Byun Y, Jacobs H A, Kim S W
Department of Pharmaceutics and Pharmaceutical Chemistry, The University of Utah, Salt Lake City 84132, USA.
Biotechnol Prog. 1996 Mar-Apr;12(2):217-25. doi: 10.1021/bp950076c.
The chemical immobilization of heparin onto polymeric materials through hydrophilic spacer groups was performed to improve the hemocompatibility of blood-contacting devices. Significant data have been gathered attesting to the biological activity of immobilized heparin in static in vitro studies (clotting times) and dynamic in vivo studies (thrombus formation). However, few studies have been performed to investigate the binding kinetics of spacer-immobilized heparin under flow (shear stress) with antithrombin III (ATIII) and thrombin. To help elucidate this binding mechanism, a mathematical model was developed which parallels experiments to measure protein binding and dissociation at the heparin immobilized surface under flow conditions. Heparinized tubing was prepared by chemically immobilizing a high-ATIII-affinity fraction of heparin onto the surface of poly(ethylene)-oxide grafted, poly(styrene-co-p-aminostyrene)-coated polyethylene tubing. ATIII was first bound onto the immobilized heparin, followed by the introduction of thrombin to interact with ATIII. The concentration of thrombin-ATIII complex (TAT) flowing from the tubing was determined, and the dissociation rate constants (kD) of TAT from immobilized heparin were calculated as a function of flow rate. The results indicate that the dissociation rate constant of TAT varied with flow rate, especially low flow rates, high flow rates, and turbulent flow. As the TAT complex dissociates from immobilized heparin, this "recovered" heparin is available for subsequent binding of more ATIII and thrombin. These in vitro mathematical results may help support mechanisms and hypotheses generated for the biological activity of spacer-immobilized heparin observed during long-term in vivo and ex vivo experiments.
通过亲水性间隔基团将肝素化学固定在聚合物材料上，以提高与血液接触装置的血液相容性。在静态体外研究（凝血时间）和动态体内研究（血栓形成）中，已经收集了大量数据证明固定化肝素的生物活性。然而，很少有研究探讨间隔基团固定化肝素在流动（剪切应力）条件下与抗凝血酶III（ATIII）和凝血酶的结合动力学。为了有助于阐明这种结合机制，开发了一个数学模型，该模型与在流动条件下测量肝素固定表面上蛋白质结合和解离的实验并行。通过将高ATIII亲和力的肝素部分化学固定在聚（环氧乙烷）接枝、聚（苯乙烯 - 对氨基苯乙烯）涂层的聚乙烯管表面上制备肝素化管。首先将ATIII结合到固定化肝素上，然后引入凝血酶与ATIII相互作用。测定从管中流出的凝血酶 - ATIII复合物（TAT）的浓度，并计算TAT从固定化肝素上的解离速率常数（kD）作为流速的函数。结果表明，TAT的解离速率常数随流速变化，特别是低流速、高流速和湍流。随着TAT复合物从固定化肝素上解离，这种“回收”的肝素可用于随后更多ATIII和凝血酶的结合。这些体外数学结果可能有助于支持在长期体内和体外实验中观察到的间隔基团固定化肝素生物活性的机制和假设。
