Loading...
Structural Determinants for Heparin Binding in Human Coagulation Factor XI
Shikov, Sergei
Shikov, Sergei
Citations
Altmetric:
Genre
Thesis/Dissertation
Date
2008
Advisor
Committee member
Group
Department
Biochemistry
Permanent link to this record
Collections
Research Projects
Organizational Units
Journal Issue
DOI
http://dx.doi.org/10.34944/dspace/3692
Abstract
Coagulation factor XI plays an important role in the consolidation phase of blood coagulation. Previous studies from our laboratory and others have demonstrated that zymogen factor XI (FXI) binds to heparin with moderate (KD~110 nM) affinity via residues (K252, K253 and K255) located in the apple 3 (A3) domain of the molecule. In contrast, the enzyme, factor XIa (FXIa), was shown to bind to heparin with significantly higher affinity (~1.5 nM by ELISA) via residues (K529, R530 and R532) within the catalytic domain (CD). The interaction between heparin and FXIa potentiates the inhibition of FXIa by protease nexin-2 by 10-fold. In addition, related polyanions heparin and dextran sulfate inhibit the catalytic activity of FXIa. The present study was designed to determine the relative contributions of positively charged residues as well as the dimeric structure of FXI in heparin binding. During this project, wtFXI, FXIR504A, FXIK505A, FXIR507A, FXIR529A, FXIR530A, FXIR532A, and FXIR586A have been expressed and purified. All mutants were homogenous and identical to wtFXI on SDS-PAGE, clotting assays and 1G5 monoclonal antibody binding studied by SPR. In addition, monomeric FXI C321S/K331A was expressed and purified. Utilizing an ELISA assay, no difference in the affinity for heparin between FXIa and FXI was found. Surface plasmon resonance (SPR) data collected for FXI clearly indicate a complex interaction which does not conform to a simple 1:1 Langmuir binding model making it difficult to obtain quantitative information. The complexity of FXI interactions with heparin is likely to arise from the multivalent nature of the binding, in which both protein and heparin have multiple binding sites. Two positively charged residues in the FXI catalytic domain, FXIR507A and FXIR532A, were found to be particularly important for interaction with heparin. The FXIR507A and FXIR532A mutants demonstrated ~ 65% and ~50% decreases respectively in total number of heparin binding sites based on ELISA. Also, the apparent dissociation constants for FXIR507A (KDapp ~13 nM) and FXIR532A (KDapp ~21 nM ) were 6 and 10-fold increased respectively compared with 2.1 nM for the wtFXI. Mutant FXIR586A also demonstrated a defect in affinity (KDapp ~ 13 nM) without an effect on the Bmax. The monomeric FXIC321S/R331A was also characterized for its ability to bind heparin compared with wtFXI. Surprisingly, the monomeric FXI displayed defective binding to heparin according to ELISA (KDapp ~ 30 nM) and SPR methods. Thus, the unique homodimeric structure of FXI in addition to the residues both in its catalytic and A3 domain chains are necessary for high-affinity heparin binding.
Description
Citation
Citation to related work
Has part
ADA compliance
For Americans with Disabilities Act (ADA) accommodation, including help with reading this content, please contact scholarshare@temple.edu