The lipoprotein-associated coagulation inhibitor that inhibits the factor VII-tissue factor complex also inhibits factor Xa: insight into its possible mechanism of action
The lipoprotein-associated coagulation inhibitor that inhibits the factor VII-tissue factor complex also inhibits factor Xa: insight into its possible mechanism of action
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抑制 VII 因子-组织因子复合物的脂蛋白相关凝血抑制剂也抑制 Xa 因子：深入了解其可能的作用机制
DOI：
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发表时间：
1988
期刊：
影响因子：
--
通讯作者：
J. Miletich
中科院分区：
文献类型：
--
作者：
G. Broze;L. A. Warren;W. Novotny;D. Higuchi;JJ Girard;J. Miletich
当血浆因子VII(a)与其必需辅因子组织因子（TF）结合并蛋白水解激活因子X和因子IX时，血液凝固开始。TF活性的进行性抑制发生在其加入血浆后。这一过程是可逆的，需要存在VII(a)、催化活性Xa、Ca2+和另一种似乎与血浆脂蛋白相关的成分——脂蛋白相关凝血抑制剂（LACI）。最近从培养的人肝细胞（HepG2）的条件培养基中分离出一种与LACI具有相同抑制特性的蛋白，LACI（HG2）。以完整LACI（HG2）免疫兔的n端序列合成肽和纯化的IgG作为抗血清，可抑制人血清中LACI的活性。在含有VIIa， Xa， Ca2+和纯化LACI（HG2）的反应混合物中，TF活性的表观半衰期（t1/2）为20秒。肝素的存在使抑制的初始速率加快了三倍。单独抗凝血酶III α没有作用，但抗凝血酶III α联合肝素可消除对TF的抑制作用。LACI（HG2）对Xa的抑制作用明显为1/2（50秒）。肝素使Xa抑制率提高2.5倍，而磷脂和Ca2+使反应减慢2.5倍。Xa对显色底物（S-2222）和生物测定均有明显的抑制作用，但通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）无法观察到Xa与LACI（HG2）之间的复合物。然而，非变性PAGE显示LACI（HG2）与Xa结合而不是与X结合，而Xa被氟磷酸二异丙基灭活。因此，LACI（HG2）似乎在其活性位点或附近与Xa结合。通过α -胰凝乳蛋白酶处理缺乏含γ -羧谷氨酸结构域BXa（-GD）的牛因子Xa，进一步研究了LACI（HG2）抑制VIIa/TF所需的Xa。LACI（HG2）与BXa（-GD）结合并抑制其对小分子底物（光谱酶Xa）的催化活性，尽管其速率比天然BXa慢约7倍。用饱和浓度的BXa（-GD）预先孵育LACI(HG2)，可显著延缓随后对BXa的抑制。在BXa（-GD）存在的情况下，VII(a)/TF复合物未被LACI（HG2）抑制，并且在添加天然Xa后，LACI（HG2）与BXa（-GD）预孵育可减缓对VIIa/TF的抑制。这一结果与先前的假设一致，即抑制VII(a)/TF涉及VIIa-TF-XA-LACI复合物的形成，该复合物需要XA的GD。（摘要删节为400字）。
Blood coagulation is initiated when plasma factor VII(a) binds to its essential cofactor tissue factor (TF) and proteolytically activates factors X and IX. Progressive inhibition of TF activity occurs upon its addition to plasma. This process is reversible and requires the presence of VII(a), catalytically active Xa, Ca2+, and another component that appears to be associated with the lipoproteins in plasma, a lipoprotein-associated coagulation inhibitor (LACI). A protein, LACI(HG2), possessing the same inhibitory properties as LACI, has recently been isolated from the conditioned media of cultured human liver cells (HepG2). Rabbit antisera raised against a synthetic peptide based on the N-terminal sequence of LACI(HG2) and purified IgG from a rabbit immunized with intact LACI(HG2) inhibit the LACI activity in human serum. In a reaction mixture containing VIIa, Xa, Ca2+, and purified LACI(HG2), the apparent half-life (t1/2) for TF activity was 20 seconds. The presence of heparin accelerated the initial rate of inhibition threefold. Antithrombin III alpha alone had no effect, but antithrombin III alpha with heparin abrogated the TF inhibition. LACI(HG2) also inhibited Xa with an apparent t1/2 of 50 seconds. Heparin enhanced the rate of Xa inhibition 2.5-fold, whereas phospholipids and Ca2+ slowed the reaction 2.5-fold. Xa inhibition was demonstrable with both chromogenic substrate (S-2222) and bioassays, but no complex between Xa and LACI(HG2) could be visualized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Nondenaturing PAGE, however, showed that LACI(HG2) bound to Xa but not to X or Xa inactivated by diisopropyl fluorophosphate. Thus, LACI(HG2) appears to bind to Xa at or near its active site. Bovine factor Xa lacking its gamma-carboxyglutamic acid-containing domain, BXa(-GD), through treatment with alpha-chymotrypsin, was used to further investigate the Xa requirement for VIIa/TF inhibition by LACI(HG2). LACI(HG2) bound to BXa(-GD) and inhibited its catalytic activity against a small molecular substrate (Spectrozyme Xa), though at a rate approximately sevenfold slower than native BXa. Preincubation of LACI(HG2) with saturating concentrations of BXa(-GD) markedly retarded the subsequent inhibition of BXa. The VII(a)/TF complex was not inhibited by LACI(HG2) in the presence of BXa(-GD), and further, preincubation of LACI(HG2) with BXa(-GD) slowed the inhibition of VIIa/TF after the addition of native Xa. The results are consistent with the hypothesis that inhibition of VII(a)/TF involves the formation of a VIIa-TF-XA-LACI complex that requires the GD of XA.(ABSTRACT TRUNCATED AT 400 WORDS).
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影响因子：
2.9
作者：
FUNAKOSHI, T;HEIMARK, RL;FUJIKAWA, K
通讯作者：
FUJIKAWA, K
XIa 因子催化激活人凝血因子 IX 的动力学。
DOI：
10.1172/jci111343
发表时间：
1984
期刊：
The Journal of clinical investigation
影响因子：
--
作者：
Walsh,PN;Bradford,H;Sinha,D;Piperno,JR;Tuszynski,GP
通讯作者：
Tuszynski,GP
抑制外源性凝血途径启动的机制研究。
影响因子：
20.3
作者：
Rao,LV;Rapaport,SI
通讯作者：
Rapaport,SI
载脂蛋白 A-II 抑制组织因子（凝血因子 III）。
DOI：
--
发表时间：
1987
期刊：
The Journal of biological chemistry
影响因子：
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作者：
Carson,SD
通讯作者：
Carson,SD
人和小鼠血浆中因子 IXa 的体外调节以及小鼠体内因子 IXa 的调节。
DOI：
10.1172/jci111377
发表时间：
1984
期刊：
The Journal of clinical investigation
影响因子：
--
作者：
Fuchs,HE;Trapp,HG;Griffith,MJ;Roberts,HR;Pizzo,SV
通讯作者：
Pizzo,SV