THROMBOSTHENIN M - PURIFICATION AND INTERACTION WITH THROMBIN

Thrombosthenin M was isolated from equine blood platelets by lysis of the platelets with n‐butanol at pH 7.0 at 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 thrombosthenin M 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 analysis of the thrombosthenin M preparation obtained (pH7.0, I= 0.6) revealed that about 90% of the protein sediments with a sedimentation coefficient of 7.2 S and about 10% with a sedimentation coefficient of 33 S. The thrombosthenin M preparation yielded a single band on disc electrophoresis; some of the protein failed, however, to penetrate the gel. Incubation of thrombosthenin M with thrombin resulted in the appearance of one mole of carboxyl terminal arginine per 105 g of thrombosthenin M. The ATPase activity of the thrombosthenin M was not affected by thrombin. Incubation of equine platelets with thrombin resulted in the disappearance of the electrophoretic band corresponding to thrombosthenin M. Antiserum against thrombosthenin M was found to inhibit the clot retraction of thrombin‐treated platelet‐rich plasma. Copyright © 1969, Wiley Blackwell. All rights reserved