Mechanisms by which soluble endothelial cell protein C receptor modulates protein C and activated protein C function
Mechanisms by which soluble endothelial cell protein C receptor modulates protein C and activated protein C function
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DOI：
10.1074/jbc.275.8.5447
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发表时间：
2000-02-25
影响因子：
4.8
通讯作者：
Esmon, CT
中科院分区：
文献类型：
--
作者：
Liaw, PCY;Neuenschwander, PF;Esmon, CT
内皮细胞蛋白C受体(EPCR)通过结合蛋白C和增强凝血酶-血栓调节蛋白复合体的激活，发挥蛋白C抗凝途径的重要调节作用。EPCR能与蛋白C和活化蛋白C(APC)高亲和力结合。一种可溶性的EPCR(SEPCR)在血浆中循环，并抑制APC的抗凝活性。在本研究中，我们研究了sEPCR调节APC功能的机制。只有在磷脂小泡存在的情况下，可溶性EPCR才能抑制APC对Va因子的失活。在3 mM的CaCl2和0.6 mM的MgCl2存在下，用流式细胞仪分析，sEPCR抑制蛋白C和APC与磷脂小泡的结合(K-I分别为40+/-7和38+/-4 nm)，而不加MgCl2，其K-I值增加约4倍。荧光素-APC和德克萨斯红-sEPCR双标记流式细胞术分析表明，APC-sEPCR复合体不与磷脂囊泡相互作用。通过表面等离子体共振，我们发现sEPCR也以剂量依赖的方式抑制蛋白C与磷脂的结合(K-I=32 nM)。为了探讨sEPCR引起APC结构变化的可能性，我们用荧光光谱学研究了sEPCR/Fl-APC的相互作用。SEPCR与Fl-APC饱和结合(K-d=27+/-13 nM)，Fl-APC荧光最大下降10.8+/-0.6%。SEPCR还能刺激APC对合成底物的氨解活性。我们的结论是，sEPCR与APC的结合阻断了磷脂相互作用并改变了APC的活性部位。
The endothelial cell protein C receptor (EPCR) functions as an important regulator of the protein C anticoagulant pathway by binding protein C and enhancing activation by the thrombin-thrombomodulin complex. EPCR binds to both protein C and activated protein C (APC) with high affinity. A soluble form of EPCR (sEPCR) circulates in plasma and inhibits APC anticoagulant activity. In this study, we investigate the mechanisms by which sEPCR modulates APC function. Soluble EPCR inhibited the inactivation of factor Va by APC only in the presence of phospholipid vesicles. By using flow cytometric analysis in the presence of 3 mM CaCl2 and 0.6 mM MgCl2, sEPCR inhibited the binding of protein C and APC to phospholipid vesicles (K-i = 40 +/- 7 and 38 +/- 4 nM, respectively), Without MgCl2 the K-i values increased approximately 4-fold. Double label flow cytometric analysis using fluorescein-APC and Texas Red-sEPCR indicated that the APC sEPCR complex does not interact with phospholipid vesicles. By using surface plasmon resonance, we found that sEPCR also inhibited binding of protein C to phospholipid in a dose dependent fashion (K-i = 32 nM). To explore the possibility that sEPCR evokes structural changes in APC, fluorescence spectroscopy studies were performed to monitor sEPCR/Fl-APC interactions. sEPCR binds saturably to Fl-APC (K-d = 27 +/- 13 nM) with a maximum decrease in Fl-APC fluorescence of 10.8 +/- 0.6%. sEPCR also stimulated the amidolytic activity of APC toward synthetic substrates. We conclude that sEPCR binding to APC blocks phospholipid interaction and alters the active site of APC.