Subjects

Abstract

Platelet activation at sites of vascular injury is essential for the arrest of bleeding; however, excessive platelet accumulation at regions of atherosclerotic plaque rupture can result in the development of arterial thrombi, precipitating diseases such as acute myocardial infarction and ischemic stroke. Rheological disturbances (high shear stress) have an important role in promoting arterial thrombosis by enhancing the adhesive and signaling function of platelet integrin αIIbβ3 (GPIIb-IIIa). In this study we have defined a key role for the Type Ia phosphoinositide 3-kinase (PI3K) p110β isoform in regulating the formation and stability of integrin αIIbβ3 adhesion bonds, necessary for shear activation of platelets. Isoform-selective PI3K p110β inhibitors have been developed which prevent formation of stable integrin αIIbβ3 adhesion contacts, leading to defective platelet thrombus formation. In vivo, these inhibitors eliminate occlusive thrombus formation but do not prolong bleeding time. These studies define PI3K p110β as an important new target for antithrombotic therapy.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

References

  1. 1.

    , & Platelet interaction with collagen fibrils in flowing blood. II. Impaired adhesion-aggregation in bleeding disorders. A comparison with subendothelium. Thromb. Haemost. 37, 17–28 (1977).

  2. 2.

    et al. Fibrinogen-independent platelet adhesion and thrombus formation on subendothelium mediated by glycoprotein IIb-IIIa complex at high shear rate. J. Clin. Invest. 83, 288–297 (1989).

  3. 3.

    et al. Increased platelet deposition on atherosclerotic coronary arteries. J. Clin. Invest. 93, 615–632 (1994).

  4. 4.

    , , & Localization of tissue factor in the normal vessel wall and in the atherosclerotic plaque. Proc. Natl. Acad. Sci. USA 86, 2839–2843 (1989).

  5. 5.

    et al. Tissue factor modulates the thrombogenicity of human atherosclerotic plaques. Circulation 95, 594–599 (1997).

  6. 6.

    , , & Distinct mechanisms of platelet aggregation as a consequence of different shearing flow conditions. J. Clin. Invest. 101, 479–486 (1998).

  7. 7.

    Mechanisms of shear-induced platelet adhesion and aggregation. Thromb. Haemost. 70, 119–123 (1993).

  8. 8.

    et al. Expression of tissue factor in high-grade carotid artery stenosis: association with plaque destabilization. Stroke 32, 850–854 (2001).

  9. 9.

    , , & Analysis of shear stress and hemodynamic factors in a model of coronary artery stenosis and thrombosis. Am. J. Physiol. 265, H1787–H1796 (1993).

  10. 10.

    Platelets in atherothrombosis. Nat. Med. 8, 1227–1234 (2002).

  11. 11.

    & Antiplatelet therapy: in search of the 'magic bullet'. Nat. Rev. Drug Discov. 2, 775–789 (2003).

  12. 12.

    et al. Essential role for phosphoinositide 3-kinase in shear-dependent signaling between platelet glycoprotein Ib/V/IX and integrin alpha(IIb)beta(3). Blood 99, 151–158 (2002).

  13. 13.

    et al. Purinergic P2Y12 receptor blockade inhibits shear-induced platelet phosphatidylinositol 3-kinase activation. Mol. Pharmacol. 63, 639–645 (2003).

  14. 14.

    , , & Phosphoinositide 3-kinases: a conserved family of signal transducers. Trends Biochem. Sci. 22, 267–272 (1997).

  15. 15.

    & Class I phosphoinositide 3-kinases. Int. J. Biochem. Cell Biol. 35, 1028–1033 (2003).

  16. 16.

    , & Phosphoinositide 3-kinases and the regulation of platelet function. Biochem. Soc. Trans. 32, 387–392 (2004).

  17. 17.

    & Signaling by distinct classes of phosphoinositide 3-kinases. Exp. Cell Res. 253, 239–254 (1999).

  18. 18.

    et al. P110delta, a novel phosphoinositide 3-kinase in leukocytes. Proc. Natl. Acad. Sci. USA 94, 4330–4335 (1997).

  19. 19.

    , & Human platelets contain p110delta phosphoinositide 3-kinase. Biochem. Biophys. Res. Commun. 296, 178–181 (2002).

  20. 20.

    et al. Functional phenotype of phosphoinositide 3-kinase p85alpha-null platelets characterized by an impaired response to GP VI stimulation. Blood 102, 541–548 (2003).

  21. 21.

    et al. Distinct glycoprotein Ib/V/IX and integrin alpha IIbbeta 3-dependent calcium signals cooperatively regulate platelet adhesion under flow. J. Biol. Chem. 277, 2965–2972 (2002).

  22. 22.

    et al. Adhesion receptor activation of phosphatidylinositol 3-kinase. von Willebrand factor stimulates the cytoskeletal association and activation of phosphatidylinositol 3-kinase and pp60c-src in human platelets. J. Biol. Chem. 269, 27093–27099 (1994).

  23. 23.

    et al. Signaling through GP Ib-IX-V activates alpha IIb beta 3 independently of other receptors. Blood 103, 3403–3411 (2004).

  24. 24.

    et al. The P2Y(12) receptor induces platelet aggregation through weak activation of the alpha(IIb)beta(3) integrin--a phosphoinositide 3-kinase-dependent mechanism. FEBS Lett. 505, 281–290 (2001).

  25. 25.

    et al. Integrin alpha IIb beta 3-dependent calcium signals regulate platelet-fibrinogen interactions under flow. Involvement of phospholipase C gamma 2. J. Biol. Chem. 278, 34812–34822 (2003).

  26. 26.

    et al. SHIP1 and Lyn Kinase Negatively Regulate Integrin alpha IIb beta 3 signaling in platelets. J. Biol. Chem. 279, 32196–32204 (2004).

  27. 27.

    , , & Importance of temporal flow gradients and integrin alpha IIb beta 3 mechanotransduction for rapid shear-activation of platelets. J. Biol. Chem. published online 8 February 2005 (10.1074/jbc.M410235200).

  28. 28.

    et al. Inhibitors of human phosphatidylinositol 3-kinase delta. US Patent No. 6,518,277 (2003).

  29. 29.

    et al. Activation of ADP receptors and platelet function. Thromb. Haemost. 78, 271–275 (1997).

  30. 30.

    et al. Relationships between Rap1b, affinity modulation of integrin alpha IIbbeta 3, and the actin cytoskeleton. J. Biol. Chem. 277, 25715–25721 (2002).

  31. 31.

    , & Intravenous nitroglycerin infusion inhibits cyclic blood flow responses caused by periodic platelet thrombus formation in stenosed canine coronary arteries. Circulation 83, 2122–2127 (1991).

  32. 32.

    & In vivo models of arterial thrombosis and thrombolysis. FASEB J. 4, 3087–3098 (1990).

  33. 33.

    , , , & Functional significance of adenosine 5 -diphosphate receptor (P2Y(12)) in platelet activation initiated by binding of von Willebrand factor to platelet GP Ibalpha induced by conditions of high shear rate. Circulation 105, 2531–2536 (2002).

  34. 34.

    et al. Distinct glycoprotein Ib/V/IX and integrin alpha IIbbeta 3-dependent calcium signals cooperatively regulate platelet adhesion under flow. J. Biol. Chem. 277, 2965–2972 (2002).

  35. 35.

    et al. The role of Rap1 in integrin-mediated cell adhesion. Biochemical Society Transactions 31, 83-6 (2003).

  36. 36.

    et al. Structural determinants of phosphoinositide 3-kinase inhibition by wortmannin, LY294002, quercetin, myricetin, and staurosporine. Mol. Cell 6, 909–919 (2000).

  37. 37.

    & Structural insight into substrate specificity and regulatory mechanisms of phosphoinositide 3-kinases. Trends Biochem. Sci. 27, 426–432 (2002).

  38. 38.

    et al. Impaired B and T cell antigen receptor signaling in p110delta PI 3-kinase mutant mice. Science 297, 1031–1034 (2002).

  39. 39.

    et al. Function of PI3Kgamma in thymocyte development, T cell activation, and neutrophil migration. Science 287, 1040–1046 (2000).

  40. 40.

    & PI3-kinase inhibition: a target for drug development? Mol. Med. Today 6, 347–357 (2000).

  41. 41.

    et al. Synergistic adhesive interactions and signaling mechanisms operating between platelet glycoprotein Ib/IX and integrin alpha IIbbeta 3. Studies in human platelets ans transfected Chinese hamster ovary cells. J. Biol. Chem. 275, 41377–41388 (2000).

  42. 42.

    et al. Preparation of morpholinyl- and pyridinyl-substituted heterobicyclic ketones as selective inhibitors of phosphoinositide 3-kinase beta for use against thrombosis. (International, WO 2003-IB4177, 2004).

  43. 43.

    et al. Tyrosine kinases regulate the cytoskeletal attachment of integrin alpha IIb beta 3 (platelet glycoprotein IIb/IIIa) and the cellular retraction of fibrin polymers. J. Biol. Chem. 269, 32479–32487 (1994).

  44. 44.

    et al. A selective role for phosphatidylinositol 3,4,5-trisphosphate in the Gi-dependent activation of platelet Rap1B. J. Biol. Chem. 278, 131–138 (2003).

  45. 45.

    , , , & Activation of Rap1B by G(i) family members in platelets. J. Biol. Chem. 277, 23382–23390 (2002).

  46. 46.

    et al. Direct effects of caffeine and theophylline on p110 delta and other phosphoinositide 3-kinases. Differential effects on lipid kinase and protein kinase activities. J. Biol. Chem. 277, 37124–37130 (2002).

  47. 47.

    , & Incomplete reversal of enoxaparin-induced bleeding by protamine sulfate. Haemostasis 21, 155–160 (1991).

  48. 48.

    , & Aprotinin reduces clopidogrel-induced prolongation of the bleeding time in the rat. Thromb. Res. 71, 433–441 (1993).

Download references

Acknowledgements

This work was supported by the National Health and Medical Research Council and the National Heart Foundation of Australia. Kinacia Pty Ltd also contributed financial support. We would like to thank L. Stephens, P. Hawkins and Z. Ruggeri for discussions, and D. Williamson, P. Mangin, K. Heel, D. Dunstan, I. Harper, and G. Currie, P. Freeman, M. Mulchandani, T. Domagala, M. Wang, N. Mistry, V. Strangis, S. Turnbull and T. Hinds for technical assistance and advice during the preparation of this manuscript.

Author information

Author notes

    • Simone M Schoenwaelder
    • , Isaac Goncalves
    • , Alan D Robertson
    •  & Hatem H Salem

    These authors contributed equally to this work. ADR and HHS are equal senior authors.

Affiliations

  1. Australian Centre for Blood Diseases, Monash University, 6th Floor Burnet Building Alfred Medical Research and Education Precinct (AMREP), 89 Commercial Road, Prahran, Victoria, Australia 3181.

    • Shaun P Jackson
    • , Simone M Schoenwaelder
    • , Isaac Goncalves
    • , Warwick S Nesbitt
    • , Cindy L Yap
    • , Vijaya Kenche
    • , Karen E Anderson
    • , Sacha M Dopheide
    • , Yuping Yuan
    • , Hishani Prabaharan
    • , Philip E Thompson
    • , Gregg D Smith
    • , Suhasini Kulkarni
    • , Belinda Abbott
    • , Dilek Saylik
    • , Simon Giuliano
    • , Sascha C Hughan
    •  & Hatem H Salem
  2. Department of Pharmacology, Level 8, Medical Building, Corner of Grattan Street and Royal Parade, University of Melbourne, Victoria, Australia 3010.

    • Christine E Wright
    • , Sharelle A Sturgeon
    • , Catherine Jones
    • , Lucy Lu
    •  & James A Angus
  3. Department of Biochemistry and Molecular Biology, University College of London, Gower Street, London, WC1E6BT, UK.

    • Peter R Shepherd
    •  & Nathalie Daniele
  4. Kinacia Pty Ltd, Cerylid Biosciences Ltd, 576 Swan Street, Richmond, Victoria, Australia 3121.

    • Vijaya Kenche
    • , Sharelle A Sturgeon
    • , Hishani Prabaharan
    • , Philip E Thompson
    • , Gregg D Smith
    • , Dilek Saylik
    • , Catherine Jones
    • , Lucy Lu
    •  & Alan D Robertson

Authors

  1. Search for Shaun P Jackson in:

  2. Search for Simone M Schoenwaelder in:

  3. Search for Isaac Goncalves in:

  4. Search for Warwick S Nesbitt in:

  5. Search for Cindy L Yap in:

  6. Search for Christine E Wright in:

  7. Search for Vijaya Kenche in:

  8. Search for Karen E Anderson in:

  9. Search for Sacha M Dopheide in:

  10. Search for Yuping Yuan in:

  11. Search for Sharelle A Sturgeon in:

  12. Search for Hishani Prabaharan in:

  13. Search for Philip E Thompson in:

  14. Search for Gregg D Smith in:

  15. Search for Peter R Shepherd in:

  16. Search for Nathalie Daniele in:

  17. Search for Suhasini Kulkarni in:

  18. Search for Belinda Abbott in:

  19. Search for Dilek Saylik in:

  20. Search for Catherine Jones in:

  21. Search for Lucy Lu in:

  22. Search for Simon Giuliano in:

  23. Search for Sascha C Hughan in:

  24. Search for James A Angus in:

  25. Search for Alan D Robertson in:

  26. Search for Hatem H Salem in:

Competing interests

Vijaya Kenche, Hishani Prabaharan, Philip E. Thompson, Gregg D. Smith, Dilek Saylik, Sharelle A. Sturgeon, Catherine Jones and Lucy Lu were full time employees of Kinacia during the performance of these studies. A number of the authors have competing financial interests, primarily as shareholders in the biotechnology company Kinacia Pty Ltd (a wholly owned subsidiary of Cerylid Biosciences Ltd), which developed the inhibitors against PI 3-kinase p110beta. None of the authors currently own more than 1% stock in Cerylid Biosciences. Gregg D. Smith, Hishani Prabaharan and Dilek Saylik are currently full-time employees of Cerylid and Alan D. Robertson, James A. Angus and Christine E. Wright have consultancy arrangements with the company. This work was partially funded by Kinacia.

Corresponding author

Correspondence to Shaun P Jackson.

Supplementary information

PDF files

  1. 1.

    Supplementary Fig. 1

    Synthesis of TGX-221, a novel inhibitor of PI 3-kinase.

  2. 2.

    Supplementary Fig. 2

    Role of PI3K p110δ and γ in shear-dependent platelet calcium flux in platelets.

  3. 3.

    Supplementary Fig. 3

    Role of PI3K p110β in promoting platelet activation in response to physiological agonists

  4. 4.

    Supplementary Fig. 4

    Effect of TGX-221 on P2Y1-dependent calcium flux and platelet shape change in ADP-stimulated platelets.

  5. 5.

    Supplementary Fig. 5

    Antithrombotic effect of TGX-221.

  6. 6.

    Supplementary Fig. 6

    Dration of the antithrombotic effect of TGX-221 following a single i.v. bolus.

  7. 7.

    Supplementary Table 1

    Relative inhibitory potency of TGX-221 against a broad panel of lipid and protein kinases.

  8. 8.

    Supplementary Methods

About this article

Publication history

Received

Accepted

Published

DOI

https://doi.org/10.1038/nm1232

Further reading