Role of the antithrombin-binding pentasaccharide in heparin acceleration of antithrombin-proteinase reactions. Resolution of the antithrombin conformational change contribution to heparin rate enhancement.
Role of the antithrombin-binding pentasaccharide in heparin acceleration of antithrombin-proteinase reactions. Resolution of the antithrombin conformational change contribution to heparin rate enhancement.
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DOI：
10.1016/s0021-9258(18)42309-5
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发表时间：
1992-06
期刊：
影响因子：
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通讯作者：
S. Olson;I. Björk;R. Sheffer;P. Craig;J. Shore;J. Choay
中科院分区：
文献类型：
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作者：
S. Olson;I. Björk;R. Sheffer;P. Craig;J. Shore;J. Choay
合成的抗凝血酶结合肝素五糖和全长肝素的约26个碱基含有这个特定的序列进行了比较，就其与抗凝血酶的相互作用和它们的能力，以促进抑制和底物反应的抗凝血酶与凝血酶和因子Xa。这些研究的目的是阐明戊糖对肝素对抗凝血酶-蛋白酶反应的加速作用的贡献。五糖和全长肝素结合抗凝血酶具有相当高的亲和力（KD值分别为36 +/- 11和10 +/- 3 nM，在I 0.15），并诱导高度相似的蛋白质荧光，紫外和圆二色性变化的抑制剂。停流荧光动力学研究的肝素结合相互作用在I0.15是一致的两个肝素的结合过程，包括一个初始的弱遇到复杂的相互作用形成类似的亲和力（KD 20-30 μ M），随后是抑制剂构象变化，具有520-700 s的不可区分的正向速率常数。1，但不同的逆转速率常数约为1 s-1的五糖和约0.2 s-1的全长肝素。二阶速率常数与凝血酶和Xa因子的抗凝血酶反应，最大限度地提高了五糖只有1.7倍的凝血酶，但一个实质性的270倍的Xa因子，在离子强度无关的方式在饱和寡糖。相比之下，全长肝素产生大的离子强度依赖性增强的二阶速率常数为4,300倍的凝血酶和580倍的因子Xa在I 0.15的抗凝血酶反应。这些增强可分解为归因于五糖的非离子组分和负责较大肝素的额外速率增加的离子组分。凝血酶和抗凝血酶灭活因子Xa的化学计量滴定以及这些反应产物的十二烷基硫酸钠-聚丙烯酰胺凝胶电泳表明，在抗凝血酶灭活因子Xa期间，五糖和全长肝素类似地促进蛋白水解修饰的抑制剂的形成，而只有全长肝素在与凝血酶反应期间有效促进抗凝血酶的这种底物反应。（400字处删节）
The synthetic antithrombin-binding heparin pentasaccharide and a full-length heparin of approximately 26 saccharides containing this specific sequence have been compared with respect to their interactions with antithrombin and their ability to promote inhibition and substrate reactions of antithrombin with thrombin and factor Xa. The aim of these studies was to elucidate the pentasaccharide contribution to heparin's accelerating effect on antithrombin-proteinase reactions. Pentasaccharide and full-length heparins bound antithrombin with comparable high affinities (KD values of 36 +/- 11 and 10 +/- 3 nM, respectively, at I 0.15) and induced highly similar protein fluorescence, ultraviolet and circular dichroism changes in the inhibitor. Stopped-flow fluorescence kinetic studies of the heparin binding interactions at I 0.15 were consistent with a two-step binding process for both heparins, involving an initial weak encounter complex interaction formed with similar affinities (KD 20-30 microM), followed by an inhibitor conformational change with indistinguishable forward rate constants of 520-700 s-1 but dissimilar reverse rate constants of approximately 1 s-1 for the pentasaccharide and approximately 0.2 s-1 for the full-length heparin. Second order rate constants for antithrombin reactions with thrombin and factor Xa were maximally enhanced by the pentasaccharide only 1.7-fold for thrombin, but a substantial 270-fold for factor Xa, in an ionic strength-independent manner at saturating oligosaccharide. In contrast, the full-length heparin produced large ionic strength-dependent enhancements in second order rate constants for both antithrombin reactions of 4,300-fold for thrombin and 580-fold for factor Xa at I 0.15. These enhancements were resolvable into a nonionic component ascribable to the pentasaccharide and an ionic component responsible for the additional rate increase of the larger heparin. Stoichiometric titrations of thrombin and factor Xa inactivation by antithrombin, as well as sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the products of these reactions, indicated that pentasaccharide and full-length heparins similarly promoted the formation of proteolytically modified inhibitor during the inactivation of factor Xa by antithrombin, whereas only the full-length heparin was effective in promoting this substrate reaction of antithrombin during the reaction with thrombin.(ABSTRACT TRUNCATED AT 400 WORDS)