Evaluation of platelet activation in vivo by quantitative measurement of myosin phosphorylation.

我们最近开发了一种新的血小板聚集仪，基于光散射，它可以灵敏地测量小尺寸的血小板聚集体。使用该仪器，我们能够检测到糖尿病患者中发生的自发聚集。由于血小板活化与肌球蛋白活化相关，因此我们尝试测量从糖尿病患者获得的血小板中肌球蛋白磷酸化的水平。为此，我们开发了一种肌球蛋白磷酸化的定量测量，通过使用单克隆抗体，该抗体仅识别肌球蛋白的磷酸化形式，而不是普通的肌球蛋白。以前测量肌球蛋白磷酸化的方法采用32P标记，其通常诱导血小板的人工活化，因此不可能评估完整的血小板。基于我们使用抗磷酸化肌球蛋白抗体的新方法，我们能够评估从糖尿病患者获得的完整血小板的肌球蛋白磷酸化水平。我们发现糖尿病患者肌球蛋白磷酸化水平显著增加，并且自发聚集水平与肌球蛋白磷酸化水平密切相关。

We recently developed a new platelet aggregometer, based on light scattering, which can sensitively measure platelet aggregates of small size. Using this instrument, we were able to detect that spontaneous aggregation occurs in patients with diabetes mellitus. Since platelet activation is associated with myosin activation, we then attempted to measure the level of myosin phosphorylation in platelets obtained from diabetes mellitus patients. For this end, we developed a quantitative measurement of myosin phosphorylation, by using an monoclonal antibody which recognizes only the phosphorylated form of myosin, but not ordinary myosin. Previous methods of measuring myosin phosphorylation employed 32P labeling, which often induced artificial activation of platelets, and thus evalution of intact platelets was not possible. Based on our new method using anti-phosphorylated myosin antibody, we were able to evaluate the level of myosin phosphorylation of intact platelets obtained from patients with diabetes mellitus. We found that the level of myosin phosphorylation is significantly increased in patients with diabetes mellitus, and that the level of spontaneous aggregation is well correlated with that of myosin phosphorylation.