Dunlop L C, Skinner M P, Bendall L J, Favaloro E J, Castaldi P A, Gorman J J, Gamble J R, Vadas M A, Berndt M C
Baker Medical Research Institute, Prahran, Victoria, Australia.
J Exp Med. 1992 Apr 1;175(4):1147-50. doi: 10.1084/jem.175.4.1147.
GMP-140 is a 140-kD granule membrane protein, found in the alpha granules of platelets and the Weibel-Palade bodies of endothelial cells, that is surface expressed on cell activation and mediates neutrophil attachment. Cloning data for GMP-140 from an endothelial library predict a soluble form of the protein, the transcription message for which is also found in platelets. In this study, we report the detection by enzyme-linked immunosorbent assay of soluble GMP-140 in plasma centrifuged for 3 h at 100,000 g (to remove platelet microparticles) and confirm its identity by purification from plasma. Plasma concentrations were found to be 0.251 +/- 0.043 micrograms/ml (means +/- SD, n = 10) in normal male controls and 0.175 +/- 0.063 micrograms/ml (means +/- SD, n = 10) in normal female controls. The purified protein had an identical molecular mass (nonreduced) to platelet membrane GMP-140 (approximately 3 kD lower, reduced) and was immunoblotted by polyclonal anti-GMP-140, and the anti-GMP-140 monoclonal antibodies AK4 and AK6. Analytical gel filtration studies indicated that the plasma GMP-140 eluted as a monomer whereas detergent-free, platelet membrane GMP-140 eluted as a tetramer consistent with plasma GMP-140 lacking a transmembrane domain. Purified plasma GMP-140 bound to the same neutrophil receptor as the membrane-bound form, and when immobilized on plastic, bound neutrophils equivalently to immobilized platelet membrane GMP-140. Since it has been shown that fluid-phase GMP-140 is antiinflammatory and downregulates CD18-dependent neutrophil adhesion and respiratory burst, its presence in plasma may be of major importance in preventing the inadvertent activation of neutrophils in the circulation.
GMP - 140是一种140千道尔顿的颗粒膜蛋白，存在于血小板的α颗粒和内皮细胞的Weibel - Palade小体中，在细胞活化时在表面表达并介导中性粒细胞附着。来自内皮细胞文库的GMP - 140克隆数据预测该蛋白存在一种可溶性形式，其转录信息在血小板中也能找到。在本研究中，我们报告了通过酶联免疫吸附测定法在以100,000克离心3小时（以去除血小板微粒）的血浆中检测到可溶性GMP - 140，并通过从血浆中纯化来确认其身份。正常男性对照组血浆浓度为0.251±0.043微克/毫升（平均值±标准差，n = 10），正常女性对照组为0.175±0.063微克/毫升（平均值±标准差，n = 10）。纯化后的蛋白与血小板膜GMP - 140具有相同的分子量（非还原状态）（还原状态下约低3千道尔顿），并且能被多克隆抗GMP - 140以及抗GMP - 140单克隆抗体AK4和AK6免疫印迹。分析性凝胶过滤研究表明，血浆中的GMP - 140以单体形式洗脱，而无去污剂的血小板膜GMP - 140以四聚体形式洗脱，这与血浆中的GMP - 140缺乏跨膜结构域一致。纯化后的血浆GMP - 140与膜结合形式的GMP - 140结合相同的中性粒细胞受体，并且固定在塑料上时，与固定的血小板膜GMP - 140等效地结合中性粒细胞。由于已经表明液相GMP - 140具有抗炎作用并下调CD18依赖性中性粒细胞黏附和呼吸爆发，其在血浆中的存在对于防止循环中中性粒细胞的意外激活可能具有重要意义。
