Tm. Odrljin et al., THROMBIN CLEAVAGE ENHANCES EXPOSURE OF A HEPARIN-BINDING DOMAIN IN THE N-TERMINUS OF THE FIBRIN BETA-CHAIN, Blood, 88(6), 1996, pp. 2050-2061
Thrombin (IIa)-cleavage of fibrinogen (FBG) to form polymerized fibrin
promotes endothelial cell spreading, proliferation, and von Willebran
d factor release, requiring the exposure of the beta 15-42 domain. Stu
dies reported here indicate that IIa-cleavage of fibrinopeptide B enha
nces exposure of a heparin binding domain at the beta 15-42 neo-N-term
inus of fibrin, Crossed immunoelectrophoresis showed heparin-induced m
obility shifts indicative of complexing with FBG and with N-terminal C
NBr fragments of FBG (NDSK) and of fibrin (IIa-NDSK), but no evidence
of heparin complexing with FBG lacking B beta 1-42 or with FBG fragmen
ts D and E was seen. Elution from heparin-agarose with a linear gradie
nt of NaCl showed that bound portions of both intact FBG and D fragmen
ts eluted below physiologic salt concentrations, whereas E(3) fragment
s lacking B beta 1-53 did not bind. NDSK bound with higher affinity th
an did intact FBG, whereas binding of IIa-NDSK was maximal in this sys
tem, Binding of fibrin(ogen) to heparin agarose was saturable as well
as inhibitable in a dose-dependent manner with both FBG and heparin, S
catchard analysis indicated a single class of binding site, with disso
ciation constants (kd) of 0.3 mu mol/L for IIa-NDSK. 0.8 mu mol/L for
NDSK, and 18 mu mol/L for FBG. Immobilized fibrin had twofold more hep
arin binding sites than did immobilized FBG and required a 5.5-fold hi
gher 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 approxima
tely threefold higher affinity in fibrin than in FBG, Synthetic peptid
e beta 15-42 showed highest binding to heparin-agarose followed by B b
eta 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 sp
ecificity of heparin binding, Basic residues within the beta 15-32 reg
ion segregate primarily to one side of an alpha-helix in a helical whe
el diagram, as is typical for authentic HBDs, Desulfated heparin and h
eparan sulfate bound more fibrin(ogen) than did other proteoglycans; h
owever, heparin bound sixfold more IIa-NDSK than NDSK. These results c
onfirm that fibrin binds to heparin with higher affinity than does FBG
and that fibrin binding is not solely dependent on charge interaction
s of beta 15-42 with the negatively charged glycosaminoglycan. (C) 199
6 by The American Society of Hematology.