Stone S R, Hofsteenge J
Friedrich Miescher-Institut, Basel, Switzerland.
Biochemistry. 1991 Apr 23;30(16):3950-5. doi: 10.1021/bi00230a021.
Partial proteolysis of human alpha-thrombin by trypsin results in the formation of beta T-thrombin and gamma T-thrombin which have a reduced affinity for the inhibitor hirudin and the cell-surface cofactor thrombomodulin as well as reduced activity with fibrinogen. The basis of the reduction in affinity of these thrombin derivatives for hirudin has been investigated by examining their kinetics of interaction with a number of hirudin mutants differing in their C-terminal charge properties as well as with a truncated form of hirudin. The results indicate that the reduced affinity of beta T-thrombin for hirudin is most likely due to a decrease in the strength of nonionic interactions between thrombin and the C-terminal region of hirudin. No decrease in the strength of ionic interactions was observed with beta T-thrombin. In contrast, the reduced affinity of gamma T-thrombin was due to a decrease in the strength of both ionic and nonionic interactions. The N-terminal core region of hirudin, which interacts predominantly with the active-site cleft of thrombin, exhibited similar affinities for alpha-, beta T-, and gamma T-thrombin, indicating that thrombin-hirudin interactions within the active site are largely preserved in beta T- and gamma T-thrombin.
用胰蛋白酶对人α-凝血酶进行部分蛋白水解会形成βT-凝血酶和γT-凝血酶，它们对抑制剂水蛭素和细胞表面辅因子血栓调节蛋白的亲和力降低，对纤维蛋白原的活性也降低。通过研究这些凝血酶衍生物与一些C端电荷性质不同的水蛭素突变体以及截短形式的水蛭素的相互作用动力学，研究了这些凝血酶衍生物对水蛭素亲和力降低的原因。结果表明，βT-凝血酶对水蛭素亲和力降低很可能是由于凝血酶与水蛭素C端区域之间非离子相互作用强度的降低。βT-凝血酶未观察到离子相互作用强度的降低。相比之下，γT-凝血酶亲和力降低是由于离子和非离子相互作用强度均降低。水蛭素的N端核心区域主要与凝血酶的活性位点裂隙相互作用，对α-、βT-和γT-凝血酶表现出相似的亲和力，表明活性位点内的凝血酶-水蛭素相互作用在βT-和γT-凝血酶中基本保持。
