Time course of recovery of endothelial cell surface thrombin receptor (PAR-1) expression

Chad A. Ellis, Chinnaswamy Tiruppathi, Raudel Sandoval, Walter D. Niles, and Asrar B. Malik

Department of Pharmacology, College of Medicine, University of Illinois, Chicago, Illinois 60612-7343

    ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

We studied dynamics of cell surface expression of proteolytically activated thrombin receptor (PAR-1) in human pulmonary artery endothelial cells (HPAEC). PAR-1 activation was measured by changes in cytosolic calcium concentration ([Ca2+]i) and HPAEC retraction response (determined by real-time transendothelial monolayer electrical resistance). [Ca2+]i increase in response to thrombin was abolished by preexposure to 25 nM thrombin for >60 min, indicating PAR-1 desensitization, but preexposure to 25 nM thrombin for only 30 min or to 10 nM thrombin for up to 2 h did not desensitize PAR-1. Exposure to 10 or 25 nM thrombin decreased monolayer electrical resistance 40-60%. Cells preexposed to 10 nM thrombin, but not those preexposed to 25 nM thrombin, remained responsive to thrombin 3 h later. Loss of cell retractility was coupled to decreased cell surface PAR-1 expression as determined by immunofluorescence. Cell surface PAR-1 disappeared upon short-term (30 min) thrombin exposure but reappeared within 90 min after incubation in thrombin-free medium. Exposure to 25 nM thrombin for >60 min prevented rapid cycloheximide-insensitive PAR-1 reappearance. Cycloheximide-sensitive recovery of cell surface PAR-1 expression required 18 h. Therefore, both duration and concentration of thrombin exposure regulate the time course of recovery of HPAEC surface PAR-1 expression. The results support the hypothesis that initial recovery of PAR-1 surface expression in endothelial cells results from a rapidly mobilizable PAR-1 pool, whereas delayed recovery results from de novo PAR-1 synthesis. We conclude that thrombin itself regulates endothelial cell surface PAR-1 expression and that decreased surface expression interferes with thrombin-induced endothelial cell activation responses.

proteolytically activated thrombin receptor; human pulmonary artery endothelial cells; endothelial monolayer resistance; cytosolic calcium concentration

    INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

ACTIVATION OF ENDOTHELIAL cells by thrombin results in cell retraction, stimulation of prostacyclin synthesis, increased cytosolic Ca2+ concentration ([Ca2+]i), expression of cell surface P-selectin and intercellular adhesion molecule-1, and increased transendothelial permeability to albumin (15). Studies have shown that proteolytically activated thrombin receptor (PAR-1) is expressed on vascular endothelial cell membranes (18). We and others have shown that thrombin-induced activation of PAR-1 in endothelial cells increases endothelial permeability (6, 7, 14), a response that results from retraction of adjacent endothelial cells, which increases the dimension of the paracellular pathway (13, 25).

The diverse effects of thrombin in endothelial cells are activated by thrombin binding to the G protein-coupled seven-transmembrane PAR-1 (3, 27). Thrombin binds to the hirudin-like domain in the extracellular amino-terminal region of PAR-1 (27) and cleaves the receptor between Arg-41 and Ser-42 (27). The newly formed amino-terminal region binds to the second extracellular loop and amino-terminal extension regions on the PAR-1 to transmit signals (7, 17, 19). Synthetic peptides corresponding to the new amino terminus are capable of activating the receptor (27). Exposure to thrombin causes rapid desensitization such that subsequent thrombin challenge produces little or no Ca2+ signaling (24). Proteolysis of PAR-1 by thrombin irreversibly cleaves the receptor, a phenomenon distinct from activation of other G protein-coupled receptors (8, 27). The internalization of the activated PAR-1 is, in part, the result of phosphorylation of the carboxy terminus of the receptor by G protein-linked receptor kinases (8, 11, 21).

The regulation of PAR-1 resensitization in endothelial cells after the internalization of the degraded receptor is not well understood. Brass and colleagues (2, 10) showed that activation of PAR-1 by thrombin in megakaryoblastic HEL and CHRF-288 cell lines (which do not have an intracellular pool of PAR-1) resulted in rapid internalization of the receptor and >90% degradation in acidic lysosomes. Horvat and Palade (9) showed by immunoelectron microscopy of human umbilical vein endothelial cells (HUVEC) the presence of preformed PAR-1 stores in intracellular vesicular compartments. Hein et al. (8) showed that this intracellularly stored PAR-1 pool (which they localized in the Golgi apparatus) could translocate to the HUVEC surface within 60 min of thrombin exposure, suggesting that endothelial cells could be rapidly reactivated by thrombin. In the present study, we investigated the kinetics of endothelial cell surface PAR-1 reexpression and the "activation potential" of the cell surface PAR-1 upon its reexpression. The results indicate that the endothelial cell surface PAR-1 can be rapidly restored even in the presence of continuous exposure to a low concentration of thrombin, but that cellular PAR-1 pools are depleted by high concentrations of thrombin and longer exposure times. We provide evidence of a direct relationship between cell surface PAR-1 expression and activation of Ca2+ signal and endothelial cell retraction.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Materials. Human alpha -thrombin was obtained from Enzyme Research Laboratories (South Bend, IN). Hanks' balanced salt solution (HBSS) and trypsin were purchased from GIBCO (Grand Island, NY). Fetal bovine serum (FBS) was purchased from HyClone (Logan, UT). Human pulmonary artery endothelial cells (HPAEC) and EBM-2 medium were from Clonetics (San Diego, CA). Electrodes for endothelial monolayer resistance measurements were purchased from Applied Biophysics (Troy, NY). Fura 2-AM (cell permeant) was purchased from Molecular Probes (Eugene, OR). Cycloheximide was purchased from United States Biochemical (Cleveland, OH). Anti-PAR-1 monoclonal antibody (MAb) raised against amino-terminal extension sequence Trp-Glu-Asp-Glu (recognizing both naive and thrombin-cleaved PAR-1) was obtained from Immunotech (Westbrook, ME).

Endothelial cell culture. HPAEC were grown in EBM-2 medium supplemented with 10% FBS. Cells were cultured on tissue culture dishes coated with 0.1% gelatin and were used between passages 4 and 8.

[Ca2+]i measurement. Thrombin-induced increase in [Ca2+]i was measured using the Ca2+-sensitive fluorescent dye fura 2 (24, 30). Cells were grown to confluence on gelatin-coated glass coverslips and then washed two times with serum-free medium and incubated for 2 h at 37°C in culture medium containing 1% FBS. After thrombin exposure, cells were washed once and loaded with 2 µM fura 2-AM for 1 h at 25°C. After the loading, cells were washed with HBSS and placed in a Sykes-Moore perfusion chamber positioned on the stage of a Nikon Diaphot microscope, which was coupled to a Deltascan microspectroflourometric system (Photon Technology International, Princeton, NJ). An optically isolated field of endothelial cells was excited alternately with wavelengths of 340 and 380 nm, and emitted light was collected at 510 nm with a photomultiplier. Background autofluorescence (in the absence of fura 2) determined at the beginning of each day of experiments was subtracted automatically during data collection. At the end of each experiment, 10 µM ionomycin was added to obtain the fluorescence of Ca2+-saturated fura 2 and 0.1 M EGTA was added to obtain the fluorescence of free fura 2. The ratios of fluorescence at 340 nm to that at 380 nm and the values for [Ca2+]i were calculated using PTI software.

Cell surface PAR-1 immunofluorescence. HPAEC were grown to confluence on glass coverslips. The cells were washed three times with serum-free medium and kept for 2 h at 37°C in medium containing 1% FBS before agonist treatment. After thrombin treatment, cells were washed three times with HBSS and fixed with 1% paraformaldehyde for 15 min at 22°C. Cells were then blocked with 5% BSA at 4°C for 60 min before addition of anti-PAR-1 MAb (5 µg/ml) for 60 min at 4°C. The anti-PAR-1 MAb binding was detected by incubating rhodamine-labeled goat anti-mouse IgG (5 µg/ml) for 30 min at 4°C. Cell surface fluorescence was visualized using digital imaging fluorescence microscopy (Diaphot 200, Nikon Instruments, Fair Lawn, NJ). Specimens were viewed with a ×100 objective. Excitation and emission wavelengths were selected with a filter set optimal for the fluorescence of tetramethylrhodamine. Images were collected for analysis with a cooled charge-coupled device array detector (Imagepoint, Photometrics, Tucson, AZ) with a spatial resolution of 768 vertical by 452 horizontal. Images were acquired by the detector with a 6-s integration period and then digitized at 8 bits/pixel brightness resolution and transferred directly to a personal computer frame memory.

Endothelial cell shape change. The time course of endothelial cell shape change (in real time) in response to thrombin was measured according to procedures described by us (25). In brief, HPAEC were grown to confluence on small gold electrodes (4.9 × 10-4 cm2). Before experiments, monolayers were washed two times with serum-free medium and incubated for 2 h in 1% serum-supplemented culture medium. The small electrode and the large electrode were connected to a phase-sensitive lock-in amplifier. A constant current of 1 µA was applied by a 1-V, 4,000-Hz alternating current connected serially to a 1-MOmega resistor between the small electrode and the larger counter electrode. The voltage change between the small electrode and the larger counter electrode was continuously monitored by the lock-in amplifier, stored, and processed on a computer. The data are presented as the change in the resistive (in-phase) portion of the impedance normalized to its initial value at time zero as described (25, 26).

    RESULTS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Desensitization of PAR-1 and its recovery in endothelial cells. Endothelial cells were pretreated with 10 nM (1.0 National Institutes of Health unit) or 25 nM thrombin for different times before measurement of the increases in [Ca2+]i. Cells were challenged for 30, 60, 90, or 120 min (as shown in Fig. 1, A and B) with 10 or 25 nM thrombin, washed with DMEM, and incubated in thrombin-free medium before the second thrombin challenge at 3 h. Pretreatment of HPAEC with either 10 or 25 nM thrombin for 30 min resulted in the same increases in the [Ca2+]i at 3 h as in control cells (Fig. 1, A and B). Cells pretreated with 10 nM thrombin for 60, 90, or 120 min remained responsive to thrombin at 3 h (Fig. 1A). In contrast, cells preexposed to 25 nM thrombin for 60, 90, or 120 min did not respond to thrombin stimulation at 3 h (Fig. 1B). Thus 25 nM thrombin exposure for >30 min fully desensitized PAR-1 in HPAEC, indicating that both concentration and duration of thrombin exposure are critical determinants of PAR-1-activated Ca2+ signaling.


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Fig. 1.   Resensitization of endothelial cells to thrombin is dependent on duration and concentration of initial thrombin exposure. Human pulmonary artery endothelial cells (HPAEC) grown to confluence on glass coverslips were washed 2 times with serum-free medium and incubated in medium containing 1% serum for 2 h at 37°C. Cells were loaded with fura 2-AM for 60 min to measure thrombin-mediated increases in cytosolic Ca2+ concentration ([Ca2+]i) as described in MATERIALS AND METHODS. In all experiments depicted, increase in [Ca2+]i was measured 3 h after initial exposure to thrombin, which varied from 30 min to 2 h, as indicated. After thrombin treatment, cells were washed and incubated with thrombin-free medium for up to 2 h at 37°C and then cells were loaded with fura 2-AM for 60 min at 25°C in thrombin-free medium before measurement of second thrombin response. A: cells were stimulated with 10 nM thrombin. Inset: thrombin response in control cells. B: cells were stimulated with 25 nM thrombin. Experiments were repeated at least 4 times. Data from representative experiments are shown, as results from experiments were similar. alpha -T, alpha -thrombin.

Loss of cell surface PAR-1 and its recovery within 60-90 min. We determined the surface expression of PAR-1 by immunofluorescence after exposing the cells to thrombin for different times. After thrombin exposure, cells were fixed, nonspecific binding was blocked, and cells were incubated with anti-PAR-1 MAb (see MATERIALS AND METHODS). Both 10 and 25 nM thrombin exposure for 30 min at 37°C prevented cell surface antibody (Ab) staining compared with control cells (Fig. 2), indicating the rapid loss of cell surface PAR-1 with both thrombin concentrations. Cells exposed to 25 nM thrombin for 60 min also showed no Ab staining (Fig. 2); however, cell surface Ab staining reappeared within this time in cells exposed to 10 nM thrombin (Fig. 2). These differences between the two thrombin concentrations were clearly evident when the thrombin pretreatment period was extended to 90 min in that cell surface PAR-1 was present only in cells exposed to 10 nM for 90 min (Fig. 2).


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Fig. 2.   Thrombin exposure alters cell surface expression of proteolytically activated thrombin receptor (PAR-1). HPAEC were incubated with 10 or 25 nM thrombin for indicated times before staining with anti-PAR-1 monoclonal antibody (MAb; see details in MATERIALS AND METHODS). Control cell surface staining is shown at top. Experiments were repeated at least 4 times. Micrographs are from representative experiments. Scale bar, 15 µm.

Cells treated continuously with 10 nM thrombin for 3 h exhibited Ab staining comparable to control cells (Fig. 2), indicating PAR-1 had translocated to the surface in these cells during this time. In contrast, cells exposed to 25 nM thrombin for 3 h failed to show Ab staining (Fig. 2), indicating that continuous exposure to the high thrombin concentration did not result in recovery of cell surface PAR-1 within this time. The lack of return of cell surface PAR-1 with prolonged exposure to 25 nM thrombin (Fig. 2) was associated with failure of thrombin to elicit the Ca2+ signal (Fig. 1B).

Because 30-min exposure to 25 nM thrombin elicited the Ca2+ signal in response to thrombin 3 h later (Fig. 1), we determined whether recovery of functional activity was associated with reexpression of cell surface PAR-1. A time course experiment was conducted in which HPAEC were incubated with 25 nM thrombin for 30 min at 37°C, washed, incubated in thrombin-free medium, and fixed at either 30 or 60 min for Ab staining. Minimal PAR-1 staining was observed at 60 min (i.e., 30-min incubation in thrombin-free medium), whereas staining was comparable to the level of control cells at 90 min (Fig. 3). These results indicate that, although the high concentration of thrombin for 30 min depleted the cell surface PAR-1, PAR-1 was replenished between 30 and 60 min by incubation of the cells in thrombin-free medium (Fig. 3).


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Fig. 3.   Time dependence of thrombin-induced translocation of intracellular pool PAR-1 to cell surface. HPAEC were incubated with 25 nM thrombin for 30 min at 37°C, washed, and incubated with thrombin-free medium before fixing and staining with anti-PAR-1 MAb (see details in MATERIALS AND METHODS). A: cells were exposed to 25 nM thrombin for 30 min and were then stained for surface expression. B: cells were incubated with 25 nM thrombin for 30 min, washed, incubated with thrombin-free medium for 30 min, and then stained. C: same as B except that thrombin-free medium incubation period was 60 min. Other details are described in MATERIALS AND METHODS. This experiment was repeated at least 4 times with similar results. Micrographs are from representative experiments.

Endothelial cells become refractory to thrombin with loss of cell surface PAR-1. We measured endothelial cell retraction response in real time by determining the effects of thrombin on HPAEC monolayer resistance (10). Addition of 10 or 25 nM thrombin caused a 40-60% decrease in resistance, whereas DMEM had no effect (Fig. 4), indicating that activation of PAR-1 in HPAEC caused the rapid retraction of confluent endothelial cells. The decreased endothelial monolayer resistance returned to the normal range within 2 h. Thrombin concentrations within the range 0.1-100 nM produced similar results (data not shown). Endothelial cell retraction was not inhibited by cycloheximide (6 µg/ml) pretreatment (data not shown), indicating that it was protein synthesis independent.


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Fig. 4.   Effect of thrombin concentration on resensitization of HPAEC. HPAEC were cultured on electrodes and grown to a confluence (see details in MATERIALS AND METHODS). Cells were washed with serum-free medium and incubated with culture medium containing 1% serum for 2 h. A and B: cells were then stimulated with either 10 (A) or 25 (B) nM thrombin. These cells were tested again for thrombin response at 3 and 5 h with same concentration of thrombin. Addition of medium alone did not influence endothelial monolayer resistance (traces at top). C: cells were first treated with 25 nM thrombin, and same cells were stimulated with 10 µM histamine 3 h after initial thrombin challenge. D: control cells were challenged with 10 µM histamine alone. A-D: arrows indicate times when cells were stimulated with thrombin or histamine. These experiments were repeated 5 times. Data represent single observations. A and B, insets: means ± SE of maximum decrease in resistance.

HPAEC grown on electrodes were stimulated with 10 or 25 nM thrombin, and in this case the effects of repeated thrombin challenges on HPAEC monolayer resistance were continuously monitored. The monolayers were challenged with the identical thrombin concentration 3 and 5 h later. HPAEC treated with 10 nM thrombin exhibited a 40-60% decrease in resistance in response to the second thrombin challenge, whereas the resistance did not decrease with the third challenge (Fig. 4A). However, preexposure to 25 nM thrombin prevented both the second and third thrombin-induced decreases in monolayer resistance (Fig. 4B). HPAEC were also stimulated with either 10 or 25 nM thrombin 8 and 15 h after the initial thrombin challenge. HPAEC did not respond to thrombin at these times (data not shown). HPAEC pretreated with 25 nM thrombin for 3 h, however, remained responsive to 10 µM histamine (Fig. 4C), and the response to histamine was comparable to that of control cells (Fig. 4D). The absence of responsivity to the second challenge with 25 nM thrombin (Fig. 4B) was associated with the failure of thrombin to produce a rise in the [Ca2+]i (Fig. 1B) as well as with the loss of cell surface PAR-1 (Fig. 2).

Long-term recovery of PAR-1 activity occurs at 18 h after PAR-1 depletion. Because continuous exposure of 25 nM thrombin for 3 h depleted PAR-1 (Fig. 2) and resulted in HPAEC becoming refractory to thrombin (Fig. 4, A and B), we determined the reversibility of this process. Cells were cultured on electrodes as described above to measure endothelial cell monolayer resistance. HPAEC were first treated with 10 or 25 nM thrombin, after which the cells were challenged with thrombin at different time intervals. After the initial 25 nM thrombin treatment, cell function was restored at 18 h after thrombin in the absence of cycloheximide (Fig. 5A). Similar results were obtained when cells were challenged with 10 nM thrombin twice at 0 and 3 h to deplete PAR-1 (data not shown). Although the endothelial cell retractile response was restored at 18 h after the initial thrombin challenge, the response did not recover following treatment with cycloheximide (Fig. 5A). Cells exposed to 25 nM thrombin for 3 h demonstrated cell surface PAR-1 expression at 18 h, whereas pretreatment of these cells with cycloheximide prevented cell surface PAR-1 expression at 18 h (Fig. 5B).


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Fig. 5.   Resensitization to thrombin 18 h after thrombin exposure. A: HPAEC grown on electrodes were continuously exposed to 25 nM thrombin (3 h) in presence and absence of cycloheximide (6 µg/ml). Cells were tested 18 h after initial thrombin challenge. Decrease in electrical resistance in control cells (-CHX) was typical of response observed in control cells, whereas response in cycloheximide (CHX)-treated cells (+CHX) was markedly reduced. Results are means ± SE for 4 experiments. B: cells were subjected to thrombin exposure conditions similar to those in A but were used for PAR-1 cell surface staining as described for Fig. 2.

Although a thrombin concentration of 10 nM was capable of activating the surface PAR-1 at 18 h, as is evident from the sharp decrease in electrical resistance (Fig. 6), interestingly, these cells, unlike the control cells (Fig. 4, A and B), did not respond to thrombin exposure at 21 h (i.e., 3 h after the first challenge; Fig. 6). On the other hand, these cells responded to histamine in a normal manner (Fig. 6, inset).


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Fig. 6.   Thrombin-induced endothelial cell retraction in response to repeated thrombin challenges in cells with restored surface PAR-1 at 18 h. HPAEC grown on electrodes were continuously exposed to 25 nM thrombin for 3 h. These cells were again challenged with 10 nM thrombin at 18 and 21 h but failed to respond to repeated thrombin challenges. Addition of medium alone did not influence endothelial monolayer resistance (trace at top). Inset: cell response to histamine 21 h after thrombin exposure (at 18 h). This experiment was repeated 4 times with similar results.

    DISCUSSION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

G protein-coupled receptors undergo homologous desensitization after short-term exposure to their agonists (1, 5). Desensitization involves a combination of sequential events: receptor phosphorylation, internalization, intracellular degradation, dephosphorylation, and recycling of internalized receptors to the cell surface (1, 5). This process has been well studied for the beta 2-adrenergic receptors (beta 2-AR) (1). However, PAR-1, a G protein-coupled receptor, is not recycled to the cell surface after its internalization because it is irreversibly modified by proteolysis (1, 27). PAR-1 is also specialized because of a mobilizable pool of the receptor in endothelial cytosol (8, 9). In the present study, we determined the characteristics of cell surface PAR-1 disappearance and its reappearance in endothelial cells. Because PAR-1 activation in vascular endothelial cells causes cell retraction, a major factor in the development of increased endothelial permeability via the paracellular pathway (15), we also determined the relationship of cell surface PAR-1 expression to activation of thrombin-induced Ca2+ signaling and retraction of endothelial cells.

Brass and colleagues (2, 10) investigated thrombin-dependent desensitization of PAR-1 in the megakaryoblastic HEL and CHRF-288 cell lines using Abs directed against the tethered-ligand domain and thrombin-binding (hirudin-like domain) region of PAR-1. Exposure to 10-20 nM thrombin for 10 min caused internalization of >85% receptors, and the receptors were subsequently degraded in acidic lysosomes (2, 10). However, some cleaved receptors (i.e., the "activated" receptors) were recycled to the cell surface but could not be activated by thrombin. Brass and colleagues (2, 10) showed that these megakaryoblastic cell lines (which exhibit platelet properties) responded to thrombin 22 h after the initial thrombin challenge and that this response was dependent on de novo protein synthesis. Hein et al. (8) expressed FLAG epitope-tagged PAR-1 and beta 2-AR in Rat1 fibroblasts and followed intracellular receptor traffic after exposure to agonists. After thrombin cleavage, the removal of epitope from receptors allowed the naive receptors to be distinguished from cleaved PAR-1 (8). In control cells, PAR-1 was localized on the cell surface and intracellular compartments; however, beta 2-AR was localized only on the cell surface. They concluded that PAR-1 (unlike beta 2-AR) is stored in a preformed state in the cytosol (8), like the N-formyl peptide receptor in neutrophils (20). Moreover, stimulation of Rat1 fibroblasts expressing both PAR-1 and beta 2-AR resulted in rapid internalization of both receptors into endosomal vesicles (8). The internalized beta 2-AR was recycled to the cell surface, whereas PAR-1 was targeted to lysosomes (8). Thrombin-challenged Rat1 fibroblasts also did not show an increase in [Ca2+]i when challenged with thrombin within 5 min after the initial thrombin exposure; however, cells responded to the second thrombin challenge at 60 min with a normal increase in [Ca2+]i, a response that was insensitive to cycloheximide (8). These results indicated that Rat1 fibroblasts have an intracellular pool of PAR-1 that upon activation can signal PAR-1 translocation to cell surface. An intracellular PAR-1 pool has also been observed in HUVEC (8, 9). In contrast, the PAR-1-expressing megakaryoblastic cell lines lacked an intracellular PAR-1 pool (2, 10).

In the present study, we determined the characteristics of PAR-1 cell surface expression using HPAEC to address the "turning off" and reactivation of PAR-1 signaling. We determined cell surface PAR-1 expression using an anti-PAR-1 MAb that recognized both the cleaved and naive cell surface PAR-1 (8, 27). The results indicate that the concentration of thrombin and exposure time are both critical determinants of loss of cell surface PAR-1. The higher concentration of thrombin (25 nM) maintained for 1-2 h depleted the cell surface PAR-1 such that the cells did not respond to thrombin for up to 18 h. The lower concentration of thrombin [10 nM, in the range of 0.1-10 nM expected after conversion of prothrombin to thrombin (12, 22)] also produced the loss of cell surface PAR-1 and Ca2+ signaling, but this response was reversible within 60-90 min after the initial thrombin challenge. These results indicate that cleavage of PAR-1 and its internalization abrogate the signaling; however, early recovery of cell surface PAR-1 can occur after short-term exposure to thrombin of ~30 min. These findings support the hypothesis that the cytosolic pool of PAR-1, which can be rapidly translocated to the cell surface (8, 28), serves an important function in enabling the endothelial cell to rapidly restore its responsiveness to thrombin after receptor inactivation by cleavage and internalization.

Exposure to 25 nM thrombin for 30 min resulted in an immediate loss of the cell surface PAR-1, which was followed by the return of PAR-1 within 90 min after thrombin treatment. These results raise the possibility that depletion of the cell surface PAR-1 itself signals translocation of cytosolic stores. Internalization of receptors and their uncoupling from heterotrimeric G proteins may signal the mobilization of the receptor to the cell surface (11). Another possibility is that the thrombin can signal translocation of cytosolic PAR-1 to the cell surface by activation of second messenger pathways (which have not been thus far defined) (8, 28).

We observed that loss of cell surface PAR-1 and Ca2+ signaling were coupled to the inactivation of the endothelial cell retraction response. We measured transendothelial electrical resistance to study retraction of endothelial cells following thrombin exposure, as this is a critical event responsible for increasing endothelial permeability (13, 25). The cells became refractory to the second stimulus after having been exposed to thrombin to deplete the cell surface PAR-1. We showed that the loss of cell surface PAR-1 and Ca2+ signaling abrogated the thrombin-induced retraction of endothelial cells. This finding is in accord with the important function of Ca2+ in signaling the thrombin-induced retraction of endothelial cells by activation of the myosin light chain kinase-dependent actin-myosin contractile machinery of endothelial cells (16, 23, 29).

Although the cell surface PAR-1 could be depleted in endothelial cells as the result of thrombin exposure and thus desensitize the receptor to further thrombin stimulation, we observed that the cells remained responsive to histamine. Histamine produced a sharp drop in electrical resistance characteristic of cell retraction (16) in cells that were not responsive to thrombin. We conclude from this observation that thrombin exposure of endothelial cells produced homologous desensitization of PAR-1.

Exposure of endothelial cells to 25 nM thrombin produced a long-lived desensitization lasting ~18 h, at which point thrombin exposure produced its characteristic decrease in electrical resistance. This response was sensitive to cycloheximide, suggesting that it required new synthesis of PAR-1. Because we have shown that thrombin can signal the activation of PAR-1 mRNA, a process regulated by the activation of tyrosine kinase pathways (4), the delayed resensitization after loss of cell surface PAR-1 appears to be distinct in endothelial cells vs. platelets because it involves de novo synthesis of PAR-1.

We observed that challenge of endothelial cells at 18 h resulted in a sharp characteristic drop in electrical resistance, suggesting that the cell surface PAR-1 had been restored. Interestingly, subsequent challenge with 10 nM thrombin, 3 h later, which normally would have elicited another decrease in resistance because of translocation of the cell surface PAR-1 to the membrane, failed to produce such an effect. This finding raises the possibility that cell surface PAR-1 is preferentially replenished at the expense of the cytosolic pool following new synthesis of PAR-1. Whether preferential targeting of the newly synthesized PAR-1 to the membrane is responsible for this effect will need to be explored.

In summary, we have shown that thrombin concentration and its exposure time regulate endothelial cell surface PAR-1 expression. Rapid resensitization occurs by replenishment of the cell surface PAR-1 by the translocation of preformed cytosolic PAR-1 to the membrane within 60-90 min of depletion of the surface PAR-1. In contrast, the delayed process of resensitization involves new PAR-1 synthesis. The finding that thrombin modulates the rapid and delayed surface expression of PAR-1 in endothelial cells suggests a novel means of preventing the increase in endothelial permeability involving the desensitization of PAR-1.

    ACKNOWLEDGEMENTS

This work was supported by National Heart, Lung, and Blood Institute Grants HL-27016, HL-45638, and T32-HL-07829.

    FOOTNOTES

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Address for reprint requests: C. Tiruppathi, Dept. of Pharmacology (M/C 868), University of Illinois at Chicago, 835 South Wolcott Ave., Chicago, IL 60612-7343.

Received 20 July 1998; accepted in final form 9 September 1998.

    REFERENCES
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

1.   Barak, L. S., and M. G. Caron. Modeling of sequestration and down regulation in cells containing beta2-adrenergic receptors. J. Recept. Signal Transduct. Res. 15: 677-690, 1995[Medline].

2.   Brass, L. F., S. Pizarro, M. Ahuja, E. Belmonte, N. Blanchard, J. M. Stadel, and J. A Hoxie. Changes in the structure and function of the human thrombin receptor during receptor activation, internalization, and recycling. J. Biol. Chem. 269: 2943-2952, 1994[Abstract/Free Full Text].

3.   Coughlin, S. R. Thrombin receptor function and cardiovascular disease. Trends Cardiovasc. Med. 4: 77-83, 1994.

4.   Ellis, C. A., C. Tiruppathi, R. Sandoval, and A. B. Malik. Time course of thrombin receptor resensitization in endothelial cells is dependent on discharge of thrombin receptor pool (Abstract). Mol. Biol. Cell 7: 663A, 1996.

5.   Ferguson, S. G. S., L. S. Barak, J. Zhang, and M. G. Caron. G-protein-coupled receptor regulation: role of G-protein-coupled receptor kinases and arrestins. Can. J. Physiol. Pharmacol. 74: 1095-1110, 1996[Medline].

6.   Garcia, J. G. N., C. E. Patterson, C. Bahler, J. Ashner, C. M. Hart, and D. English. Thrombin receptor activating peptides induce Ca2+ mobilization, barrier dysfunction, prostaglandin synthesis, and platelet-derived growth factor mRNA expression in cultured endothelium. J. Cell. Physiol. 156: 541-549, 1993[Medline].

7.   Gerstzen, R. E., J. I. Chen, M. Ishii, L. Wang, T. Nanevlcz, C. W. Turck, T. K. H. Vu, and S. R. Coughlin. Specificity of the thrombin receptor for agonist peptide is defined by its extracellular surface. Nature 368: 648-651, 1994[Medline].

8.   Hein, L., K. Ishii, S. R. Coughlin, and B. K. Kobilka. Intracellular targeting and trafficking of thrombin receptors. A novel mechanism for resensitization of a G protein-coupled receptor. J. Biol. Chem. 269: 27719-27726, 1994[Abstract/Free Full Text].

9.   Horvat, R., and G. E. Palade. The functional thrombin receptor is associated with the plasmalemma and a large endosomal network in cultured human umbilical vein endothelial cells. J. Cell Sci. 108: 1155-1164, 1995[Abstract/Free Full Text].

10.   Hoxie, J. A., M. Ahuja, E. Belmonte, S. Pizarro, R. Parton, and L. F. Brass. Internalization and recycling of activated thrombin receptors. J. Biol. Chem. 268: 13756-13763, 1993[Abstract/Free Full Text].

11.   Ishii, K., J. Chen, M. Ishii, W. J. Koch, N. J. Freedman, R. J. Lefkowitz, and S. R. Coughlin. Inhibition of thrombin receptor signaling by a G-protein coupled receptor kinase. Functional specificity among G-protein coupled receptor kinases. J. Biol. Chem. 269: 1125-1130, 1994[Abstract/Free Full Text].

12.   Ishii, K., L. Hein, B. Kobilka, and S. R. Coughlin. Kinetics of thrombin receptor cleavage on intact cells. J. Biol. Chem. 268: 9780-9786, 1993[Abstract/Free Full Text].

13.   Laposata, M., D. K. Dovnarsky, and H. S. Shin. Thrombin-induced gap formation in confluent endothelial cell monolayers in vitro. Blood 62: 549-556, 1983[Abstract].

14.   Lum, H., T. T. Andersen, A. Siflinger-Birnboim, C. Tiruppathi, M. S. Goligorsky, and A. B Malik. Thrombin receptor peptide inhibits thrombin-induced increases in endothelial permeability by receptor desensitization. J. Cell Biol. 120: 1491-1499, 1993[Abstract].

15.   Malik, A. B., and J. W. Fenton II. Thrombin-mediated increase in vascular endothelial permeability. Semin. Thromb. Hemost. 18: 193-199, 1992[Medline].

16.   Moy, A. B., J. V. Engelenhoven, J. Bodmer, J. Kamath, C. R. Keese, I. Giaever, S. Shasby, and D. M. Shasby. Histamine and thrombin modulate endothelial focal adhesion through centrifugal forces. J. Clin. Invest. 97: 1020-1027, 1996[Abstract/Free Full Text].

17.   Nanevicz, T., M. Ishii, L. Wang, M. Chen, C. W. Turck, F. E. Cohen, and S. R. Coughlin. Mechanisms of thrombin receptor agonist specificity. Chimeric receptors and complementary mutations identify an agonist recognition site. J. Biol. Chem. 270: 21619-21625, 1995[Abstract/Free Full Text].

18.   Nelken, N. A., S. J. Soifer, J. O'Keefe, T. K. H. Vu, I. F. Charo, and S. R. Coughlin. Thrombin receptor expression in normal and atherosclerotic human arteries. J. Clin. Invest. 90: 1614-1621, 1992[Medline].

19.   Nguyen, L. T., H. Lum, C. Tiruppathi, and A. B. Malik. Site-specific thrombin receptor antibodies inhibit Ca2+ signaling and increased endothelial permeability. Am. J. Physiol. 273 (Cell Physiol. 42): C1756-C1763, 1997[Abstract/Free Full Text].

20.   Sengelov, H., F. Boulay, L. Kjeldsen, and N. Borregaard. Subcellular localization and translocation of the receptor for N-formylmethionyl-leucyl-phenylalanine in human neutrophils. Biochem. J. 299: 473-479, 1994[Medline].

21.   Shapiro, M. J., J. Trejo, D. Zeng, and S. R. Coughlin. Role of the thrombin receptor's cytoplasmic tail in intracellular trafficking. J. Biol. Chem. 271: 32874-32880, 1996[Abstract/Free Full Text].

22.   Shuman, M. A. Thrombin-cellular interactions. Ann. NY Acad. Sci. 485: 228-239, 1986[Abstract].

23.   Stasek, J. E., C. E. Patterson, and J. G. N. Garcia. Protein kinase C phosphorylates caldesmon and vimentin and enhances albumin permeability across cultured bovine pulmonary artery endothelial cell monolayers. J. Cell. Physiol. 153: 62-75, 1992[Medline].

24.   Tiruppathi, C., H. Lum, T. T. Andersen, J. W. Fenton II, and A. B. Malik. Thrombin receptor 14-amino acid peptide binds to endothelial cells and stimulates calcium transients. Am. J. Physiol. 263 (Lung Cell. Mol. Physiol. 7): L595-L601, 1992[Abstract/Free Full Text].

25.   Tiruppathi, C., A. B. Malik, P. J. Del Vecchio, C. R. Keese, and I. Giaever. Electrical method for detection of endothelial cell shape change in real time: assessment of endothelial barrier function. Proc. Natl. Acad. Sci. USA 89: 7919-7923, 1992[Abstract].

26.   Tiruppathi, C., W. Song, M. Bergenfeldt, P. Sass, and A. B. Malik. Gp60 activation mediates albumin transcytosis in endothelial cells by tyrosine kinase-dependent pathway. J. Biol. Chem. 272: 25968-25975, 1997[Abstract/Free Full Text].

27.   Vu, T. K. H., 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 64: 1057-1068, 1991[Medline].

28.   Woolkalis, M. J., T. M. DeMelfi, Jr., N. Blanchard, J. A. Hoxie, and L. F. Brass. Regulation of thrombin receptors on human umbilical vein endothelial cells. J. Biol. Chem. 270: 9868-9875, 1995[Abstract/Free Full Text].

29.   Wysolmerski, R. B, and D. Lagunoff. Regulation of permeabilized endothelial cell retraction by myosin phosphorylation. Am. J. Physiol. 261 (Cell Physiol. 30): C32-C40, 1991[Abstract/Free Full Text].

30.   Yan, W., C. Tiruppathi, H. Lum, R. Qiao, and A. B. Malik. Protein kinase Cbeta regulates heterologous desensitization of thrombin receptor (PAR-1) in endothelial cells. Am. J. Physiol. 274 (Cell Physiol. 43): C387-C395, 1998[Abstract/Free Full Text].


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