Hull York Medical School, University of Hull, Castle Hill Hospital, Castle Road, Cottingham HU67RX, UK.
Platelets. 2010;21(6):421-8. doi: 10.3109/09537104.2010.483296.
Peroxynitrite is a potent nitrating and oxidizing agent that exerts differential effects on platelets. In the present study we investigated the influence of peroxynitrite on vasodilator-stimulated phosphoprotein (VASP), a protein that plays a key role in inhibition of platelet adhesion and spreading. In platelets, VASP is a substrate for protein kinase A (PKA), PKC and PKG and phosphorylation by these kinases is thought to block VASP-mediated actin cytoskeletal rearrangement. In the present study, we demonstrate that peroxynitrite phosphorylates VASP by a PKC-dependent mechanism. Peroxynitrite (0-100 microM) induced a concentration and time-dependent increase in phosphorylation of VASP at serine(157) (Ser(157)) and Ser(239). Inhibition of soluble guanylyl cyclase (sGC) did not significantly reduce peroxynitrite-mediated phosphorylation, indicating a cGMP-independent pathway for VASP phosphorylation. In contrast nitric oxide-mediated VASP phosphorylation was abolished under conditions of sGC inhibition. Further exploration of the mechanisms underlying VASP phosphorylation indicated a requirement for Ca2+ mobilization, but was independent of protein kinase A, Src kinases and protein nitration. Consistent with previous reports phorbol 12-myristate 13-acetate (PMA; 300 nM) induced phosphorylation of VASP at Ser(157), but not Ser(239), which was blocked by general protein kinase C (PKC) inhibitors, Ro31-8220 and Bisindolylmaleimide I (BIM-1), and Gö6976, an inhibitor of conventional PKC isoforms. Interestingly, treatment of platelets with these PKC inhibitors significantly reduced peroxynitrite-mediated phosphorylation of both sites, indicating that phosphorylation occurred through PKC-dependent mechanism. Consistent with these findings peroxynitrite caused a small increase in PKC activity as evidenced by increased phosphorylation of PKC substrates. Together these data indicate that peroxynitrite may inhibit platelet function by inducing the phosphorylation of VASP through a mechanism that requires the activation of PKC.
过氧亚硝酸盐是一种强效的硝化和氧化试剂，对血小板产生不同的影响。在本研究中，我们研究了过氧亚硝酸盐对血管扩张刺激磷蛋白（VASP）的影响，VASP 是一种在抑制血小板黏附和铺展中起关键作用的蛋白质。在血小板中，VASP 是蛋白激酶 A（PKA）、蛋白激酶 C（PKC）和蛋白激酶 G（PKG）的底物，这些激酶的磷酸化被认为可阻断 VASP 介导的肌动蛋白细胞骨架重排。在本研究中，我们证明过氧亚硝酸盐通过 PKC 依赖性机制使 VASP 磷酸化。过氧亚硝酸盐（0-100μM）诱导 VASP 丝氨酸（Ser）157 和 Ser239 位点的磷酸化呈浓度和时间依赖性增加。可溶性鸟苷酸环化酶（sGC）抑制并未显著减少过氧亚硝酸盐介导的磷酸化，表明 VASP 磷酸化存在 cGMP 非依赖性途径。相反，在 sGC 抑制的条件下，一氧化氮介导的 VASP 磷酸化被消除。对 VASP 磷酸化机制的进一步探索表明需要动员 Ca2+，但与蛋白激酶 A、Src 激酶和蛋白硝化无关。与先前的报道一致，佛波醇 12-肉豆蔻酸 13-乙酸酯（PMA；300nM）诱导 VASP 在 Ser157 位点磷酸化，但不诱导 Ser239 位点磷酸化，这被 PKC 通用抑制剂 Ro31-8220 和双吲哚马来酰亚胺 I（BIM-1）和常规 PKC 同工型抑制剂 Gö6976 阻断。有趣的是，用这些 PKC 抑制剂处理血小板可显著减少过氧亚硝酸盐介导的两个位点的磷酸化，表明磷酸化是通过 PKC 依赖性机制发生的。与这些发现一致，过氧亚硝酸盐引起 PKC 活性的轻微增加，这表现为 PKC 底物的磷酸化增加。总之，这些数据表明，过氧亚硝酸盐可能通过需要激活 PKC 的机制诱导 VASP 磷酸化，从而抑制血小板功能。
