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 c haracterized. Factor Xa bound to immobilized fibrin with a concentrati on at half-maximal binding (C-50) of 100 nM. The 4-carboxyglutamic aci d (Gla) domain of factor Xa is important in factor Xa binding to fibri n monomer, based on the following observations; the binding requires C a2+; 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 fla voviridis which specifically binds to the Gla domain of human factors IX and X. Factor Xa also bound to fibrinogen, fibrinogen degradation p roducts (FDP)-D acid FDP-E, with a similar affinity (C-50 = 75-131 nM) . In a solution-phase equilibrated binding assay, approximately 0.76 m ol factor Xa bound to 1 mol fibrinogen with a dissociation constant of 180 nM. The binding of I-125-labeled factor Xa to the fibrin monomer was inhibited markedly by unlabeled factor Xa, but only slightly by th rombin, suggesting that the binding site of factor Xa on fibrin monome r differs from that of thrombin. We localized the binding site of fact or Xa on fibrinogen: factor Xa bound strongly to the A alpha chain, bu t weakly to the B beta and gamma chains of fibrinogen. The A alpha cha in 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 c entral E domain and the terminal D domain of fibrinogen and is apparen tly distinct from the reported thrombin-binding site.