Abstract
The stimulation of human platelets with physiological agonists results in the incorporation of several proteins into the cytoskeleton, fibrinogen binding, and platelet aggregation. We recently demonstrated that the Ras-related low molecular weight GTP-binding protein Rap2B associates with the cytoskeleton in activated platelets and that this interaction requires platelet aggregation. In the present study we demonstrate that agonist-induced actin polymerization is necessary for the translocation of Rap2B to the cytoskeleton, suggesting that Rap2B interacts with the newly formed actin filaments. Moreover, the association of Rap2B with Triton X-100-insoluble material from platelets was totally blocked by treatment of intact platelets with monoclonal antibodies against the fibrinogen receptor glycoprotein IIb-IIIa. Platelets from patients affected by Glanzmann thrombastenia, a genetic disorder in which platelet plasma membranes lack glycoprotein IIb-IIIa but possess normal levels of Ras-related proteins, failed to incorporate Rap2B into the cytoskeleton upon activation by thrombin. Comparative immunoblotting revealed that the translocation of Rap2B to the cytoskeleton during platelet aggregation was accompanied by the simultaneous translocation of glycoprotein IIb-IIIa. Moreover, the cytoskeleton from aggregated platelets contained Rap2B and glycoprotein IIb-IIIa in comparable amounts. These results demonstrate the association of Rap2B and glycoprotein IIb-IIIa and their translocation to the cytoskeleton in aggregated human platelets.
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- Andrieux A., Rabiet M. J., Chapel A., Concord E., Marguerie G. A highly conserved sequence of the Arg-Gly-Asp-binding domain of the integrin beta 3 subunit is sensitive to stimulation. J Biol Chem. 1991 Aug 5;266(22):14202–14207. [PubMed] [Google Scholar]
- Banga H. S., Simons E. R., Brass L. F., Rittenhouse S. E. Activation of phospholipases A and C in human platelets exposed to epinephrine: role of glycoproteins IIb/IIIa and dual role of epinephrine. Proc Natl Acad Sci U S A. 1986 Dec;83(23):9197–9201. doi: 10.1073/pnas.83.23.9197. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Bennett J. S., Vilaire G. Exposure of platelet fibrinogen receptors by ADP and epinephrine. J Clin Invest. 1979 Nov;64(5):1393–1401. doi: 10.1172/JCI109597. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Campa M. J., Farrell F. X., Lapetina E. G., Chang K. J. Microinjection of Rap2B protein or RNA induces rearrangement of pigment granules in Xenopus oocytes. Biochem J. 1993 May 15;292(Pt 1):231–236. doi: 10.1042/bj2920231. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Farrell F. X., Ohmstede C. A., Reep B. R., Lapetina E. G. cDNA sequence of a new ras-related gene (rap2b) isolated from human platelets with sequence homology to rap2. Nucleic Acids Res. 1990 Jul 25;18(14):4281–4281. doi: 10.1093/nar/18.14.4281. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Farrell F. X., Yamamoto K., Lapetina E. G. Prenyl group identification of rap2 proteins: a ras superfamily member other than ras that is farnesylated. Biochem J. 1993 Jan 15;289(Pt 2):349–355. doi: 10.1042/bj2890349. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Ferrell J. E., Jr, Martin G. S. Tyrosine-specific protein phosphorylation is regulated by glycoprotein IIb-IIIa in platelets. Proc Natl Acad Sci U S A. 1989 Apr;86(7):2234–2238. doi: 10.1073/pnas.86.7.2234. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Fischer T. H., Gatling M. N., Lacal J. C., White G. C., 2nd rap1B, a cAMP-dependent protein kinase substrate, associates with the platelet cytoskeleton. J Biol Chem. 1990 Nov 15;265(32):19405–19408. [PubMed] [Google Scholar]
- Fitzgerald L. A., Leung B., Phillips D. R. A method for purifying the platelet membrane glycoprotein IIb-IIIa complex. Anal Biochem. 1985 Nov 15;151(1):169–177. doi: 10.1016/0003-2697(85)90067-3. [DOI] [PubMed] [Google Scholar]
- Frech M., John J., Pizon V., Chardin P., Tavitian A., Clark R., McCormick F., Wittinghofer A. Inhibition of GTPase activating protein stimulation of Ras-p21 GTPase by the Krev-1 gene product. Science. 1990 Jul 13;249(4965):169–171. doi: 10.1126/science.2164710. [DOI] [PubMed] [Google Scholar]
- Frelinger A. L., 3rd, Cohen I., Plow E. F., Smith M. A., Roberts J., Lam S. C., Ginsberg M. H. Selective inhibition of integrin function by antibodies specific for ligand-occupied receptor conformers. J Biol Chem. 1990 Apr 15;265(11):6346–6352. [PubMed] [Google Scholar]
- Frelinger A. L., 3rd, Lam S. C., Plow E. F., Smith M. A., Loftus J. C., Ginsberg M. H. Occupancy of an adhesive glycoprotein receptor modulates expression of an antigenic site involved in cell adhesion. J Biol Chem. 1988 Sep 5;263(25):12397–12402. [PubMed] [Google Scholar]
- Gartner T. K., Bennett J. S. The tetrapeptide analogue of the cell attachment site of fibronectin inhibits platelet aggregation and fibrinogen binding to activated platelets. J Biol Chem. 1985 Oct 5;260(22):11891–11894. [PubMed] [Google Scholar]
- Golden A., Brugge J. S., Shattil S. J. Role of platelet membrane glycoprotein IIb-IIIa in agonist-induced tyrosine phosphorylation of platelet proteins. J Cell Biol. 1990 Dec;111(6 Pt 2):3117–3127. doi: 10.1083/jcb.111.6.3117. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Grondin P., Plantavid M., Sultan C., Breton M., Mauco G., Chap H. Interaction of pp60c-src, phospholipase C, inositol-lipid, and diacyglycerol kinases with the cytoskeletons of thrombin-stimulated platelets. J Biol Chem. 1991 Aug 25;266(24):15705–15709. [PubMed] [Google Scholar]
- Horvath A. R., Muszbek L., Kellie S. Translocation of pp60c-src to the cytoskeleton during platelet aggregation. EMBO J. 1992 Mar;11(3):855–861. doi: 10.1002/j.1460-2075.1992.tb05123.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Hynes R. O. Integrins: a family of cell surface receptors. Cell. 1987 Feb 27;48(4):549–554. doi: 10.1016/0092-8674(87)90233-9. [DOI] [PubMed] [Google Scholar]
- Isenberg W. M., McEver R. P., Phillips D. R., Shuman M. A., Bainton D. F. The platelet fibrinogen receptor: an immunogold-surface replica study of agonist-induced ligand binding and receptor clustering. J Cell Biol. 1987 Jun;104(6):1655–1663. doi: 10.1083/jcb.104.6.1655. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Janoueix-Lerosey I., Polakis P., Tavitian A., de Gunzburg J. Regulation of the GTPase activity of the ras-related rap2 protein. Biochem Biophys Res Commun. 1992 Nov 30;189(1):455–464. doi: 10.1016/0006-291x(92)91580-j. [DOI] [PubMed] [Google Scholar]
- Klinz F. J., Seifert R., Schwaner I., Gausepohl H., Frank R., Schultz G. Generation of specific antibodies against the rap1A, rap1B and rap2 small GTP-binding proteins. Analysis of rap and ras proteins in membranes from mammalian cells. Eur J Biochem. 1992 Jul 1;207(1):207–213. doi: 10.1111/j.1432-1033.1992.tb17039.x. [DOI] [PubMed] [Google Scholar]
- Kloczewiak M., Timmons S., Lukas T. J., Hawiger J. Platelet receptor recognition site on human fibrinogen. Synthesis and structure-function relationship of peptides corresponding to the carboxy-terminal segment of the gamma chain. Biochemistry. 1984 Apr 10;23(8):1767–1774. doi: 10.1021/bi00303a028. [DOI] [PubMed] [Google Scholar]
- Kouns W. C., Fox C. F., Lamoreaux W. J., Coons L. B., Jennings L. K. The effect of glycoprotein IIb-IIIa receptor occupancy on the cytoskeleton of resting and activated platelets. J Biol Chem. 1991 Jul 25;266(21):13891–13900. [PubMed] [Google Scholar]
- Kouns W. C., Wall C. D., White M. M., Fox C. F., Jennings L. K. A conformation-dependent epitope of human platelet glycoprotein IIIa. J Biol Chem. 1990 Nov 25;265(33):20594–20601. [PubMed] [Google Scholar]
- Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
- Lapetina E. G., Lacal J. C., Reep B. R., Molina y Vedia L. A ras-related protein is phosphorylated and translocated by agonists that increase cAMP levels in human platelets. Proc Natl Acad Sci U S A. 1989 May;86(9):3131–3134. doi: 10.1073/pnas.86.9.3131. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Lerosey I., Pizon V., Tavitian A., de Gunzburg J. The cAMP-dependent protein kinase phosphorylates the rap1 protein in vitro as well as in intact fibroblasts, but not the closely related rap2 protein. Biochem Biophys Res Commun. 1991 Mar 15;175(2):430–436. doi: 10.1016/0006-291x(91)91582-w. [DOI] [PubMed] [Google Scholar]
- Maridonneau-Parini I., de Gunzburg J. Association of rap1 and rap2 proteins with the specific granules of human neutrophils. Translocation to the plasma membrane during cell activation. J Biol Chem. 1992 Mar 25;267(9):6396–6402. [PubMed] [Google Scholar]
- Molina y Vedia L., Ohmstede C. A., Lapetina E. G. Properties of the exchange rate of guanine nucleotides to the novel rap-2B protein. Biochem Biophys Res Commun. 1990 Aug 31;171(1):319–324. doi: 10.1016/0006-291x(90)91395-9. [DOI] [PubMed] [Google Scholar]
- Newman P. J., Hillery C. A., Albrecht R., Parise L. V., Berndt M. C., Mazurov A. V., Dunlop L. C., Zhang J., Rittenhouse S. E. Activation-dependent changes in human platelet PECAM-1: phosphorylation, cytoskeletal association, and surface membrane redistribution. J Cell Biol. 1992 Oct;119(1):239–246. doi: 10.1083/jcb.119.1.239. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Oda A., Druker B. J., Smith M., Salzman E. W. Association of pp60src with Triton X-100-insoluble residue in human blood platelets requires platelet aggregation and actin polymerization. J Biol Chem. 1992 Oct 5;267(28):20075–20081. [PubMed] [Google Scholar]
- Ohmstede C. A., Farrell F. X., Reep B. R., Clemetson K. J., Lapetina E. G. RAP2B: a RAS-related GTP-binding protein from platelets. Proc Natl Acad Sci U S A. 1990 Sep;87(17):6527–6531. doi: 10.1073/pnas.87.17.6527. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Parise L. V., Helgerson S. L., Steiner B., Nannizzi L., Phillips D. R. Synthetic peptides derived from fibrinogen and fibronectin change the conformation of purified platelet glycoprotein IIb-IIIa. J Biol Chem. 1987 Sep 15;262(26):12597–12602. [PubMed] [Google Scholar]
- Phillips D. R., Charo I. F., Parise L. V., Fitzgerald L. A. The platelet membrane glycoprotein IIb-IIIa complex. Blood. 1988 Apr;71(4):831–843. [PubMed] [Google Scholar]
- Phillips D. R., Jennings L. K., Edwards H. H. Identification of membrane proteins mediating the interaction of human platelets. J Cell Biol. 1980 Jul;86(1):77–86. doi: 10.1083/jcb.86.1.77. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Plow E. F., Pierschbacher M. D., Ruoslahti E., Marguerie G. A., Ginsberg M. H. The effect of Arg-Gly-Asp-containing peptides on fibrinogen and von Willebrand factor binding to platelets. Proc Natl Acad Sci U S A. 1985 Dec;82(23):8057–8061. doi: 10.1073/pnas.82.23.8057. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Ramaschi G., Balduini C., Torti M., Sinigaglia F. Effect of cAMP on the association of small GTP-binding proteins with the cytoskeleton of human platelets. Biochim Biophys Acta. 1994 Jan 5;1199(1):20–26. doi: 10.1016/0304-4165(94)90091-4. [DOI] [PubMed] [Google Scholar]
- Shattil S. J., Hoxie J. A., Cunningham M., Brass L. F. Changes in the platelet membrane glycoprotein IIb.IIIa complex during platelet activation. J Biol Chem. 1985 Sep 15;260(20):11107–11114. [PubMed] [Google Scholar]
- Siess W., Winegar D. A., Lapetina E. G. Rap1-B is phosphorylated by protein kinase A in intact human platelets. Biochem Biophys Res Commun. 1990 Jul 31;170(2):944–950. doi: 10.1016/0006-291x(90)92182-y. [DOI] [PubMed] [Google Scholar]
- Sims P. J., Ginsberg M. H., Plow E. F., Shattil S. J. Effect of platelet activation on the conformation of the plasma membrane glycoprotein IIb-IIIa complex. J Biol Chem. 1991 Apr 25;266(12):7345–7352. [PubMed] [Google Scholar]
- Sinigaglia F., Bisio A., Torti M., Balduini C. L., Bertolino G., Balduini C. Effect of GPIIb-IIIa complex ligands on calcium ion movement and cytoskeleton organization in activated platelets. Biochem Biophys Res Commun. 1988 Jul 15;154(1):258–264. doi: 10.1016/0006-291x(88)90678-x. [DOI] [PubMed] [Google Scholar]
- Sinigaglia F., Torti M., Ramaschi G., Balduini C. Fibronectin modulates the activation of human platelets. Biochem Biophys Res Commun. 1989 Dec 29;165(3):966–972. doi: 10.1016/0006-291x(89)92697-1. [DOI] [PubMed] [Google Scholar]
- Sinigaglia F., Torti M., Ramaschi G., Balduini C. The occupancy of glycoprotein IIb-IIIa complex modulates thrombin activation of human platelets. Biochim Biophys Acta. 1989 Sep 4;984(2):225–230. doi: 10.1016/0005-2736(89)90220-4. [DOI] [PubMed] [Google Scholar]
- Sultan C., Plantavid M., Bachelot C., Grondin P., Breton M., Mauco G., Lévy-Toledano S., Caen J. P., Chap H. Involvement of platelet glycoprotein IIb-IIIa (alpha IIb-beta 3 integrin) in thrombin-induced synthesis of phosphatidylinositol 3',4'-bisphosphate. J Biol Chem. 1991 Dec 15;266(35):23554–23557. [PubMed] [Google Scholar]
- Särndahl E., Lindroth M., Bengtsson T., Fällman M., Gustavsson J., Stendahl O., Andersson T. Association of ligand-receptor complexes with actin filaments in human neutrophils: a possible regulatory role for a G-protein. J Cell Biol. 1989 Dec;109(6 Pt 1):2791–2799. doi: 10.1083/jcb.109.6.2791. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Torti M., Lapetina E. G. Role of rap1B and p21ras GTPase-activating protein in the regulation of phospholipase C-gamma 1 in human platelets. Proc Natl Acad Sci U S A. 1992 Aug 15;89(16):7796–7800. doi: 10.1073/pnas.89.16.7796. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Torti M., Marti K. B., Altschuler D., Yamamoto K., Lapetina E. G. Erythropoietin induces p21ras activation and p120GAP tyrosine phosphorylation in human erythroleukemia cells. J Biol Chem. 1992 Apr 25;267(12):8293–8298. [PubMed] [Google Scholar]
- Torti M., Ramaschi G., Sinigaglia F., Lapetina E. G., Balduini C. Association of the low molecular weight GTP-binding protein rap2B with the cytoskeleton during platelet aggregation. Proc Natl Acad Sci U S A. 1993 Aug 15;90(16):7553–7557. doi: 10.1073/pnas.90.16.7553. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Torti M., Sinigaglia F., Ramaschi G., Balduini C. Platelet glycoprotein IIb-IIIa is associated with 21-kDa GTP-binding protein. Biochim Biophys Acta. 1991 Nov 18;1070(1):20–26. doi: 10.1016/0005-2736(91)90141-t. [DOI] [PubMed] [Google Scholar]
- Towbin H., Staehelin T., Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350–4354. doi: 10.1073/pnas.76.9.4350. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Wheeler M. E., Cox A. C., Carroll R. C. Retention of the glycoprotein IIb-IIIa complex in the isolated platelet cytoskeleton. Effects of separable assembly of platelet pseudopodal and contractile cytoskeletons. J Clin Invest. 1984 Sep;74(3):1080–1089. doi: 10.1172/JCI111475. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Winegar D. A., Molina y Vedia L., Lapetina E. G. Isoprenylation of rap2 proteins in platelets and human erythroleukemia cells. J Biol Chem. 1991 Mar 5;266(7):4381–4386. [PubMed] [Google Scholar]
- Winegar D. A., Ohmstede C. A., Chu L., Reep B., Lapetina E. G. Antisera specific for rap 1 proteins distinguish between processed and nonprocessed rap 1b. J Biol Chem. 1991 Mar 5;266(7):4375–4380. [PubMed] [Google Scholar]
- Yamaguchi A., Yamamoto N., Kitagawa H., Tanoue K., Yamazaki H. Ca2+ influx mediated through the GPIIb/IIIa complex during platelet activation. FEBS Lett. 1987 Dec 10;225(1-2):228–232. doi: 10.1016/0014-5793(87)81163-8. [DOI] [PubMed] [Google Scholar]
- Zhang J., Fry M. J., Waterfield M. D., Jaken S., Liao L., Fox J. E., Rittenhouse S. E. Activated phosphoinositide 3-kinase associates with membrane skeleton in thrombin-exposed platelets. J Biol Chem. 1992 Mar 5;267(7):4686–4692. [PubMed] [Google Scholar]
- Zhang J., King W. G., Dillon S., Hall A., Feig L., Rittenhouse S. E. Activation of platelet phosphatidylinositide 3-kinase requires the small GTP-binding protein Rho. J Biol Chem. 1993 Oct 25;268(30):22251–22254. [PubMed] [Google Scholar]