Identification of a Binding Site for Glycoprotein Ibα in the Apple 3 Domain of Factor XI

Frank A Baglia,David Gailani,José A López,Peter N Walsh

doi:10.1074/jbc.m406727200

Abstract

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.

Highlights

Factor XI (FXI)1 is a 160,000-dalton disulfide-linked homodimeric coagulation protein that exists in plasma in a complex with high molecular weight kininogen (HK) [1,2,3,4,5,6,7]
We have previously shown that specific amino acids in the A3 domain (Ser248, Arg250, Lys255, Phe260, and Gln263) mediate the binding of FXI to platelets [21, 24], whereas amino acids juxtaposed to the plateletbinding site (Lys252 and Lys253) are important for binding heparin [22]
ZnCl2-dependent Binding of Factor XI to Glycocalicin—The FXI-binding site on platelets is postulated to reside within the GPIb␣ subunit of the GPIb-IX-V complex [15], because Bernard-Soulier platelets deficient in this complex are deficient in FXI binding, because two GPIb␣ ligands, SZ-2 and bovine von Willebrand factor, both inhibit FXI binding to platelets, and because FXI was shown to bind to glycocalicin in a Zn2ϩ-dependent fashion (Kd app ϭ ϳ52 nM) by surface plasmon resonance

Summary

Introduction

Factor XI (FXI) is a 160,000-dalton disulfide-linked homodimeric coagulation protein that exists in plasma in a complex with high molecular weight kininogen (HK) [1,2,3,4,5,6,7]. We have previously demonstrated that in the presence of Zn2ϩ ions alone (i.e. in the absence of added HK), FXI binds to about half the number of sites on activated platelets [19, 21] that are observed in the presence of both Zn2ϩ ions and HK. It is not clear whether the A3 domain mediates FXI binding to GPIb␣ and which specific FXI residues are involved in this interaction. The aim of the present study is to identify the amino acid sequences in FXI that interact with GPIb␣ under the conditions of Zn2ϩ-dependent and HK and Zn2ϩ-dependent binding of FXI to the activated platelet

Objectives

Results

Conclusion