Bennett J S, Zigmond S, Vilaire G, Cunningham M E, Bednar B
Department of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA.
J Biol Chem. 1999 Sep 3;274(36):25301-7. doi: 10.1074/jbc.274.36.25301.
Agonist-generated inside-out signals enable the platelet integrin alpha(IIb)beta(3) to bind soluble ligands such as fibrinogen. We found that inhibiting actin polymerization in unstimulated platelets with cytochalasin D or latrunculin A mimics the effects of platelet agonists by inducing fibrinogen binding to alpha(IIb)beta(3). By contrast, stabilizing actin filaments with jasplakinolide prevented cytochalasin D-, latrunculin A-, and ADP-induced fibrinogen binding. Cytochalasin D- and latrunculin A-induced fibrinogen was inhibited by ADP scavengers, suggesting that subthreshold concentrations of ADP provided the stimulus for the actin filament turnover required to see cytochalasin D and latrunculin A effects. Gelsolin, which severs actin filaments, is activated by calcium, whereas the actin disassembly factor cofilin is inhibited by serine phosphorylation. Consistent with a role for these factors in regulating alpha(IIb)beta(3) function, cytochalasin D- and latrunculin A-induced fibrinogen binding was inhibited by the intracellular calcium chelators 1,2-bis(2-aminophenoxy)ethane-N,N,N', N'-tetraacetic acid acetoxymethyl ester and EGTA acetoxymethyl ester and the Ser/Thr phosphatase inhibitors okadaic acid and calyculin A. Our results suggest that the actin cytoskeleton in unstimulated platelets constrains alpha(IIb)beta(3) in a low affinity state. We propose that agonist-stimulated increases in platelet cytosolic calcium initiate actin filament turnover. Increased actin filament turnover then relieves cytoskeletal constraints on alpha(IIb)beta(3), allowing it to assume the high affinity conformation required for soluble ligand binding.
激动剂产生的外向内信号使血小板整合素α(IIb)β(3)能够结合可溶性配体，如纤维蛋白原。我们发现，用细胞松弛素D或拉特罗毒素A抑制未刺激血小板中的肌动蛋白聚合，通过诱导纤维蛋白原与α(IIb)β(3)结合来模拟血小板激动剂的作用。相比之下，用茉莉酮酸稳定肌动蛋白丝可防止细胞松弛素D、拉特罗毒素A和ADP诱导的纤维蛋白原结合。ADP清除剂可抑制细胞松弛素D和拉特罗毒素A诱导的纤维蛋白原结合，这表明亚阈值浓度的ADP为观察细胞松弛素D和拉特罗毒素A作用所需的肌动蛋白丝周转提供了刺激。凝溶胶蛋白可切断肌动蛋白丝，它被钙激活，而肌动蛋白解聚因子丝切蛋白则被丝氨酸磷酸化抑制。与这些因子在调节α(IIb)β(3)功能中的作用一致，细胞松弛素D和拉特罗毒素A诱导的纤维蛋白原结合被细胞内钙螯合剂1,2-双(2-氨基苯氧基)乙烷-N,N,N',N'-四乙酸乙酰甲酯和EGTA乙酰甲酯以及丝氨酸/苏氨酸磷酸酶抑制剂冈田酸和花萼海绵诱癌素A抑制。我们的结果表明，未刺激血小板中的肌动蛋白细胞骨架将α(IIb)β(3)限制在低亲和力状态。我们提出，激动剂刺激引起的血小板胞质钙增加启动了肌动蛋白丝周转。肌动蛋白丝周转增加随后减轻了细胞骨架对α(IIb)β(3)的限制，使其能够呈现可溶性配体结合所需的高亲和力构象。
