Domain V of β2-Glycoprotein I Binds Factor XI/XIa and Is Cleaved at Lys317-Thr318

Tong Shi,Bill Giannakopoulos,G Michael Iverson,Keith A Cockerill,Matthew D Linnik,Steven A Krilis

doi:10.1074/jbc.m410291200

Tong Shi, Bill Giannakopoulos + Show 4 more

Open Access

https://doi.org/10.1074/jbc.m410291200

Copy DOI

Abstract

The fifth domain (DV) of beta2-glycoprotein I (beta2GPI) is important for binding a number of ligands including phospholipids and factor XI (FXI). Beta2GPI is proteolytically cleaved in DV by plasmin but not by thrombin, VIIa, tissue plasminogen activator, or uPA. Following proteolytic cleavage of DV by plasmin, beta2GPI retains binding to FXI but not to phospholipids. Native beta2GPI, but not cleaved beta2GPI, inhibits activation of FXI by thrombin and factor XIIa, attenuating a positive feedback mechanism for additional thrombin generation. In this report, we have defined the FXI/FXIa binding site on beta2GPI using site-directed mutagenesis. We show that the positively charged residues Lys284, Lys286, and Lys287 in DV are essential for the interaction of beta2GPI with FXI/FXIa. We also demonstrate that FXIa proteolytically cleaves beta2GPI at Lys317-Thr318 in DV. Thus, FXIa cleavage of beta2GPI in vivo during thrombus formation may accelerate FXI activation by decreasing the inhibitory effect of beta2GPI.

Highlights

The fifth domain (DV) of ␤2-glycoprotein I (␤2GPI) is important for binding a number of ligands including phospholipids and factor XI (FXI). ␤2GPI is proteolytically cleaved in DV by plasmin but not by thrombin, VIIa, tissue plasminogen activator, or uPA
FXI/factor XIa (FXIa) Bind ␤2GPI via Lysine Residues in DV—To investigate the FXI binding site on ␤2GPI, we measured 125I-FXI binding to ␤2GPI and various mutants coated on microplate wells. 125I-FXI bound to n␤2GPI, Recombinant full-length human ␤2GPI (rh␤2GPI) [27], domain deletion mutants DV, and DII-V but not to DI, DI-II, DI-III, or DI-IV (Fig. 1)
We recently reported that ␤2GPI binds FXI and inhibits its activation by thrombin and factor XIIa in the presence of dextran sulfate at concentrations lower than those normally found in human plasma [27]

Summary

EXPERIMENTAL PROCEDURES

Materials—Plasma-derived FXI, FXIa, and HK were purchased from Calbiochem-Novabiochem. 100 ␮l of 125I-FXI (0.28 nM) in 0.5% BSA/PBS was added to individual wells and incubated for 4 –5 h at 25 °C. Competitive Inhibition Assays—The effect of native recombinant fulllength and domain deletion mutants of ␤2GPI on the binding of 125I-FXI to rh␤2GPI was studied using Lockwell microtiter plates, the wells of which were coated with 100 ␮l of rh␤2GPI (50 nM) by incubation overnight at 4 °C. The plate was treated as described above before 50 ␮l of 125I-FXI (0.56 nM), and 50 ␮l of various ␤2GPI preparations or BSA (1.3 nM-4 ␮M) in 0.5% BSA/PBS was added to the wells and incubated for 4 –5 h at 25 °C. The plate was treated as described above and 100 ␮l of FXIa (10 nM) in 0.5% BSA/PBS was added to individual wells and incubated for 3 h at 37 °C. Differences between the groups were evaluated using Student’s t test

RESULTS

Amino acids time

DISCUSSION