Phillips J E, Shirk R A, Whinna H C, Henriksen R A, Church F C
Center for Thrombosis and Hemostasis, University of North Carolina School of Medicine, Chapel Hill 27599-7035.
J Biol Chem. 1993 Feb 15;268(5):3321-7.
Heparin cofactor II and antithrombin are plasma serine proteinase inhibitors whose ability to inhibit alpha-thrombin is accelerated by glycosaminoglycans. Dysfunctional thrombin mutants Quick I (Arg67-->Cys) and Quick II (Gly226-->Val) were used to further compare heparin cofactor II and antithrombin interactions. Quick I, Quick II, and alpha-thrombin were eluted at the same salt concentration from heparin-Sepharose suggesting that the putative heparin-binding site (also termed anion binding exosite-II) is functional. Antithrombin yielded similar inhibition rates for Quick I and alpha-thrombin in the absence or presence of various amounts of heparin. Also, Quick I was inhibited similarly to alpha-thrombin by heparin cofactor II in the absence of glycosaminoglycan. In contrast, glycosaminoglycan-accelerated Quick I inhibition by heparin cofactor II was greatly reduced indicating that anion binding exosite-I (where the mutation occurs in Quick I) is critical for increased inhibition by heparin cofactor II. We also found that heparin cofactor II formed a SDS-resistant bimolecular complex with Quick II and alpha-thrombin at similar rates and the rate of complex formation was accelerated in the presence of glycosaminoglycans. A three-dimensional molecular model of the Quick II active site compared to alpha-thrombin suggested that the heparin cofactor II Leu-Ser-reactive site sequence (P1-P1') is a compatible "pseudosubstrate" in contrast to the Arg-Ser sequence found in antithrombin. The importance of heparin cofactor II as a thrombin regulator will depend upon its ability to interact with glycosaminoglycans and the functional availability of thrombin exosites.
肝素辅因子II和抗凝血酶是血浆丝氨酸蛋白酶抑制剂，其抑制α-凝血酶的能力可被糖胺聚糖加速。功能失调的凝血酶突变体Quick I（Arg67→Cys）和Quick II（Gly226→Val）被用于进一步比较肝素辅因子II和抗凝血酶的相互作用。Quick I、Quick II和α-凝血酶在相同盐浓度下从肝素-琼脂糖凝胶上洗脱，这表明假定的肝素结合位点（也称为阴离子结合外位点-II）是有功能的。在不存在或存在不同量肝素的情况下，抗凝血酶对Quick I和α-凝血酶产生相似的抑制率。此外，在不存在糖胺聚糖的情况下，肝素辅因子II对Quick I的抑制作用与对α-凝血酶的抑制作用相似。相比之下，肝素辅因子II对Quick I的糖胺聚糖加速抑制作用大大降低，这表明阴离子结合外位点-I（Quick I中发生突变的位点）对于肝素辅因子II增强抑制作用至关重要。我们还发现，肝素辅因子II以相似的速率与Quick II和α-凝血酶形成抗SDS的双分子复合物，并且在存在糖胺聚糖的情况下复合物形成速率加快。与α-凝血酶相比，Quick II活性位点的三维分子模型表明，肝素辅因子II的亮氨酸-丝氨酸反应位点序列（P1-P1'）是一种相容的“假底物”，这与抗凝血酶中的精氨酸-丝氨酸序列不同。肝素辅因子II作为凝血酶调节剂的重要性将取决于其与糖胺聚糖相互作用的能力以及凝血酶外位点的功能可用性。
