Thromb Haemost 1976; 35(03): 635-642
DOI: 10.1055/s-0038-1647962
Original Article
Schattauer GmbH

Effect of Thrombin on Phosphorylation of Platelet Membrane Proteins

M Steiner
1   Division of Hematologic Research, The Memorial Hospital, Pawtucket and Brown University, Providence, R. I., U.S.A.
› Author Affiliations
Further Information

Publication History

Received 12 August 1975

Accepted 16 December 1975

Publication Date:
02 July 2018 (online)

Summary

The effect of thrombin on the phosphorylating activity of platelet membranes was compared to that of trypsin. Preincubation of non-32P phosphorylated platelet membranes with or without either of these two enzymes resulted in a considerable loss of membrane protein kinase activity which was most severe when trypsin was used. Protein kinase activity and endogenous protein acceptors decreased in parallel. 32P-phosphorylated membranes showed a slow but progressive loss of label which was accelerated by trypsin. Thrombin under these conditions prevented the loss of 32P-phosphate. These results are interpreted to indicate a thrombin-induced destruction of a phosphoprotein phosphatase. The protein kinase activity of phosphorylated platelet membranes using endogenous or exogenous protein substrates showed a significant reduction compared to non-phosphorylated membranes suggesting a deactivation of protein kinase by phosphorylation of platelet membranes. Neither thrombin nor trypsin caused a qualitative change in the membrane polypeptides accepting 32P-phosphate but resulted in quantitative alterations of their ability to become phosphorylated.

 
  • References

  • 1 Ando A, Steiner M. 1973; Sulfhydryl and disulfide groups of platelet membranes. I. Determination of sulfhydryl groups. Biochimica et Biophysica Acta 311: 26.
  • 2 Baenziger N. L, Brodie G. N, Majerus P. W. 1971; A thrombin-sensitive protein of human platelet membranes. Proceedings of the National Academy of Sciences of the United States of America 68: 240.
  • 3 Baenziger N. L, Brodie G. N, Majerus P. W. 1972; Isolation and properties of a thrombin-sensitive protein of human platelets. Journal of Biological Chemistry 247: 2723.
  • 4 Brodie G. N, Baenziger N. L, Chase L. R, Majerus P. W. 1972; The effects of thrombin on adenyl cyclase activity and a membrane protein from human platelets. Journal of Clinical Investigation 51: 81.
  • 5 Detwiler T. C, Feinman R. D. 1973; Kinetics of the thrombin-induced release of calcium (II) by platelets. Biochemistry 12: 282.
  • 6 Ganguly P. 1974; Binding of thrombin to human platelets. Nature 247: 306.
  • 7 Lowry O. H, Rosenbrough N. J, Farr A. L, Randall R. J. 1951; Protein measurement with the Folin phenol reagent. Journal of Biological Chemistry 193: 265.
  • 8 Lundblad R. L. 1971; A rapid method for the purification of bovine thrombin and the inhibition of the purified enzyme with phenyl methylsulfonyl fluoride. Biochemistry 10: 2501.
  • 9 Miyamoto E, Kuo J. F, Greengard P. 1969; Cyclic nucleotide-dependent protein kinases. II. Purification and properties of adenosine 3’, 5’-monophosphate-dependent protein kinase from bovine brain. Journal of Biological Chemistry 244: 6395.
  • 10 Phillips D. R, Agin P. P. 1973; Identification of a thrombin labile glycoprotein on the surface of the human platelet plasma membrane. Federation Proceedings 32: 219.
  • 11 Steiner M. 1973; Effect of thrombin on the platelet membrane. Biochimica et Biophysica Acta 323: 653.
  • 12 Steiner M. 1975 Endogenous phosphorylation of platelet membrane proteins. Archives of Biochemistry and Biophysics. (in the press).
  • 13 Tollefsen D. M, Feagler J. R, Majerus P. W. 1974; The binding of thrombin to the surface of human platelets. Journal of Biological Chemistry 249: 2646.