van den Eijnden-Schrauwen Y, Atsma D E, Lupu F, de Vries R E, Kooistra T, Emeis J J
Gaubius Laboratory TNO-PG, Leiden, The Netherlands.
Arterioscler Thromb Vasc Biol. 1997 Oct;17(10):2177-87. doi: 10.1161/01.atv.17.10.2177.
In this study, we investigated the role of Ca2+ and G proteins in thrombin-induced acute release (regulated secretion) of tissue-type plasminogen activator (TPA) and von Willebrand factor (vWF), using a previously described system of primary human umbilical vein endothelial cells (HUVECs). The acute release of TPA and vWF, as induced by alpha-thrombin, was almost zero after chelation of Ca2+i, showing that an increase in [Ca2+]i was required. It did not matter whether the increase in [Ca2+]i came from an intracellular or extracellular Ca2+ source. Thrombin-induced release of TPA and vWF already started at low [Ca2+]i, around 100 nmol/L. Half-maximal release was found at a [Ca2+]i, of 261 nmol/L for TPA and at 222 nmol/L for vWF. The Ca2+ signal was transduced to calmodulin, as calmodulin inhibitors inhibited TPA and vWF release. The Ca2+ ionophore ionomycin dose dependently released vWF; half-maximal vWF release occurred at a [Ca2+]i of 311 nmol/L. In contrast, no TPA release was found at all below a [Ca2+]i of 500 nmol/L. Thus, below 500 nmol/L [Ca2+]i, an increase in [Ca2+]i alone was sufficient to induce vWF release but not sufficient to induce TPA release. Protein kinase C did not appear to be involved in TPA or vWF release, as neither an activator nor an inhibitor of protein kinase C significantly influenced release. Inhibition of phospholipase A2 also did not reduce thrombin-induced TPA and vWF release. The involvement of G proteins was studied by using both saponin-permeabilized and intact cells. GDP-beta-S, which inhibits heterotrimeric and small G proteins, significantly inhibited thrombin-induced vWF and TPA release from permeabilized cells. AlF-4, which activates heterotrimeric G proteins, induced TPA and vWF release in both intact and permeabilized HUVECs. Preincubation of HUVECs with pertussis toxin significantly inhibited thrombin-induced vWF release, due to inhibition of thrombin-induced Ca2+ influx. Pertussis toxin did not affect ionomycin-induced release. The inhibitory effect of pertussis toxin was less obvious in thrombin-induced TPA release, because it was counterbalanced by a positive effect of the toxin on TPA release. Thus, both inhibitory and stimulatory (pertussis toxin-sensitive) G proteins were involved in TPA release. Therefore, thrombin-induced acute release of TPA and vWF differed in two respects. First, below a [Ca2+]i of 500 nmol/L, an increase in Ca2+ was sufficient for vWF release but not for TPA release. Second, pertussis toxin-sensitive G proteins were differentially involved in acute TPA and vWF release.
在本研究中，我们使用先前描述的原代人脐静脉内皮细胞（HUVECs）系统，研究了Ca2+和G蛋白在凝血酶诱导的组织型纤溶酶原激活剂（TPA）和血管性血友病因子（vWF）急性释放（调节性分泌）中的作用。α-凝血酶诱导的TPA和vWF急性释放在螯合细胞内Ca2+后几乎为零，表明需要细胞内Ca2+浓度（[Ca2+]i）升高。[Ca2+]i的升高是来自细胞内还是细胞外Ca2+源并不重要。凝血酶诱导的TPA和vWF释放在低[Ca2+]i（约100 nmol/L）时就已开始。TPA的半最大释放浓度为[Ca2+]i 261 nmol/L，vWF为222 nmol/L。Ca2+信号转导至钙调蛋白，因为钙调蛋白抑制剂抑制了TPA和vWF的释放。Ca2+离子载体离子霉素能剂量依赖性地释放vWF；vWF的半最大释放发生在[Ca2+]i为311 nmol/L时。相比之下，在[Ca2+]i低于500 nmol/L时未发现TPA释放。因此，在[Ca2+]i低于500 nmol/L时，单独的[Ca2+]i升高足以诱导vWF释放，但不足以诱导TPA释放。蛋白激酶C似乎不参与TPA或vWF的释放，因为蛋白激酶C的激活剂或抑制剂均未显著影响释放。磷脂酶A2的抑制也未降低凝血酶诱导的TPA和vWF释放。通过使用皂素通透细胞和完整细胞研究了G蛋白的参与情况。抑制异三聚体和小G蛋白的GDP-β-S显著抑制了凝血酶诱导的通透细胞中vWF和TPA的释放。激活异三聚体G蛋白的AlF-4在完整和通透的HUVECs中均诱导了TPA和vWF的释放。用百日咳毒素预孵育HUVECs可显著抑制凝血酶诱导的vWF释放，这是由于抑制了凝血酶诱导的Ca2+内流。百日咳毒素不影响离子霉素诱导的释放。百日咳毒素对凝血酶诱导的TPA释放的抑制作用不太明显，因为其对TPA释放的正向作用抵消了这种抑制。因此，抑制性和刺激性（百日咳毒素敏感）G蛋白均参与了TPA释放。因此，凝血酶诱导的TPA和vWF急性释放在两个方面存在差异。首先，在[Ca2+]i低于500 nmol/L时，Ca2+升高足以诱导vWF释放，但不足以诱导TPA释放。其次，百日咳毒素敏感的G蛋白在TPA和vWF急性释放中的参与情况不同。
