Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Letter
  • Published:

CalDAG-GEFI integrates signaling for platelet aggregation and thrombus formation

Nature Medicine volume 10, pages 982–986 (2004)Cite this article

An Erratum to this article was published on 01 October 2004

Abstract

Signaling through the second messengers calcium and diacylglycerol (DAG) is a critical element in many biological systems. Integration of calcium and DAG signals has been suggested to occur primarily through protein kinase C family members, which bind both calcium and DAG. However, an alternative pathway may involve members of the CalDAG-GEF/RasGRP protein family, which have structural features (calcium-binding EF hands and DAG-binding C1 domains) that suggest they can function in calcium and DAG signal integration1,2. To gain insight into the signaling systems that may be regulated by CalDAG-GEF/RasGRP family members, we have focused on CalDAG-GEFI, which is expressed preferentially in the brain and blood1. Through genetic ablation in the mouse, we have found that CalDAG-GEFI is crucial for signal integration in platelets. Mouse platelets that lack CalDAG-GEFI are severely compromised in integrin-dependent aggregation as a consequence of their inability to signal through CalDAG-GEFI to its target, the small GTPase Rap1. These results suggest that analogous signaling defects are likely to occur in the central nervous system when CalDAG-GEFI is absent or compromised in function.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Loss of CalDAG-GEFI expression in platelets and megakaryocytes of CalDAG-GEFI knockout mice.
Figure 2: Impaired platelet function in CalDAG-GEFI knockouts.
Figure 3: Expression of CalDAG-GEFI in the hematopoietic system.
Figure 4: Impaired Rap1 activation in CalDAG-GEFI knockout mice.

Similar content being viewed by others

References

  1. Kawasaki, H. et al. A Rap guanine nucleotide exchange factor enriched highly in the basal ganglia. Proc. Natl. Acad. Sci. USA 95, 13278–13283 (1998).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Ebinu, J.O. et al. RasGRP, a Ras guanyl nucleotide-releasing protein with calcium- and diacylglycerol-binding motifs. Science 280, 1082–1086 (1998).

    Article  CAS  PubMed  Google Scholar 

  3. Bos, J.L. et al. The role of Rap1 in integrin-mediated cell adhesion. Biochem. Soc. Trans. 31, 83–86 (2003).

    Article  CAS  PubMed  Google Scholar 

  4. Bertoni, A. et al. Relationships between Rap1b, affinity modulation of integrin αIIbβ3, and the actin cytoskeleton. J. Biol. Chem. 277, 25715–25721 (2002).

    Article  CAS  PubMed  Google Scholar 

  5. Eto, K. et al. Megakaryocytes derived from embryonic stem cells implicate CalDAG-GEFI in integrin signaling. Proc. Natl. Acad. Sci. USA 99, 12819–12824 (2002).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. de Bruyn, K.M., Zwartkruis, F.J., de Rooij, J., Akkerman, J.W. & Bos, J.L. The small GTPase Rap1 is activated by turbulence and is involved in integrin αIIbβ3-mediated cell adhesion in human megakaryocytes. J. Biol. Chem. 278, 22412–22417 (2003).

    Article  CAS  PubMed  Google Scholar 

  7. Franke, B., Akkerman, J.W. & Bos, J.L. Rapid Ca2+-mediated activation of Rap1 in human platelets. EMBO J. 16, 252–259 (1997).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Franke, B. et al. Sequential regulation of the small GTPase Rap1 in human platelets. Mol. Cell. Biol. 20, 779–785 (2000).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Newman, P.J., Seligsohn, U., Lyman, S. & Coller, B.S. The molecular genetic basis of Glanzmann thrombasthenia in the Iraqi-Jewish and Arab populations in Israel. Proc. Natl. Acad. Sci. USA 88, 3160–3164 (1991).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Bergmeier, W. et al. Flow cytometric detection of activated mouse integrin αIIbβ3 with a novel monoclonal antibody. Cytometry 48, 80–86 (2002).

    Article  CAS  PubMed  Google Scholar 

  11. Klages, B., Brandt, U., Simon, M.I., Schultz, G. & Offermanns, S. Activation of G12/G13 results in shape change and Rho/Rho-kinase-mediated myosin light chain phosphorylation in mouse platelets. J. Cell Biol. 144, 745–754 (1999).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Nieswandt, B. et al. Glycoprotein VI but not α2β1 integrin is essential for platelet interaction with collagen. EMBO J. 20, 2120–2130 (2001).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Savage, B., Almus-Jacobs, F. & Ruggeri, Z.M. Specific synergy of multiple substrate-receptor interactions in platelet thrombus formation under flow. Cell 94, 657–666 (1998).

    Article  CAS  PubMed  Google Scholar 

  14. Davis, R.E., Stenberg, P.E., Levin, J. & Beckstead, J.H. Localization of megakaryocytes in normal mice and following administration of platelet antiserum, 5-fluorouracil, or radiostrontium: evidence for the site of platelet production. Exp. Hematol. 25, 638–648 (1997).

    CAS  PubMed  Google Scholar 

  15. Ebinu, J.O. et al. RasGRP links T-cell receptor signaling to Ras. Blood 95, 3199–3203 (2000).

    CAS  PubMed  Google Scholar 

  16. Teixeira, C., Stang, S.L., Zheng, Y., Beswick, N.S. & Stone, J.C. Integration of DAG signaling systems mediated by PKC-dependent phosphorylation of RasGRP3. Blood 102, 1414–1420 (2003).

    Article  CAS  PubMed  Google Scholar 

  17. Yamashita, S. et al. CalDAG-GEFIII activation of Ras, R-ras, and Rap1. J. Biol. Chem. 275, 25488–25493 (2000).

    Article  CAS  PubMed  Google Scholar 

  18. Yang, Y. et al. RasGRP4, a new mast cell-restricted Ras guanine nucleotide-releasing protein with calcium- and diacylglycerol-binding motifs. Identification of defective variants of this signaling protein in asthma, mastocytosis, and mast cell leukemia patients and demonstration of the importance of RasGRP4 in mast cell development and function. J. Biol. Chem. 277, 25756–25774 (2002).

    Article  CAS  PubMed  Google Scholar 

  19. Lova, P., Paganini, S., Sinigaglia, F., Balduini, C. & Torti, M. A Gi-dependent pathway is required for activation of the small GTPase Rap1B in human platelets. J. Biol. Chem. 277, 12009–12015 (2002).

    Article  CAS  PubMed  Google Scholar 

  20. Woulfe, D., Jiang, H., Mortensen, R., Yang, J. & Brass, L.F. Activation of Rap1B by Gi family members in platelets. J. Biol. Chem. 277, 23382–23390 (2002).

    Article  CAS  PubMed  Google Scholar 

  21. Larson, M.K. et al. Identification of P2Y12-dependent and -independent mechanisms of glycoprotein VI-mediated Rap1 activation in platelets. Blood 101, 1409–1415 (2003).

    Article  CAS  PubMed  Google Scholar 

  22. Herrmann, C., Horn, G., Spaargaren, M. & Wittinghofer, A. Differential interaction of the ras family GTP-binding proteins H-Ras, Rap1A, and R-Ras with the putative effector molecules Raf kinase and Ral-guanine nucleotide exchange factor. J. Biol. Chem. 271, 6794–6800 (1996).

    Article  CAS  PubMed  Google Scholar 

  23. Katagiri, K., Maeda, A., Shimonaka, M. & Kinashi, T. RAPL, a Rap1-binding molecule that mediates Rap1-induced adhesion through spatial regulation of LFA-1. Nat. Immunol. 4, 741–748 (2003).

    Article  CAS  PubMed  Google Scholar 

  24. Bergmeier, W. et al. Metalloproteinase inhibitors improve the recovery and hemostatic function of in vitro-aged or -injured mouse platelets. Blood 102, 4229–4235 (2003).

    Article  CAS  PubMed  Google Scholar 

  25. Guo, F., Kumahara, E. & Saffen, D. A CalDAG-GEFI/Rap1/B-Raf cassette couples M1 muscarinic acetylcholine receptors to the activation of ERK1/2. J. Biol. Chem. 276, 25568–25581 (2001).

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

We are grateful to B. Roe for providing a BAC clone of CalDAG-GEFI; T. Kinashi for providing antibodies to RapL; M. Yaffe for critical reading of the manuscript; R. Shivdasani and R. Hynes for advice; and P. Harlan for technical support. This work was funded by National Institute of Child Health and Development grant R01-HD28341 (A.M.G.); the James and Pat Poitras Research Fund (A.M.G.); the Grace B. Kerr Fund (A.M.G.); the Angus & Monaise MacDonald Fund (A.M.G.); National Institutes of Mental Health grant F32-MH065815 (J.R.C.); a grant from the Deutsche Forschungsgemeinschaft (W.B.); and the National Heart, Lung, and Blood Institute of the National Institutes of Health grants P01-HL56949 (D.D.W.) and HL66105 (D.E.H.).

Author information

Author notes

  1. Jill R Crittenden and Wolfgang Bergmeier: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Brain and Cognitive Sciences, and McGovern Institute for Brain Research, Massachusetts Institute of Technology, 45 Carleton Street, E25-618, Cambridge, 02139, Massachusetts, USA

    Jill R Crittenden, Yanyu Zhang & Ann M Graybiel

  2. The CBR Institute for Biomedical Research and the Department of Pathology, Harvard Medical School, Boston, 02115, Massachusetts, USA

    Wolfgang Bergmeier, Crystal L Piffath & Denisa D Wagner

  3. Department of Immunology, University of Washington, Box 357370, Seattle, 98195, Washington, USA

    Yuqiong Liang

  4. Center for Cancer Research, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, 02139, Massachusetts, USA

    Jill R Crittenden & David E Housman

Authors
  1. Jill R Crittenden
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Wolfgang Bergmeier
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Yanyu Zhang
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Crystal L Piffath
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Yuqiong Liang
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Denisa D Wagner
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. David E Housman
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Ann M Graybiel
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Ann M Graybiel.

Ethics declarations

Competing interests

The Massachusetts Institute of Technology has filed a patent application on behalf of J.R.C., A.M.G. and D.E.H., “The Use of Protein Inhibitors as Antithrombotic Agents,” case no. 10198 filed with the US Patent Office on June 2, 2003. The protein referred to in the patent application is that described in this manuscript, CalDAG-GEFI.

Supplementary information

Supplementary Fig. 1

Aggregation assays in response to calcium ionophore A23187. (PDF 643 kb)

Supplementary Table 1

Clotting factor assays in CalDAG-GEFT−/− mice. (PDF 17 kb)

Supplementary Table 2

Blood cell counts in CalDAG-GEFT−/− mice. (PDF 24 kb)

Supplementary Video 1

Platelets in whole blood from CalDAG-GEFI knockout mice do not form thrombi on a collagen surface. Real-time movies of platelets perfused over a collagen plate. a, wild-type calcein-labeled platelets, and b, CalDAG-GEFI−/− calcein-labeled platelets. (MOV 2658 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Crittenden, J., Bergmeier, W., Zhang, Y. et al. CalDAG-GEFI integrates signaling for platelet aggregation and thrombus formation. Nat Med 10, 982–986 (2004). https://doi.org/10.1038/nm1098

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nm1098

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing