凝血因子XI苹果1结构域中凝血酶原kringle II结构域的结合位点。

A binding site for the kringle II domain of prothrombin in the apple 1 domain of factor XI.

作者信息

Baglia F A, Badellino K O, Ho D H, Dasari V R, Walsh P N

机构信息

Sol Sherry Thrombosis Research Center, Departments of Medicine and Biochemistry, Temple University School of Medicine, Philadelphia, Pennsylvania 19140, USA.

出版信息

J Biol Chem. 2000 Oct 13;275(41):31954-62. doi: 10.1074/jbc.M005465200.

DOI:10.1074/jbc.M005465200

PMID:10924522

Abstract

Previously we defined binding sites for high molecular weight kininogen (HK) and thrombin in the Apple 1 (A1) domain of factor XI (FXI). Since prothrombin (and Ca(2+)) can bind FXI and can substitute for HK (and Zn(2+)) as a cofactor for FXI binding to platelets, we have attempted to identify a prothrombin-binding site in FXI. The recombinant A1 domain (rA1, Glu(1)-Ser(90)) inhibited the saturable, specific and reversible binding of prothrombin to FXI, whereas neither the rA2 domain (Ser(90)-Ala(181)), rA3 domain (Ala(181)-Val(271)), nor rA4 domain (Phe(272)-Glu(361)) inhibited prothrombin binding to FXI. Kinetic binding studies using surface plasmon resonance showed binding of FXI (K(d) approximately 71 nm) and the rA1 domain (K(d) approximately 239 nm) but not rA2, rA3, or rA4 to immobilized prothrombin. Reciprocal binding studies revealed that synthetic peptides (encompassing residues Ala(45)-Ser(86)) containing both HK- and thrombin-binding sites, inhibit (125)I-rA1 (Glu(1)-Ser(90)) binding to prothrombin, (125)I-prothrombin binding to FXI, and (125)I-prothrombin fragment 2 (Ser(156)-Arg(271)) binding to FXI. However, homologous prekallikrein-derived peptides (encompassing Pro(45)-Gly(86)) did not inhibit FXI rA1 binding to prothrombin. The peptides Ala(45)-Arg(54), Phe(56)-Val(71), and Asp(72)-Ser(86), derived from sequences of the A1 domain of FXI, acted synergistically to inhibit (125)I-rA1 binding to prothrombin. Mutant rA1 peptides (V64A and I77A), which did not inhibit FXI binding to HK, retained full capacity to inhibit rA1 domain binding to prothrombin, and mutant rA1 peptides Ala(45)-Ala(54) (D51A) and Val(59)-Arg(70) (E66A), which did not inhibit FXI binding to thrombin, retained full capacity to inhibit rA1 domain binding to prothrombin. Thus, these experiments demonstrate that a prothrombin binding site exists in the A1 domain of FXI spanning residues Ala(45)-Ser(86) that is contiguous with but separate and distinct from the HK- and thrombin-binding sites and that this interaction occurs through the kringle II domain of prothrombin.

摘要

此前我们已确定了因子 XI（FXI）苹果 1（A1）结构域中高分子量激肽原（HK）和凝血酶的结合位点。由于凝血酶原（以及 Ca²⁺）可结合 FXI，并可替代 HK（以及 Zn²⁺）作为 FXI 与血小板结合的辅因子，因此我们试图在 FXI 中鉴定凝血酶原结合位点。重组 A1 结构域（rA1，Glu¹ - Ser⁹⁰）可抑制凝血酶原与 FXI 的饱和、特异性和可逆性结合，而 rA2 结构域（Ser⁹⁰ - Ala¹⁸¹）、rA3 结构域（Ala¹⁸¹ - Val²⁷¹）或 rA4 结构域（Phe²⁷² - Glu³⁶¹）均不能抑制凝血酶原与 FXI 的结合。使用表面等离子体共振进行的动力学结合研究表明，FXI（Kd 约为 71 nM）和 rA1 结构域（Kd 约为 239 nM）可与固定化的凝血酶原结合，而 rA2、rA3 或 rA4 则不能。相互结合研究表明，包含 HK 和凝血酶结合位点的合成肽（涵盖 Ala⁴⁵ - Ser⁸⁶ 残基）可抑制¹²⁵I - rA1（Glu¹ - Ser⁹⁰）与凝血酶原的结合、¹²⁵I - 凝血酶原与 FXI 的结合以及¹²⁵I - 凝血酶原片段 2（Ser¹⁵⁶ - Arg²⁷¹）与 FXI 的结合。然而，源自前激肽释放酶的同源肽（涵盖 Pro⁴⁵ - Gly⁸⁶）不能抑制 FXI rA1 与凝血酶原的结合。源自 FXI A1 结构域序列的肽 Ala⁴⁵ - Arg⁵⁴、Phe⁵⁶ - Val⁷¹ 和 Asp⁷² - Ser⁸⁶ 协同作用可抑制¹²⁵I - rA1 与凝血酶原的结合。不抑制 FXI 与 HK 结合的突变 rA1 肽（V64A 和 I77A）保留了完全抑制 rA1 结构域与凝血酶原结合的能力，而不抑制 FXI 与凝血酶结合的突变 rA1 肽 Ala⁴⁵ - Ala⁵⁴（D51A）和 Val⁵⁹ - Arg⁷⁰（E66A）也保留了完全抑制 rA1 结构域与凝血酶原结合的能力。因此，这些实验表明，FXI 的 A1 结构域中存在一个凝血酶原结合位点，其跨越 Ala⁴⁵ - Ser⁸⁶ 残基，与 HK 和凝血酶结合位点相邻但分开且不同，并且这种相互作用通过凝血酶原的kringle II 结构域发生。