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

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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.