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. 1980 Jul 1;86(1):77–86. doi: 10.1083/jcb.86.1.77

Identification of membrane proteins mediating the interaction of human platelets

D Phillips, L Jennings, H Edwards
PMCID: PMC2110644  PMID: 6893455

Abstract

Membrane glycoproteins that mediate platelet-platelet interactions were investigated by identifying those associated with the cytoskeletal structures from aggregated platelets. The cytoskeletal structures from washed platelets, thrombin-activated platelets (platelets incubated with thrombin in the presence of mM EDTA to prevent aggregation) and thrombin- aggregated platelets (platelets activated in the presence of mM Ca(++) were prepared by first treating platelet suspensions with 1 percent Triton X-100 and 5 mM EGTA and then isolating the insoluble residue by centrifugation. The readily identifiable structures in electron micrographs of the residue from washed platelets had the shape and dimensions of actin filaments. Analysis of this residue from washed platelets had the shape and dimensions of actin filaments. Analysis of this residue by SDS gel electrophoresis showed that it consisted primarily of three proteins: actin (mol wt = 43,000), myosin (mol wt = 200,000) and a high molecular weight polypeptide (mol wt = 255,000) which had properties indentical to actin-binding protein (filamin). When platelets are activated with thrombin in the presence of EDTA to prevent aggregation, there was a marked increase in the amount of insoluble precipitate in the subsequent Triton extraction. Transmission electron microscopy showed that this residue not only contained the random array of actin filaments as seen above, but also organized structures from individual platelets which appeared as balls of electron-dense filamentous material approximately 1mum in diameter. SDS polyacrylamide gel analysis of the Triton residue of activated platelets showed that this preparation contained more actin, myosin and actin-binding protein than that from washed platelets plus polypeptides with mol wt of 56,000 and 90,000 and other minor polypeptides. Thus, thrombin activation appeared to increase polymerization of actin in association with other cytoskeletal proteins into structures that are observable after Triton extraction. The cytoskeletal structures from thrombin-aggregated platelets were similar to those from thrombin-activated platelets, except that the structural elements from individual platelets remained aggregated rather than randomly dispersed in the actin filaments. This suggested that the membrane components that mediate the direct interaction of platelets were in Triton residue from aggregated platelets. Only a small percentage of the membrane surface proteins and glycoproteins were found in the cytoskeletal structures from either washed platelets or thrombin-activated platelets. In contrast, the aggregated cytoskeletal structures from thrombin-aggregated platelets contained membrane glycoproteins IIb (26 percent of the total in pre-extracted platelets) and III (14 percent), suggesting that one or both of these glycoproteins participate in the direct interaction of platelets during aggregation.

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Selected References

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  1. Ames G. F., Nikaido K. Two-dimensional gel electrophoresis of membrane proteins. Biochemistry. 1976 Feb 10;15(3):616–623. doi: 10.1021/bi00648a026. [DOI] [PubMed] [Google Scholar]
  2. Andersson L. C., Gahmberg C. G. Surface glycoproteins of human white blood cells. Analysis by surface labeling. Blood. 1978 Jul;52(1):57–67. [PubMed] [Google Scholar]
  3. Begg D. A., Rodewald R., Rebhun L. I. The visualization of actin filament polarity in thin sections. Evidence for the uniform polarity of membrane-associated filaments. J Cell Biol. 1978 Dec;79(3):846–852. doi: 10.1083/jcb.79.3.846. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Blikstad I., Markey F., Carlsson L., Persson T., Lindberg U. Selective assay of monomeric and filamentous actin in cell extracts, using inhibition of deoxyribonuclease I. Cell. 1978 Nov;15(3):935–943. doi: 10.1016/0092-8674(78)90277-5. [DOI] [PubMed] [Google Scholar]
  5. Bray D., Thomas C. Unpolymerized actin in fibroblasts and brain. J Mol Biol. 1976 Aug 25;105(4):527–544. doi: 10.1016/0022-2836(76)90233-3. [DOI] [PubMed] [Google Scholar]
  6. Brown S., Levinson W., Spudich J. A. Cytoskeletal elements of chick embryo fibroblasts revealed by detergent extraction. J Supramol Struct. 1976;5(2):119–130. doi: 10.1002/jss.400050203. [DOI] [PubMed] [Google Scholar]
  7. Clemetson K. J., Capitanio A., Lüscher E. F. High resolution two-dimensional gel electrophoresis of the proteins and glycoproteins of human blood platelets and platelet membranes. Biochim Biophys Acta. 1979 May 3;553(1):11–24. doi: 10.1016/0005-2736(79)90027-0. [DOI] [PubMed] [Google Scholar]
  8. Condeelis J. Isolation of concanavalin A caps during various stages of formation and their association with actin and myosin. J Cell Biol. 1979 Mar;80(3):751–758. doi: 10.1083/jcb.80.3.751. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Davies P. J., Wallach D., Willingham M. C., Pastan I., Yamaguchi M., Robson R. M. Filamin-actin interaction. Dissociation of binding from gelation by Ca2+-activated proteolysis. J Biol Chem. 1978 Jun 10;253(11):4036–4042. [PubMed] [Google Scholar]
  10. Dulley J. R., Grieve P. A. A simple technique for eliminating interference by detergents in the Lowry method of protein determination. Anal Biochem. 1975 Mar;64(1):136–141. doi: 10.1016/0003-2697(75)90415-7. [DOI] [PubMed] [Google Scholar]
  11. Fisher K. A. "Half" membrane enrichment: verification by electron microscopy. Science. 1975 Dec 5;190(4218):983–985. doi: 10.1126/science.1188378. [DOI] [PubMed] [Google Scholar]
  12. Flanagan J., Koch G. L. Cross-linked surface Ig attaches to actin. Nature. 1978 May 25;273(5660):278–281. doi: 10.1038/273278a0. [DOI] [PubMed] [Google Scholar]
  13. Gartner T. K., Williams D. C., Minion F. C., Phillips D. R. Thrombin-induced platelet aggregation is mediated by a platelet plasma membrane-bound lectin. Science. 1978 Jun 16;200(4347):1281–1283. doi: 10.1126/science.663608. [DOI] [PubMed] [Google Scholar]
  14. George J. N. Direct assessment of platelet adhesion to glass: a study of the forces of interaction and the effects of plasma and serum factors, platelet function, and modification of the glass surface. Blood. 1972 Dec;40(6):862–874. [PubMed] [Google Scholar]
  15. Gerrard J. M., Schollmeyer J. V., Phillips D. R., White J. G. alpha-Actinin deficiency in thrombasthenia: possible identity of alpha-actinin and glycoprotein III. Am J Pathol. 1979 Mar;94(3):509–528. [PMC free article] [PubMed] [Google Scholar]
  16. Gonen A., Weisman-Shomer P., Fry M. Cell adhesion and acquisition of detergent resistance by the cytoskeleton of cultured chick fibroblasts. Biochim Biophys Acta. 1979 Apr 4;552(2):307–321. doi: 10.1016/0005-2736(79)90285-2. [DOI] [PubMed] [Google Scholar]
  17. Gordon D. J., Boyer J. L., Korn E. D. Comparative biochemistry of non-muscle actins. J Biol Chem. 1977 Nov 25;252(22):8300–8309. [PubMed] [Google Scholar]
  18. HUXLEY H. E. ELECTRON MICROSCOPE STUDIES ON THE STRUCTURE OF NATURAL AND SYNTHETIC PROTEIN FILAMENTS FROM STRIATED MUSCLE. J Mol Biol. 1963 Sep;7:281–308. doi: 10.1016/s0022-2836(63)80008-x. [DOI] [PubMed] [Google Scholar]
  19. Hartwig J. H., Stossel T. P. Isolation and properties of actin, myosin, and a new actinbinding protein in rabbit alveolar macrophages. J Biol Chem. 1975 Jul 25;250(14):5696–5705. [PubMed] [Google Scholar]
  20. Kane R. E. Actin polymerization and interaction with other proteins in temperature-induced gelation of sea urchin egg extracts. J Cell Biol. 1976 Dec;71(3):704–714. doi: 10.1083/jcb.71.3.704. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Koch G. L., Smith M. J. An association between actin and the major histocompatibility antigen H-2. Nature. 1978 May 25;273(5660):274–278. doi: 10.1038/273274a0. [DOI] [PubMed] [Google Scholar]
  22. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  23. 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]
  24. Lazarides E., Lindberg U. Actin is the naturally occurring inhibitor of deoxyribonuclease I. Proc Natl Acad Sci U S A. 1974 Dec;71(12):4742–4746. doi: 10.1073/pnas.71.12.4742. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Majerus P. W., Brodie G. N. The binding of phytohemagglutinins to human platelet plasma membranes. J Biol Chem. 1972 Jul 10;247(13):4253–4257. [PubMed] [Google Scholar]
  26. Massini P., Metcalf L. C., Näf U., Lüscher E. F. Induction of aggregation and of the release reaction in human platelets by polylysine. Haemostasis. 1974;3(1):8–19. doi: 10.1159/000214038. [DOI] [PubMed] [Google Scholar]
  27. Maupin-Szamier P., Pollard T. D. Actin filament destruction by osmium tetroxide. J Cell Biol. 1978 Jun;77(3):837–852. doi: 10.1083/jcb.77.3.837. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Miletich J. P., Jackson C. M., Majerus P. W. Interaction of coagulation factor Xa with human platelets. Proc Natl Acad Sci U S A. 1977 Sep;74(9):4033–4036. doi: 10.1073/pnas.74.9.4033. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Mustard J. F., Packham M. A. Factors influencing platelet function: adhesion, release, and aggregation. Pharmacol Rev. 1970 Jun;22(2):97–187. [PubMed] [Google Scholar]
  30. Nachmias V. T., Sullender J. S., Fallon J. R. Effects of local anesthetics on human platelets: filopodial suppression and endogenous proteolysis. Blood. 1979 Jan;53(1):63–72. [PubMed] [Google Scholar]
  31. Nachmias V., Sullender J., Asch A. Shape and cytoplasmic filaments in control and lidocaine-treated human platelets. Blood. 1977 Jul;50(1):39–53. [PubMed] [Google Scholar]
  32. O'Farrell P. H. High resolution two-dimensional electrophoresis of proteins. J Biol Chem. 1975 May 25;250(10):4007–4021. [PMC free article] [PubMed] [Google Scholar]
  33. Okumura T., Hasitz M., Jamieson G. A. Platelet glycocalicin. Interaction with thrombin and role as thrombin receptor of the platelet surface. J Biol Chem. 1978 May 25;253(10):3435–3443. [PubMed] [Google Scholar]
  34. Osborn M., Weber K. The detertent-resistant cytoskeleton of tissue culture cells includes the nucleus and the microfilament bundles. Exp Cell Res. 1977 May;106(2):339–349. doi: 10.1016/0014-4827(77)90179-3. [DOI] [PubMed] [Google Scholar]
  35. Phillips D. R., Agin P. P. Platelet membrane defects in Glanzmann's thrombasthenia. Evidence for decreased amounts of two major glycoproteins. J Clin Invest. 1977 Sep;60(3):535–545. doi: 10.1172/JCI108805. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Phillips D. R., Agin P. P. Platelet plasma membrane glycoproteins. Evidence for the presence of nonequivalent disulfide bonds using nonreduced-reduced two-dimensional gel electrophoresis. J Biol Chem. 1977 Mar 25;252(6):2121–2126. [PubMed] [Google Scholar]
  37. Phillips D. R., Agin P. P. Platelet plasma membrane glycoproteins. Identification of a proteolytic substrate for thrombin. Biochem Biophys Res Commun. 1977 Apr 25;75(4):940–947. doi: 10.1016/0006-291x(77)91473-5. [DOI] [PubMed] [Google Scholar]
  38. Phillips D. R., Agin P. P. Thrombin substrates and the proteolytic site of thrombin action on human-platelet plasma membranes. Biochim Biophys Acta. 1974 Jun 13;352(2):218–227. doi: 10.1016/0005-2736(74)90213-2. [DOI] [PubMed] [Google Scholar]
  39. Phillips D. R., Agin P. P. Thrombin-induced alterations in the surface structure of the human platelet plasma membrane. Ser Haematol. 1973;6(3):292–310. [PubMed] [Google Scholar]
  40. Phillips D. R. Effect of trypsin on the exposed polypeptides and glycoproteins in the human platelet membrane. Biochemistry. 1972 Nov 21;11(24):4582–4588. doi: 10.1021/bi00774a025. [DOI] [PubMed] [Google Scholar]
  41. Phillips D. R., Jakábová M. Ca2+-dependent protease in human platelets. Specific cleavage of platelet polypeptides in the presence of added Ca2+. J Biol Chem. 1977 Aug 25;252(16):5602–5605. [PubMed] [Google Scholar]
  42. Pollard T. D., Thomas S. M., Niederman R. Human platelet myosin. I. Purification by a rapid method applicable to other nonmuscle cells. Anal Biochem. 1974 Jul;60(1):258–266. doi: 10.1016/0003-2697(74)90152-3. [DOI] [PubMed] [Google Scholar]
  43. Rubin R. W., Goldstein L., Ko C. Differences between nucleus and cytoplasm in the degree of actin polymerization. J Cell Biol. 1978 Jun;77(3):698–701. doi: 10.1083/jcb.77.3.698. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Shizuta Y., Shizuta H., Gallo M., Davies P., Pastan I. Purification and properties of filamin, and actin binding protein from chicken gizzard. J Biol Chem. 1976 Nov 10;251(21):6562–6567. [PubMed] [Google Scholar]
  45. Stossel T. P., Hartwig J. H. Interactions of actin, myosin, and a new actin-binding protein of rabbit pulmonary macrophages. II. Role in cytoplasmic movement and phagocytosis. J Cell Biol. 1976 Mar;68(3):602–619. doi: 10.1083/jcb.68.3.602. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Tollefsen D. M., Feagler J. R., Majerus P. W. The binding of thrombin to the surface of human platelets. J Biol Chem. 1974 Apr 25;249(8):2646–2651. [PubMed] [Google Scholar]
  47. Trotter J. A., Foerder B. A., Keller J. M. Intracellular fibres in cultured cells: analysis by scanning and transmission electron microscopy and by SDS-polyacrylamide gel electrophoresis. J Cell Sci. 1978 Jun;31:369–392. doi: 10.1242/jcs.31.1.369. [DOI] [PubMed] [Google Scholar]
  48. Tschopp T. B., Weiss H. J., Baumgartner H. R. Interaction of thrombasthenic platelets with subendothelium: normal adhesion, absent aggregation. Experientia. 1975 Jan 15;31(1):113–116. doi: 10.1007/BF01924709. [DOI] [PubMed] [Google Scholar]
  49. Wang K., Singer S. J. Interaction of filamin with f-actin in solution. Proc Natl Acad Sci U S A. 1977 May;74(5):2021–2025. doi: 10.1073/pnas.74.5.2021. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. White J. G. Fine structural alterations induced in platelets by adenosine diphosphate. Blood. 1968 May;31(5):604–622. [PubMed] [Google Scholar]
  51. Zucker-Franklin D., Nachman R. L., Marcus A. J. Ultrastructure of thrombosthenin, the contractile protein of human blood platelets. Science. 1967 Aug 25;157(3791):945–946. doi: 10.1126/science.157.3791.945. [DOI] [PubMed] [Google Scholar]
  52. Zucker M. B., Pert J. H., Hilgartner M. W. Platelet function in a patient with thrombasthenia. Blood. 1966 Oct;28(4):524–534. [PubMed] [Google Scholar]

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