Bizios R, Lai L C, Cooper J A, Del Vecchio P J, Malik A B
Department of Physiology, Albany Medical College of Union University, New York 12208.
J Cell Physiol. 1988 Feb;134(2):275-80. doi: 10.1002/jcp.1041340214.
We examined the effects of alpha-thrombin on the adherence of neutrophils to endothelial cell monolayers. Endothelial cells derived from the ovine pulmonary artery and ovine neutrophils were used. Thrombin (10(-8) M) resulted in a time-dependent increase in neutrophil adherence to the endothelium. The response was concentration-dependent with a maximal response at 10(-8) M. Thrombin did not induce neutrophil adherence either to plastic or to endothelial cell-derived matrix. The adherence response was inhibited in the presence of alpha-thrombin that had been inactivated with anti-thrombin III (1U:1U) or with hirudin (1 U/ml). However, the addition of either anti-thrombin III or hirudin simultaneously with alpha-thrombin to the cultured endothelial monolayers did not prevent neutrophil adherence. The monoclonal antibody MoAb 60.3, which precipitates a complex of four neutrophil surface glycoproteins (CDw18) was used to further characterize the reaction. MoAb 60.3 decreased the thrombin-induced adherence of neutrophils to the endothelial monolayer. Addition of 10(-8) M thrombin to the endothelial monolayer for 60 min, followed by washing the endothelium with fresh medium, caused resting neutrophils to adhere to the endothelial monolayers. MoAb 60.3 decreased neutrophil adherence to the washed endothelium. The factor(s) responsible for adherence was partially transferable. Medium obtained from incubating endothelial monolayers with thrombin (10(-8) M) for 60 min, adding hirudin to the medium to inactivate thrombin, and transferring it to untreated endothelial monolayers, elicited neutrophil adherence. The response was less than that obtained with thrombin alone (22.9 +/- 2.3% vs. 12.9 +/- 3.3%). The results indicate that the catalytic site of the thrombin molecule is responsible for the adherent activity. Thrombin elicits a rapid activation of endothelial cells with a response that involves the expression of endothelial adhesion sites and sites that interact with the neutrophil CDw18 adhesive glycoprotein complex. In addition, soluble transferable factor(s) which are generated by the endothelium also contribute to thrombin-induced neutrophil adherence.
我们研究了α-凝血酶对中性粒细胞与内皮细胞单层黏附的影响。使用了源自绵羊肺动脉的内皮细胞和绵羊中性粒细胞。凝血酶（10⁻⁸ M）导致中性粒细胞与内皮细胞的黏附呈时间依赖性增加。该反应呈浓度依赖性，在10⁻⁸ M时达到最大反应。凝血酶既不诱导中性粒细胞黏附于塑料表面，也不诱导其黏附于内皮细胞衍生的基质。在存在用抗凝血酶III（1U:1U）或水蛭素（1 U/ml）灭活的α-凝血酶的情况下，黏附反应受到抑制。然而，将抗凝血酶III或水蛭素与α-凝血酶同时添加到培养的内皮细胞单层中并不能阻止中性粒细胞黏附。单克隆抗体MoAb 60.3可沉淀四种中性粒细胞表面糖蛋白（CDw18）的复合物，用于进一步表征该反应。MoAb 60.3降低了凝血酶诱导的中性粒细胞与内皮细胞单层的黏附。向内皮细胞单层中添加10⁻⁸ M凝血酶60分钟，然后用新鲜培养基洗涤内皮细胞，导致静息中性粒细胞黏附于内皮细胞单层。MoAb 60.3降低了中性粒细胞对洗涤后内皮细胞的黏附。负责黏附的因子具有部分可转移性。将内皮细胞单层与凝血酶（10⁻⁸ M）孵育60分钟，向培养基中添加水蛭素以灭活凝血酶，然后将其转移至未处理的内皮细胞单层，所获得的培养基可引发中性粒细胞黏附。该反应小于单独使用凝血酶时获得的反应（22.9±2.3%对12.9±3.3%）。结果表明，凝血酶分子的催化位点负责黏附活性。凝血酶引发内皮细胞的快速激活，其反应涉及内皮黏附位点以及与中性粒细胞CDw18黏附糖蛋白复合物相互作用的位点的表达。此外，内皮细胞产生的可溶性可转移因子也有助于凝血酶诱导的中性粒细胞黏附。
