Human neutrophils synthesize thrombomodulin that does not promote thrombin-dependent protein C activation.
Human neutrophils synthesize thrombomodulin that does not promote thrombin-dependent protein C activation.
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人类中性粒细胞合成的血栓调节蛋白不会促进凝血酶依赖性蛋白 C 的激活。
作者：
Edward M. Conway;Barbara E. Nowakowski;M. Steiner‐Mosonyi
血栓调节蛋白（TM）是一种表面糖蛋白，与凝血酶形成1：1的复合物，从而相互作用形成主要生理相关的天然抗凝机制的基础。虽然最初被描述为血管内皮细胞受体，但据报道TM存在于其他几种细胞中，包括巨核细胞、血小板、单核细胞和几种培养细胞。其他研究者报道，中性粒细胞（PMN）可能通过支持凝血酶原转化为凝血酶而在止血机制中发挥作用。为了确定PMN是否可能进一步促进凝血系统的调节，我们评估了这些细胞的TM表达。通过标准技术分离大量人白细胞，并提取PMN部分，显示不含血小板和单核细胞。在抗-TM-Affigel-10基质上亲和纯化膜制备物，并通过Western印迹、十二烷基硫酸钠-聚丙烯酰胺凝胶电泳和银染色检查洗脱的材料。纯化的材料在表观分子量上与来自人胎盘和人脐静脉内皮细胞（HUVEC）的TM相同。使用灵敏和特异的免疫测定，我们估计每个PMN至少有5，220 +/-1，658个TM分子，而HUVEC中有超过50，000个。对中性粒细胞RNA的北方分析表明，TM的特异性信使RNA（由单一3.8 kb条带鉴定）与HUVEC的相同，从而证实中性粒细胞也可以合成该受体。TM在中性粒细胞的本地化，试图通过免疫荧光，和受体是可视化的，只有在透化的中性粒细胞，但没有看到在nonpermeabilized细胞的表面上。流式细胞术也被使用，并可以检测TM在10%至15%的nonpermeabilized中性粒细胞，而抗原存在于大于80%的透性细胞。生物功能的PMN衍生TM，测试凝血酶依赖性激活蛋白C，是不存在的。我们的研究结果表明，TM是由中性粒细胞合成的，但在非刺激条件下，该蛋白质在很大程度上被排除在膜表面，缺乏促进凝血酶激活蛋白C的能力。来自PMN的TM可能提供炎症和血栓形成之间的进一步联系，也可能是血浆TM的重要来源。
Thrombomodulin (TM) is a surface glycoprotein that forms a 1:1 complex with thrombin, thereby interacting to form the basis of a major physiologically relevant natural anticoagulant mechanism. Although initially described as a vascular endothelial cell receptor, TM has been reported to be present in several other cells, including megakaryocytes, platelets, monocytes, and several cultured cells. Other investigators have reported that neutrophils (PMN) may play a role in the hemostatic mechanism by supporting transformation of prothrombin to thrombin. To determine whether PMN might contribute further to the regulation of the coagulation system, we have evaluated these cells for the expression of TM. Large numbers of human leukocytes were isolated by standard techniques, and the PMN fraction was extracted and shown to be free of platelets and monocytes. Membrane preparations were affinity purified on an anti-TM-Affigel-10 matrix and the eluted material was examined by Western blotting, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and silver staining. The purified material was identical in apparent molecular weight to TM from human placenta and human umbilical vein endothelial cells (HUVEC). Using a sensitive and specific immunoassay, we estimated that there are a minimum of 5,220 +/- 1,658 molecules of TM per PMN, as compared with more than 50,000 in HUVEC. Northern analysis of RNA from PMN indicates that specific messenger RNA for TM, as identified by a single 3.8-kb band, is identical to that from HUVEC, and thereby confirms that PMN can also synthesize the receptor. Localization of TM in PMN was attempted by immunofluorescence, and the receptor was visualized only in permeabilized PMN, but was not seen on the surface of nonpermeabilized cells. Flow cytometry was also used, and could detect TM in 10% to 15% of nonpermeabilized PMN, whereas the antigen was present in greater than 80% of permeabilized cells. Biologic function of the PMN-derived TM, as tested by thrombin-dependent activation of protein C, was absent. Our results suggest that TM is synthesized by PMN, but under nonstimulated conditions, the protein is largely excluded from the membrane surface, and lacks the ability to promote activation of protein C by thrombin. TM from PMN may provide a further link between inflammation and thrombosis and may also be a significant source of plasma TM.
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