Itoh K, Hara T, Yamada F, Shibata N
Division of Molecular Cardiology, Center for Adult Diseases, Osaka, Japan.
Biochim Biophys Acta. 1992 Jul 22;1136(1):52-6. doi: 10.1016/0167-4889(92)90084-o.
We prepared anti-platelet 20-kDa myosin light chain (MLC-20) antibody and demonstrated diphosphorylation of MLC-20 in platelets ex vivo in the initial phase of activation by thrombin. Our results are as follows. (1) By Western blotting, using anti-MLC-20 antibody, both mono- and diphosphorylated myosin were seen in the initial phase of aggregation of platelets by thrombin. The peak of the diphosphorylation was later than that of monophosphorylation and the degree of both mono- and diphosphorylation reduced in the process of aggregation. (2) ML-7 (a synthetic inhibitor of MLCK) inhibited both mono- and diphosphorylation of myosin and also blocked aggregation of thrombin-activated platelets. However, H-7 (an inhibitor of protein kinase C) had little effect on either the (di)phosphorylation of myosin or the aggregation of thrombin-activated platelets. (3) Arg-Gly-Asp-Ser (RGDS) peptide, a synthetic anti-adhesive peptide, inhibited aggregation of thrombin-activated platelets in a dose-dependent manner (100-200 microM). However, it had little effect on either mono- or diphosphorylation of myosin in the process of the platelet aggregation stimulated by thrombin. From these results, we conclude that mono- and diphosphorylation of myosin by MLCK play a role in the initial phase of activation of thrombin-stimulated platelets in vivo and that mono- and diphosphorylation of myosin by MLCK precedes the secondary signal mediated by GPIIb/IIIa.
我们制备了抗血小板20-kDa肌球蛋白轻链（MLC-20）抗体，并证实在凝血酶激活的初始阶段，血小板中的MLC-20会发生双磷酸化。我们的结果如下。（1）通过蛋白质免疫印迹法，使用抗MLC-20抗体，在凝血酶诱导的血小板聚集初始阶段可观察到单磷酸化和双磷酸化的肌球蛋白。双磷酸化的峰值晚于单磷酸化，并且在聚集过程中，单磷酸化和双磷酸化的程度均降低。（2）ML-7（一种肌球蛋白轻链激酶的合成抑制剂）可抑制肌球蛋白的单磷酸化和双磷酸化，还能阻断凝血酶激活的血小板聚集。然而，H-7（一种蛋白激酶C抑制剂）对肌球蛋白的（双）磷酸化或凝血酶激活的血小板聚集几乎没有影响。（3）Arg-Gly-Asp-Ser（RGDS）肽，一种合成的抗黏附肽，以剂量依赖方式（100 - 200 microM）抑制凝血酶激活的血小板聚集。然而，在凝血酶刺激的血小板聚集过程中，它对肌球蛋白的单磷酸化或双磷酸化几乎没有影响。从这些结果中，我们得出结论，肌球蛋白轻链激酶介导的肌球蛋白单磷酸化和双磷酸化在体内凝血酶刺激的血小板激活初始阶段发挥作用，并且肌球蛋白轻链激酶介导的肌球蛋白单磷酸化和双磷酸化先于由糖蛋白IIb/IIIa介导的次级信号。
