Agonist-induced Actin Polymerization Is Required for the Irreversibility of Platelet Aggregation

Summary

Cytochalasin D was used to investigate the role of intracellular cyto-skeleton in the stabilization of platelet aggregation induced by strong platelet agonists. Incubation of gel-filtered platelets with increasing concentrations of cytochalasin D resulted in a dose-dependent inhibition of actin polymerization and association of actin-binding proteins with the Triton X-100-insoluble material induced by the thromboxane analogue, U46619, and the thrombin receptor activating peptide, TRAP. The same concentrations of cytochalasin D did not significantly inhibit platelet aggregation promoted by the two agonists. The addition of the chelating agent EDTA to fully aggregated platelets, that had been treated with cytochalasin D, resulted in the rapid and almost complete disaggregation. EDTA did not cause disaggregation of control, solvent-treated, aggregated platelets. The degree of platelet disaggregation induced by EDTA was dependent on the dose of cytochalasin D used, and was correlated with the inhibition of the cytoskeletal reorganization. Aggregation of cytochalasin D-treated platelets stimulated with U46619 or TRAP was also reverted by the addition of the tetrapeptide RGDS or the fibrinogen y-chain dodecapeptide, which competitively interfere with fibrinogen binding to the glycoprotein Ilb-IIIa complex. These results indicate that the intracellular cytoskeleton plays an essential role in the stabilization of the fibrinogen-platelet interaction, and is necessary for the irreversibility of platelet aggregation induced by strong agonists.

• References

• 1 Siess W. Molecular mechanisms of platelet activation. Physiol Rev 1989; 69: 58-178
• 2 Phillips DR, Charo IF, Parise LV, Fitzgerald LA. The platelet membrane glycoprotein IIb-IIIa complex. Blood 1988; 71: 831-843
• 3 Gartner TK, Bennett JS. The tetrapeptide analogue of the cell attachment site of fibronectin inhibits platelet aggregation and fibrinogen binding to activated platelets. J Biol Chem 1985; 260: 11891-11894
• 4 Koczewiak M, Timmons S, Lukas TJ, Hawiger J. Platelet recognition site on human fibrinogen. Synthesis and structure-function relationship of peptides corresponding to the carboxy-terminal segment of the γ-chain. Biochemistry 1984; 23: 1767-1774
• 5 Bennett JS, Vilaire G. Exposure of platelet fibrinogen receptor by ADP and epinephrine. J Clin Invest 1979; 64: 1393-1401
• 6 Peerschke El, Zucker MB, Grant RA, Egan JJ, Johnson MM. Correlation between fibrinogen binding to human platelets and platelet aggregability. Blood 1980; 55: 841-847
• 7 Fox JEB. The platelet cytoskeleton. Thromb Haemost 1993; 70: 884-893
• 8 Sinigaglia F, Bisio A, Torti M, Balduini CL, Bertolino G, Balduini C. Effect of GPIIb-IIIa complex ligands on calcium ion movement and cytoskeleton organization in activated platelets. Biochem Biophys Res Com-mun 1988; 154: 258-264
• 9 Phillips DR, Jennings LK, Edwards HH. Identification of membrane proteins mediating the interaction of human platelets. J Cell Biol 1980; 86: 77-86
• 10 Newman PJ, Hillery CA, Albrecht R, Parise LV, Bemdt MC, Mazurov AV, Dunlop LC, Zhang J, Rittenhouse SE. Activation-dependent changes in human platelet PECAM-1: phosphorylation, cytoskeletal association, and surface membrane redistribution. J Cell Biol 1992; 119: 239-246
• 11 Grondin P, Plantavid M, Sultan C, Breton M, Mauco G, Chap H. Interaction of pp60c src, phospholipase C, inositol-lipid, and diacylglycerol kinases with the cytoskeleton of thrombin-stimulated platelets. J Biol Chem 1991; 266: 15705-15709
• 12 Horvath AR, Muszbek L, Kellie S. Translocation of pp60^c-src to the cytoskeleton during platelet aggregation. EMBO J 1992; 11: 855-861
• 13 Fischer TH, Gatling MN, Lacal JC, White GC II. RaplB, a cAMP-dependent protein kinase substrate, associates with the platelet cytoskeleton. J Biol Chem 1990; 265: 19405-19408
• 14 Torti M, Ramaschi G, Sinigaglia F, Lapetina EG, Balduini C. Association of the low molecular weight GTP-binding protein rap2B with the cytoskeleton during platelet aggregation. Proc Natl Acad Sci USA 1993; 90: 7553-7557
• 15 Fox JEB, Phillips DR. Inhibition of actin polymerization in blood platelets by cytochalasins. Nature 1981; 292: 650-652
• 16 Casella JF, Flanagan MD, Lin S. Cytochalasin D inhibits actin polymerization of actin filaments formed during platelet shape change. Nature 1981; 293: 302-305
• 17 Levebvre P, White JG, Krumwiede MD, Cohen I. Role of actin in platelet function. Eur J Cell Biol 1993; 62: 194-204
• 18 Kirkpatrick Z, Mclntire LV, Moake JL, Cimo PL. Differential effects of cytochalasin B on platelet release, aggregation and contractility: evidence against a contractile mechanism for release of platelet granular contents. Thromb Haemost 1980; 42: 1483-1489
• 19 Siess W, Lapetina EG, Cuatrecasas P. Cytochalasins inhibit arachidonic acid metabolism in thrombin-stimulated platelets. Proc Natl Acad Sci USA 1982; 79: 7709-7713
• 20 Peerschke El, Zucker MB. Relationship of ADP-induced fibrinogen binding to platelet shape change and aggregation elucidated by the use of colchicine and cytochalasin B. Thromb Haemost 1980; 43: 58-60
• 21 Peerschke EIB, Wainer JA. Examination of irreversible platelet-fibrinogen interactions. Amer J Physiol 1985; 248: C466-C472
• 22 Peerschke EL Observations on the effects of cytochalasin B and D on ADP-and chymotrypsin-treated platelets. Proc Soc Exp Biol and Med 1984; 175: 109-115
• 23 Yamaguchi A, Tanoue K, Yamazaki H. Secondary signals mediated by GPIIb-IIIa in thrombin-activated platelets. Biochim Biophys Acta 1990; 1054: 8-13
• 24 Haslam RJ, Davidson MML, McClenaghan MD. Cytochalasin B, the blood platelet reaction and cyclic GMP. Nature 1975; 253: 455-457
• 25 Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970; 227: 680-685
• 26 Marguerie GA, Edgington TS, PLow EF. Interaction of fibrinogen with its platelet receptor as part of a multistep reaction in ADP-induced platelet aggregation. J Biol Chem 1980; 255: 154-161
• 27 Golden A, Brugge JS, Shattil SJ. Role of platelet membrane glycoprotein IIb-IIIa in agonist-induced tyrosine phosphorylation of platelet proteins. J Cell Biol 1990; 111: 3117-3127
• 28 Fox JEB, Taylor RG, Taffarel M, Boyles JK, Goll DE. Evidence that activation of platelet calpain is induced as a consequence of binding of adhesive ligand to the integrin glycoprotein IIb-IIIa. J Cell Biol 1993; 120: 1501-1507
• 29 Sultan C, Plantavid M, Bachelot C, Grondin P, Breton M, Mauco G, Levy-Toledano C, Caen JP, Chap H. Involvement of platelet glycoprotein IIb-IIIa (αIIb-β₃ integrin) in thrombin-induced synthesis of phosphatidylinositol 3′,4′-bisphosphate. J Biol Chem 1991; 266: 23554-23557
• 30 Schoenwaelder SM, Jackson SP, Yuan Y, Teasdale MS, Salem HH, Mitchell CA. Tyrosine kinases regulate the cytoskeletal attachment of integrin allb-b3 (platelet glycoprotein Ilb-IIIa) and the cellular retraction of fibrin polymers. J Biol Chem 1994; 269: 32479-32487