Calcium ion control of platelet thrombosthenin ATPase activity

This study demonstrates that Ca²⁺ regulates thrombosthenin ATPase activity, likening the control of platelet contraction to that of cardiac and skeletal muscle. Thrombosthenin, the platelet contractile protein, was isolated by repeated low ionic strength and isoelectric precipitation. Thrombosthenin superprecipitation and ATPase activity were measured in 10^-4 M CaCl₂ (high ionized Ca²⁺) and 0.25 mM ethylene glycol bis-(β-aminoethyl ether)-N,N′-tetraacetic acid (EGTA) (low ionized Ca²⁺). In both high and low Ca²⁺, superprecipitation, measured as an increase in turbidity, ocurred shortly after addition of ATP. ATP hydrolysis by thrombosthenin, which proceeded linearly for several hours, was greater in high Ca²⁺ (approx. 2.3 nmoles·mg^-1·min^-1) than in low Ca²⁺ (approx. 1.8 nmoles·mg^-1·min^-1). This difference, when analyzed by the Student's t-test for paired samples was highly significant (P < 0.001). Thrombosthenin ATPase activity was not significantly altered by azide, an inhibitor of mitochondrial ATPase, nor by ouabain, an inhibitor of (Na⁺ + K⁺)-activated ATPase. The dependence of thrombosthenin activation on ionized Ca²⁺, measured with the use of CaEGTA buffers, was studied. The Ca²⁺-dependent portion of thrombosthenin ATPase was half maximal at 4.5·10^-7 M Ca²⁺. This corresponds to an apparent binding constant of 2.2·10⁶ M^-1, a value that is comparable to that of skeletal and cardiac muscle. These data suggest that a Ca²⁺ control mechanism similar to that of the troponin-tropomyosin complex of muscle exists in the platelet.