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 (
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 [
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 [
A pathophysiological role has been recognized for the plasminogen-activating system in exacerbating intracranial bleeding, excitotoxicity and cell death in neurons or white matter, ischemia reperfusion injury, and increased permeability of the blood-brain barrier (BBB) [
PAR receptors share a common activation mechanism, whereby proteolytic cleavage unmasks a hexapeptide ligand sequence in the exodomain of the receptor, which can then dock intramolecularly and transmit G-protein-coupled signals into the cell [
Although plasmin and PAR1 have independently been implicated in pathways of cerebral injury, a plasmin/PAR1 axis remains to be identified in cells of the central nervous system. In mice, genetic deletion of the PAR1 homolog or deletion of tPA confers neuroprotection in a model of transient cerebral ischemia [
Plasmin, plasminogen, streptokinase, aprotinin, and
The human glioma cell line A172 was purchased from the American Type Culture Collection (ATCC; Manassas, VA). Cells were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM) enriched with 10% fetal calf serum, 2 mmoles/L L-glutamine, 100 U/mL penicillin, and 100
PAR1 receptor expression and cleavage was carried out by flow cytometry as previously described [
Cells were observed and photographed at 0 min, 30 min, 4 h, and 24 h using a Leica DM IL inverted microscope (Leica Microsystems, Wetzlar, Germany) at ×40 or ×100 magnification. Morphological changes were scored on a 6-point ordinal scale according to the scheme: 1 = fully confluent lawn of cells; 2 = cellular pseudopodia have started to retract from one area; 3 = cell processes has started to retract from multiple areas; 4 = cell lawns show visible detachment from well substrata and flapping; 5 = cells have completely detached from well substrata to form a floating island; 6 = floating island of cells with shrivelled appearance.
Apoptosis of A172 cells was monitored flow cytometrically by staining with Annexin V FITC Apoptosis Detection Kit according to the manufacturer’s instructions (Sigma-Aldrich). The percentage of apoptosis was calculated by determining the area under the histogram greater than background staining in resting cells. Serum starvation was used as a positive control for apoptosis.
Relative flourescent intensity and the morphological change scale were summarised using robust measures: median and interquartile range. Group differences were plotted using boxplots and assessed formally using the Wilcoxon-Mann-Whitney test for two-group comparisons. Statistical analyses used exact algorithms performed in Stata 10 (Stata Corp., College Station, TX).
Initial experiments examined whether PAR1 was expressed on resting A172 glioma cells. A representative flow cytometric histogram illustrates expression of PAR1 detected with a pan-receptor antibody WEDE15 (Figure
Expression of PAR1 epitopes. Flow cytometric histogram depicting expression of WEDE15 (a pan-receptor antibody) and SPAN12 (an activation-dependent antibody) on human A172 glioma cells. The filled histogram represents background staining with control antibody of the same isotype (IgG1).
Effect of plasmin on PAR1 receptor activation. Proteolytic activation of PAR1 at 5 minutes was monitored flow cytometrically using antibody SPAN12 to detect intact (i.e., unactivated) receptor. Results were expressed in units of relative fluorescent intensity (RFI), calculated by dividing the mean fluorescent staining intensity obtained with SPAN12 antibody by the staining intensity obtained with a class matched (IgG1) control antibody. Results were expressed as the median ± interquartile range (IQR) from
A timecourse of photomicrographs taken at 30 minutes, 4 hours and 24 hours after plasmin activation illustrates remarkable morphological transformation of A172 glioma cells (Figures
Effect of plasmin on cell morphology. (a) Time 0: confluent lawn of resting A172 glial cells. (b) 30 minutes after addition of plasmin: cell processes have started retracting from basal substrata, and flaps of detached cells were observed, although cell-to-cell contacts were maintained. (c) 4 hours: A172 cells fully detached into a floating island. (d) 24 hours: shrivelled floating cell mass. (e) 24 hours: aprotinin (200 KIU/mL) preserved the confluent monolayer of A172 cells in the face of plasmin activation. (f) 24 hours: a PAR1 agonist peptide SFLLRN (25
Effect of plasmin or PAR1 agonist peptide on glial cell detachment. (a) A172 cells were stimulated with plasmin (5 U/mL) for the length of time indicated, and cell detachment was quantified as defined in the Materials and Methods. (b) Effect of PAR1 specific activating peptide SFLLRN or inactive control peptide FSLLRN (both 25
Effect of plasmin on glial cell apoptosis. A172 cells were stimulated with plasmin (5 U/mL) for the length of time indicated and monitored flow cytometrically for apoptosis (programmed cell death) by staining with Annexin V. Representative flow cytometric histograms depict the effect of plasmin (open histogram) versus resting cells (filled histogram) on Annexin V expression. Serum starvation was used as a positive control for apoptosis.
The effect of antifibrinolytics on glial cell morphology was determined in cultures in which plasmin was generated
Effect of antifibrinolytics on plasmin induced glial cell detachment. A172 cells were stimulated with plasmin generated
The present study has proven the principle that plasmin can activate human glial cells via proteolytic cleavage of PAR1. The type of cell-lawn detachment observed was similar to that previously described in A172 cells treated with an integrin antagonist, that also caused separation from basal substratum and accumulation of cells into floating spheroids [
These findings add to a growing literature that morphological transformation of astroglial cells can take place in a pathway involving the plasminogen activating system and components of the focal cell adhesion machinery [
Most studies investigating the effect of serine proteases on neuronal PAR1 have focused on thrombin, which can either be neuroprotective or, conversely, induce apoptosis in neurons, depending on concentration and length of exposure to thrombin [
Cell lawn detachment may represent a manifestation of natural morphological processes, such as cell migration or astrocyte stellation. Reversal of the stellate phenotype by thrombin or changes in astrocyte morphology described for tPA may utilize the same cellular pathway, since both are regulated by ROCK [
The morphological changes described here may be most relevant in the setting of neurotrauma or neuroinjury secondary to BBB breakdown, when zymogens such as plasmin that are normally confined to the systemic circulation can enter the brain [
There are some limitations to this study. The cell culture system may fail adequately to model the homeostatic environment of the brain, which is endowed not only with serine proteases and their zymogens but also naturally occurring serine protease inhibitors (serpins) [
In conclusion, we have identified a PAR1 axis of glial cell activation triggered by plasmin or the plasminogen system. This may be especially relevant under conditions of BBB breakdown or intracranial hemorrhage when serine proteases gain access to the neurovascular unit.
R. C. Landis discloses unrestricted research grants from the Barbados Diabetes Foundation and Bayer Pharmaceuticals Inc. which supported this work. The authors declare that they have no conflict of interests.