Changes in G-Actin after Platelet Activation in Platelet Rich Plasma

Summary

We have used the DNase I inhibition assay to study changes in G-actin after platelet activation in platelet-rich plasma (PRP) induced by ADP. Because of problems associated with depolymerization of F-actin after lysis of ADP-activated platelets in the presence of plasma, G-actin was measured using a lysis buffer that contained formaldehyde to prevent any depolymerization of F-actin.

Different patterns of response were seen depending on the concentration of ADP used, and these were modified by avoiding aggregation by either not stirring the sample or by adding EDTA. The results show rapid conversion of G-actin to F-actin in association with shape change, and there is a further decrease in G-actin associated with irreversible platelet aggregation. Thus evidence is presented that actin polymerization occurs in two phases after ADP stimulation.

• REFERENCES

• 1 Fox JEB. The platelet cytoskeleton. In: Thrombosis and Haemostasis 1987. Verstraete M, Vermylen J, Lijnen R, Arnout J. (eds) Leuven University Press, Leuven: 1987. pp 175-225
• 2 Carlsson L, Markey F, Blikstad I, Persson T, Lindberg U. Reorganization of actin in platelets stimulated by thrombin as measured by DNase I inhibition. Proc Natl Acad Sci USA 1979; 76: 6376-6380
• 3 Jennings LK, Fox JEB, Edwards HH, Phillips DR. Changes in the cytoskeletal structures of human platelets following thrombin activation. J Biol Chem 1981; 256: 6927-6932
• 4 Fox JEB, Phillips DR. Inhibition of actin polymerization in blood platelets by cytochalasins. Nature 1981; 292: 650-652
• 5 Casella JF, Flanagan MD, Lin S. Cytochalasin D inhibits actin polymerization formed during platelet shape change. Nature 1981; 293: 302-305
• 6 Pribluda V, Laub F, Rotman A. The state of actin in activated human platelets. Eur J Biochem 1981; 116: 293-296
• 7 Pribluda V, Rotman A. Dynamics of membrane-cytoskeleton interactions in activated blood platelets. Biochemistry 1982; 21: 2825-2832
• 8 Fox JEB, Phillips DR. Polymerization and organization of actin filaments within platelets. Semin Hematol 1983; 20: 243-260
• 9 Fox JEB, Boyles J, Reynolds C, Phillips DR. Actin filament content and organization in unstimulated platelets. J Cell Biol 1984; 98: 1985-1991
• 10 Fox JEB, Berndt MC. Cyclic AMP-dependent phosphorylation of glycoprotein lb inhibits collagen-induced polymerization of actin in platelets. J Biol Chem 1989; 264: 9520-9526
• 11 Spangenberg P, Till U. Actin filament content in platelets – a sensitive index of cellular reactivity. Biosc Rep 1989; 9: 307-313
• 12 Holme T, Kellie S, Wyke JA, Crawford N. Effect of transformation by Rous sarcoma virus on the character and distribution of actin in rat-1 fibroblasts: a biochemical and microscopical study. Br J Cancer 1986; 53: 465-476
• 13 Heptinstall S, Fox SC. Human blood platelet behaviour after inhibition of thromboxane synthetase. Br J Clin Pharmacol 1983; 15: 31S-37S
• 14 Blikstad I, Markey F, Carlsson L, Persson T, Lindberg U. Selective assay of monomeric and filamentous actin in extracts. Cell 1978; 15: 935-943
• 15 Carroll RC, Butler RG, Morris PA, Gerrard JM. Separable assembly of platelet pseudopodial and contractile cytoskeletons. Cell 1982; 30: 385-393