Doré M, Hawkins H K, Entman M L, Smith C W
Speros P. Martel Laboratory of Leukocyte Biology, Department of Pediatrics, Baylor College of Medicine, Houston, TX.
Vet Pathol. 1993 May;30(3):213-22. doi: 10.1177/030098589303000301.
Rapid upregulation of the adhesion molecule GMP-140 (P-selectin) on endothelial cells is believed to play an important role in the initial binding of leukocytes to endothelium, a very early step in the inflammatory response. Activated platelets that are involved in the coagulation system and in inflammatory processes also express GMP-140 on their surfaces. The objectives of the present study were to develop a monoclonal antibody against this adhesion molecule in the dog and to use this antibody to study platelet-neutrophil interactions in whole blood and to characterize the in vivo localization of GMP-140 in canine tissues. Five Balb/c mice were immunized with thrombin-stimulated dog platelets, and clones were screened using an enzyme-linked immunosorbent assay. The clone MD3 (IgG1) showed preferential binding to activated as compared with resting platelets. Flow cytometric analysis using MD3 revealed that 27% of circulating neutrophils in unstimulated blood had platelets bound to their surfaces; stimulation with platelet activating factor increased this percentage to 85%. Immunoblot analysis of solubilized dog platelets resolved by sodium dodecyl sulfate polyacrylamide gel electrophoresis indicated that the antibody MD3 recognized an approximately 140-kd protein. Immunohistochemical study of normal dog tissues with MD3 revealed that the antigen was present in endothelial cells of arteries, capillaries, and veins, depending on the specific tissue examined. Blood vessels staining positively with MD3 were most abundant in the digestive system (liver, stomach, small and large intestines), moderate in the lungs, kidneys, spleen, lymph nodes, and endocrine glands, and minimal in the brain, myocardium, skeletal system, and skin. Based on its presence on stimulated but not resting platelets, its molecular weight, and its vascular distribution, the antigen recognized by MD3 appears to be the selectin GMP-140 of the dog. This study documents that the cellular and tissue distribution of GMP-140 in dogs is very similar to that in human beings.
内皮细胞上粘附分子GMP-140（P-选择素）的快速上调被认为在白细胞与内皮的初始结合中起重要作用，这是炎症反应中非常早期的一步。参与凝血系统和炎症过程的活化血小板在其表面也表达GMP-140。本研究的目的是制备针对犬类这种粘附分子的单克隆抗体，并使用该抗体研究全血中血小板与中性粒细胞的相互作用，以及确定GMP-140在犬类组织中的体内定位。用凝血酶刺激的犬血小板免疫5只Balb/c小鼠，并使用酶联免疫吸附测定法筛选克隆。与静息血小板相比，克隆MD3（IgG1）显示出对活化血小板的优先结合。使用MD3进行的流式细胞术分析显示，未刺激血液中27%的循环中性粒细胞表面有血小板结合；用血小板活化因子刺激后，这一比例增加到85%。通过十二烷基硫酸钠聚丙烯酰胺凝胶电泳分离的可溶性犬血小板的免疫印迹分析表明，抗体MD3识别一种约140-kd的蛋白质。用MD3对正常犬组织进行免疫组织化学研究表明，根据所检查的特定组织，抗原存在于动脉、毛细血管和静脉的内皮细胞中。MD3染色呈阳性的血管在消化系统（肝脏、胃、小肠和大肠）中最为丰富，在肺、肾、脾、淋巴结和内分泌腺中中等丰富，在脑、心肌、骨骼系统和皮肤中最少。基于其在活化而非静息血小板上的存在、分子量及其血管分布，MD3识别的抗原似乎是犬类的选择素GMP-140。这项研究证明，犬类中GMP-140的细胞和组织分布与人类非常相似。
