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Neurochemical Research

, Volume 40, Issue 11, pp 2220–2229 | Cite as

Concentration-Dependent Dual Role of Thrombin in Protection of Cultured Rat Cortical Neurons

  • Paul S. GarcíaEmail author
  • Vincent T. Ciavatta
  • Jonathan A. Fidler
  • Anna Woodbury
  • Jerrold H. Levy
  • William R. Tyor
Original Paper

Abstract

Thrombin’s role in the nervous system is not well understood. Under conditions of blood–brain barrier compromise (e.g., neurosurgery or stroke), thrombin can result in neuroapoptosis and the formation of glial scars. Despite this, preconditioning with thrombin has been found to be neuroprotective in models of cerebral ischemia and intracerebral hemorrhage. We investigated the effects of physiologically relevant concentrations of thrombin on cortical neurons using two culture-based assays. We examined thrombin’s effect on neurites by quantitative analysis of fluorescently labeled neurons. To characterize thrombin’s effects on neuron survival, we spectrophotometrically measured changes in enzymatic activity. Using receptor agonists and thrombin inhibitors, we separately examined the role of thrombin and its receptor in neuroprotection. We found that low concentrations of thrombin (1 nM) enhances neurite growth and branching, neuron viability, and protects against excitotoxic damage. In contrast, higher concentrations of thrombin (100 nM) are potentially detrimental to neuronal health as evidenced by inhibition of neurite growth. Lower concentrations of thrombin resulted in equivalent neuroprotection as the antifibrinolytic, aprotinin, and the direct thrombin inhibitor, argatroban. Interestingly, exogenous application of the species-specific thrombin inhibitor, antithrombin III, was detrimental to neuronal health; suggesting that some endogenous thrombin is necessary for optimal neuron health in our culture system. Activation of the thrombin receptor, protease-activated receptor-1 (PAR-1), via micromolar concentrations of the thrombin receptor agonist peptide, TRAP, did not adversely affect neuronal viability. An optimal concentration of thrombin exists to enhance neuronal health. Neurotoxic effects of thrombin do not involve activation of PAR receptors and thus separate pharmacologic manipulation of thrombin’s receptor in the setting of direct thrombin inhibitors could be a potential neuroprotective strategy.

Keywords

Thrombin Cortical neuron culture Neurotoxicity 

Notes

Acknowledgments

This work was supported by a Grant (GRC 001) from the Atlanta Research and Education Foundation (AREF) and from departmental funds. We gratefully acknowledge funding for the project from a Career Development Award awarded by the Department of Veterans Affairs (PSG, CDA BX001677). We gratefully acknowledge training in confocal microscopy by Shaojin You, MD, PhD; Director of Histology Core, Research Division, Atlanta VA Medical Center, Atlanta, Georgia. We also gratefully acknowledge technical help from Mayuri Maddi, Britany Raymond, and Cari Fritz-French Lessing.

Compliance with Ethical Standards

Conflict of interest

The authors declare that they have no conflict of interest.

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Copyright information

© Springer Science+Business Media New York (outside the USA) 2015

Authors and Affiliations

  • Paul S. García
    • 1
    • 5
    Email author
  • Vincent T. Ciavatta
    • 1
    • 2
    • 5
  • Jonathan A. Fidler
    • 1
    • 5
  • Anna Woodbury
    • 1
    • 5
  • Jerrold H. Levy
    • 3
  • William R. Tyor
    • 1
    • 4
  1. 1.Research DivisionAtlanta VA Medical CenterDecaturUSA
  2. 2.Department of OphthalmologyEmory University School of MedicineAtlantaUSA
  3. 3.Cardiothoracic ICU, Department of AnesthesiologyDuke UniversityDurhamUSA
  4. 4.Department of NeurologyEmory University School of MedicineAtlantaUSA
  5. 5.Neuroanesthesia Laboratory, Department of AnesthesiologyEmory University School of MedicineAtlantaUSA

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