Taneda H, Andoh K, Nishioka J, Takeya H, Suzuki K
Department of Molecular Biology on Genetic Disease, Mie University School of Medicine.
J Biochem. 1994 Sep;116(3):589-97. doi: 10.1093/oxfordjournals.jbchem.a124565.
Prothrombin is a vitamin K-dependent plasma protein composed of several functional domains, which is proteolytically activated into thrombin by factor Xa in the presence of factor Va, Ca2+, and phospholipids. During the activation, prothrombin is cleaved into three fragments: fragment 1, containing a domain rich in gamma-carboxyglutamic acid residues and kringle 1 domain; fragment 2, containing the kringle 2 domain; and a protease catalytic domain, thrombin. Here we studied the interaction site for factor Xa in human prothrombin during the activation. The isolated fragment 2 inhibited the activation of prothrombin by either prothrombinase complex or factor Xa alone in a dose-dependent manner, whereas fragment 1 and diisopropylphosphate (DIP)-thrombin did not. Factor Xa directly bound to fragment 2 immobilized to microwell plates with a Kd of 9.0 x 10(-8) M, but not to fragment 1 or DIP-thrombin. Factor Xa also bound to immobilized prothrombin and prethrombin 1 with Kds of 2.0 x 10(-7) and 1.5 x 10(-7) M, respectively, suggesting that factor Xa interacts with the kringle 2 domain in these molecules. The binding of factor Xa to immobilized fragment 2 was Ca(2+)-dependent with an optimal concentration at 6 mM. In the presence of Ca2+, the interaction was enhanced by phospholipids in a concentration-dependent manner. To localize the factor Xa-binding site in the kringle 2 domain, fragment 2 was digested with lysyl endopeptidase and then trypsin after reduction and S-carboxymethylation. The resulting peptides were immobilized to microwell plates and assayed for factor Xa binding ability. The amino acid sequence of the peptide positive in the assay was determined to be residues His205 to Arg220. Factor Xa bound to a synthetic peptide corresponding to the residues His205 to Arg220 immobilized to microwell plates. The peptide inhibited factor Xa-catalyzed activation of prothrombin, but a peptide with the reversed sequence of His205 to Arg220 did not. These findings indicate that factor Xa interacts with at least a linear sequence, His205 to Arg220, in the kringle 2 domain of prothrombin during its activation into thrombin.
凝血酶原是一种维生素K依赖的血浆蛋白，由几个功能结构域组成，在因子Va、Ca2+和磷脂存在的情况下，被因子Xa蛋白水解激活为凝血酶。在激活过程中，凝血酶原被切割成三个片段：片段1，含有富含γ-羧基谷氨酸残基的结构域和kringle 1结构域；片段2，含有kringle 2结构域；以及一个蛋白酶催化结构域，即凝血酶。在此，我们研究了人凝血酶原激活过程中因子Xa的相互作用位点。分离的片段2以剂量依赖的方式抑制凝血酶原酶复合物或单独的因子Xa对凝血酶原的激活，而片段1和二异丙基磷酸（DIP）-凝血酶则无此作用。因子Xa直接与固定在微孔板上的片段2结合，解离常数（Kd）为9.0×10（-8）M，但不与片段1或DIP-凝血酶结合。因子Xa也分别以2.0×10（-7）和1.5×10（-7）M的Kd与固定的凝血酶原和凝血酶原1结合，这表明因子Xa与这些分子中的kringle 2结构域相互作用。因子Xa与固定的片段2的结合是Ca（2+）依赖性的，最佳浓度为6 mM。在Ca2+存在的情况下，磷脂以浓度依赖的方式增强这种相互作用。为了在kringle 2结构域中定位因子Xa结合位点，片段2在还原和S-羧甲基化后先用赖氨酰内肽酶消化，然后用胰蛋白酶消化。将所得肽段固定在微孔板上，并检测其因子Xa结合能力。检测呈阳性的肽段的氨基酸序列确定为His205至Arg220残基。因子Xa与固定在微孔板上的对应于His205至Arg220残基的合成肽结合。该肽抑制因子Xa催化的凝血酶原激活，但His205至Arg220反向序列的肽则无此作用。这些发现表明，因子Xa在激活为凝血酶的过程中，至少与凝血酶原kringle 2结构域中的线性序列His205至Arg220相互作用。
