Thrombosthenin M was isolated from equine blood platelets by lysis of the platelets with n-butanol a t pH 7.0 a t low ionic strength ( I = 0.05), separation of the insoluble thrombosthenin by centrifugation, and extraction of thrombosthenin M from the sediment with 0.02 M pyrophosphate buffer pH 9.5. Contaminating ribonucleic acid was digested with ribonuclease and the protein finally purified by gel filtration on Sephadex G-200. The purified thrombostheninM was found to be free of thrombosthenin A and of fibrinogen. Thrombosthenin M, similarly to actin-free muscle myosin, possesses a Ca++-activated but not a Mg++-activated ATPase activity, and shows neither a drop in viscosity nor superprecipitation on addition of ATP. Ultracentrifugal A new procedure for the isolation of thrombosthenin M from blood platelets is described in the following. It is patterned after the method described by Baril et al. [7] for the isolation of chick musclemyosin by extraction with pyrophosphate a t pH 9.5. The procedure adopted yielded a thrombosthenin M preparation free of thrombosthenin A and of fibrinogen. Its composition, its immunological, and some of its physicochemical properties are described.The availability of a purified thrombosthenin M preparation enabled us to investigate its interaction with thrombin. This reaction is of interest since thrombin triggers clot retraction by interacting with metabolically active platelets [8]. Several authors have indicated that a protein constituent of the blood platelet different from fibrinogen might be involved in this reaction [4,9-151. Bettex-Galland and Luscher [6] suggested that thrombosthenin might be the contractile element responsible for the retraction process since some of the compounds which inhibit the ATPase activity and the superprecipitation of thrombosthenin also inhibit clot retraction. This suggestion is supported by the observation of Nachman [l] that clot retraction is inhibited by antiserum to thrombosthenin.
MATERIALS;AND METHODS
Reagents
*Adenosine 5'-triphosphate disodium salt was purchased from Sigma ; bovine pancreatic ribonuclease (5 x crystallized, lot 45483) and 1-chloro-3-tosylami-
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