Skip to main content
NCBI home page
The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1991 Mar 1;112(5):903–913. doi: 10.1083/jcb.112.5.903

Formation and contraction of a microfilamentous shell in saponin- permeabilized platelets

PMCID: PMC2288883  PMID: 1900299

Abstract

To study the mechanism of granule centralization in platelets, we permeabilized with saponin in either EGTA (5 mM) or calcium (1 or 10 microM). Under all conditions, platelets retained 40-50% of their total actin and greater than 70% of their actin-binding protein (ABP) but lost greater than 80% of talin and myosin to the supernatant. Thin sections of platelets permeabilized in EGTA showed a microfilament network under the residual plasma membrane and throughout the cytoplasm. Platelets permeabilized in calcium contained a microfilament shell partly separated from the residual membrane. The shell stained brightly for F-actin. A less dense microfilament shell was also seen in sections of ADP-stimulated intact platelets subsequently permeabilized in EGTA. In the presence of 1 mM ATP gamma S and calcium, myosin was retained (70%) and was localized by indirect immunofluorescence in bright central spots that also stained intensely for F-actin. Electron micrographs showed centralized granules surrounded by a closely packed mass of microfilaments much like the structures seen in thrombin- stimulated intact platelets subsequently permeabilized in EGTA. Permeabilization in calcium, ATP, and okadaic acid, produced the same configuration of centralized granules and packed microfilaments; myosin was retained and the myosin regulatory light chain became phosphorylated. Microtubule coil disassembly before permeabilization did not inhibit granule centralization. These results suggest a possible mechanism for granule centralization in these models. The cytoskeletal network first separates from some of its connections to the plasma membrane by a calcium-dependent mechanism not involving ABP proteolysis. Phosphorylated myosin interacts with the microfilaments to contract the shell moving the granules to the platelet's center.

Full Text

The Full Text of this article is available as a PDF (3.3 MB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Adelstein R. S., Conti M. A. Phosphorylation of platelet myosin increases actin-activated myosin ATPase activity. Nature. 1975 Aug 14;256(5518):597–598. doi: 10.1038/256597a0. [DOI] [PubMed] [Google Scholar]
  2. Authi K. S., Hornby E. J., Evenden B. J., Crawford N. Inositol 1,4,5-trisphosphate (IP3) induced rapid formation of thromboxane B2 in saponin-permeabilised human platelets: mechanism of IP3 action. FEBS Lett. 1987 Mar 9;213(1):95–101. doi: 10.1016/0014-5793(87)81471-0. [DOI] [PubMed] [Google Scholar]
  3. Behnke O. Effects of some chemicals on blood platelet microtubules, platelet shape and some platelet functions in vitro. Scand J Haematol. 1970;7(2):123–140. doi: 10.1111/j.1600-0609.1970.tb01878.x. [DOI] [PubMed] [Google Scholar]
  4. Bialojan C., Rüegg J. C., Takai A. Effects of okadaic acid on isometric tension and myosin phosphorylation of chemically skinned guinea-pig taenia coli. J Physiol. 1988 Apr;398:81–95. doi: 10.1113/jphysiol.1988.sp017030. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Born G. V., Dearnley R., Foulks J. G., Sharp D. E. Quantification of the morphological reaction of platelets to aggregating agents and of its reversal by aggregation inhibitors. J Physiol. 1978 Jul;280:193–212. doi: 10.1113/jphysiol.1978.sp012380. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Brass L. F., Joseph S. K. A role for inositol triphosphate in intracellular Ca2+ mobilization and granule secretion in platelets. J Biol Chem. 1985 Dec 5;260(28):15172–15179. [PubMed] [Google Scholar]
  7. Carroll R. C., Butler R. G., Morris P. A., Gerrard J. M. Separable assembly of platelet pseudopodal and contractile cytoskeletons. Cell. 1982 Sep;30(2):385–393. doi: 10.1016/0092-8674(82)90236-7. [DOI] [PubMed] [Google Scholar]
  8. Chantler P. D., Sellers J. R., Szent-Györgyi A. G. Cooperativity in scallop myosin. Biochemistry. 1981 Jan 6;20(1):210–216. doi: 10.1021/bi00504a035. [DOI] [PubMed] [Google Scholar]
  9. Cox A. C., Carroll R. C., White J. G., Rao G. H. Recycling of platelet phosphorylation and cytoskeletal assembly. J Cell Biol. 1984 Jan;98(1):8–15. doi: 10.1083/jcb.98.1.8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Dabrowska R., Hartshorne D. J. A Ca2+-and modulator-dependent myosin light chain kinase from non-muscle cells. Biochem Biophys Res Commun. 1978 Dec 29;85(4):1352–1359. doi: 10.1016/0006-291x(78)91152-x. [DOI] [PubMed] [Google Scholar]
  11. Daniel J. L., Molish I. R., Rigmaiden M., Stewart G. Evidence for a role of myosin phosphorylation in the initiation of the platelet shape change response. J Biol Chem. 1984 Aug 10;259(15):9826–9831. [PubMed] [Google Scholar]
  12. Fox J. E., Boyles J. K., Reynolds C. C., Phillips D. R. Actin filament content and organization in unstimulated platelets. J Cell Biol. 1984 Jun;98(6):1985–1991. doi: 10.1083/jcb.98.6.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Fox J. E., Dockter M. E., Phillips D. R. An improved method for determining the actin filament content of nonmuscle cells by the DNase I inhibition assay. Anal Biochem. 1981 Oct;117(1):170–177. doi: 10.1016/0003-2697(81)90707-7. [DOI] [PubMed] [Google Scholar]
  14. Fox J. E., Goll D. E., Reynolds C. C., Phillips D. R. Identification of two proteins (actin-binding protein and P235) that are hydrolyzed by endogenous Ca2+-dependent protease during platelet aggregation. J Biol Chem. 1985 Jan 25;260(2):1060–1066. [PubMed] [Google Scholar]
  15. Fox J. E. Identification of actin-binding protein as the protein linking the membrane skeleton to glycoproteins on platelet plasma membranes. J Biol Chem. 1985 Oct 5;260(22):11970–11977. [PubMed] [Google Scholar]
  16. Fox J. E., Phillips D. R. Role of phosphorylation in mediating the association of myosin with the cytoskeletal structures of human platelets. J Biol Chem. 1982 Apr 25;257(8):4120–4126. [PubMed] [Google Scholar]
  17. Gerrard J. M., White J. G. The structure and function of platelets, with emphasis on their contractile nature. Pathobiol Annu. 1976;6:31–59. [PubMed] [Google Scholar]
  18. Hallam T. J., Sanchez A., Rink T. J. Stimulus-response coupling in human platelets. Changes evoked by platelet-activating factor in cytoplasmic free calcium monitored with the fluorescent calcium indicator quin2. Biochem J. 1984 Mar 15;218(3):819–827. doi: 10.1042/bj2180819. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Hartwig J. H., Chambers K. A., Stossel T. P. Association of gelsolin with actin filaments and cell membranes of macrophages and platelets. J Cell Biol. 1989 Feb;108(2):467–479. doi: 10.1083/jcb.108.2.467. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Hartwig J. H., Stossel T. P. Structure of macrophage actin-binding protein molecules in solution and interacting with actin filaments. J Mol Biol. 1981 Jan 25;145(3):563–581. doi: 10.1016/0022-2836(81)90545-3. [DOI] [PubMed] [Google Scholar]
  21. Hathaway D. R., Adelstein R. S. Human platelet myosin light chain kinase requires the calcium-binding protein calmodulin for activity. Proc Natl Acad Sci U S A. 1979 Apr;76(4):1653–1657. doi: 10.1073/pnas.76.4.1653. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Janmey P. A., Stossel T. P. Modulation of gelsolin function by phosphatidylinositol 4,5-bisphosphate. Nature. 1987 Jan 22;325(6102):362–364. doi: 10.1038/325362a0. [DOI] [PubMed] [Google Scholar]
  23. Jennings L. K., Fox J. E., Edwards H. H., Phillips D. R. Changes in the cytoskeletal structure of human platelets following thrombin activation. J Biol Chem. 1981 Jul 10;256(13):6927–6932. [PubMed] [Google Scholar]
  24. Kenney D. M., Linck R. W. The cystoskeleton of unstimulated blood platelets: structure and composition of the isolated marginal microtubular band. J Cell Sci. 1985 Oct;78:1–22. doi: 10.1242/jcs.78.1.1. [DOI] [PubMed] [Google Scholar]
  25. Lebowitz E. A., Cooke R. Contractile properties of actomyosin from human blood platelets. J Biol Chem. 1978 Aug 10;253(15):5443–5447. [PubMed] [Google Scholar]
  26. Lind S. E., Janmey P. A., Chaponnier C., Herbert T. J., Stossel T. P. Reversible binding of actin to gelsolin and profilin in human platelet extracts. J Cell Biol. 1987 Aug;105(2):833–842. doi: 10.1083/jcb.105.2.833. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Loftus J. C., Choate J., Albrecht R. M. Platelet activation and cytoskeletal reorganization: high voltage electron microscopic examination of intact and Triton-extracted whole mounts. J Cell Biol. 1984 Jun;98(6):2019–2025. doi: 10.1083/jcb.98.6.2019. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Morgan M., Perry S. V., Ottaway J. Myosin light-chain phosphatase. Biochem J. 1976 Sep 1;157(3):687–697. doi: 10.1042/bj1570687. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Nachmias V. T. Cytoskeleton of human platelets at rest and after spreading. J Cell Biol. 1980 Sep;86(3):795–802. doi: 10.1083/jcb.86.3.795. [DOI] [PMC free article] [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. Nakata T., Hirokawa N. Cytoskeletal reorganization of human platelets after stimulation revealed by the quick-freeze deep-etch technique. J Cell Biol. 1987 Oct;105(4):1771–1780. doi: 10.1083/jcb.105.4.1771. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. O'Halloran T., Beckerle M. C., Burridge K. Identification of talin as a major cytoplasmic protein implicated in platelet activation. Nature. 1985 Oct 3;317(6036):449–451. doi: 10.1038/317449a0. [DOI] [PubMed] [Google Scholar]
  33. Okita J. R., Pidard D., Newman P. J., Montgomery R. R., Kunicki T. J. On the association of glycoprotein Ib and actin-binding protein in human platelets. J Cell Biol. 1985 Jan;100(1):317–321. doi: 10.1083/jcb.100.1.317. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Painter R. G., Ginsberg M. H. Centripetal myosin redistribution in thrombin-stimulated platelets. Relationship to platelet Factor 4 secretion. Exp Cell Res. 1984 Nov;155(1):198–212. doi: 10.1016/0014-4827(84)90781-x. [DOI] [PubMed] [Google Scholar]
  35. 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]
  36. Rosenberg S., Stracher A., Lucas R. C. Isolation and characterization of actin and actin-binding protein from human platelets. J Cell Biol. 1981 Oct;91(1):201–211. doi: 10.1083/jcb.91.1.201. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. 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]
  38. Wang L. L., Bryan J. Isolation of calcium-dependent platelet proteins that interact with actin. Cell. 1981 Sep;25(3):637–649. doi: 10.1016/0092-8674(81)90171-9. [DOI] [PubMed] [Google Scholar]
  39. White J. G. Effects of colchicine and vinca alkaloids on human platelets. 3. Influence on primary internal contraction and secondary aggregation. Am J Pathol. 1969 Mar;54(3):467–478. [PMC free article] [PubMed] [Google Scholar]
  40. Wysolmerski R. B., Lagunoff D. Involvement of myosin light-chain kinase in endothelial cell retraction. Proc Natl Acad Sci U S A. 1990 Jan;87(1):16–20. doi: 10.1073/pnas.87.1.16. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Yin H. L., Stossel T. P. Control of cytoplasmic actin gel-sol transformation by gelsolin, a calcium-dependent regulatory protein. Nature. 1979 Oct 18;281(5732):583–586. doi: 10.1038/281583a0. [DOI] [PubMed] [Google Scholar]
  42. Yoshida K., Dubyak G., Nachmias V. T. Rapid effects of phorbol ester on platelet shape change, cytoskeleton and calcium transient. FEBS Lett. 1986 Oct 6;206(2):273–278. doi: 10.1016/0014-5793(86)80995-4. [DOI] [PubMed] [Google Scholar]
  43. Yoshida K., Nachmias V. T. Phorbol ester stimulates calcium sequestration in saponized human platelets. J Biol Chem. 1987 Nov 25;262(33):16048–16054. [PubMed] [Google Scholar]
  44. Yoshida K., Stark F., Nachmias V. T. Comparison of the effects of phorbol 12-myristate 13-acetate and prostaglandin E1 on calcium regulation in human platelets. Biochem J. 1988 Jan 15;249(2):487–493. doi: 10.1042/bj2490487. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Zavoico G. B., Feinstein M. B. Cytoplasmic Ca2+ in platelets is controlled by cyclic AMP: antagonism between stimulators and inhibitors of adenylate cyclase. Biochem Biophys Res Commun. 1984 Apr 30;120(2):579–585. doi: 10.1016/0006-291x(84)91294-4. [DOI] [PubMed] [Google Scholar]

Articles from The Journal of Cell Biology are provided here courtesy of The Rockefeller University Press

RESOURCES