Cell lines

N27 cells derived from E12 rat mesencephalic tissue

Preparation Method

AA and lipid hydroperoxides content was assayed from the N27 cells 24 h after thrombin (0.5 U/mL) treatment.

Reaction Conditions

24 h ,thrombin (0.5 U/ml)

Applications

Thrombin dose-dependently induced N27 neuronal cell death, which accompanied elevated AA and lipid hydroperoxides, and smaller mitochondria with increased membrane density. Thrombin did not affect the intracellular level of ferrous iron

Animal models

Male Sprague-Dawley rats, each weighing between 250 and 350 g

Preparation Method

Solutions containing 10 U of thrombin in 10 μl saline were infused into the brain over a period of 1 minute using a Harvard pump.

Dosage form

10 U of thrombin in 10 μl saline for 1 minute

Applications

AIB(a marker of BBB opening) has increased BBB permeability. Thrombin causes disruption of the BBB in the ipsilateral hemisphere

Thrombin (MW 37kDa) is a Na+-activated, allosteric serine protease. Thrombin induces the activation of ERK1 and ERK2^[1]. Thrombin recognition sequence and can be used to digest GST-tagged proteins^[2].Thrombin could activate the immune system by directly cleaving pro-interleukin-1α to the active form (IL-1α)^[3]. Thrombin may be “a gas pedal” driving the innate immune system^[4].

Thrombin induces neuronal ferroptosis, in N27 cells, Thrombin dose-dependently induced N27 neuronal cell death, which accompanied elevated AA and lipid hydroperoxides, and smaller mitochondria with increased membrane density. Thrombin did not affect the intracellular level of ferrous iron^[5]. Thrombin elicits rapid and full activation of cPLA2 not only by promoting a rise in cytosolic free Ca²⁺but also by inducing phosphorylation of cPLA2 thereby improving its catalytic activity^[6].Low concentrations of thrombin and thrombin preconditioning yield the potential of rescuing cells and to induce survival of neurons and astrocytes exposed to various ischemic insults[7].Thrombin also has roles in brain cell death and survival as well as neuroinflammation, predominantly via the cellular protease-activated receptor (PAR) activation and downstream signaling pathways^[5]

In vivo，thrombin induces BBB disruption as well as death of parenchymal cells, whereas CBF and vasoreactivity are not altered. Cell toxicity and BBB disruption by thrombin are triggering mechanisms for the edema formation that follows intracerebral hemorrhage[9].In the primary hemostatic processis. Thrombin activates platelets, and also in the secondary hemostasis, it mediates the conversion of fibrinogen to fibrin. Thus, thrombin contributes to thrombus formation that stops bleeding and hematoma formation after blood enters the brain parenchyma [10]. Intracerebral administration of exogenous thrombin (at a dose that is non-toxic to normal brain), markedly exacerbated brain edema after transient focal cerebral ischemia. Extravascular thrombin inhibition may be a new therapeutic target for cerebral ischemia^[6]

References:
[1]. Kramer RM, Roberts EF, et,al. Differential activation of cytosolic phospholipase A2 (cPLA2) by thrombin and thrombin receptor agonist peptide in human platelets. Evidence for activation of cPLA2 independent of the mitogen-activated protein kinases ERK1/2. J Biol Chem. 1995 Jun 16;270(24):14816-23. doi: 10.1074/jbc.270.24.14816. PMID: 7782348.
[2]. Golderman V, Shavit-Stein E, et,al. Thrombin and the Protease-Activated Receptor-1 in Organophosphate-Induced Status Epilepticus. J Mol Neurosci. 2019 Feb;67(2):227-234. doi: 10.1007/s12031-018-1228-6. Epub 2018 Dec 4. PMID: 30515700.
[3]. Burzynski LC, Humphry M, et,al. The Coagulation and Immune Systems Are Directly Linked through the Activation of Interleukin-1α by Thrombin. Immunity. 2019 Apr 16;50(4):1033-1042.e6. doi: 10.1016/j.immuni.2019.03.003. Epub 2019 Mar 26. PMID: 30926232; PMCID: PMC6476404.
[4]. Petzold T, Massberg S. Thrombin: A Gas Pedal Driving Innate Immunity. Immunity. 2019 Apr 16;50(4):1024-1026. doi: 10.1016/j.immuni.2019.03.006. PMID: 30995493.
[5]. Tuo QZ, Liu Y, et,al. Thrombin induces ACSL4-dependent ferroptosis during cerebral ischemia/reperfusion. Signal Transduct Target Ther. 2022 Feb 23;7(1):59. doi: 10.1038/s41392-022-00917-z. PMID: 35197442; PMCID: PMC8866433.
[6]. Kramer RM, Roberts EF, et,al. Thrombin-induced phosphorylation and activation of Ca(2+)-sensitive cytosolic phospholipase A2 in human platelets. J Biol Chem. 1993 Dec 15;268(35):26796-804. PMID: 8253817.
[7]. Donovan FM, Pike CJ, et,al. Thrombin induces apoptosis in cultured neurons and astrocytes via a pathway requiring tyrosine kinase and RhoA activities. J Neurosci. 1997 Jul 15;17(14):5316-26. doi: 10.1523/JNEUROSCI.17-14-05316.1997. PMID: 9204916; PMCID: PMC6793831.
[8]. Delvaeye M, Conway EM. Coagulation and innate immune responses: can we view them separately? Blood. 2009 Sep 17;114(12):2367-74. doi: 10.1182/blood-2009-05-199208. Epub 2009 Jul 7. PMID: 19584396.
[9]. Lee KR, Kawai N, et,al. Mechanisms of edema formation after intracerebral hemorrhage: effects of thrombin on cerebral blood flow, blood-brain barrier permeability, and cell survival in a rat model. J Neurosurg. 1997 Feb;86(2):272-8. doi: 10.3171/jns.1997.86.2.0272. PMID: 9010429.
[10]. Goldsack NR, Chambers RC, et,al. Thrombin. Int J Biochem Cell Biol. 1998 Jun;30(6):641-6. doi: 10.1016/s1357-2725(98)00011-9. PMID: 9695019.
[11]. Hua Y, Wu J, et,al. Thrombin exacerbates brain edema in focal cerebral ischemia. Acta Neurochir Suppl. 2003;86:163-6. doi: 10.1007/978-3-7091-0651-8_34. PMID: 14753426.