Influence of membrane composition on the enhancement of factor VIIa/tissue factor activity by magnesium ions.
Influence of membrane composition on the enhancement of factor VIIa/tissue factor activity by magnesium ions.
复制标题
膜组成对镁离子增强因子 VIIa/组织因子活性的影响。
DOI：
10.1160/th13-07-0628
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发表时间：
2014
影响因子：
6.7
通讯作者：
Morrissey,JamesH
中科院分区：
文献类型：
--
作者：
Tavoosi,Narjes;Morrissey,JamesH
许多研究表明，维生素k依赖性凝血蛋白的γ-羧谷氨酸丰富（GLA）结构域需要Ca2+才能正确折叠并结合到膜上（1,2）。虽然血浆中含有约1.25 mM游离Ca2+和0.5 mM Mg2+(3)，但凝血因子功能的体外测定通常采用超生理Ca2+浓度（2.5-5 mM Ca2+），而不含Mg2+。Sekiya等人发现Mg2+结合生理Ca2+浓度可增强因子IX （fIX）的结构和功能（4,5）。随后的报道表明，在Ca2+的血浆浓度存在下，Mg2+增强了与组织因子（TF）结合的VIIa因子（fVIIa）的活性（6-10）。这个概念是，当它是唯一的二价金属离子存在时（在超生理Ca2+浓度下），GLA结构域通常结合7或8个Ca2+，但在Ca2+和Mg2+的血浆浓度下，这些“钙”结合位点中的2或3个实际上被Mg2+占据，具有功能后果（11）。在没有Ca2+的情况下，Mg2+不支持凝血反应（12），这也与只有GLA结构域的一部分金属离子结合位点可以被Mg2+有效占据的观点一致。许多关于TF活性的研究都采用了磷脂酰丝氨酸（PS）和磷脂酰胆碱（PC）的二元混合物。我们之前报道过，磷脂酰乙醇胺（PE）在PE/PC脂质体中支持很少的促凝活性，但PE显著降低了对TF: fVIIa活性的PS需求（13,14）。最近，我们报道了因子VIIa优先与磷脂酸（PA）结合，并且PA通过单独的fVIIa或与分离的组织因子外结构域（sTF）复合来提高fX的激活率（15）。然而，这些磷脂协同作用的研究是在饱和Ca2+浓度下进行的，没有Mg2+。此外，先前的研究表明Mg2+增强GLA结构域功能通常是通过PS/PC脂质体或细胞膜进行的。因此，Mg2+增强fVIIa激活fX的能力尚未被系统地探讨作为磷脂组成的函数。我们现在研究Mg2+在PE或PA存在下增强fVIIa功能的能力。重组膜锚定TF （memitf）重组成不同磷脂组成的脂质体（棕榈酰油酰PC、棕榈酰油酰PS、二油酰PE和/或棕榈酰油酰PA），并用于量化fX的激活率，如前所述（15）。我们首先用fVIIa测定了由二元PS/PC混合物组成的tf -脂质体对fX的激活。与上述研究一致，在测试的PS成分范围内，二价金属离子的生理浓度（1.25 mM Ca2+ + 0.5 mM Mg2+）比超生理Ca2+浓度（2.5 mM Ca2+不含Mg2+）支持稍高的fX激活率，比1.25 mM Ca2+不含Mg2+的激活率高得多（▶图1a，开放钻石，圆圈和正方形，分别）。我们重新
A number of studies have shown that the γ-carboxyglutamate-rich (GLA) domains of vitamin K-dependent clotting proteins require Ca2+ to fold properly and bind to membranes (1, 2). Although plasma contains about 1.25 mM free Ca2+ and 0.5 mM Mg2+(3), in vitro assays of clotting factor function often employ supraphysiologic Ca2+ concentrations (2.5-5 mM Ca2+), and no Mg2+. Sekiya et al. showed that Mg2+ enhances factor IX (fIX) structure and function in combination with physiologic Ca2+ concentrations (4, 5). Subsequent reports showed that, in the presence of plasma concentrations of Ca2+, Mg2+ enhances the activity of factor VIIa (fVIIa) bound to tissue factor (TF)(6–10). The concept is that GLA domains typically bind seven or eight Ca2+ when it is the only divalent metal ion present (at supraphysiologic Ca2+ concentrations), but at plasma concentrations of Ca2+ and Mg2+, two or three of these “calcium” binding sites are actually occupied by Mg2+, with functional consequences (11). Mg2+ does not support clotting reactions in the absence of Ca2+(12), also consistent with the notion that only a subset of the metal ion binding sites in GLA domains can be productively occupied by Mg2+.Many studies of TF activity have employed binary mixtures of phosphatidylserine (PS) and phosphatidylcholine (PC). We previously reported that phosphatidylethanolamine (PE) supports little procoagulant activity in PE/PC liposomes, but that PE dramatically reduces the PS requirement for TF: fVIIa activity (13, 14). More recently, we reported that factor VIIa binds preferentially to phosphatidic acid (PA) and that PA enhances rates of fX activation by fVIIa alone or in complex with the isolated tissue factor ectodomain (sTF)(15). However, these studies of phospholipid synergy were conducted at saturating Ca2+ concentrations without Mg2+. Furthermore, the previous studies showing that Mg2+ enhances GLA domain function were typically performed with PS/PC liposomes or cell membranes. Therefore, the ability of Mg2+ to enhance fX activation by fVIIa has not been explored systematically as a function of phospholipid composition. We now examine the ability of Mg2+ to enhance fVIIa function in the presence of PE or PA. Recombinant membrane-anchored TF (membTF) was reconstituted into liposomes of varying phospholipid composition (palmitoyl-oleoyl PC, palmitoyl-oleoyl PS, dioleoyl PE, and/or palmitoyl-oleoyl PA) and used to quantify rates of fX activation as previously described (15). We first measured fX activation by fVIIa on TF-liposomes composed of binary PS/PC mixtures. Consistent with studies cited above, physiologic concentrations of divalent metal ions (1.25 mM Ca2+ plus 0.5 mM Mg2+) supported slightly higher rates of fX activation by membTF: fVIIa than did supraphysiologic Ca2+ concentrations (2.5 mM Ca2+ without Mg2+) and substantially higher rates than with 1.25 mM Ca2+ without Mg2+(▶ Figure 1 A, open diamonds, circles and squares, respectively), over the range of PS compositions tested. We re-