Nurden P, Humbert M, Piotrowicz R S, Bihour C, Poujol C, Nurden A T, Kunicki T J
URA 1464 CNRS, Hopital Cardiologique, Pessac, France.
Blood. 1996 Aug 1;88(3):887-99.
The sequence beta (3)203-228 is involved, in a yet undetermined manner, in alpha IIb beta 3 function. We now show that murine monoclonal antibody (MoAb) AP6, specific for beta (3)211-221, binds to alpha IIb beta 3 on adenosine diphosphate (ADP)-activated platelets only when the receptor is occupied by intact fibrinogen. The ligand-induced binding-site reported by AP6 is unique in that it is not expressed following occupancy by either RGD peptides or the gamma-chain carboxy-terminal dodecapeptide. Binding of AP6 to platelets coincides temporally with the binding of the MoAb 9F9, specific for a receptor-induced binding site on fibrinogen. Thus, AP6 reports the binding of fibrinogen to the recognition pocket of alpha IIb beta 3. Its binding to thrombin-stimulated washed platelets correlates with secretion as determined using an MoAb to P-selectin. When ultrathin sections of nonactivated platelets were examined by immunogold staining and electron microscopy, AP6 identified a pool of alpha IIb beta 3 colocalizing with P-selectin and suggesting the presence of alpha IIb beta 3-ligand complexes in the alpha-granule membrane. There was little binding of AP6 to surface alpha IIb beta 3 of unstimulated platelets. After ADP-induced activation, AP6 was abundantly distributed over the entire platelet surface, including pseudopods, but only when fibrinogen was present in the medium. ADP had little effect on AP6 reactivity within platelets. This contrasted with washed platelets and thrombin, where extensive AP6 binding was observed within internal membrane pools as early as 10 to 15 seconds after stimulation. Surface labeling with AP6 followed slower kinetics. Flow cytometry on Triton X-100 permeabilized fixed platelets confirmed AP6 binding to alpha IIb beta 3 within the platelet. Thus, our results provide evidence of (1) a pool of alpha-granule alpha IIb beta 3 occupied by ligand in nonactivated platelets; (2) thrombin-induced activation of alpha IIb beta 3 within the platelet, and (3) thrombin-induced mobilization of ligand-bound alpha IIb beta 3 to the surface.
β(3)203 - 228序列以一种尚未明确的方式参与αIIbβ3的功能。我们现在表明，针对β(3)211 - 221的鼠单克隆抗体（MoAb）AP6，仅在完整纤维蛋白原占据受体时才与二磷酸腺苷（ADP）激活的血小板上的αIIbβ3结合。AP6报道的配体诱导结合位点是独特的，因为在被RGD肽或γ链羧基末端十二肽占据后它不表达。AP6与血小板的结合在时间上与针对纤维蛋白原上受体诱导结合位点的单克隆抗体9F9的结合一致。因此，AP6报道了纤维蛋白原与αIIbβ3识别口袋的结合。它与凝血酶刺激的洗涤血小板的结合与使用抗P - 选择素单克隆抗体测定的分泌相关。当通过免疫金染色和电子显微镜检查未激活血小板的超薄切片时，AP6鉴定出与P - 选择素共定位的αIIbβ3池，提示α颗粒膜中存在αIIbβ3 - 配体复合物。未刺激血小板表面的αIIbβ3与AP6几乎没有结合。ADP诱导激活后，AP6大量分布在整个血小板表面，包括伪足，但仅当培养基中存在纤维蛋白原时才会如此。ADP对血小板内的AP6反应性影响很小。这与洗涤血小板和凝血酶形成对比，在凝血酶刺激后早在10至15秒内就观察到内膜池中广泛的AP6结合。用AP6进行表面标记的动力学较慢。对用Triton X - 100通透处理的固定血小板进行流式细胞术证实了AP6与血小板内的αIIbβ3结合。因此，我们的结果提供了以下证据：（1）未激活血小板中被配体占据的α颗粒αIIbβ3池；（2）凝血酶诱导血小板内αIIbβ3的激活；（3）凝血酶诱导配体结合型αIIbβ3向表面的动员。
