Sugama Y, Tiruppathi C, offakidevi K, Andersen T T, Fenton J W, Malik A B
Department of Physiology, Albany Medical College, New York 12208.
J Cell Biol. 1992 Nov;119(4):935-44. doi: 10.1083/jcb.119.4.935.
Thrombin-induced expression of endothelial adhesivity toward neutrophils (PMN) was studied using human umbilical vein endothelial cells (HUVEC). HUVEC were challenged with human alpha-thrombin for varying durations up to 120 min, after which the cells were fixed with 1% paraformaldehyde and 51Cr-labeled human PMN were added to determine PMN adhesion. Endothelial adhesivity increased within 15 min after alpha-thrombin exposure, and the response persisted up to 120 min. Expression of endothelial adhesion proteins, P-selectin (GMP-140, PADGEM, CD62), and intercellular adhesion molecule-1 (ICAM-1; CD54) on the endothelial surface was quantitated by increase in the specific binding of anti-P-selectin mAb G1 and anti-ICAM-1 mAb RR1/1 labeled with 125I. P-selectin expression was maximal at 5-15 min alpha-thrombin exposure and decayed to basal levels within 90 min. In contrast, ICAM-1 activity increased at 30 min and remained elevated for 120 min after alpha-thrombin challenge. The initial endothelial adhesivity was dependent on P-selectin expression since PMN adhesion occurring within the first 30 min after alpha-thrombin challenge was inhibited by mAb G1. The later prolonged PMN adhesion was ICAM-1 dependent since this response was inhibited by mAb RR1/1 and to the same degree by the anti-CD18 mAb IB4. Anti-ELAM-1 mAb BB11 had no effect on adhesion of PMN to the alpha-thrombin-challenged cells. The initial P-selectin expression and PMN adhesion responses were reproduced by the 14-amino peptide (SFLLRNPNDKYEPF) (thrombin-receptor activity peptide; TRP-14) which comprised the NH2 terminus created by thrombin's proteolytic action on its receptors. However, TRP-14-induced PMN adhesion was transient, and TRP-14 did not cause ICAM-1 expression. The ICAM-1-dependent PMN adhesion mediated by alpha-thrombin was protein synthesis independent since ICAM-1 expression and PMN adhesion were not inhibited by cycloheximide pretreatment of HUVEC. Moreover, Northern blot analysis indicated absence of ICAM-1 mRNA signal up to 180 min after alpha-thrombin challenge. In conclusion, thrombin-induced endothelial adhesivity involves early- and late-phase responses. The initial reversible PMN adhesion is mediated by rapid P-selectin expression via TRP-14 generation. Thrombin-induced PMN adhesion is stabilized by a protein synthesis-independent upregulation of the constitutive ICAM-1 activity which enables the interaction of ICAM-1 with the CD18 beta 2 integrin on PMN.
利用人脐静脉内皮细胞（HUVEC）研究了凝血酶诱导的内皮细胞对中性粒细胞（PMN）黏附性的表达。用人类α-凝血酶对HUVEC进行长达120分钟的不同时长刺激，之后用1%多聚甲醛固定细胞，并加入51Cr标记的人类PMN以测定PMN黏附情况。α-凝血酶暴露后15分钟内内皮细胞黏附性增加，且该反应持续至120分钟。通过增加用125I标记的抗P-选择素单克隆抗体G1和抗细胞间黏附分子-1（ICAM-1；CD54）单克隆抗体RR1/1的特异性结合来定量内皮细胞表面内皮黏附蛋白P-选择素（GMP-140、PADGEM、CD62）和细胞间黏附分子-1的表达。P-选择素表达在α-凝血酶暴露5 - 15分钟时达到最大值，并在90分钟内衰减至基础水平。相比之下，ICAM-1活性在30分钟时增加，并在α-凝血酶刺激后120分钟内保持升高。最初的内皮细胞黏附性依赖于P-选择素的表达，因为α-凝血酶刺激后最初30分钟内发生的PMN黏附受到单克隆抗体G1的抑制。随后延长的PMN黏附依赖于ICAM-1，因为该反应受到单克隆抗体RR1/1的抑制，且抗CD18单克隆抗体IB4对其抑制程度相同。抗内皮白细胞黏附分子-1单克隆抗体BB11对PMN与α-凝血酶刺激细胞的黏附没有影响。由14个氨基酸组成的肽（SFLLRNPNDKYEPF）（凝血酶受体活性肽；TRP-14）可重现最初的P-选择素表达和PMN黏附反应，该肽包含凝血酶对其受体进行蛋白水解作用产生的NH2末端。然而，TRP-14诱导的PMN黏附是短暂的，且TRP-14不会引起ICAM-1表达。α-凝血酶介导的依赖于ICAM-1的PMN黏附与蛋白质合成无关，因为HUVEC经环己酰亚胺预处理后ICAM-1表达和PMN黏附并未受到抑制。此外，Northern印迹分析表明，α-凝血酶刺激后长达180分钟均未检测到ICAM-1 mRNA信号。总之，凝血酶诱导的内皮细胞黏附涉及早期和晚期反应。最初的可逆性PMN黏附由通过TRP-14生成快速表达P-选择素介导。凝血酶诱导的PMN黏附通过组成型ICAM-1活性的蛋白质合成非依赖性上调得以稳定，这使得ICAM-1与PMN上的CD18β2整合素相互作用。
