Skip to main content

Advertisement

SpringerLink
Log in

Quantitative Detection of Thrombin Activity in an Ischemic Stroke Model

  • Published:
Journal of Molecular Neuroscience Aims and scope Submit manuscript

Abstract

Thrombin, a central factor in thrombogenesis, affects cells in the brain through protease activated receptors. Low levels of thrombin activity are neuroprotective while higher levels are deleterious, and we have therefore developed a new method for its direct quantitative measurement in brain slices following stroke. Thrombin activity was measured by a fluorescent substrate on fresh coronal slices taken from the ipsilateral and contralateral hemispheres 24–72 h following permanent right middle cerebral artery occlusion. Prolyl endopeptidase and aminopeptidases were inhibited as a critical step to insure the specificity of the assay for thrombin detection. Infarct volume was assessed using TTC staining. Thrombin activity in the right ischemic hemisphere was significantly higher compared to the contralateral hemisphere (32 ± 6 and 27 ± 10 mU/ml, mean ± SE in the two most affected slices from the ischemic hemisphere vs. 21 ± 6 and 8 ± 2 mU/ml in corresponding contralateral slices; p < 0.05). Thrombin levels in the ischemic and contralateral hemispheres were significantly higher compared to healthy control mice and were above the range known to be protective to brain cells. A significant correlation was found between thrombin activity in the ischemic hemisphere and the infarct volume. Results of studies based on this method may translate into potential thrombin based therapies.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4

Similar content being viewed by others

References

  • Bar-Shavit R, Hruska KA, Kahn AJ, Wilner GD (1986) Hormone-like activity of human thrombin. Ann N Y Acad Sci 485:335–348

    Article  PubMed  CAS  Google Scholar 

  • Beilin O, Gurwitz D, Korczyn AD, Chapman J (2001) Quantitative measurements of mouse brain thrombin-like and thrombin inhibition activities. Neuroreport 12:2347–2351

    Article  PubMed  CAS  Google Scholar 

  • Belayev L, Busto R, Zhao W, Fernandez G, Ginsberg MD (1999) Middle cerebral artery occlusion in the mouse by intraluminal suture coated with poly-L-lysine: neurological and histological validation. Brain Res 833:181–190

    Article  PubMed  CAS  Google Scholar 

  • Camilo O, Goldstein LB (2004) Seizures and epilepsy after ischemic stroke. Stroke 35:1769–1775

    Article  PubMed  Google Scholar 

  • Chen B, Cheng Q, Yang K, Lyden PD (2010) Thrombin mediates severe neurovascular injury during ischemia. Stroke 41:2348–2352

    Article  PubMed  CAS  Google Scholar 

  • Chen B, Friedman B, Whitney MA et al (2012) Thrombin activity associated with neuronal damage during acute focal ischemia. J Neurosci 32:7622–7631

    Article  PubMed  CAS  Google Scholar 

  • Coughlin SR (2000) Thrombin signalling and protease-activated receptors. Nature 407:258–264

    Article  PubMed  CAS  Google Scholar 

  • Desmond DW, Moroney JT, Sano M, Stern Y (2002) Incidence of dementia after ischemic stroke: results of a longitudinal study. Stroke 33:2254–2260

    Article  PubMed  Google Scholar 

  • Festoff BW, Smirnova IV, Ma J, Citron BA (1996) Thrombin, its receptor and protease nexin I, its potent serpin, in the nervous system. Semin Thromb Hemost 22:267–271

    Article  PubMed  CAS  Google Scholar 

  • Gingrich MB, Traynelis SF (2000) Serine proteases and brain damage—is there a link? Trends Neurosci 23:399–407

    Article  PubMed  CAS  Google Scholar 

  • Henrich-Noack P, Striggow F, Reiser G, Reymann KG (2006) Preconditioning with thrombin can be protective or worsen damage after endothelin-1-induced focal ischemia in rats. J Neurosci Res 83:469–475

    Article  PubMed  CAS  Google Scholar 

  • Hua Y, Wu J, Keep RF, Hoff JT, Xi G (2003) Thrombin exacerbates brain edema in focal cerebral ischemia. Acta Neurochir Suppl 86:163–166

    PubMed  CAS  Google Scholar 

  • Junge CE, Sugawara T, Mannaioni G et al (2003) The contribution of protease-activated receptor 1 to neuronal damage caused by transient focal cerebral ischemia. Proc Natl Acad Sci U S A 100:13019–13024

    Article  PubMed  CAS  Google Scholar 

  • Kameda K, Kikkawa Y, Hirano M, Matsuo S, Sasaki T, Hirano K (2012) Combined argatroban and anti-oxidative agents prevents increased vascular contractility to thrombin and other ligands after subarachnoid haemorrhage. Br J Pharmacol 165:106–119

    Article  PubMed  CAS  Google Scholar 

  • Kawabata S, Miura T, Morita T et al (1988) Highly sensitive peptide-4-methylcoumaryl-7-amide substrates for blood-clotting proteases and trypsin. Eur J Biochem 172:17–25

    Article  PubMed  CAS  Google Scholar 

  • Longa EZ, Weinstein PR, Carlson S, Cummins R (1989) Reversible middle cerebral artery occlusion without craniectomy in rats. Stroke 20:84–91

    Article  PubMed  CAS  Google Scholar 

  • Maggio N, Shavit E, Chapman J, Segal M (2008) Thrombin induces long-term potentiation of reactivity to afferent stimulation and facilitates epileptic seizures in rat hippocampal slices: toward understanding the functional consequences of cerebrovascular insults. J Neurosci 28:732–736

    Article  PubMed  CAS  Google Scholar 

  • Maggio N, Itsekson Z, Dominissini D et al (2013) Thrombin regulation of synaptic plasticity: Implications for physiology and pathology. Exp Neurol

  • Masada T, Xi G, Hua Y, Keep RF (2000) The effects of thrombin preconditioning on focal cerebral ischemia in rats. Brain Res 867:173–179

    Article  PubMed  CAS  Google Scholar 

  • Nicholson CCK, Hrabĕtová S, Tao L (2000) Diffusion of molecules in brain extracellular space: theory and experiment. Prog Brain Res 125:129–154

    Article  PubMed  CAS  Google Scholar 

  • Noorbakhsh F, Vergnolle N, Hollenberg MD, Power C (2003) Proteinase-activated receptors in the nervous system. Nat Rev Neurosci 4:981–990

    Article  PubMed  CAS  Google Scholar 

  • Papadopoulos Marios CDKB, Verkman AS (2005) Enhanced macromolecular diffusion in brain extracellular space in mouse models of vasogenic edema measured by cortical surface photobleaching. FASEB J 19:425–427

    PubMed  CAS  Google Scholar 

  • Shah Zahoor Ahmad NK, Klaus J, Kibler K, Doré S (2006) Use of an optimized transient occlusion of the middle cerebral artery protocol for the mouse stroke model. J Stroke Cerebrovasc Dis 15:133–138

    Article  PubMed  CAS  Google Scholar 

  • Shavit E, Michaelson DM, Chapman J (2011) Anatomical localization of protease-activated receptor-1 and protease-mediated neuroglial crosstalk on peri-synaptic astrocytic endfeet. J Neurochem 119:460–473

    Article  PubMed  CAS  Google Scholar 

  • Striggow F, Riek M, Breder J, Henrich-Noack P, Reymann KG, Reiser G (2000) The protease thrombin is an endogenous mediator of hippocampal neuroprotection against ischemia at low concentrations but causes degeneration at high concentrations. Proc Natl Acad Sci U S A 97:2264–2269

    Article  PubMed  CAS  Google Scholar 

  • Weinstein JR, Gold SJ, Cunningham DD, Gall CM (1995) Cellular localization of thrombin receptor mRNA in rat brain: expression by mesencephalic dopaminergic neurons and codistribution with prothrombin mRNA. J Neurosci 15:2906–2919

    PubMed  CAS  Google Scholar 

  • Xi G, Keep RF, Hua Y, Xiang J, Hoff JT (1999) Attenuation of thrombin-induced brain edema by cerebral thrombin preconditioning. Stroke 30:1247–1255

    Article  PubMed  CAS  Google Scholar 

  • Xi G, Reiser G, Keep RF (2003) The role of thrombin and thrombin receptors in ischemic, hemorrhagic and traumatic brain injury: deleterious or protective? J Neurochem 84:3–9

    Article  PubMed  CAS  Google Scholar 

  • Xue M, Del Bigio MR (2001) Acute tissue damage after injections of thrombin and plasmin into rat striatum. Stroke 32:2164–2169

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgments

We would like to thank Mr. Sami Sagol for his scholarship support.

Author information

Authors and Affiliations

  1. Comprehensive Stroke Center, the Department of Neurology and Joseph Sagol Neuroscience Center, Sheba Medical Center, Tel-Hashomer, Israel

    Doron Bushi, Joab Chapman, Aviva Katzav, Efrat Shavit-Stein, Noa Molshatzki, Nicola Maggio & David Tanne

  2. Department of Physiology and Pharmacology, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel

    Doron Bushi & Joab Chapman

  3. Department of Neurology, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel

    Joab Chapman & David Tanne

  4. Talpiot Medical Leadership Program, Sheba Medical Center, Tel-Hashomer, Israel

    Nicola Maggio

Authors
  1. Doron Bushi
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Joab Chapman
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Aviva Katzav
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Efrat Shavit-Stein
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Noa Molshatzki
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Nicola Maggio
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. David Tanne
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Doron Bushi.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Bushi, D., Chapman, J., Katzav, A. et al. Quantitative Detection of Thrombin Activity in an Ischemic Stroke Model. J Mol Neurosci 51, 844–850 (2013). https://doi.org/10.1007/s12031-013-0072-y

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s12031-013-0072-y

Keywords

Search

Navigation