活化蛋白C使因子Va和因子VaR506Q失活过程中的肽键断裂及功能活性丧失。
Peptide bond cleavages and loss of functional activity during inactivation of factor Va and factor VaR506Q by activated protein C.
作者信息
Nicolaes G A, Tans G, Thomassen M C, Hemker H C, Pabinger I, Varadi K, Schwarz H P, Rosing J
机构信息
Cardiovascular Research Institute Maastricht, University of Limburg, The Netherlands.
出版信息
J Biol Chem. 1995 Sep 8;270(36):21158-66. doi: 10.1074/jbc.270.36.21158.
Factor V was purified from the plasma of an activated protein C (APC)-resistant patient who is homozygous for the mutation Arg506-->Gln (factor VR506Q). Factor VR506Q was converted by thrombin into factor Va which was further purified yielding a factor Va preparation that had the same cofactor activity in prothrombin activation as normal factor Va. Inactivation of low concentrations of normal factor Va (< 5 nM) by 0.15 nM APC in the presence of phospholipid vesicles proceeded via a biphasic reaction that consisted of a rapid phase (k = 4.3 x 10(7) M-1s-1), yielding a reaction intermediate with reduced cofactor activity that was fully inactivated during the subsequent slow phase (k = 2.3 x 10(6) M-1s-1). Inactivation of factor VaR506Q proceeded via a monophasic reaction (k = 1.7 x 10(6) M-1s-1). Immunoblot analysis showed that APC-catalyzed inactivation of factor Va occurred via peptide bond cleavages in the heavy chain. The rapid phase of inactivation of normal factor Va was associated with cleavage at Arg506 and full inactivation of factor Va required subsequent cleavage at Arg306. The slow monophasic inactivation of factor VaR506Q correlated with cleavage at Arg306. Cleavage at Arg506 in normal factor Va resulted in accumulation of a reaction intermediate that exhibited 40% cofactor activity in prothrombin activation mixtures that contained a high factor Xa concentration (5 nM). Compared with native factor Va, the reaction intermediate retained virtually no cofactor activity at low factor Xa concentrations (0.3 nM). This demonstrates that factor Va that is cleaved at Arg506 is impaired in its ability to interact with factor Xa. Michaelis-Menten kinetic analysis showed that cleavage at Arg506 in membrane-bound factor Va was characterized by a low Km for factor Va (20 nM) and kcat = 0.96 s-1. For cleavage at Arg306 in factor VaR506Q the kinetic parameters were Km = 196 nM and kcat = 0.37 s-1. This means that differences between APC-catalyzed inactivation of factors Va and VaR506Q become much less pronounced at high factor Va concentrations. When factor VaR506Q was inactivated by APC in the absence of phospholipids, cleavage at Arg679 of the heavy chain also contributed to factor Va inactivation. Comparison of rate constants for APC-catalyzed cleavage at Arg306, Arg506, and Arg679 in the absence and presence of phospholipids indicated that phospholipids accelerated these cleavages to a different extent.(ABSTRACT TRUNCATED AT 400 WORDS)
因子V从一名对活化蛋白C（APC）耐药患者的血浆中纯化得到，该患者为Arg506→Gln突变的纯合子（因子VR506Q）。因子VR506Q被凝血酶转化为因子Va，进一步纯化后得到一种因子Va制剂，其在凝血酶原激活中的辅因子活性与正常因子Va相同。在磷脂囊泡存在的情况下，0.15 nM的APC对低浓度（<5 nM）的正常因子Va的灭活通过双相反应进行，该反应包括一个快速阶段（k = 4.3×10⁷ M⁻¹s⁻¹），产生一种辅因子活性降低的反应中间体，该中间体在随后的缓慢阶段（k = 2.3×10⁶ M⁻¹s⁻¹）被完全灭活。因子VaR506Q的灭活通过单相反应（k = 1.7×10⁶ M⁻¹s⁻¹）进行。免疫印迹分析表明，APC催化的因子Va灭活是通过重链中的肽键断裂发生的。正常因子Va灭活的快速阶段与Arg506处的切割有关，而因子Va的完全灭活需要随后在Arg306处的切割。因子VaR506Q的缓慢单相灭活与Arg306处的切割相关。正常因子Va中Arg506处的切割导致一种反应中间体的积累，该中间体在含有高浓度因子Xa（5 nM）的凝血酶原激活混合物中表现出40%的辅因子活性。与天然因子Va相比，该反应中间体在低因子Xa浓度（0.3 nM）下几乎没有保留辅因子活性。这表明在Arg506处被切割的因子Va与因子Xa相互作用的能力受损。米氏动力学分析表明，膜结合因子Va中Arg506处的切割的特征是因子Va的Km较低（20 nM），kcat = 0.96 s⁻¹。对于因子VaR506Q中Arg306处的切割，动力学参数为Km = 196 nM，kcat = 0.37 s⁻¹。这意味着在高因子Va浓度下，APC催化的因子Va和VaR506Q灭活之间的差异变得不那么明显。当因子VaR506Q在没有磷脂的情况下被APC灭活时，重链Arg679处的切割也有助于因子Va的灭活。在有和没有磷脂的情况下，APC催化的Arg306、Arg506和Arg679处切割的速率常数比较表明，磷脂对这些切割的加速程度不同。（摘要截断于400字）
Zotero 插件