The objective of this study was to determine whether plasmin could induce morphological changes in human glial cells via PAR1. Human glioblastoma A172 cells were cultured in the presence of plasmin or the PAR1 specific activating hexapeptide, SFLLRN. Cells were monitored by flow cytometry to detect proteolytic activation of PAR1 receptor. Morphological changes were recorded by photomicroscopy and apoptosis was measured by annexinV staining. Plasmin cleaved the PAR1 receptor on glial cells at 5 minutes ( ). After 30 minutes, cellular processes had begun to retract from the basal substratum and by 4 hours glial cells had become detached. Similar results were obtained by generating plasmin de novo from plasminogen. Morphological transformation was blocked by plasmin inhibitors aprotinin or epsilon-aminocaproic acid ( ). Cell viability was unimpaired during early morphological changes, but by 24 hours following plasmin treatment 22% of glial cells were apoptotic. PAR1 activating peptide SFLLRN (but not inactive isomer FSLLRN) promoted analogous glial cell detachment ( ), proving the role for PAR1 in this process. This study has identified a plasmin/PAR1 axis of glial cell activation, linked to changes in glial cell morophology. This adds to our understanding of pathophysiological disease mechanisms of plasmin and the plasminogen system in neuroinjury. 1. Introduction Plasmin is a serine protease best known for its thrombolytic properties in the coagulation system. However, it can also act on cells that bear receptors belonging to the protease-activated receptor (PAR) family to cause secretion of inflammatory cytokines, oxidative radicals, matrix metalloproteinases, proliferation, cell migration, and platelet aggregation [1–6]. PARs are widely expressed in the central nervous system [7]. Plasmin is generated from plasminogen, by proteolytic cleavage with either tissue-type plasminogen activator (tPA), urinary plasminogen activator (uPA), or bacterial streptokinase. It catalyzes the breakdown of fibrin into D-dimers, hence acting as a brake on coagulation. Antifibrinolytics are in clinical use to limit bleeding in cardiac surgery and intracranial bleeding in traumatic brain injury [8, 9]. Antifibrinolytics fall into two categories: lysine analogues that prevent plasmin generation from plasminogen, (e.g., ε-aminocaproic acid), or active site inhibitors (e.g., serine protease inhibitor aprotinin [10]). A pathophysiological role has been recognized for the plasminogen-activating system in exacerbating intracranial bleeding, excitotoxicity and cell death in
References
[1]
I. Clemmensen and R. B. Andersen, “The fibrinolytic system and its relation to inflammatory diseases,” Seminars in Arthritis and Rheumatism, vol. 11, no. 4, pp. 390–398, 1982.
[2]
N. Kamio, H. Hashizume, S. Nakao, K. Matsushima, and H. Sugiya, “Plasmin is involved in inflammation via protease-activated receptor-1 activation in human dental pulp,” Biochemical Pharmacology, vol. 75, no. 10, pp. 1974–1980, 2008.
[3]
T. M. Quinton, S. Kim, C. K. Derian, J. Jin, and S. P. Kunapuli, “Plasmin-mediated activation of platelets occurs by cleavage of protease-activated receptor 4,” The Journal of Biological Chemistry, vol. 279, no. 18, pp. 18434–18439, 2004.
[4]
V. Lukic-Panin, K. Deguchi, T. Yamashita et al., “Free radical scavenger edaravone administration protects against tissue plasminogen activator induced oxidative stress and blood brain barrier damage,” Current Neurovascular Research, vol. 7, no. 4, pp. 319–329, 2010.
[5]
S. L. Raza, L. C. Nehring, S. D. Shapiro, and L. A. Cornelius, “Proteinase-activated receptor-1 regulation of macrophage elastase (MMP-12) secretion by serine proteinases,” The Journal of Biological Chemistry, vol. 275, no. 52, pp. 41243–41250, 2000.
[6]
S. C. Even-Ram, M. Maoz, E. Pokroy et al., “Tumor cell invasion is promoted by activation of protease activated receptor-1 in cooperation with the alpha vbeta 5 integrin,” The Journal of Biological Chemistry, vol. 276, no. 14, pp. 10952–10962, 2001.
[7]
E. Sokolova and G. Reiser, “Prothrombin/thrombin and the thrombin receptors PAR-1 and PAR-4 in the brain: localization, expression and participation in neurodegenerative diseases,” Thrombosis and Haemostasis, vol. 100, no. 4, pp. 576–581, 2008.
[8]
V. J. Marder, “Historical perspective and future direction of thrombolysis research: the re-discovery of plasmin,” Journal of Thrombosis and Haemostasis, vol. 9, no. 1, pp. 364–373, 2011.
[9]
F. Olldashi, M. Ker？i, T. Zhurda et al., “The importance of early treatment with tranexamic acid in bleeding trauma patients: an exploratory analysis of the CRASH-2 randomised controlled trial,” The Lancet, vol. 377, no. 9771, pp. 1096–1101, 1101.e1-1101.e2, 2011.
[10]
D. A. Henry, A. J. Moxey, P. A. Carless et al., “Anti-fibrinolytic use for minimising perioperative allogeneic blood transfusion,” Cochrane Database of Systematic Reviews, no. 1, Article ID CD001886, 2001.
[11]
S. Fujimoto, H. Katsuki, M. Ohnishi, M. Takagi, T. Kume, and A. Akaike, “Plasminogen potentiates thrombin cytotoxicity and contributes to pathology of intracerebral hemorrhage in rats,” Journal of Cerebral Blood Flow and Metabolism, vol. 28, no. 3, pp. 506–515, 2008.
[12]
J. J. Sheehan, C. Zhou, I. Gravanis et al., “Proteolytic activation of monocyte chemoattractant protein-1 by plasmin underlies excitotoxic neurodegeneration in mice,” Journal of Neuroscience, vol. 27, no. 7, pp. 1738–1745, 2007.
[13]
S. E. Tsirka, T. H. Bugge, J. L. Degen, and S. Strickland, “Neuronal death in the central nervous system demonstrates a non-fibrin substrate for plasmin,” Proceedings of the National Academy of Sciences of the United States of America, vol. 94, no. 18, pp. 9779–9781, 1997.
[14]
S. E. Tsirka, A. D. Rogove, T. H. Bugge, J. L. Degen, and S. Strickland, “An extracellular proteolytic cascade promotes neuronal degeneration in the mouse hippocampus,” Journal of Neuroscience, vol. 17, no. 2, pp. 543–552, 1997.
[15]
B. Niego, R. Freeman, T. B. Puschmann, A. M. Turnley, and R. L. Medcalf, “t-PA-specific modulation of a human blood-brain barrier model involves plasmin-mediated activation of the Rho kinase pathway in astrocytes,” Blood, vol. 119, no. 20, pp. 4752–4761, 2012.
[16]
Y. F. Wang, S. E. Tsirka, S. Strickland, P. E. Stieg, S. G. Soriano, and S. A. Lipton, “Tissue plasminogen activator (tPA) increases neuronal damage after focal cerebral ischemia in wild-type and tPA-deficient mice,” Nature Medicine, vol. 4, no. 2, pp. 228–231, 1998.
[17]
F. Adhami, D. Yu, W. Yin et al., “Deleterious effects of plasminogen activators in neonatal cerebral hypoxia-ischemia,” American Journal of Pathology, vol. 172, no. 6, pp. 1704–1716, 2008.
[18]
B. Reinsfelt, S.-E. Ricksten, H. Zetterberg, K. Blennow, J. Fredén-Lindqvist, and A. Westerlind, “Cerebrospinal fluid markers of brain injury, inflammation, and blood-brain barrier dysfunction in cardiac surgery,” Annals of Thoracic Surgery, vol. 94, no. 2, pp. 549–555, 2012.
[19]
T. K. H. Vu, V. I. Wheaton, D. T. Hung, I. Charo, and S. R. Coughlin, “Domains specifying thrombin-receptor interaction,” Nature, vol. 353, no. 6345, pp. 674–677, 1991.
[20]
T. K. H. Vu, D. T. Hung, V. I. Wheaton, and S. R. Coughlin, “Molecular cloning of a functional thrombin receptor reveals a novel proteolytic mechanism of receptor activation,” Cell, vol. 64, no. 6, pp. 1057–1068, 1991.
[21]
M. Poullis, R. Manning, M. Laffan, D. O. Haskard, K. M. Taylor, and R. C. Landis, “The antithrombotic effect of aprotinin: actions mediated via the protease-activated receptor 1,” Journal of Thoracic and Cardiovascular Surgery, vol. 120, no. 2, pp. 370–378, 2000.
[22]
C. E. Junge, T. Sugawara, G. Mannaioni et al., “The contribution of protease-activated receptor 1 to neuronal damage caused by transient focal cerebral ischemia,” Proceedings of the National Academy of Sciences of the United States of America, vol. 100, no. 22, pp. 13019–13024, 2003.
[23]
E. E. Olson, P. Lyuboslavsky, S. F. Traynelis, and R. J. McKeon, “PAR-1 deficiency protects against neuronal damage and neurologic deficits after unilateral cerebral hypoxia/ischemia,” Journal of Cerebral Blood Flow and Metabolism, vol. 24, no. 9, pp. 964–971, 2004.
[24]
Y. Wang, W. Luo, and G. Reiser, “Activation of protease-activated receptors in astrocytes evokes a novel neuroprotective pathway through release of chemokines of the growth-regulated oncogene/cytokine-induced neutrophil chemoattractant family,” European Journal of Neuroscience, vol. 26, no. 11, pp. 3159–3168, 2007.
[25]
F. M. Donovan and D. D. Cunningham, “Signaling pathways involved in thrombin-induced cell protection,” The Journal of Biological Chemistry, vol. 273, no. 21, pp. 12746–12752, 1998.
[26]
M. B. Gingrich, C. E. Junge, P. Lyuboslavsky, and S. F. Traynelis, “Potentiation of NMDA receptor function by the serine protease thrombin,” Journal of Neuroscience, vol. 20, no. 12, pp. 4582–4595, 2000.
[27]
G. Mannaioni, A. G. Orr, C. E. Hamill et al., “Plasmin potentiates synaptic N-methyl-D-aspartate receptor function in hippocampal neurons through activation of protease-activated receptor-1,” The Journal of Biological Chemistry, vol. 283, no. 29, pp. 20600–20611, 2008.
[28]
L. J. Houenou, “Thrombin perturbs neurite outgrowth and induces apoptotic cell death in enriched chick spinal motoneuron cultures through caspase activation,” Journal of Neuroscience, vol. 18, no. 17, pp. 6882–6891, 1998.
[29]
J. R. S. Day, K. M. Taylor, E. A. Lidington et al., “Aprotinin inhibits proinflammatory activation of endothelial cells by thrombin through the protease-activated receptor 1,” Journal of Thoracic and Cardiovascular Surgery, vol. 131, no. 1, pp. 21–27, 2006.
[30]
L. F. Brass, S. Pizarro, M. Ahuja et al., “Changes in the structure and function of the human thrombin receptor during receptor activation, internalization, and recycling,” The Journal of Biological Chemistry, vol. 269, no. 4, pp. 2943–2952, 1994.
[31]
A. Maglott, P. Bartik, S. Cosgun et al., “The small α5β1 integrin antagonist, SJ749, reduces proliferation and clonogenicity of human astrocytoma cells,” Cancer Research, vol. 66, no. 12, pp. 6002–6007, 2006.
[32]
Y. Fukushima, M. Tamura, H. Nakagawa, and K. Itoh, “Induction of glioma cell migration by vitronectin in human serum and cerebrospinal fluid,” Journal of Neurosurgery, vol. 107, no. 3, pp. 578–585, 2007.
[33]
Z. Suo, B. A. Citron, and B. W. Festoff, “Thrombin: a potential proinflammatory mediator in neurotrauma and neurodegenerative disorders,” Current Drug Targets, vol. 3, no. 1, pp. 105–114, 2004.
[34]
R. C. Landis and K. D. Hall, “Plasmin but not thrombin activates A172 glial cells via serine proteolytic cleavage of preotease-activated receptor (PAR)1,” Heart Surgery Forum, vol. 11, no. 5, article 278, 2008.
[35]
C. E. Hamill, G. Mannaioni, P. Lyuboslavsky, A. A. Sastre, and S. F. Traynelis, “Protease-activated receptor 1-dependent neuronal damage involves NMDA receptor function,” Experimental Neurology, vol. 217, no. 1, pp. 136–146, 2009.
[36]
H. S. Suidan, C. D. Nobes, A. Hall, and D. Monard, “Astrocyte spreading in response to thrombin and lysophosphatidic acid is dependent on the Rho GTPase,” GLIA, vol. 21, no. 2, pp. 244–252, 1997.
[37]
O. Nicole, A. Goldshmidt, C. E. Hamill et al., “Activation of protease-activated receptor-1 triggers astrogliosis after brain injury,” Journal of Neuroscience, vol. 25, no. 17, pp. 4319–4329, 2005.
[38]
K. P. Cavanaugh, D. Gurwitz, D. D. Cunningham, and R. A. Bradshaw, “Reciprocal modulation of astrocyte stellation by thrombin and protease nexin-1,” Journal of Neurochemistry, vol. 54, no. 5, pp. 1735–1743, 1990.
[39]
D. D. Cunningham and D. Gurwitz, “Proteolytic regulation of neurite outgrowth from neuroblastoma cells by thrombin and protease nexin-1,” Journal of Cellular Biochemistry, vol. 39, no. 1, pp. 55–64, 1989.
[40]
S. F. Traynelis and J. Trejo, “Protease-activated receptor signaling: new roles and regulatory mechanisms,” Current Opinion in Hematology, vol. 14, no. 3, pp. 230–235, 2007.
[41]
T. Okamoto, M. Nishibori, K. Sawada et al., “The effects of stimulating protease-activated receptor-1 and -2 in A172 human glioblastoma,” Journal of Neural Transmission, vol. 108, no. 2, pp. 125–140, 2001.
[42]
M. B. Gingrich and S. F. Traynelis, “Serine proteases and brain damage—is there a link?” Trends in Neurosciences, vol. 23, no. 9, pp. 399–407, 2000.
[43]
C. Landis, “Why the inflammatory response is important to the cardiac surgical patient,” Journal of Extra-Corporeal Technology, vol. 39, no. 4, pp. 281–284, 2007.
[44]
D. A. Stump, “Deformable emboli and inflammation: temporary or permanent damage?” Journal of Extra-Corporeal Technology, vol. 39, no. 4, pp. 289–290, 2007.
[45]
A. M. Schuller, J. Windolf, R. Blaheta et al., “Degradation of microvascular brain endothelial cell β-catenin after co-culture with activated neutrophils from patients undergoing cardiac surgery with prolonged cardiopulmonary bypass,” Biochemical and Biophysical Research Communications, vol. 329, no. 2, pp. 616–623, 2005.
[46]
M. Scholz, J. Cinatl, M. Sch？del-H？pfner, and J. Windolf, “Neutrophils and the blood-brain barrier dysfunction after trauma,” Medicinal Research Reviews, vol. 27, no. 3, pp. 401–416, 2007.
[47]
P. Tricoci, Z. Huang, C. Held et al., “Thrombin-receptor antagonist vorapaxar in acute coronary syndromes,” The New England Journal of Medicine, vol. 366, no. 1, pp. 20–33, 2012.
[48]
C. E. Junge, C. J. Lee, K. B. Hubbard et al., “Protease-activated receptor-1 in human brain: localization and functional expression in astrocytes,” Experimental Neurology, vol. 188, no. 1, pp. 94–103, 2004.
[49]
O. Eser, E. Kalkan, M. Cosar et al., “The effect of aprotinin on brain ischemic-reperfusion injury after hemorrhagic shock in rats: an experimental study,” Journal of Trauma, vol. 63, no. 2, pp. 373–378, 2007.
[50]
K. Durgut, K. Hosgor, N. Gormus, U. Ozergin, and H. Solak, “The cerebroprotective effects of pentoxifylline and aprotinin during cardiopulmonary bypass in dogs,” Perfusion, vol. 19, no. 2, pp. 101–106, 2004.
[51]
V. Anttila, I. Hagino, Y. Iwata et al., “Aprotinin improves cerebral protection: evidence from a survival porcine model,” Journal of Thoracic and Cardiovascular Surgery, vol. 132, no. 4, pp. 948–953, 2006.
[52]
V. L. Turgeon and L. J. Houenou, “The role of thrombin-like (serine) proteases in the development, plasticity and pathology of the nervous system,” Brain Research Reviews, vol. 25, no. 1, pp. 85–95, 1997.
[53]
P. Rossignol, B. Ho-Tin-Noé, R. Vranckx et al., “Protease nexin-1 inhibits plasminogen activation-induced apoptosis of adherent cells,” The Journal of Biological Chemistry, vol. 279, no. 11, pp. 10346–10356, 2004.
