Department of Medicine/Hematology-Oncology, University of Wisconsin-Madison, Madison, Wisconsin 53706, United States.
Biochemistry. 2010 Nov 23;49(46):9997-10005. doi: 10.1021/bi100906q. Epub 2010 Oct 27.
Supersulfated low molecular weight heparin (ssLMWH) inhibits the intrinsic tenase (factor IXa-factor VIIIa) complex in an antithrombin-independent manner. Recombinant factor IXa with alanine substitutions in the protease domain (K126A, N129A, K132A, R165A, R170A, N178A, R233A) was assessed with regard to heparin affinity in solution and ability to regulate protease activity within the factor IXa-phospholipid (PL) and intrinsic tenase complexes. In a soluble binding assay, factor IXa K126A, K132A, and R233A dramatically (10-20-fold) reduced ssLMWH affinity, while factor IXa N129A and R165A moderately (5-fold) reduced affinity relative to wild type. In the factor IXa-PL complex, binding affinity for ssLMWH was increased 4-fold, and factor X activation was inhibited with a potency 7-fold higher than predicted for wild-type protease-ssLMWH affinity in solution. In the intrinsic tenase complex, ssLMWH inhibited factor X activation with a 4-fold decrease in potency relative to wild-type factor IXa-PL. The mutations increased resistance to inhibition by ssLMWH in a similar fashion for both enzyme complexes (R233A > K126A > K132A/R165A > N129A/N178A/wild type) except for factor IXa R170A. This protease had ssLMWH affinity and potency for the factor IXa-PL complex similar to wild-type protease but was moderately resistant (6-fold) to inhibition in the intrinsic tenase complex based on increased cofactor affinity. These results are consistent with conformational regulation of the heparin-binding exosite and macromolecular substrate catalysis by factor IXa. An extensive overlap exists between the heparin and factor VIIIa binding sites on the protease domain, with residues K126 and R233 dominating the heparin interaction and R165 dominating the cofactor interaction.
超硫酸化低分子量肝素（ssLMWH）以抗凝血酶非依赖性方式抑制内在凝血酶原复合物（因子 IXa-因子 VIIIa）。用丙氨酸取代蛋白酶结构域中的重组因子 IXa（K126A、N129A、K132A、R165A、R170A、N178A、R233A），评估其在溶液中的肝素亲和力以及在因子 IXa-磷脂（PL）和内在凝血酶原复合物内调节蛋白酶活性的能力。在可溶性结合测定中，因子 IXa K126A、K132A 和 R233A 极大地（10-20 倍）降低了 ssLMWH 的亲和力，而因子 IXa N129A 和 R165A 则适度地（5 倍）降低了亲和力。在因子 IXa-PL 复合物中，ssLMWH 的结合亲和力增加了 4 倍，因子 X 的激活被抑制，其效力比预测的在溶液中野生型蛋白酶-ssLMWH 亲和力高 7 倍。在内在凝血酶原复合物中，ssLMWH 抑制因子 X 的激活，其效力比野生型因子 IXa-PL 复合物降低了 4 倍。这些突变以类似的方式增加了对两种酶复合物的 ssLMWH 抑制的抗性（R233A>K126A>K132A/R165A>N129A/N178A/野生型），除了因子 IXa R170A。这种蛋白酶对因子 IXa-PL 复合物的 ssLMWH 亲和力和效力与野生型蛋白酶相似，但由于辅助因子亲和力增加，对内在凝血酶原复合物的抑制具有中度抗性（6 倍）。这些结果与因子 IXa 的肝素结合外位和大分子底物催化的构象调节一致。蛋白酶结构域上的肝素和因子 VIIIa 结合位点有很大的重叠，残基 K126 和 R233 主导肝素相互作用，而残基 R165 主导辅助因子相互作用。
