Byun Y, Jacobs H A, Feijen J, Kim S W
Department of Pharmaceutics and Pharmaceutical Chemistry, University of Utah, Salt Lake City 84112, USA.
J Biomed Mater Res. 1996 Jan;30(1):95-100. doi: 10.1002/(SICI)1097-4636(199601)30:1<95::AID-JBM12>3.0.CO;2-P.
An objective of this research is to verify the mechanism of anticoagulant 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 vitro. However, the strong ionic character of heparin leads to its specific and nonspecific binding with many other plasma proteins. Most notably, fibronectin contains six active binding sites for heparin which may interfere with the subsequent binding of heparin with ATIII or thrombin. Heparin was covalently immobilized through polyethylene oxide (PEO) hydrophilic spacer groups onto a model surface synthesized by random copolymerization of styrene and p-aminostyrene. The binding interaction of immobilized heparin with ATIII was then determined in the presence 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 surface immobilized heparin with ATIII and fibronectin. The extent of ATIII binding to heparin in each experiment was assayed using a chromogenic substrate for ATIII, S-2238. The results of this study demonstrate that the displacement of ATIII from immobilized heparin was proportional 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.
本研究的一个目标是验证在存在血浆蛋白的情况下表面固定化肝素的抗凝活性机制。已在体外描述了固定化肝素与抗凝血酶III（ATIII）/凝血酶之间的竞争和结合相互作用。然而，肝素的强离子特性导致其与许多其他血浆蛋白发生特异性和非特异性结合。最值得注意的是，纤连蛋白含有六个肝素活性结合位点，这可能会干扰肝素随后与ATIII或凝血酶的结合。通过聚环氧乙烷（PEO）亲水间隔基团将肝素共价固定在由苯乙烯和对氨基苯乙烯无规共聚合成的模型表面上。然后在不同纤连蛋白浓度存在的情况下测定固定化肝素与ATIII的结合相互作用。通过首先将固定化肝素与ATIII结合，然后引入纤连蛋白；肝素与纤连蛋白结合，然后与ATIII孵育，以及表面固定化肝素与ATIII和纤连蛋白同时孵育来研究结合相互作用。在每个实验中，使用ATIII的显色底物S-2238测定ATIII与肝素的结合程度。本研究结果表明，ATIII从固定化肝素上的置换与纤连蛋白浓度成正比，并且是可逆的。此外，结合顺序对结合到固定化肝素上的ATIII的最终浓度没有影响。
