Characterization of the binding of factor Xa to fibrinogen/fibrin derivatives and localization of the factor Xa binding site on fibrinogen.
The binding of human factor Xa to fibrinogen and its derivatives was characterized. Factor Xa bound to immobilized fibrin with a concentration at half-maximal binding (C50) of 100 nM. The 4-carboxyglutamic acid (Gla) domain of factor Xa is important in factor Xa binding to fibrin monomer, based on the following observations; the binding requires Ca2+; Gla-domain-lacking factor Xa could not bind to fibrin; factor Xa binding was significantly reduced by prior treatment of factor Xa with factor IX/factor-X-binding protein from the venom of Trimeresurus flavoviridis which specifically binds to the Gla domain of human factors IX and X. Factor Xa also bound to fibrinogen, fibrinogen degradation products (FDP)-D and FDP-E, with a similar affinity (C50 = 75-131 nM). In a solution-phase equilibrated binding assay, approximately 0.76 mol factor Xa bound to 1 mol fibrinogen with a dissociation constant of 180 nM. The binding of 125I-labeled factor Xa to the fibrin monomer was inhibited markedly by unlabeled factor Xa, but only slightly by thrombin, suggesting that the binding site of factor Xa on fibrin monomer differs from that of thrombin. We localized the binding site of factor Xa on fibrinogen: factor Xa bound strongly to the A alpha chain, but weakly to the B beta and gamma chains of fibrinogen. The A alpha chain was then digested with lysyl endopeptidase and separated by reverse-phase HPLC. Among resulting peptides, factor Xa bound specifically to a peptide corresponding to residues Asp82-Lys123 of the A alpha chain. This factor-Xa-binding site is located in the boundary between the central E domain and the terminal D domain of fibrinogen and is apparently distinct from the reported thrombin-binding site.
Iino
M
M
Department of Molecular Pathobiology, Mie University School of Medicine, Japan.
Takeya
H
H
Takemitsu
T
T
Nakagaki
T
T
Gabazza
E C
EC
Suzuki
K
K
D001665:Binding Sites; D015951:Factor Xa / Q000378:metabolism*; D005338:Fibrin Fibrinogen Degradation Products / Q000378:metabolism*; D005340:Fibrinogen / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D011869:Radioligand Assay
D005338:Fibrin Fibrinogen Degradation Products; D005340:Fibrinogen; D015951:Factor Xa