Huang M M, Lipfert L, Cunningham M, Brugge J S, Ginsberg M H, Shattil S J
Ariad Pharmaceuticals Inc., Cambridge, Massachusetts 02139.
J Cell Biol. 1993 Jul;122(2):473-83. doi: 10.1083/jcb.122.2.473.
Tyrosine phosphorylation of multiple platelet proteins is stimulated by thrombin and other agonists that cause platelet aggregation and secretion. The phosphorylation of a subset of these proteins, including a protein tyrosine kinase, pp125FAK, is dependent on the platelet aggregation that follows fibrinogen binding to integrin alpha IIb beta 3. In this report, we examined whether fibrinogen binding, per se, triggers a process of tyrosine phosphorylation in the absence of exogenous agonists. Binding of soluble fibrinogen was induced with Fab fragments of an anti-beta 3 antibody (anti-LIBS6) that directly exposes the fibrinogen binding site in alpha IIb beta3. Proteins of 50-68 KD and 140 kD became phosphorylated on tyrosine residues in a fibrinogen-dependent manner. This response did not require prostaglandin synthesis, an increase in cytosolic free calcium, platelet aggregation or granule secretion, nor was it associated with tyrosine phosphorylation of pp125FAK. Tyrosine phosphorylation of the 50-68-kD and 140-kD proteins was also observed when (a) fibrinogen binding was stimulated by agonists such as epinephrine, ADP, or thrombin instead of by anti-LIBS6; (b) fragment X, a dimeric plasmin-derived fragment of fibrinogen was used instead of fibrinogen; or (c) alpha IIb beta 3 complexes were cross-linked by antibodies, even in the absence of fibrinogen. In contrast, no tyrosine phosphorylation was observed when the ligand consisted of monomeric cell recognition peptides derived from fibrinogen (RGDS or gamma 400-411). Fibrinogen-dependent tyrosine phosphorylation was inhibited by cytochalasin D. These studies demonstrate that fibrinogen binding to alpha IIb beta 3 initiates a process of tyrosine phosphorylation that precedes platelet aggregation and the phosphorylation of pp125FAK. This reaction may depend on the oligomerization of integrin receptors and on the state of actin polymerization, organizational processes that may juxtapose tyrosine kinases with their substrates.
凝血酶和其他能引起血小板聚集及分泌的激动剂可刺激多种血小板蛋白的酪氨酸磷酸化。这些蛋白中的一部分，包括一种蛋白酪氨酸激酶pp125FAK，其磷酸化依赖于纤维蛋白原与整合素αIIbβ3结合后引发的血小板聚集。在本报告中，我们研究了在无外源性激动剂的情况下，纤维蛋白原结合本身是否会引发酪氨酸磷酸化过程。用直接暴露αIIbβ3中纤维蛋白原结合位点的抗β3抗体（抗-LIBS6）的Fab片段诱导可溶性纤维蛋白原的结合。50 - 68KD和140kD的蛋白以纤维蛋白原依赖的方式在酪氨酸残基上发生磷酸化。这种反应不需要前列腺素合成、胞质游离钙增加、血小板聚集或颗粒分泌，也与pp125FAK的酪氨酸磷酸化无关。当（a）用肾上腺素、ADP或凝血酶等激动剂而非抗-LIBS6刺激纤维蛋白原结合时；（b）用纤维蛋白原的二聚体纤溶酶衍生片段X代替纤维蛋白原时；或（c）即使在无纤维蛋白原的情况下用抗体交联αIIbβ3复合物时，也观察到了50 - 68KD和140kD蛋白的酪氨酸磷酸化。相反，当配体由源自纤维蛋白原的单体细胞识别肽（RGDS或γ400 - 411）组成时，未观察到酪氨酸磷酸化。细胞松弛素D可抑制纤维蛋白原依赖的酪氨酸磷酸化。这些研究表明，纤维蛋白原与αIIbβ3的结合启动了一个在血小板聚集和pp125FAK磷酸化之前的酪氨酸磷酸化过程。该反应可能依赖于整合素受体的寡聚化以及肌动蛋白聚合状态，这些组织过程可能使酪氨酸激酶与其底物并列。
