Skip to main content
PMC home page
The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1992 Sep 2;118(6):1421–1442. doi: 10.1083/jcb.118.6.1421

Mechanisms of actin rearrangements mediating platelet activation

PMCID: PMC2289606  PMID: 1325975

Abstract

The detergent-insoluble cytoskeleton of the resting human blood platelet contains approximately 2,000 actin filaments approximately 1 micron in length crosslinked at high angles by actin-binding protein and which bind to a spectrin-rich submembrane lamina (Fox, J., J. Boyles, M. Berndt, P. Steffen, and L. Anderson. 1988. J. Cell Biol. 106:1525-1538; Hartwig, J., and M. DeSisto. 1991. J. Cell Biol. 112:407- 425). Activation of the platelets by contact with glass results within 30 s in a doubling of the polymerized actin content of the cytoskeleton and the appearance of two distinct new actin structures: bundles of long filaments within filopodia that end at the filopodial tips (filopodial bundles) and a circumferential zone of orthogonally arrayed short filaments within lamellipodia (lamellipodial network). Neither of these structures appears in cells exposed to glass with cytochalasin B present; instead the cytoskeletons have numerous 0.1-0.3-microns-long actin filament fragments attached to the membrane lamina. With the same time course as the glass-induced morphological changes, cytochalasin- sensitive actin nucleating activity, initially low in cytoskeletons of resting platelets, increases 10-fold in cytoskeletons of thrombin- activated platelets. This activity decays with a time course consistent with depolymerization of 0.1-0.3-microns-long actin filaments, and phalloidin inhibits this decay. Cytochalasin-insensitive and calcium- dependent nucleation activity also increases markedly in platelet extracts after thrombin activation of the cells. Prevention of the rise in cytosolic Ca2+ normally associated with platelet activation with the permeant Ca2+ chelator, Quin-2, inhibits formation of lamellipodial networks but not filopodial bundles after glass contact and reduces the cytochalasin B-sensitive nucleation activity by 60% after thrombin treatment. The filopodial bundles, however, are abnormal in that they do not end at the filopodial tips but form loops and return to the cell body. Addition of calcium to chelated cells restores lamellipodial networks, and calcium plus A23187 results in cytoskeletons with highly fragmented actin filaments within seconds. Immunogold labeling with antibodies against gelsolin reveals gelsolin molecules at the ends of filaments attached to the submembrane lamina of resting cytoskeletons and at the ends of some filaments in the lamellipodial networks and filopodial bundles of activated cytoskeletons. Addition of monomeric actin to myosin subfragment 1-labeled activated cytoskeletons leads to new (undecorated) filament growth off the ends of filaments in the filopodial bundles and the lamellipodial network. The simplest explanation for these findings is that gelsolin caps the barbed ends of the filaments in the resting platelet. Uncapping some of these filaments after activation leads to filopodial bundles.(ABSTRACT TRUNCATED AT 400 WORDS)

Full Text

The Full Text of this article is available as a PDF (14.8 MB).

Selected References

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

  1. Allen R. D., Zacharski L. R., Widirstky S. T., Rosenstein R., Zaitlin L. M., Burgess D. R. Transformation and motility of human platelets: details of the shape change and release reaction observed by optical and electron microscopy. J Cell Biol. 1979 Oct;83(1):126–142. doi: 10.1083/jcb.83.1.126. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Boyles J., Fox J. E., Phillips D. R., Stenberg P. E. Organization of the cytoskeleton in resting, discoid platelets: preservation of actin filaments by a modified fixation that prevents osmium damage. J Cell Biol. 1985 Oct;101(4):1463–1472. doi: 10.1083/jcb.101.4.1463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Casella J. F., Flanagan M. D., Lin S. Cytochalasin D inhibits actin polymerization and induces depolymerization of actin filaments formed during platelet shape change. Nature. 1981 Sep 24;293(5830):302–305. doi: 10.1038/293302a0. [DOI] [PubMed] [Google Scholar]
  4. Davies G. E., Cohen C. M. Platelets contain proteins immunologically related to red cell spectrin and protein 4.1. Blood. 1985 Jan;65(1):52–59. [PubMed] [Google Scholar]
  5. Davies T. A., Drotts D. L., Weil G. J., Simons E. R. Cytoplasmic Ca2+ is necessary for thrombin-induced platelet activation. J Biol Chem. 1989 Nov 25;264(33):19600–19606. [PubMed] [Google Scholar]
  6. Di Virgilio F., Meyer B. C., Greenberg S., Silverstein S. C. Fc receptor-mediated phagocytosis occurs in macrophages at exceedingly low cytosolic Ca2+ levels. J Cell Biol. 1988 Mar;106(3):657–666. doi: 10.1083/jcb.106.3.657. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. DiNubile M. J., Southwick F. S. Effects of macrophage profilin on actin in the presence and absence of acumentin and gelsolin. J Biol Chem. 1985 Jun 25;260(12):7402–7409. [PubMed] [Google Scholar]
  8. Fox J. E., Boyles J. K., Berndt M. C., Steffen P. K., Anderson L. K. Identification of a membrane skeleton in platelets. J Cell Biol. 1988 May;106(5):1525–1538. doi: 10.1083/jcb.106.5.1525. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. 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]
  10. Fox J. E., Phillips D. R. Inhibition of actin polymerization in blood platelets by cytochalasins. Nature. 1981 Aug 13;292(5824):650–652. doi: 10.1038/292650a0. [DOI] [PubMed] [Google Scholar]
  11. Fox J. E., Reynolds C. C., Morrow J. S., Phillips D. R. Spectrin is associated with membrane-bound actin filaments in platelets and is hydrolyzed by the Ca2+-dependent protease during platelet activation. Blood. 1987 Feb;69(2):537–545. [PubMed] [Google Scholar]
  12. Hartwig J. H., Chambers K. A., Hopcia K. L., Kwiatkowski D. J. Association of profilin with filament-free regions of human leukocyte and platelet membranes and reversible membrane binding during platelet activation. J Cell Biol. 1989 Oct;109(4 Pt 1):1571–1579. doi: 10.1083/jcb.109.4.1571. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. 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]
  14. Hartwig J. H., DeSisto M. The cytoskeleton of the resting human blood platelet: structure of the membrane skeleton and its attachment to actin filaments. J Cell Biol. 1991 Feb;112(3):407–425. doi: 10.1083/jcb.112.3.407. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Hartwig J. H., Janmey P. A. Stimulation of a calcium-dependent actin nucleation activity by phorbol 12-myristate 13-acetate in rabbit macrophage cytoskeletons. Biochim Biophys Acta. 1989 Jan 17;1010(1):64–71. doi: 10.1016/0167-4889(89)90185-7. [DOI] [PubMed] [Google Scholar]
  16. Hartwig J. H., Shevlin P. The architecture of actin filaments and the ultrastructural location of actin-binding protein in the periphery of lung macrophages. J Cell Biol. 1986 Sep;103(3):1007–1020. doi: 10.1083/jcb.103.3.1007. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Janmey P. A., Chaponnier C., Lind S. E., Zaner K. S., Stossel T. P., Yin H. L. Interactions of gelsolin and gelsolin-actin complexes with actin. Effects of calcium on actin nucleation, filament severing, and end blocking. Biochemistry. 1985 Jul 2;24(14):3714–3723. doi: 10.1021/bi00335a046. [DOI] [PubMed] [Google Scholar]
  18. Janmey P. A., Iida K., Yin H. L., Stossel T. P. Polyphosphoinositide micelles and polyphosphoinositide-containing vesicles dissociate endogenous gelsolin-actin complexes and promote actin assembly from the fast-growing end of actin filaments blocked by gelsolin. J Biol Chem. 1987 Sep 5;262(25):12228–12236. [PubMed] [Google Scholar]
  19. Janmey P. A., Stossel T. P. Gelsolin-polyphosphoinositide interaction. Full expression of gelsolin-inhibiting function by polyphosphoinositides in vesicular form and inactivation by dilution, aggregation, or masking of the inositol head group. J Biol Chem. 1989 Mar 25;264(9):4825–4831. [PubMed] [Google Scholar]
  20. Janmey P. A., Stossel T. P. Kinetics of actin monomer exchange at the slow growing ends of actin filaments and their relation to the elongation of filaments shortened by gelsolin. J Muscle Res Cell Motil. 1986 Oct;7(5):446–454. doi: 10.1007/BF01753587. [DOI] [PubMed] [Google Scholar]
  21. 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]
  22. Karlsson R., Lassing I., Höglund A. S., Lindberg U. The organization of microfilaments in spreading platelets: a comparison with fibroblasts and glial cells. J Cell Physiol. 1984 Oct;121(1):96–113. doi: 10.1002/jcp.1041210113. [DOI] [PubMed] [Google Scholar]
  23. Kurth M. C., Bryan J. Platelet activation induces the formation of a stable gelsolin-actin complex from monomeric gelsolin. J Biol Chem. 1984 Jun 25;259(12):7473–7479. [PubMed] [Google Scholar]
  24. Kurth M. C., Wang L. L., Dingus J., Bryan J. Purification and characterization of a gelsolin-actin complex from human platelets. Evidence for Ca2+-insensitive functions. J Biol Chem. 1983 Sep 25;258(18):10895–10903. [PubMed] [Google Scholar]
  25. 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]
  26. Lind S. E., Yin H. L., Stossel T. P. Human platelets contain gelsolin. A regulator of actin filament length. J Clin Invest. 1982 Jun;69(6):1384–1387. doi: 10.1172/JCI110578. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Maekawa S., Sakai H. Inhibition of actin regulatory activity of the 74-kDa protein from bovine adrenal medulla (adseverin) by some phospholipids. J Biol Chem. 1990 Jul 5;265(19):10940–10942. [PubMed] [Google Scholar]
  28. Markey F., Lindberg U., Eriksson L. Human platelets contain profilin, a potential regulator of actin polymerisability. FEBS Lett. 1978 Apr 1;88(1):75–79. doi: 10.1016/0014-5793(78)80610-3. [DOI] [PubMed] [Google Scholar]
  29. Markey F., Persson T., Lindberg U. Characterization of platelet extracts before and after stimulation with respect to the possible role of profilactin as microfilament precursor. Cell. 1981 Jan;23(1):145–153. doi: 10.1016/0092-8674(81)90279-8. [DOI] [PubMed] [Google Scholar]
  30. 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]
  31. Nachmias V. T., Sullender J., Fallon J., Asch A. Observations on the "cytoskeleton" of human platelets. Thromb Haemost. 1980 Feb 29;42(5):1661–1666. [PubMed] [Google Scholar]
  32. 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]
  33. Oda A., Daley J. F., Kang J., Smith M., Ware J. A., Salzman E. W. Quasi-simultaneous measurement of ionized calcium and alpha-granule release in individual platelets. Am J Physiol. 1991 Feb;260(2 Pt 1):C242–C248. doi: 10.1152/ajpcell.1991.260.2.C242. [DOI] [PubMed] [Google Scholar]
  34. 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]
  35. Safer D., Elzinga M., Nachmias V. T. Thymosin beta 4 and Fx, an actin-sequestering peptide, are indistinguishable. J Biol Chem. 1991 Mar 5;266(7):4029–4032. [PubMed] [Google Scholar]
  36. Safer D., Golla R., Nachmias V. T. Isolation of a 5-kilodalton actin-sequestering peptide from human blood platelets. Proc Natl Acad Sci U S A. 1990 Apr;87(7):2536–2540. doi: 10.1073/pnas.87.7.2536. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Sage S. O., Rink T. J. The kinetics of changes in intracellular calcium concentration in fura-2-loaded human platelets. J Biol Chem. 1987 Dec 5;262(34):16364–16369. [PubMed] [Google Scholar]
  38. Schoepper B., Wegner A. Rate constants and equilibrium constants for binding of actin to the 1:1 gelsolin-actin complex. Eur J Biochem. 1991 Dec 18;202(3):1127–1131. doi: 10.1111/j.1432-1033.1991.tb16480.x. [DOI] [PubMed] [Google Scholar]
  39. Selve N., Wegner A. Rate constants and equilibrium constants for binding of the gelsolin-actin complex to the barbed ends of actin filaments in the presence and absence of calcium. Eur J Biochem. 1986 Oct 15;160(2):379–387. doi: 10.1111/j.1432-1033.1986.tb09982.x. [DOI] [PubMed] [Google Scholar]
  40. Shariff A., Luna E. J. Diacylglycerol-stimulated formation of actin nucleation sites at plasma membranes. Science. 1992 Apr 10;256(5054):245–247. doi: 10.1126/science.1373523. [DOI] [PubMed] [Google Scholar]
  41. Slot J. W., Geuze H. J. A new method of preparing gold probes for multiple-labeling cytochemistry. Eur J Cell Biol. 1985 Jul;38(1):87–93. [PubMed] [Google Scholar]
  42. Stossel T. P. From signal to pseudopod. How cells control cytoplasmic actin assembly. J Biol Chem. 1989 Nov 5;264(31):18261–18264. [PubMed] [Google Scholar]
  43. Tchakarov L., Vitale M. L., Jeyapragasan M., Rodriguez Del Castillo A., Trifaró J. M. Expression of scinderin, an actin filament-severing protein, in different tissues. FEBS Lett. 1990 Jul 30;268(1):209–212. doi: 10.1016/0014-5793(90)81010-l. [DOI] [PubMed] [Google Scholar]
  44. 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]
  45. Ware J. A., Johnson P. C., Smith M., Salzman E. W. Effect of common agonists on cytoplasmic ionized calcium concentration in platelets. Measurement with 2-methyl-6-methoxy 8-nitroquinoline (quin2) and aequorin. J Clin Invest. 1986 Mar;77(3):878–886. doi: 10.1172/JCI112385. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. White J. G. Arrangements of actin filaments in the cytoskeleton of human platelets. Am J Pathol. 1984 Nov;117(2):207–217. [PMC free article] [PubMed] [Google Scholar]
  47. Yin H. L. Gelsolin: calcium- and polyphosphoinositide-regulated actin-modulating protein. Bioessays. 1987 Oct;7(4):176–179. doi: 10.1002/bies.950070409. [DOI] [PubMed] [Google Scholar]

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

RESOURCES