Concentration-Dependent Dual Role of Thrombin in Protection of Cultured Rat Cortical Neurons.
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.
Research Division, Atlanta VA Medical Center, Decatur, GA, 30033, USA. pgarcia@emory.edu.|Neuroanesthesia Laboratory, Department of Anesthesiology, Emory University School of Medicine, Atlanta, GA, 30332, USA. pgarcia@emory.edu.
Paul S
PS
García
Research Division, Atlanta VA Medical Center, Decatur, GA, 30033, USA.|Department of Ophthalmology, Emory University School of Medicine, Atlanta, GA, USA.|Neuroanesthesia Laboratory, Department of Anesthesiology, Emory University School of Medicine, Atlanta, GA, 30332, USA.
Vincent T
VT
Ciavatta
Research Division, Atlanta VA Medical Center, Decatur, GA, 30033, USA.|Neuroanesthesia Laboratory, Department of Anesthesiology, Emory University School of Medicine, Atlanta, GA, 30332, USA.
Jonathan A
JA
Fidler
Research Division, Atlanta VA Medical Center, Decatur, GA, 30033, USA.|Neuroanesthesia Laboratory, Department of Anesthesiology, Emory University School of Medicine, Atlanta, GA, 30332, USA.
Anna
A
Woodbury
Cardiothoracic ICU, Department of Anesthesiology, Duke University, Durham, NC, USA.
Jerrold H
JH
Levy
Research Division, Atlanta VA Medical Center, Decatur, GA, 30033, USA.|Department of Neurology, Emory University School of Medicine, Atlanta, GA, USA.
William R
WR
Tyor
D000818:Animals; D000990:Antithrombin III / Q000494:pharmacology; D007611:Aprotinin / Q000494:pharmacology; D001120:Arginine / Q000031:analogs & derivatives; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D002540:Cerebral Cortex / Q000166:cytology* / Q000187:drug effects*; D016501:Neurites / Q000187:drug effects; D009474:Neurons / Q000187:drug effects*; D018696:Neuroprotective Agents / Q000494:pharmacology*; D010446:Peptide Fragments / Q000494:pharmacology; D010875:Pipecolic Acids / Q000494:pharmacology; D051381:Rats; D017207:Rats, Sprague-Dawley; D044463:Receptor, PAR-1 / Q000819:agonists; D018179:Receptors, Thrombin / Q000187:drug effects; D013449:Sulfonamides; D013917:Thrombin / Q000037:antagonists & inhibitors / Q000494:pharmacology*
D018696:Neuroprotective Agents; D010446:Peptide Fragments; D010875:Pipecolic Acids; D044463:Receptor, PAR-1; D018179:Receptors, Thrombin; D013449:Sulfonamides; C086309:thrombin receptor peptide SFLLRNP; D000990:Antithrombin III; D007611:Aprotinin; D001120:Arginine; D013917:Thrombin; C031942:argatroban
Cortical neuron culture; Neurotoxicity; Thrombin