Theunissen H J, Dijkema R, Grootenhuis P D, Swinkels J C, de Poorter T L, Carati P, Visser A
Department of Biotechnology and Biochemistry, Organon International bv, Oss, The Netherlands.
J Biol Chem. 1993 Apr 25;268(12):9035-40.
Antithrombin-III (AT-III) is a heparin-dependent inhibitor of thrombin and Factor Xa, two serine proteases that are crucial for blood coagulation. In order to assess whether it would be possible to target AT-III only towards Factor Xa, we replaced parts of the reactive site, or P region, of AT-III by sequences present in prothrombin, a substrate of Factor Xa in the coagulation cascade. We show that replacement of the P3 to P3' region generates the hypothesized phenotype. In fact, point mutation of the P1' site from Ser (present in AT-III) to Ile (present in prothrombin) is sufficient to dissociate heparin-dependent thrombin and Factor Xa inhibitory activities. Interestingly, a combined mutation at P3 and P3' brings about the same dissociation. We show that besides Ile, other amino acids at P1' can lead to the dissociation in inhibitory activity. Amino acids with small side chains (Gly, Ser, Ala, and Thr) have only a marginal effect on the inhibitory activity against either protease. However, larger residues at the P1' position abolish the heparin-dependent anti-thrombin activity, whereas the heparin-dependent anti-Factor Xa activity is not at all or only moderately affected. These results can be rationalized by a comparison of the x-ray structure and a three-dimensional model of the S1' binding pockets of thrombin and Factor Xa, respectively. It appears that the S1' pocket of Factor Xa leaves much more space for the P1' residue of AT-III than the S1' pocket of thrombin.
抗凝血酶III（AT-III）是一种依赖肝素的凝血酶和Xa因子抑制剂，这两种丝氨酸蛋白酶对血液凝固至关重要。为了评估是否有可能使AT-III仅作用于Xa因子，我们用凝血酶原（凝血级联反应中Xa因子的底物）中存在的序列替换了AT-III反应位点或P区的部分区域。我们发现，替换P3至P3'区域会产生假设的表型。事实上，将P1'位点的丝氨酸（存在于AT-III中）点突变为异亮氨酸（存在于凝血酶原中）足以使肝素依赖的凝血酶和Xa因子抑制活性解离。有趣的是，P3和P3'处的联合突变也会导致同样的解离。我们发现，除了异亮氨酸，P1'处的其他氨基酸也会导致抑制活性的解离。侧链较小的氨基酸（甘氨酸、丝氨酸、丙氨酸和苏氨酸）对两种蛋白酶的抑制活性影响很小。然而，P1'位置较大的残基会消除肝素依赖的抗凝血酶活性，而肝素依赖的抗Xa因子活性则不受影响或仅受到中度影响。通过分别比较凝血酶和Xa因子S1'结合口袋的X射线结构和三维模型，可以解释这些结果。似乎Xa因子的S1'口袋比凝血酶的S1'口袋为AT-III的P1'残基留下了更多空间。
