Grau E, Moroz L A
Harry Webster Thorp Laboratories, Division of Clinical Immunology, McGill University Clinic, Royal Victoria Hospital, Montreal, Quebec, Canada.
Thromb Res. 1989 Jan 15;53(2):145-62. doi: 10.1016/0049-3848(89)90375-7.
Neutrophils (PMN) are important in the cellular phase of blood fibrino lytic activity (FA). The contribution of monocytes (MC), which have FA, is unclear. To determine the relative roles of these cells to activity in normal blood, we examined, by solid phase radiofibrin assay, FA of normal blood and plasma, and of purified PMN and MC, with and without plasminogen (PLG), mini-plasminogen (mPLG), the other major elastase fragment of PLG, or autologous plasma. PMN alone (0.5 x 10(6)/ml) had striking activity (292 +/- 25 SEM ng fibrin lysed/h; n = 10 normal subjects) while MC alone (0.5 x 10(6)/ml) had mean FA of 32 +/- 4 ng/h, which could be accounted for by contaminating PMN (36 +/- 8 ng/h). Thus, in a 1 h assay (when cellular FA accounts for 70-80% of FA in whole blood), normal numbers of MC (0.5 x 10(6)/ml) had no detectable FA when assayed with PLG or normal plasma. With longer assay times (2-6 h), PLG-dependent (plasminogen activator, PA) activity was demonstrated with mixtures of MC and PLG or plasma. This PA activity was released into the medium and required prior contact of MC and an intact, soluble PLG molecule for PA activity to be detected in medium (suggesting a PLG-MC triggering mechanism), since activity was reduced or absent when MC were exposed to mPLG, the other major elastase fragment of PLG, or solid phase PLG. Exposure of MC to solid phase fibrin did not result in PA release. MC PA activity was little affected by cycloheximide pretreatment, indicating preformed rather than newly synthesized PA. By SDS-PAGE and fibrin zymography, MC extracts revealed a single PA band with features of pro-urokinase (single chain urinary-type PA): Mr 55,000, inhibition by antiurokinase antibody (but not by anti-tPA), and resistance to inhibition by DFP. By ELISA assay, approximate normal monocyte content of this PA (as Mr 55,000 urokinase) was 0.03 fg (3.3 x 10(8) molecules) per cell.
中性粒细胞（PMN）在血液纤维蛋白溶解活性（FA）的细胞阶段中起重要作用。具有纤维蛋白溶解活性的单核细胞（MC）的作用尚不清楚。为了确定这些细胞在正常血液中对活性的相对作用，我们通过固相放射纤维蛋白测定法，检测了正常血液和血浆、纯化的PMN和MC在有或无纤溶酶原（PLG）、微型纤溶酶原（mPLG，PLG的另一个主要弹性蛋白酶片段）或自体血浆情况下的纤维蛋白溶解活性。单独的PMN（0.5×10⁶/ml）具有显著活性（292±25 SEM ng纤维蛋白溶解/h；n = 10名正常受试者），而单独的MC（0.5×10⁶/ml）的平均纤维蛋白溶解活性为32±4 ng/h，这可能是由污染的PMN（36±8 ng/h）导致的。因此，在1小时的检测中（此时细胞纤维蛋白溶解活性占全血纤维蛋白溶解活性的70 - 80%），用PLG或正常血浆检测时，正常数量的MC（0.5×10⁶/ml）没有可检测到的纤维蛋白溶解活性。在更长的检测时间（2 - 6小时）下，MC与PLG或血浆的混合物表现出依赖PLG的（纤溶酶原激活物，PA）活性。这种PA活性释放到培养基中，并且需要MC与完整的可溶性PLG分子预先接触才能在培养基中检测到PA活性（提示一种PLG - MC触发机制），因为当MC暴露于mPLG（PLG的另一个主要弹性蛋白酶片段）或固相PLG时，活性降低或消失。MC暴露于固相纤维蛋白不会导致PA释放。MC的PA活性受环己酰亚胺预处理的影响很小，表明是预先形成的而非新合成的PA。通过SDS - PAGE和纤维蛋白凝块酶谱分析，MC提取物显示出一条具有尿激酶原（单链尿激酶型PA）特征的单一PA条带：分子量55,000，被抗尿激酶抗体抑制（但不被抗tPA抑制），并且对DFP抑制具有抗性。通过ELISA测定，每个细胞中这种PA（作为分子量55,000的尿激酶）的单核细胞含量约为正常水平，即0.03 fg（3.3×10⁸个分子）。
