Clemetson K J, Kocher M, von Tscharner V
Theodor Kocher Institute, University of Berne, Switzerland.
Adv Exp Med Biol. 1993;344:119-28. doi: 10.1007/978-1-4615-2994-1_9.
Although the importance of protein kinases in platelet activation, particularly protein kinase C (PKC), is well established there remain many problems regarding the various phosphorylation cascades, the role of phosphatases and the importance of other serine/threonine and tyrosine kinases. A particular problem is the mechanism of activation of the fibrinogen receptor, GPIIb/IIIa, a critical step in aggregation. Although GPIIIa is phosphorylated (on threonine) neither the stoichiometry nor the minor changes on activation seem adequate to explain the response. Relatively unspecific inhibitors of PKC such as staurosporine prevent PO4 incorporation into most kinase substrates but only inhibit platelet aggregation partially. However, staurosporine does induce activation and then inhibits several renaturable serine/threonine kinases, probably via phosphatases. Staurosporine did not, however, inhibit the platelet Ca2+ signal in response to thrombin but rather enhanced it. 17-Hydroxywortmannin (HWT), a fungal metabolite, has been shown to inhibit respiratory burst in neutrophils and causes haemorrhages. It was recently reported to be a myosin light chain kinase (MLCK) inhibitor and to inhibit PKC only at much higher concentrations. In platelets, HWT inhibits aggregation and partially inhibits phosphorylation of myosin light chain and P47 in thrombin-activated platelets. It also allows the discrimination of an early and a late phase in the cytoplasmic Ca2+ signal since at lower concentrations it only inhibits the late phase. The late phase of ATP release was also inhibited in a dose-dependent manner. The activation of most of the renaturable serine/threonine kinases was also inhibited by HWT. These results support earlier conclusions that the early phase of the Ca2+ signal is phospholipase C dependent but indicate that other mechanisms must be responsible for the late phase. The relative specificity of HWT for MLCK might indicate that this has an unexpected major role in controlling these late phase reactions including activation of GPIIb/IIIa or its clustering. However, staurosporine completely inhibits phosphorylation of myosin light chain by its kinase (as well as other kinases) and has the opposite effect on Ca2+ signals. Clearly, the interactions and feed-back mechanisms between these kinases are very complex but the results suggest that phosphatases acting together with their complementary kinases should also be considered as important platelet activation regulators. P47, long considered a major PKC substrate, may also be phosphorylated by MLCK.
尽管蛋白激酶，尤其是蛋白激酶C（PKC）在血小板激活中的重要性已得到充分证实，但在各种磷酸化级联反应、磷酸酶的作用以及其他丝氨酸/苏氨酸激酶和酪氨酸激酶的重要性方面，仍存在许多问题。一个特殊的问题是纤维蛋白原受体GPIIb/IIIa的激活机制，这是聚集过程中的关键步骤。尽管GPIIIa（苏氨酸位点）会发生磷酸化，但磷酸化的化学计量以及激活时的微小变化似乎都不足以解释其反应。相对非特异性的PKC抑制剂，如星形孢菌素，可阻止磷酸根掺入大多数激酶底物，但只能部分抑制血小板聚集。然而，星形孢菌素确实会诱导激活，然后可能通过磷酸酶抑制几种可复性丝氨酸/苏氨酸激酶。不过，星形孢菌素并未抑制血小板对凝血酶的Ca2+信号反应，反而增强了该信号。17-羟基渥曼青霉素（HWT）是一种真菌代谢产物，已被证明可抑制中性粒细胞的呼吸爆发并导致出血。最近有报道称它是肌球蛋白轻链激酶（MLCK）抑制剂，只有在高得多的浓度下才会抑制PKC。在血小板中，HWT抑制聚集，并部分抑制凝血酶激活的血小板中肌球蛋白轻链和P47的磷酸化。它还能区分细胞质Ca2+信号的早期和晚期阶段，因为在较低浓度下它只抑制晚期阶段。ATP释放的晚期阶段也以剂量依赖的方式受到抑制。HWT还抑制了大多数可复性丝氨酸/苏氨酸激酶的激活。这些结果支持了早期的结论，即Ca2+信号的早期阶段依赖于磷脂酶C，但表明其他机制必定负责晚期阶段。HWT对MLCK的相对特异性可能表明，它在控制这些晚期反应（包括GPIIb/IIIa的激活或其聚集）中具有意想不到的主要作用。然而，星形孢菌素完全抑制了其激酶（以及其他激酶）对肌球蛋白轻链的磷酸化，并且对Ca2+信号有相反的作用。显然，这些激酶之间的相互作用和反馈机制非常复杂，但结果表明，与其互补激酶共同作用的磷酸酶也应被视为重要的血小板激活调节因子。长期以来被认为是主要PKC底物的P47，也可能被MLCK磷酸化。
