Characterization of the H-kininogen-binding site on factor XI: a comparison of factor XI and plasma prekallikrein.
Factor XI (FXI), the zymogen of the blood coagulation protease FXIa, and the structurally homologous protein plasma prekallikrein circulate in plasma in noncovalent complexes with H-kininogen (HK). HK binds to the heavy chains of FXI and of prekallikrein. Each chain contains four apple domains (F1-F4 for FXI and P1-P4 for prekallikrein). Previous studies indicated that the HK-binding site on FXI is located in F1, whereas the major HK-binding site on prekallikrein is in P2. To determine the contribution of each FXI apple domain to HK-FXI complex formation, we examined binding of recombinant single apple domain-tissue plasminogen activator fusion proteins to HK. The order of affinity from highest to lowest is F2 F4 > F1 F3. Monoclonal antibodies against F2 are superior to F4 or F1 antibodies as inhibitors of HK binding to FXI. Antibody alphaP2, raised against prekallikrein, cross-reacts with FXI F2 and inhibits FXI-HK binding with an IC(50) of 8 nm. HK binding to a platelet-specific FXI variant lacking the N-terminal half of F2 is reduced > 5-fold compared with full-length FXI. A chimeric FXI molecule in which F2 is replaced by P2 is cleaved within P2 during activation by factor XIIa, resulting in greatly reduced HK binding capacity. In contrast, wild-type FXI is not cleaved within F2, and its binding capacity for HK is unaffected by factor XIIa. Our data show that HK binding to FXI involves multiple apple domains, with F2 being most important. The findings demonstrate a similarity in mechanism for FXI and prekallikrein binding to HK.
Renné
Thomas
T
Institute for Biochemistry II, Johann Wolfgang Goethe-University of Frankfurt, Theodor-Stern-Kai 7, D-60590 Frankfurt, Germany.
Gailani
David
D
Meijers
Joost C M
JC
Müller-Esterl
Werner
W
D017398:Alternative Splicing; D000818:Animals; D001665:Binding Sites; D015153:Blotting, Western; D002460:Cell Line; D006224:Cricetinae; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D005172:Factor XI / Q000737:chemistry* / Q000378:metabolism*; D006801:Humans; D020128:Inhibitory Concentration 50; D019679:Kininogen, High-Molecular-Weight / Q000737:chemistry* / Q000378:metabolism*; D011233:Precipitin Tests; D011288:Prekallikrein / Q000737:chemistry*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000378:metabolism
D019679:Kininogen, High-Molecular-Weight; D011993:Recombinant Fusion Proteins; D005172:Factor XI; D011288:Prekallikrein