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Thrombin cleavage enhances exposure of a heparin binding domain in the
N-terminus of the fibrin beta chain
TM Odrljin, JR Shainoff, SO Lawrence and PJ Simpson-Haidaris
Department of Medicine, University of Rochester, School of Medicine and
Dentistry, NY, USA.
Thrombin (IIa)-cleavage of fibrinogen (FBG) to form polymerized fibrin
promotes endothelial cell spreading, proliferation, and von Willebrand
factor release, requiring the exposure of the beta 15-42 domain. Studies
reported here indicate that IIa-cleavage of fibrinopeptide B enhances
exposure of a heparin binding domain at the beta 15-42 neo-N- terminus of
fibrin. Crossed immunoelectrophoresis showed heparin- induced mobility
shifts indicative of complexing with FBG and with N- terminal CNBr
fragments of FBG (NDSK) and of fibrin (IIa-NDSK), but not evidence of
heparin complexing with FBG lacking B beta 1-42 or with FBG fragments D and
E was seen. Elution from heparin-agarose with a linear gradient of NaCl
showed that bound portions of both intact FBG and D fragments eluted below
physiologic salt concentrations, whereas E3 fragments lacking B beta 1-53
did not bind. NDSK bound with higher affinity than did intact FBG, whereas
binding of IIa-NDSK was maximal in this system. Binding of fibrin(ogen) to
heparin agarose was saturable as well as inhibitable in a dose-dependent
manner with both FBG and heparin. Scatchard analysis indicated a single
class of binding site, with dissociation constants (kd) of 0.3 mumol/L for
IIa-NDSK, 0.8 mumol/L for NDSK, and 18 mumol/L for FBG. Immobilized fibrin
had twofold more heparin binding sites than did immobilized FBG and
required a 5.5-fold higher concentration of heparin to inhibit by 50% the
binding of labeled heparin. Together, the results indicate that IIa-
cleavage results in enhanced exposure of two heparin binding domains (HBDs)
with approximately threefold higher affinity in fibrin than in FBG.
Synthetic peptide beta 15-42 showed highest binding to heparin- agarose
followed by B beta 1-42, whereas peptides beta 18-31, beta 18- 27, and beta
24-42 did not bind. Thus, the primary structure of beta 15- 42 is required
for specificity of heparin binding. Basic residues within the beta 15-32
region segregate primarily to one side of an alpha-helix in a helical wheel
diagram, as is typical for authentic HBDs. Desulfated heparin and heparan
sulfate bound more fibrin(ogen) than did other proteoglycans; however,
heparin bound sixfold more Ila- NDSK than NDSK. These results confirm that
fibrin binds to heparin with higher affinity than does FBG and that fibrin
binding is not solely dependent on charge interactions of beta 15-42 with
the negatively charged glycosaminoglycan.
Volume 88,
Issue 6,
pp. 2050-2061,
09/15/1996
Copyright © 1996 by The American Society of Hematology
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