Human plasma kallikrein releases neutrophil elastase during blood coagulation.
Human plasma kallikrein releases neutrophil elastase during blood coagulation.
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DOI：
10.1172/jci111126
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发表时间：
1983-11
期刊：
影响因子：
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通讯作者：
Y. Wachtfogel;U. Kucich;H. James;C. Scott;M. Schapira;M. Zimmerman;A. Cohen;R. Colman
中科院分区：
文献类型：
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作者：
Y. Wachtfogel;U. Kucich;H. James;C. Scott;M. Schapira;M. Zimmerman;A. Cohen;R. Colman
在血液凝固的早期事件中，人类中性粒细胞释放弹性蛋白酶。人血浆激肽释放酶已被证明可以刺激中性粒细胞趋化、聚集和耗氧。因此，研究了激肽释放酶释放中性粒细胞弹性蛋白酶的能力。通过葡聚糖沉降分离中性粒细胞，并通过酶联免疫吸附测定和使用叔丁氧基-羰基-Ala-Ala-Pro-Val-氨基甲基香豆素作为底物的酶测定测量弹性蛋白酶释放。将激肽释放酶，0.1-1.0 U/ml（0.045-0.45 µM）与用细胞松弛素 B（5 µg/ml）预孵育的中性粒细胞一起孵育。发现弹性蛋白酶的释放与激肽释放酶浓度成正比。通过在非离子去污剂 Triton X-100 中溶解中性粒细胞来测量，激肽释放酶最多释放总弹性蛋白酶含量的 34%。进行了一系列实验以确定激肽释放酶是否是参与血液凝固过程中中性粒细胞弹性蛋白酶释放的主要酶。当 1000 万个中性粒细胞在 1 ml 正常血浆中、30 mM CaCl2 存在下孵育 90 分钟时，释放出 2.75 微克弹性蛋白酶。相反，与正常血浆相比，在缺乏前激肽释放酶或缺乏因子XII的血浆中孵育的中性粒细胞释放的弹性蛋白酶不到一半。向缺乏前激肽释放酶的血浆中添加纯化的前激肽释放酶可使中性粒细胞弹性蛋白酶释放恢复至正常水平。此外，在缺乏 C1 抑制剂（激肽释放酶的主要血浆抑制剂）的血浆中，弹性蛋白酶的释放增强。这种释放不依赖于凝血途径中的进一步步骤或C5a，因为在缺乏因子XI或C5的血浆中释放的弹性蛋白酶的水平与正常血浆中的相似，并且C5的抗体不能抑制弹性蛋白酶的释放。这些数据表明激肽释放酶可能是血液凝固过程中负责释放弹性蛋白酶的主要酶。
Elastase is released from human neutrophils during the early events of blood coagulation. Human plasma kallikrein has been shown to stimulate neutrophil chemotaxis, aggregation, and oxygen consumption. Therefore, the ability of kallikrein to release neutrophil elastase was investigated. Neutrophils were isolated by dextran sedimentation, and elastase release was measured by both an enzyme-linked immunosorbent assay, and an enzymatic assay using t-butoxy-carbonyl-Ala-Ala-Pro-Val-amino methyl coumarin as the substrate. Kallikrein, 0.1-1.0 U/ml, (0.045-0.45 microM), was incubated with neutrophils that were preincubated with cytochalasin B (5 micrograms/ml). The release of elastase was found to be proportional to the kallikrein concentration. Kallikrein released a maximum of 34% of the total elastase content, as measured by solubilizing the neutrophils in the nonionic detergent Triton X-100. A series of experiments was carried out to determine if kallikrein was a major enzyme involved in neutrophil elastase release during blood coagulation. When 10 million neutrophils were incubated in 1 ml of normal plasma in the presence of 30 mM CaCl2 for 90 min, 2.75 micrograms of elastase was released. In contrast, neutrophils incubated in prekallikrein-deficient or Factor XII-deficient plasma released less than half of the elastase, as compared with normal plasma. The addition of purified prekallikrein to prekallikrein-deficient plasma restored neutrophil elastase release to normal levels. Moreover, release of elastase was enhanced in plasma deficient in C1-inhibitor, the major plasma inhibitor of kallikrein. This release was not dependent upon further steps in the coagulation pathway, or on C5a, since levels of elastase, released in Factor XI- or C5-deficient plasma, were similar to that in normal plasma, and an antibody to C5 failed to inhibit elastase release. These data suggest that kallikrein may be a major enzyme responsible for the release of elastase during blood coagulation.