Dackiw A P, Grinstein S, Brisseau G F, McGilvray I D, Nathens A B, McGuire J A, Romanek R, Cheung P Y, Rotstein O D
Department of Surgery, Toronto Hospital and the University of Toronto, Ontario, Canada.
Infect Immun. 1997 Jun;65(6):2362-70. doi: 10.1128/iai.65.6.2362-2370.1997.
The expression of surface procoagulants by exudative macrophages represents an important mechanism underlying local fibrin deposition at sites of extravascular inflammation. The present studies investigated the contribution of tyrosine phosphorylation to the generation of macrophage procoagulant activity (PCA) and tissue factor expression in response to proinflammatory stimuli. Both lipopolysaccharide (LPS) and zymosan rapidly stimulated tyrosine phosphorylation in elicited murine peritoneal macrophages. This effect was prevented by the tyrosine kinase inhibitors genistein and herbimycin and augmented by the addition of the phosphotyrosine phosphatase inhibitor vanadate. The vanadate-mediated rise in phosphotyrosine accumulation was abrogated by the use of diphenylene iodonium, an inhibitor of the respiratory burst oxidase, suggesting a role for peroxides of vanadate as contributors to the tyrosine phosphorylation. This notion was supported by the finding that vanadyl hydroperoxide markedly increased the accumulation of phosphotyrosine residues. To define the role of tyrosine phosphorylation in the induction of macrophage PCA by LPS, the effects of tyrosine kinase inhibition by genistein and herbimycin were investigated. Both agents inhibited the expression of macrophage PCA. Further, Northern blot analysis with the cDNA probe for murine tissue factor indicated that the inhibition occurred at the mRNA level or earlier. Since vanadate augmented phosphotyrosine accumulation, it was hypothesized that it might enhance generation of macrophage products. However, vanadate reduced induction of PCA in response to LPS. By contrast, vanadate augmented basal prostaglandin E2 (PGE2) release and stimulated PGE2 release by macrophages. Indomethacin prevented the increase in PGE2 but only partially restored normal levels of PCA. The effect of vanadate on tissue factor expression appeared to be posttranscriptional. These studies thus demonstrate, by functional Western blotting and Northern blotting techniques, that tyrosine phosphorylation plays a role in the regulation of macrophage PCA and tissue factor expression in response to proinflammatory stimuli.
渗出性巨噬细胞表面促凝血剂的表达是血管外炎症部位局部纤维蛋白沉积的重要机制。本研究调查了酪氨酸磷酸化对巨噬细胞促凝血活性（PCA）生成及对促炎刺激反应中组织因子表达的作用。脂多糖（LPS）和酵母聚糖均可迅速刺激诱导的小鼠腹腔巨噬细胞中的酪氨酸磷酸化。酪氨酸激酶抑制剂染料木黄酮和除莠霉素可阻止这种效应，而磷酸酪氨酸磷酸酶抑制剂钒酸盐则可增强该效应。使用呼吸爆发氧化酶抑制剂二亚苯基碘鎓可消除钒酸盐介导的磷酸酪氨酸积累增加，这表明钒酸盐的过氧化物在酪氨酸磷酸化过程中发挥作用。过氧化钒酰可显著增加磷酸酪氨酸残基的积累，这一发现支持了上述观点。为明确酪氨酸磷酸化在LPS诱导巨噬细胞PCA中的作用，研究了染料木黄酮和除莠霉素对酪氨酸激酶的抑制作用。这两种药物均抑制巨噬细胞PCA的表达。此外，用小鼠组织因子的cDNA探针进行的Northern印迹分析表明，这种抑制发生在mRNA水平或更早阶段。由于钒酸盐可增加磷酸酪氨酸积累，因此推测它可能增强巨噬细胞产物的生成。然而，钒酸盐可降低LPS诱导的PCA。相反，钒酸盐可增加基础前列腺素E2（PGE2）的释放，并刺激巨噬细胞释放PGE2。吲哚美辛可阻止PGE2的增加，但只能部分恢复PCA的正常水平。钒酸盐对组织因子表达的影响似乎是转录后水平的。因此，这些研究通过功能Western印迹和Northern印迹技术证明，酪氨酸磷酸化在促炎刺激反应中对巨噬细胞PCA和组织因子表达的调节中发挥作用。
