CHARACTERIZATION OF THE MOLECULAR DEFECT IN FACTOR V-R506Q
CHARACTERIZATION OF THE MOLECULAR DEFECT IN FACTOR V-R506Q
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DOI：
10.1074/jbc.270.8.4053
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发表时间：
1995-02-24
影响因子：
4.8
通讯作者：
MANN, KG
中科院分区：
文献类型：
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作者：
KALAFATIS, M;BERTINA, RM;MANN, KG
血浆对活化蛋白C的不良抗凝反应与因子V分子中的单一突变（Arg（506）→ Gln）相关。（患者I，因子V-I，和患者II，因子V-II），其对于该突变是纯合的，当在基于凝固时间的测定和测量α-凝血酶形成的测定中与从正常血浆分离的因子V相比时，来自两个患者的因子V分子具有正常的促凝血活性，α-凝血酶激活的膜结合辅因子的裂解和随后的活化蛋白C（APC）失活在正常的因子Va中，在Arg（506）处的切割产生M（r）= 75，000的片段和M（r）= 28，000/26，000的双联体，并且对于随后在Arg（306）和Arg（679）处切割的位点的最佳暴露是必需的，在这些位点处的蛋白水解导致M（r）= 45，000和30，000的片段和M（r）= 22，000/20，000的双联体的出现。Arg（306）处的切割是膜依赖性的，并且是完全失活所需的，与APC孵育5分钟后（5.4 nM）膜结合正常因子Va（280 nM）实际上没有辅因子活性，而在类似的实验条件下，因子Va（I）和因子Va（II）保留其初始活性的约50%。因子Va（I）保留其初始辅因子活性的20%而因子Va（II）具有10%的剩余辅因子活性，辅因子活性的初始损失在失活反应的前10分钟期间，膜结合因子Va（I）和因子Va（II）的减少（类似于70%）与Arg（306）处的切割和M（r）= 45的出现相关，随后，M（r）= 62，000/60，000的双联体在Arg（679）处被切割以产生M（r）= 56，000/54，000的双联体，导致辅因子活性的完全丧失。在Arg（306）和Arg（679）处切割后，两种原辅因子，因子V-I和因子V-II均失活，APC失活率与正常因子V的失活率相当。我们的数据表明：1）Arg（506）的切割对于Arg（306）和Arg（679）处切割位点的最佳暴露和膜结合因子Va的快速失活是必需的;和2）APC在膜结合因子V上的Arg（306）处的切割在正常和APC抗性个体中以相同的速率发生。因此，Arg（306）和Arg（679）处的切割以及随后膜结合原辅因子因子（因子V）的失活不需要在Arg（506）处的预先切割以获得最佳暴露。
A poor anticoagulant response of plasma to activated protein C is correlated with a single mutation in the factor V molecule (Arg(506) --> Gln), Factor V was purified to homogeneity from plasma of two unrelated patients (patient I, factor V-I, and patient II, factor V-II), who are homozygous for this mutation, The factor V molecule from both patients has normal procoagulant activity when compared with factor V isolated from normal plasma in both a clotting time-based assay and in an assay measuring alpha-thrombin formation, The cleavage and subsequent inactivation by activated protein C (APC) of the alpha-thrombin-activated membrane-bound cofactor (factor Va) from both patients were analyzed and compared with the cleavage and inactivation of normal human factor Va, In normal factor Va, cleavage at Arg(506) generates a M(r) = 75,000 fragment and a M(r) = 28,000/26,000 doublet and is necessary for the optimum exposure of the sites for subsequent cleavage at Arg(306) and Arg(679), Proteolysis at these sites leads to the appearance of M(r) = 45,000 and 30,000 fragments and a M(r) = 22,000/20,000 doublet. Cleavage at Arg(306) is membrane-dependent and is required for complete inactivation, Following 5 min of incubation with APC (5.4 nM) membrane-bound normal factor Va (280 nM) has virtually no cofactor activity whereas under similar experimental conditions factor Va(I) and factor Va(II) retain approximately 50% of their initial activity, After 1 h of incubation with APC, factor Va(I) retains 20% of its initial cofactor activity whereas factor Va(II) has 10% remaining cofactor activity, The initial loss in cofactor activity (similar to 70%) of membrane-bound factor Va(I) and factor Va(II) during the first 10 min of the inactivation reaction is correlated with cleavage at Arg(306) and appearance of a M(r) = 45,000 fragment and a M(r) = 62,000/60,000 doublet, Subsequently, the M(r) = 62,000/60,000 doublet is cleaved at Arg(679) to generate a M(r) = 56,000/54,000 doublet resulting in complete loss of cofactor activity. Both procofactors, factor V-I and factor V-II were inactivated following cleavage at Arg(306) and Arg(679), With APC inactivation rates equivalent to those observed for normal factor V. Our data demonstrate that; 1) cleavage of Arg(506) is required for optimum exposure of the cleavage sites at Arg(306) and Arg(679) and rapid inactivation of membrane-bound factor Va; and 2) cleavage at Arg(306) by APC On membrane-bound factor V occurs at the same rate in both normal and APC-resistant individuals, Thus cleavage at Arg(306) and Arg(679) and subsequent inactivation of the membrane-bound procofactor, factor V, does not require prior cleavage at Arg(506) for optimum exposure.