Ahmad S S, Wong M Y, Rawala R, Jameson B A, Walsh P N
Sol Sherry Thrombosis Research Center, Temple University School of Medicine, Philadelphia, Pennsylvania 19140, USA.
Biochemistry. 1998 Feb 10;37(6):1671-9. doi: 10.1021/bi971591h.
High-affinity, specific factor IX/IXa binding to platelets is mediated at least in part by amino acids (G4-Q11) exposed on the surface of the gamma-carboxyglutamic acid (Gla) domain. Rationally designed, conformationally constrained synthetic peptides were screened for their capacity to inhibit factor IXa binding to platelets. Each of these peptides (G4-Q11, S3-L6, and F9-Q11) acted alone to inhibit factor IXa binding to approximately 50% of the 500-600 sites/platelet with Ki values of 2.9 nM (G4-Q11), 24 nM (S3-L6), and 240 nM (F9-Q11), compared with native factor IXa (Ki approximately 2.5 nM). The two peptides S3-L6 and F9-Q11 added together at equimolar concentration demonstrated approximately 50-fold synergism (Ki = 2.4 nM). Although both factor IX and the Gla peptide (G4-Q11) displaced 100% of bound factor IX and approximately 50% of bound factor IXa, factor IX was ineffective (at > 1000-fold molar excess) and the Gla domain peptide (G4-Q11) was relatively ineffective (Ki = 165 microM) in inhibiting platelet receptor-mediated factor X activation by factor IXa. We conclude that the Gla domain (G4-Q11) of factor IXa contains two conformationally constrained loop structures that mediate binding of factor IX/IXa to a shared site on activated human platelets which is separate and distinct from the site used by the enzyme, factor IXa, for assembly of the factor X activating complex.
高亲和力、特异性的因子IX/IXa与血小板的结合至少部分是由γ-羧基谷氨酸（Gla）结构域表面暴露的氨基酸（G4-Q11）介导的。对经过合理设计、构象受限的合成肽进行筛选，以评估其抑制因子IXa与血小板结合的能力。这些肽（G4-Q11、S3-L6和F9-Q11）单独作用时，可抑制因子IXa与血小板上约500-600个位点中的约50%结合，其Ki值分别为2.9 nM（G4-Q11）、24 nM（S3-L6）和240 nM（F9-Q11），而天然因子IXa的Ki值约为2.5 nM。等摩尔浓度的两种肽S3-L6和F9-Q11一起添加时表现出约50倍的协同作用（Ki = 2.4 nM）。尽管因子IX和Gla肽（G4-Q11）均可置换100%结合的因子IX和约50%结合的因子IXa，但因子IX无效（摩尔过量>1000倍时），Gla结构域肽（G4-Q11）在抑制血小板受体介导的因子IXa激活因子X方面相对无效（Ki = 165 μM）。我们得出结论，因子IXa的Gla结构域（G4-Q11）包含两个构象受限的环结构，它们介导因子IX/IXa与活化的人血小板上的一个共享位点结合，该位点与酶因子IXa用于组装因子X激活复合物的位点不同且相互独立。
