Baglia Frank A, Gailani David, López José A, Walsh Peter N
Sol Sherry Thrombosis Research Center, Temple University School oof Medicine, Philadelphia, PA 19140, USA.
J Biol Chem. 2004 Oct 29;279(44):45470-6. doi: 10.1074/jbc.M406727200. Epub 2004 Aug 17.
Factor XI (FXI) is a homodimeric plasma zymogen that is cleaved at two internal Arg(369)-Ile(370) bonds by thrombin, factor XIIa, or factor XIa. FXI circulates as a complex with the glycoprotein high molecular weight kininogen (HK). FXI binds to specific sites (K(d) = approximately 10 nM, B(max) = approximately 1,500/platelet) on the surface of stimulated platelets, where it is efficiently activated by thrombin. The FXI Apple 3 (A3) domain mediates binding to platelets in the presence of HK and zinc ions (Zn(2+)) or prothrombin and calcium ions. The platelet glycoprotein (GP) Ib-IX-V complex is the receptor for FXI. Using surface plasmon resonance, we determined that FXI binds specifically to glycocalicin, the extracellular domain of GPIbalpha, in a Zn(2+)-dependent fashion (K(d) = approximately 52 nM). We now show that recombinant FXI A3 domain inhibits FXI inbinding to glycocalicin in the presence of Zn(2+), whereas the recombinant FXI A1, A2, or A4 domains have no effect. Experiments with full-length recombinant FXI mutants show that, in the presence of Zn(2+), glycocalicin binds FXI at a heparin-binding site in A3 (Lys(252) and Lys(253)) and not by amino acids previously shown to be required for platelet binding (Ser(248), Arg(250), Lys(255), Phe(260), and Gln(263)). However, binding in the presence of HK and Zn(2+) requires Ser(248), Arg(250), Lys(255), Phe(260), and GLn(263) and not Lys(252) and Lys(253). Thus, binding of FXI to GPIbalpha is mediated by amino acids in the A3 domain in the presence or absence of HK. This interaction is important for the initiation of the consolidation phase of blood coagulation and the generation of thrombin at sites of platelet thrombus formation.
因子 XI（FXI）是一种同源二聚体血浆酶原，可被凝血酶、因子 XIIa 或因子 XIa 在两个内部 Arg(369)-Ile(370)键处切割。FXI 以与糖蛋白高分子量激肽原（HK）形成的复合物形式循环。FXI 与活化血小板表面的特定位点（解离常数 K(d)约为 10 nM，最大结合量 B(max)约为 1,500/血小板）结合，在该位点它可被凝血酶有效激活。FXI 的苹果 3（A3）结构域在存在 HK 和锌离子（Zn(2+)）或凝血酶原和钙离子的情况下介导与血小板的结合。血小板糖蛋白（GP）Ib-IX-V 复合物是 FXI 的受体。利用表面等离子体共振技术，我们确定 FXI 以 Zn(2+)依赖的方式特异性结合糖萼蛋白，即 GPIbalpha 的细胞外结构域（K(d)约为 52 nM）。我们现在表明，重组 FXI A3 结构域在存在 Zn(2+)的情况下抑制 FXI 与糖萼蛋白的结合，而重组 FXI A1、A2 或 A4 结构域则无此作用。对全长重组 FXI 突变体的实验表明，在存在 Zn(2+)的情况下，糖萼蛋白在 A3 结构域的肝素结合位点（Lys(252)和 Lys(253)）结合 FXI，而不是通过先前显示的血小板结合所需氨基酸（Ser(248)、Arg(250)、Lys(255)、Phe(第260)和 Gln(263)）。然而，在存在 HK 和 Zn(2+)的情况下结合需要 Ser(248)、Arg(250)、Lys(255)、Phe(260)和 GLn(263)，而不是 Lys(252)和 Lys(253)。因此,FXI 与 GPIbalpha 的结合在有无 HK 的情况下均由 A3 结构域中的氨基酸介导。这种相互作用对于血液凝固巩固阶段的启动以及血小板血栓形成部位凝血酶的产生很重要。
