Woods V L, Wolff L E, Keller D M
J Biol Chem. 1986 Nov 15;261(32):15242-51.
Recent evidence suggests the presence in resting platelets of centrally located compartments of glycoprotein (GP) IIb-IIIa. We have employed an experimental procedure which dissociates and antigenically denatures the surface compartment of GP IIb-IIIa and allows internal compartments of GP IIb-IIIa to be studied immunochemically and functionally in intact platelets. When gel-filtered platelets are incubated with 0.25 mM EGTA at 37 degrees C for 30 min, and then supplemented for 30 min with 5 mM calcium, they lose their ability to bind GP IIb-IIIa complex-specific monoclonal antibody Fab fragments. However, when such platelets are subsequently stimulated with thrombin, GP IIb-IIIa-specific Fabs are again able to bind in large amounts to the platelet surface, in concert with the appearance of substantial amounts of receptors for fibrinogen and fibronectin. In immunoprecipitation experiments, we have found that this thrombin-displayed pool of GP IIb-IIIa originates from a pool that is not labeled by lactoperoxidase-catalyzed radioiodination of intact resting platelets. In immunofluorescence experiments, we have found that EGTA-incubated platelets contain a large sequestered internal pool of GP IIb-IIIa which upon thrombin stimulation is translocated to the platelet surface. Additional experiments suggest that this centrally located compartment may be surface connected in resting platelets and that it is accessible to some extracellular proteins, but not others.
最近的证据表明，静息血小板中存在位于中央的糖蛋白（GP）IIb-IIIa区室。我们采用了一种实验方法，该方法可使GP IIb-IIIa的表面区室解离并进行抗原性变性，并允许在完整血小板中对GP IIb-IIIa的内部区室进行免疫化学和功能研究。当凝胶过滤的血小板在37℃下与0.25 mM EGTA孵育30分钟，然后补充5 mM钙30分钟时，它们失去结合GP IIb-IIIa复合物特异性单克隆抗体Fab片段的能力。然而，当随后用凝血酶刺激这些血小板时，GP IIb-IIIa特异性Fabs又能够大量结合到血小板表面，同时出现大量纤维蛋白原和纤连蛋白受体。在免疫沉淀实验中，我们发现这种凝血酶诱导显示的GP IIb-IIIa池源自一个未被完整静息血小板的乳过氧化物酶催化放射性碘化标记的池。在免疫荧光实验中，我们发现用EGTA孵育的血小板含有大量隔离的内部GP IIb-IIIa池，在凝血酶刺激后，该池会转移到血小板表面。其他实验表明，这个位于中央的区室在静息血小板中可能与表面相连，并且一些细胞外蛋白可以接触到它，而其他蛋白则不能。
