Equilibrium binding of thrombin to platelets.
Equilibrium binding of thrombin to platelets.
复制标题
凝血酶与血小板的平衡结合。
DOI：
10.1021/bi00667a021
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发表时间：
1976
期刊：
影响因子：
2.9
通讯作者：
T. Detwiler
中科院分区：
文献类型：
--
作者：
B. M. Martin;W. W. Wasiewski;J. Fenton;T. Detwiler
本文研究了人[~(125)I]凝血酶与洗涤的人血小板的结合，以分析凝血酶刺激血小板的非酶方面。经乳过氧化物酶标记的高纯度α-凝血酶保持了完全的凝血酶和酯酶活性以及对血小板的全部活性，经十二烷基硫酸钠-聚丙烯酰胺凝胶电泳法和凝胶层析法与天然凝血酶没有区别，与未标记的凝血酶结合在一起。用油从悬浮介质中快速分离血小板后，测定结合的和游离的[125I]凝血酶。最大结合时间在15 S以内，这是测得的最短时间。当凝血酶的浓度足以引起小于最大的血小板刺激时，总凝血酶的90%在悬浮液中是游离的。建立了平衡结合，游离凝血酶和游离血小板都保持了活性，并在稀释或添加未标记凝血酶后迅速重新平衡。平衡是复杂的，表观结合位点数和解离常数取决于凝血酶浓度。结合凝血酶与凝血酶浓度的关系通过双倒数和Scatchard图分析，发现300-400个高亲和力位点(Kdiss=1.8-2 nM)；这些与凝血酶刺激钙分泌有关，在1.5 nM凝血酶时作用一半最大，当每个血小板结合500-600个凝血酶时作用最大。
Binding of human [125I]thrombin to washed human platelets was studied in order to analyze the nonenzymic aspects of the thrombin stimulation of platelets. Highly purified alpha-thrombin that was iodinated with lactoperoxidase retained full clotting and esterase activities and full activity toward platelets, was not distinguished from native thrombin by sodium dodecyl sulfate-polyacrylamide gel electrophoresis or gel chromatography, and bound to platelets the same as unlabeled thrombin. Bound and free [125I]thrombin were measured after rapid separation of platelets from the suspending medium by centrifugation through oil. Maximum binding was within 15 s, the shortest time measured. At concentrations of thrombin sufficient to cause less than maximal platelet stimulation, 90% of the total thrombin was free in the suspending solutions. Equilibrium binding was established, with both free thrombin and free platelets retaining activity, and with rapid reequilibration after dilution or addition of unlabeled thrombin. The equilibrium was complex, with the apparent number of binding sites and dissociation constants dependent on thrombin concentration. Analysis of bound thrombin as a function of thrombin concentration by double-reciprocal and Scatchard plots indicated 300-400 high affinity sites (Kdiss = 1.8-2 nM); these correlate with thrombin stimulation of Ca2+ secretion, which shows half maximal effect at 1.5 nM thrombin and maximal effect with 500-600 thrombins bound per platelet.