylamide-gel electrophoresis of the reduced, purified protein, demonstrated three polypeptides with apparent Mr values of 31,500, 29,000 and 28,000 and four polypeptides were resolved on acid-gel electrophoresis. Purified cathepsin G from neutrophils cross-reacted with anti-(cathepsin G) serum in a double immunodiffusion assay and elicited platelet calcium mobilization, 5-hydroxytryptamine secretion and aggregation. Calcium mobilization and secretion induced by low concentrations of cathepsin G were partially dependent on arachidonic acid metabolites and ADP, while stimulation by higher enzyme concentrations was independent of amplification pathways, indicating that cathepsin G is a strong platelet agonist. These results suggest that pathological processes which stimulate neutrophils and release cathepsin G can in turn result in the recruitment and activation of platelets.
本研究旨在鉴定由N-甲酰-L-蛋氨酰-L-亮氨酰-L-苯丙氨酸（fMet-Leu-Phe）刺激的中性粒细胞释放的一种丝氨酸蛋白酶，该蛋白酶能迅速诱导血小板钙动员、分泌和聚集。与该蛋白酶相关的生物活性不受亮抑酶肽影响，仅被N-对甲苯磺酰-L-赖氨酸氯甲基酮轻微减弱，但被α1-抗胰蛋白酶、大豆胰蛋白酶抑制剂、N-对甲苯磺酰-L-苯丙氨酸氯甲基酮和苄氧羰基-Gly-Leu-Phe-氯甲基酮（Z-Gly-Leu-PheCH2Cl）强烈抑制。这些观察结果表明，中性粒细胞上清液的生物活性可归因于一种类似胰凝乳蛋白酶的酶，如组织蛋白酶G。此外，发现fMet-Leu-Phe刺激的中性粒细胞的无细胞上清液诱导的血小板聚集和5-羟色胺释放被抗组织蛋白酶G血清以浓度依赖的方式阻断，但不受抗弹性蛋白酶血清的影响。在抑肽酶-琼脂糖亲和层析和羧甲基-葡聚糖凝胶层析过程中，中性粒细胞上清液中存在的生物活性与组织蛋白酶G的酶活性共纯化。还原的纯化蛋白的SDS/聚丙烯酰胺凝胶电泳显示出三条表观分子量分别为31,500、29,000和28,000的多肽，在酸性凝胶电泳上分离出四条多肽。从中性粒细胞纯化的组织蛋白酶G在双向免疫扩散试验中与抗（组织蛋白酶G）血清发生交叉反应，并引起血小板钙动员、5-羟色胺分泌和聚集。低浓度组织蛋白酶G诱导的钙动员和分泌部分依赖于花生四烯酸代谢产物和ADP，而较高酶浓度的刺激则不依赖于放大途径，表明组织蛋白酶G是一种强血小板激动剂。这些结果表明，刺激中性粒细胞并释放组织蛋白酶G的病理过程反过来可导致血小板的募集和激活。
