Interaction of fibrin(ogen) with heparin: further characterization and localization of the heparin-binding site.
The beta chain 15-42 sequence of the fibrin(ogen) E region was implicated in heparin binding [Odrljin et al. (1996) Blood 88, 2050-2061]; whether heparin binds to other fibrin(ogen) regions remains to be clarified. To address this question, we studied the interaction of heparin with fibrinogen, fibrin, and their major fragments D(1), D-D, E(1), E(3), and alphaC, which together cover the entire structure of the molecule, by ligand blotting, surface plasmon resonance, and fluorescence. All three techniques revealed that at physiological ionic conditions only fibrin(ogen) and the E(1) fragment bind heparin, indicating that the only physiologically relevant heparin-binding site of fibrin(ogen) is located in its E region. To test whether the beta15-42 sequence is sufficient to form this site or some additional sequences are also involved, we tested the interaction of heparin with a number of beta15-42-containing fragments. The synthetic beta15-42 peptide bound heparin weakly (K(d) = 44.5 microM) while the recombinant beta15-57 and beta15-64 fragments exhibited almost 7-fold higher affinity (K(d) = 6.4 and 7.1 microM, respectively), indicating that the beta43-57 region is also important for heparin binding. At the same time the recombinant dimeric disulfide-linked (beta15-66)(2) fragment which mimics the dimeric arrangement of the beta chains in fibrin bound heparin with high affinity (K(d) = 66 nM), almost 100-fold higher than that for the monomeric fragments. This affinity was similar to those determined for fibrin and the E(1) fragment (K(d) = 72 and 70 nM, respectively) suggesting that (beta15-66)(2) mimics well the heparin-binding properties of the latter two. Altogether, these results indicate that the only heparin-binding site in fibrin(ogen) is formed by NH(2)-terminal portions of the beta chains, including residues beta15-57, and that dimerization is essential for high-affinity binding.
Yakovlev
Sergiy
S
Biochemistry Department, Jerome H. Holland Laboratory for the Biomedical Sciences, American Red Cross, 15601 Crabbs Branch Way, Rockville, Maryland 20855, USA.
Gorlatov
Sergey
S
Ingham
Kenneth
K
Medved
Leonid
L
D001665:Binding Sites; D005338:Fibrin Fibrinogen Degradation Products / Q000378:metabolism; D005340:Fibrinogen / Q000378:metabolism*; D005456:Fluorescent Dyes; D006493:Heparin / Q000378:metabolism*; D007700:Kinetics; D011485:Protein Binding; D013194:Staining and Labeling
D005338:Fibrin Fibrinogen Degradation Products; D005456:Fluorescent Dyes; C018543:fibrinogen fragment E; D005340:Fibrinogen; D006493:Heparin