THROMBIN CLEAVAGE ENHANCES EXPOSURE OF A HEPARIN-BINDING DOMAIN IN THE N-TERMINUS OF THE FIBRIN BETA-CHAIN

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.