EFFECT OF FIBRONECTIN ON THE BINDING OF ANTITHROMBIN-III TO IMMOBILIZED HEPARIN

An objective of this research is to verify the mechanism of anticoagul ant activity of surface-immobilized heparin in the presence of plasma proteins. The competition and binding interaction between immobilized heparin and antithrombin III (ATIII)/thrombin have been described in v itro. However, the strong ionic character of heparin leads to its spec ific and nonspecific binding with many other plasma proteins. Most not ably, fibronectin contains six active binding sites for heparin which may interfere with the subsequent binding of heparin with ATIII or thr ombin. Heparin was covalently immobilized through polyethylene oxide ( PEG) hydrophilic spacer groups onto a model surface synthesized by ran dom copolymerization of styrene and p-aminostyrene. The binding intera ction of immobilized heparin with ATIII was then determined in the pre sence of different fibronectin concentrations. The binding interaction was studied by first binding immobilized heparin with ATIII, followed by the introduction of fibronectin; heparin binding with fibronectin, followed by incubation with ATIII, and simultaneous incubation of sur face immobilized heparin with ATIII and fibronectin. The extent of ATI II binding to heparin in each experiment was assayed using a chromogen ic substrate for ATIII, S-2238. The results of this study demonstrate that the displacement of ATIII from immobilized heparin was proportion al to the fibronectin concentration, and was reversible. Furthermore, the binding sequence did not play a role in the final concentration of ATIII bound to immobilized heparin. (C) 1996 John Wiley & Sons, Inc.