Noé G, Hofsteenge J, Rovelli G, Stone S R
Friedrich Miescher Institut, Basel, Switzerland.
J Biol Chem. 1988 Aug 25;263(24):11729-35.
The peptide comprising residues 62-73 of the B-chain of human alpha-thrombin was synthesized and polyclonal antibodies raised against it. These antibodies were found to bind to the synthetic peptide, a CNBr fragment, and a proteolytic subfragment containing this sequence, as well as the entire thrombin molecule. The purified antibodies had no effect on the hydrolysis by thrombin of D-Phe-pipecolyl-Arg-p-nitroanilide and caused only a minimal decrease (20%) in the second-order rate constant for inactivation by antithrombin III. On the other hand, the antibodies competitively inhibited the binding of hirudin over the concentration range tested (0-43 nM), and a dissociation constant of 3.4 +/- 0.5 nM was found for the antibodies. The release of fibrinopeptide A from the A alpha-chain of fibrinogen by thrombin was competitively inhibited with an inhibition constant of 11.7 +/- 0.4 nM. The activation of protein C by thrombin in the presence of thrombomodulin was also inhibited by the antibodies, and an apparent inhibition constant of 10.7 +/- 1.5 nM was found. In contrast, the antibodies had no effect on the activation of protein C in the absence of thrombomodulin. These results are discussed in relation to data obtained recently on the interaction of well defined proteolytic derivatives of human alpha-thrombin with the ligands described above.
合成了包含人α-凝血酶B链62 - 73位残基的肽段，并制备了针对该肽段的多克隆抗体。发现这些抗体可与合成肽段、一个溴化氰片段、一个包含该序列的蛋白水解亚片段以及整个凝血酶分子结合。纯化后的抗体对凝血酶水解D - 苯丙氨酸 - 哌啶基 - 精氨酸 - 对硝基苯胺没有影响，并且对抗凝血酶III灭活的二级速率常数仅引起极小的降低（20%）。另一方面，在测试的浓度范围内（0 - 43 nM），这些抗体竞争性抑制水蛭素的结合，并且发现抗体的解离常数为3.4±0.5 nM。凝血酶从纤维蛋白原Aα链释放纤维蛋白肽A受到竞争性抑制，抑制常数为11.7±0.4 nM。在血栓调节蛋白存在的情况下，凝血酶对蛋白C的激活也受到这些抗体的抑制，表观抑制常数为10.7±1.5 nM。相比之下，在没有血栓调节蛋白的情况下，这些抗体对蛋白C的激活没有影响。结合最近关于人α-凝血酶明确的蛋白水解衍生物与上述配体相互作用获得的数据对这些结果进行了讨论。
