Relationship between cytoplasmic free calcium and myosin light chain phosphorylation in intact platelets

Human platelets were prepared and loaded with the fluorescent Ca2+ indicator quin2. The relation between cytoplasmic free calcium concentration, [Ca2+]i, and the extent of the phosphorylation of myosin light chains of Mr 20 000 could then be examined. When the calcium ionophore ionomycin is used to stimulate platelets, little phosphorylation is seen until [Ca2+]i exceeds 400 nM; half-maximal response occurs at 600 nM with a full response at about 1 microM-[Ca2+]i. Under optimal conditions, physiological stimuli such as platelet-activating factor and thrombin can increase [Ca2+]i to sufficiently high levels [Rink, Smith & Tsien (1982) FEBS Lett. 148, 21-26; Hallam, Sanchez & Rink (1984) Biochem. J. 218, 819-827] that Ca2+ ions could be the trigger for the myosin phosphorylation evoked by these agonists. However, in this paper we show that, in the absence of external calcium, platelet-activating factor and thrombin can stimulate myosin phosphorylation while [Ca2+]i remains at levels which are well below those needed when the calcium ionophore is the stimulus. This observation suggests that myosin light chain phosphorylation may be controlled by an additional pathway.