Loike J D, Silverstein R, Cao L, Solomon L, Weitz J, Haber E, Matsueda G R, Bernatowicz M S, Silverstein S C
Department of Physiology and Cellular Biophysics, College of Physicians and Surgeons of Columbia University, New York, New York 10032.
J Cell Biol. 1993 May;121(4):945-55. doi: 10.1083/jcb.121.4.945.
Leukocytes form zones of close apposition when they adhere to ligand-coated surfaces. Because plasma proteins are excluded from these contact zones, we have termed them protected zones of adhesion. To determine whether platelets form similar protected zones of adhesion, gel-filtered platelets stimulated with thrombin or ADP were allowed to adhere to fibrinogen- or fibronectin-coated surfaces. The protein-coated surfaces with platelets attached were stained with either fluorochrome-conjugated goat anti-human fibrinogen or anti-human fibronectin antibodies, or with rhodamine-conjugated polyethylene glycol polymers. Fluorescence microscopy revealed that F(ab')2 anti-fibrinogen (100 kD) did not penetrate into the contact zones between stimulated platelets and the underlying fibrinogen-coated surface, while Fab antifibrinogen (50 kD) and 10 kD polyethylene glycol readily penetrated and stained the substrate beneath the platelets. Thrombin- or ADP-stimulated platelets also formed protected zones of adhesion on fibronectin-coated surfaces. F(ab')2 anti-fibronectin and 10 kD polyethylene glycol were excluded from these adhesion zones, indicating that they are much less permeable than those formed by platelets on fibrinogen-coated surfaces. The permeability properties of protected zones of adhesion formed by stimulated platelets on surfaces coated with both fibrinogen and fibronectin were similar to the zones of adhesion formed on fibronectin alone. mAb 7E3, directed against the alpha IIb beta 3 integrin blocked the formation of protected adhesion zones between thrombin-stimulated platelets and fibrinogen or fibronectin coated surfaces. mAb C13 is directed against the alpha 5 beta 1 integrin on platelets. Stimulated platelets treated with this mAb formed protected zones of adhesion on surfaces coated with fibronectin. These protected zones were impermeable to F(ab')2 antifibronectin but were permeable to 10 kD polyethylene glycol. These results show that activated platelets form protected zones of adhesion and that the size of molecules excluded from these zones depends upon the composition of the matrix proteins to which the platelets adhere. They also show that formation of protected zones of adhesion by platelets requires alpha IIb beta 3 integrins while the permeability properties of these zones of adhesion are regulated by both alpha IIb beta 3 and alpha 5 beta 1 integrins.
白细胞粘附于配体包被的表面时会形成紧密贴合区域。由于血浆蛋白被排除在这些接触区域之外，我们将其称为粘附保护区。为了确定血小板是否形成类似的粘附保护区，将用凝血酶或二磷酸腺苷（ADP）刺激的凝胶过滤血小板使其粘附于纤维蛋白原或纤连蛋白包被的表面。附着有血小板的蛋白包被表面用荧光染料偶联的山羊抗人纤维蛋白原或抗人纤连蛋白抗体，或罗丹明偶联的聚乙二醇聚合物进行染色。荧光显微镜检查显示，F(ab')2抗纤维蛋白原（100 kD）未渗透到受刺激血小板与下层纤维蛋白原包被表面之间的接触区域，而Fab抗纤维蛋白原（50 kD）和10 kD聚乙二醇很容易渗透并对血小板下方的底物进行染色。凝血酶或ADP刺激的血小板在纤连蛋白包被的表面上也形成了粘附保护区。F(ab')2抗纤连蛋白和10 kD聚乙二醇被排除在这些粘附区域之外，表明它们的渗透性远低于血小板在纤维蛋白原包被表面上形成的区域。受刺激血小板在同时包被有纤维蛋白原和纤连蛋白的表面上形成的粘附保护区的渗透特性与仅在纤连蛋白上形成的粘附区域相似。针对αIIbβ3整合素的单克隆抗体7E3可阻断凝血酶刺激的血小板与纤维蛋白原或纤连蛋白包被表面之间的保护性粘附区域的形成。单克隆抗体C13针对血小板上的α5β1整合素。用该单克隆抗体处理的受刺激血小板在包被有纤连蛋白的表面上形成了粘附保护区。这些保护区对F(ab')2抗纤连蛋白不可渗透，但对10 kD聚乙二醇可渗透。这些结果表明，活化的血小板形成了粘附保护区，并且被排除在这些区域之外的分子大小取决于血小板所粘附的基质蛋白的组成。它们还表明，血小板形成粘附保护区需要αIIbβ3整合素，而这些粘附区域的渗透特性受αIIbβ3和α5β1整合素共同调节。
