©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Regulation of Thrombin Receptors on Human Umbilical Vein Endothelial Cells (*)

Marilyn J. Woolkalis (1)(§), Thomas M. DeMelfiJr. (1), Nadine Blanchard (2), James A. Hoxie (2), Lawrence F. Brass (2)

From the (1) Department of Physiology, Thomas Jefferson University, Philadelphia, Pennsylvania 19107 and the (2) Departments of Medicine and Pathology, University of Pennsylvania, Philadelphia, Pennsylvania 19104

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Activated thrombin receptors on human umbilical vein endothelial cells rapidly undergo homologous desensitization, leaving the cells unable to respond to thrombin. The present studies examine the fate of activated thrombin receptors on endothelial cells and the mechanisms that restore intact receptors to the cell surface. The results show that: 1) at biologically relevant concentrations, thrombin rapidly cleaves all of its receptors on the cell surface. 2) The cleaved receptors are cleared from the cell surface in a two-phase process, with 60% being internalized within 10 min, the remainder requiring several hours. 3) The restoration of intact, thrombin-responsive receptors on the cell surface initially occurs from an intracellular pool of receptors in a process that is independent of protein synthesis. 4) Recycling of cleaved receptors either does not occur on endothelial cells or is masked by receptor clearance. 5) Subconfluent endothelial cells re-express intact receptors on the cell surface at a slower rate than confluent cells. 6) The agonist peptide, SFLLRN, also causes receptor internalization, although at concentrations greater than those required for receptor activation and desensitization. These results are distinctly different from those observed with megakaryoblastic cell lines, where >90% of the activated thrombin receptors are internalized rapidly, up to 40% of the cleaved receptors are recycled, and no intracellular pool of intact receptors has been detected. Since the primary structure of the thrombin receptor is the same in all the cell types studied, these results demonstrate that there can be substantial differences between cell types in the mechanisms involved in the clearance of activated receptors and the re-expression on the cell surface of intact receptors capable of responding to thrombin.


INTRODUCTION

Thrombin is a potent agonist for a number of cells including endothelial cells, platelets, vascular smooth muscle cells, and fibroblasts. In all of these cells, thrombin elicits responses by cleaving the N terminus of a high affinity G protein-coupled receptor (1, 2) . To date, only a single subtype of the thrombin receptor has been detected and cloning studies suggest that its primary structure is identical in different cells within a given animal species. Thrombin is thought to bind to the human form of the receptor via its anion-binding exosite, cleaving the receptor between residues Argand Serand exposing a new N terminus that serves as a tethered ligand (1, 2) . Peptides corresponding to at least the first five residues of the tethered ligand sequence (SFLLR) can, when added to thrombin-responsive cells, elicit many of the effects of thrombin. In the case of endothelial cells, such effects include activation of phospholipases Aand C (3, 4, 5) , and regulation of cAMP formation.()

Even before the sequence and mechanism of activation of the thrombin receptor had been determined, it had been noted in a variety of cell types that thrombin responses are subject to homologous desensitization (4, 6, 7, 8, 9) . In the megakaryoblastic HEL and CHRF-288 cell lines, desensitization occurs whether the receptors are activated proteolytically with thrombin or non-proteolytically with an agonist peptide, such as SFLLRN, and activation by either agonist prevents a subsequent response to the other (9, 10) . As with other G protein-coupled receptors, desensitization of signaling through thrombin receptors is thought to be due in part to receptor phosphorylation by a member of the receptor kinase family, such as -adrenergic receptor kinase 2, a kinase that has been shown to phosphorylate thrombin receptors when co-expressed in Xenopus oocytes (11) . In addition, thrombin receptors that have been activated by thrombin are left with a cleaved N terminus which, under most circumstances, precludes a second response to thrombin (10, 12) . Activated thrombin receptors are also subject to sequestration into intracellular compartments, irrespective of whether they were activated by thrombin or an agonist peptide. In the case of HEL and CHRF-288 cells, >90% of the thrombin receptors are cleared from the cell surface via coated pits within 5 min of activation (13) . Once internalized, most of the receptors are routed through endosomes to lysosomes, ultimately to be degraded. However, a substantial population (25-40%) returns to the cell surface. These ``recycled'' receptors are no longer desensitized, suggesting that they have been dephosphorylated by endosomal phosphatases, although this has not been demonstrated. Lacking an intact N terminus, they can be activated by SFLLRN, but not by thrombin (13) . Full recovery of the thrombin response requires the synthesis and expression on the cell surface of new receptors over a period of many hours.

This model of receptor desensitization, internalization, and recycling is based upon studies in megakaryoblastic cell lines. That it may not be universal is suggested by studies with human thrombin receptors over-expressed in Rat-1 fibroblasts, in which the loss of receptors from the cell surface was incomplete (14) . In the present studies, we have examined the fate of activated thrombin receptors on early passage human umbilical vein endothelial cells (HUVEC),() non-transformed cells that are exposed to thrombin under normal physiological conditions. The results demonstrate that all of the thrombin receptors on endothelial cells are cleaved upon thrombin addition. However, although many of the cleaved receptors are internalized rapidly, the remainder are internalized slowly, in sharp contrast to the megakaryoblastic cell lines. Furthermore, the reappearance of thrombin-responsive receptors on the surface of endothelial cells is detectable within 30 min and is due to the presence within these cells of an intracellular reserve of intact receptors that is not present in the megakaryoblastic cell lines. The rate of receptor mobilization from this reserve is affected by the proliferative state of the endothelial cells, occurring more rapidly when the cells are confluent than when they are subconfluent.


EXPERIMENTAL PROCEDURES

Materials

The peptide-directed IgGmonoclonal antibodies SPAN12, WEDE15, and ATAP2 are directed against sequences within the N terminus of the human thrombin receptor (10) . Antibody SPAN12 recognizes an epitope that is lost when thrombin cleaves the receptor. Antibodies WEDE15 and ATAP2 recognize epitopes that are retained following receptor cleavage, allowing these antibodies to bind to both intact and cleaved receptors (13) . Monoclonal antibody SSA6 is directed against human glycoprotein IIIa (integrin ; Ref. 15). Antibody EH1, used as a isotype-matched negative control, is reactive with the HIV-1 Nef protein (13) . Highly purified human -thrombin (3000 units/mg) was provided by Dr. J. Fenton (New York State Department of Health, Albany, NY). Hirudin was obtained from Sigma or Calbiochem.

Culture of Endothelial Cells

Endothelial cells were isolated from human umbilical cord veins by the procedure of Jaffe et al. (16) and were cultured on fibronectin-coated dishes in complete medium, Medium 199 containing 10% fetal calf serum (Hyclone), 100 units/ml penicillin, 100 µg/ml streptomycin, 0.6 mM glutamine, 12 units/ml heparin, and 200 µg/ml crude endothelial cell growth factor at 37 °C under 5% CO. The cells were routinely passaged with trypsin-EDTA and used for experiments at passages 1-6. Since trypsin can cleave thrombin receptors (1) , exposure of the cells to trypsin was avoided for at least 2 days prior to the experiments.

Cytosolic Calcium Measurements

Cells were washed twice with phosphate-buffered saline, then loaded with 5 µM Fura-2/AM (Molecular Probes) in phenol red-free RPMI 1640 medium for 60 min at 37 °C. The endothelial cells were released from culture dishes by incubation at 37 °C for 5-15 min in phosphate-buffered saline containing 5 mM EGTA and 1 mM EDTA. The cells were resuspended at 1 10cells/ml in phenol red-free RPMI 1640 medium and allowed to equilibrate 30 min, washed, and resuspended in fresh medium at 2 10cells/ml. Fluorescence was detected in a SLM/Aminco AB2 spectrophotometer and approximate values for [Ca]were calculated using an assumed kof 224 nM.

Flow Cytometry

Cell suspensions (2 10cells/ml in RPMI 1640 medium) were prepared as described above. Samples were treated with agonists at 37 °C. Cells (2 10) were incubated with undiluted hybridoma supernatant or affinity purified monoclonal antibodies (10-20 µg/ml) for 30 min at 4 °C, washed with staining buffer (phosphate-buffered saline, 0.02% sodium azide, 0.1% bovine serum albumin, pH 7.4), then incubated with a 1:40 dilution of fluorescein isothiocyanate-labeled goat anti-mouse IgG (TAGO) for an additional 30 min at 4 °C. Cells were washed and resuspended in staining buffer. Antibody binding was analyzed on a FACScan flow cytometer (Becton Dickinson). When the monoclonal antibodies directed against the thrombin receptor were preincubated with the appropriate immunizing peptide, binding to HUVEC decreased to the level of the negative control as measured by flow cytometry. Preincubation of the antibodies with an irrelevant peptide had no effect upon their binding. For thrombin receptor recovery experiments, cell monolayers were treated with thrombin in RPMI, then allowed to recover for different periods of time in complete medium containing hirudin. Cell suspensions were then prepared, incubated with antibodies, and processed for flow cytometry as described above.

Immunofluorescence

The endothelial cells were cultured on fibronectin-coated 35-mm dishes. For surface staining of intact cells, cell monolayers were washed in Medium 199, incubated in the presence or absence of agonist 10 min at 37 °C, placed on ice, and washed with ice-cold staining buffer. Cells were incubated with undiluted hybridoma supernatant or affinity purified monoclonal antibodies (10-20 µg/ml) for 30 min at 4 °C, washed with staining buffer, then incubated with 1:100 dilution rhodamine-labeled donkey anti-mouse IgG (Jackson Immunoresearch Laboratories) for 30 min at 4 °C. Cell monolayers were washed with staining buffer, fixed in 1% formalin (Polysciences, Inc.) for 10 min, washed with staining buffer, then distilled water, and coverslips mounted with Elvanol.


RESULTS

As has also been noted by other investigators (4, 17) , the addition of thrombin to human umbilical vein endothelial cells caused a rapid, but transient increase in the cytosolic free Caconcentration (Fig. 1 A). When maximally-effective thrombin concentrations were used, the initial increase in the intracellular calcium concentration subsided after 3-4 min, approaching a plateau somewhat greater than baseline. A second addition of thrombin after the initial spike produced no response. However, the addition of SFLLRN after the initial thrombin spike resulted in a second transient increase in Cawhose magnitude was 50% of the initial response, even when the initial thrombin concentration was more than sufficient to cleave all of the receptors on the cell surface (see below). Fig. 1 B shows the response of endothelial cells to SFLLRN. Like thrombin, SFLLRN caused a transient increase in the cytosolic Caconcentration and a second addition of SFLLRN had little effect. Addition of thrombin after SFLLRN also had no effect unless the cells were washed to removed the agonist peptide, in which case a diminished second response to both thrombin (Fig. 1 C) and SFLLRN (not shown) was observed. The magnitude of this second response was similar to that seen when the cells were initially exposed to thrombin and then restimulated with SFLLRN (Fig. 1 A).


Figure 1: Desensitization of the Caresponse in endothelial cells. Early passage human umbilical vein endothelial cells were loaded with Fura-2/AM, incubated in the absence ( A and B) or presence of 100 µM SFLLRN ( C), washed briefly, and then stimulated with thrombin (5 units/ml) or SFLLRN (100 µM), as indicated. These results are representative of two studies.



These results support observations made in other cell systems that thrombin receptor desensitization follows receptor activation by either thrombin or SFLLRN and, therefore, does not require receptor cleavage. However, the capability, although attenuated, of thrombin-activated cells to respond to SFLLRN and SFLLRN-activated cells to respond to both SFLLRN and thrombin after washing suggests that some of the receptors were either never desensitized or were rapidly resensitized after an initial desensitization. This result is consistent with studies of transfected Rat-1 fibroblasts, but is distinctly different from the results obtained with the megakaryoblastic cell lines where the cross-desensitization between thrombin and SFLLRN is essentially complete (10, 13, 14) .

Receptor Cleavage and Internalization

Three monoclonal antibodies directed at defined epitopes within the thrombin receptor N terminus (Fig. 2) were used to discriminate intact from cleaved receptors and to examine the fate of activated receptors on endothelial cells in greater detail. Antibody SPAN12, which was prepared against a peptide whose sequence spans the receptor cleavage site, binds only to intact receptors. Antibody ATAP2, which was prepared against a peptide analogous to the tethered ligand domain, binds to cleaved as well as intact receptors, as does antibody WEDE15, whose epitope includes the portion of the receptor that interacts with thrombin's anion-binding exosite. Flow cytometry was used to detect antibody binding. In the studies shown in Fig. 3 A, endothelial cells were incubated with different concentrations of thrombin for 10 min, after which the thrombin was inactivated with an excess of hirudin. Binding of the cleavage-sensitive antibody SPAN12 decreased as the thrombin concentration increased, with little binding detected at thrombin concentrations 0.3 units/ml. Exposure of the endothelial cells to increasing concentrations of thrombin also caused diminished binding of the cleavage-insensitive antibodies, ATAP2 and WEDE15. However, in contrast to the results with antibody SPAN12, the binding of antibodies WEDE15 (Fig. 3 A) and ATAP2 (not shown) plateaued at 40-50% of control values at thrombin concentrations 1 unit/ml. The loss of SPAN12 binding sites was complete within 3 min, while the decrease in WEDE15 binding sites was complete after 10 min (Fig. 3 B). These results suggest that thrombin rapidly cleaves essentially all of its receptors on the surface of endothelial cells, after which approximately half of the cleaved receptors are internalized.


Figure 2: N terminus of the human thrombin receptor. The approximate location of the epitope for each of the monoclonal antibodies used in the present studies is indicated by the antibody designations. The solid bars indicate the peptides used for the production of the corresponding antibody. Antibody SPAN12 binds only to intact receptors. Antibodies ATAP2 and WEDE15 bind to both intact and cleaved receptors.




Figure 3: Loss of binding sites for thrombin receptor antibodies following incubation with thrombin. A, the binding of antibodies WEDE15 and SPAN12 to HUVEC was measured by flow cytometry after incubating the cells for 10 min with thrombin and then adding 5-fold excess hirudin. The results shown are the mean ± S.E. of four studies and are expressed as percentage of the mean fluorescence obtained in the absence of thrombin. B, the endothelial cells were incubated with thrombin (1 unit/ml) for up to 30 min, after which hirudin (5 units/ml) was added. The results shown are the mean ± S.E. of three studies.



Similar studies were performed with SFLLRN to see whether the loss of receptors requires only activation, as opposed to proteolysis. The results are shown in Fig. 4. Like thrombin, SFLLRN caused a time- and concentration-dependent loss of antibody binding sites. As would be expected given the peptide's ability to activate the receptor without cleaving it, there was a comparable loss of binding sites for antibodies WEDE15 (Fig. 4) and SPAN12 (not shown). Both SFLLRN and thrombin caused a decrease in WEDE15 binding over a similar time course, but only at concentrations of SFLLRN 2 mM were the majority of the thrombin receptors lost from the cell surface. In contrast, the Caresponse to SFLLRN was maximal at 100 µM SFLLRN (not shown). This suggests that SFLLRN, in contrast to thrombin, is less effective at causing receptor internalization than it is at causing receptor activation.


Figure 4: Loss of binding sites for thrombin receptor antibodies following incubation with SFLLRN. A, the binding of antibody WEDE15 was measured after incubating endothelial cells with SFLLRN for 15 min. The results shown are the mean ± S.E. of three studies. B, the time course of the loss of WEDE15 binding sites following the addition of SFLLRN. The results shown are the mean ± S.E. of five studies.



Restoration of Thrombin Receptors on the Cell Surface

In order to study the recovery of thrombin receptors on the cell surface following activation, endothelial cells were incubated with 1 unit/ml thrombin for 10 min, sufficient to cleave all of the receptors, after which the medium was removed and replaced with fresh medium containing excess hirudin. At intervals up to 5 h, the total number of receptors on the cell surface was determined with antibody WEDE15 and the number of intact receptors was determined with antibody SPAN12. The difference between the total and intact receptor numbers reflects the number of cleaved receptors remaining on the cell surface at any given time. The studies shown in Fig. 5 A were performed with confluent monolayers of endothelial cells. At the end of the 10-min incubation with thrombin, SPAN12 binding was 10% of control and WEDE15 binding was 40% of control. By 30 min there was a detectable increase in the number of SPAN12 binding sites, which rose to 60% of control by 2 h and 90% of control by 5 h. This process was unaffected by the protein synthesis inhibitor, cycloheximide (not shown). Binding sites for antibody WEDE15 also increased from 40% relative to control immediately after thrombin exposure to 90% of control by 5 h. As new receptors appeared on the cell surface, the number of cleaved receptors gradually decreased and was undetectable by 5 h (Fig. 5 C).


Figure 5: Recovery of antibody binding sites after exposure to thrombin. Confluent ( A) or subconfluent ( B) endothelial cells were incubated with thrombin for 10 min, after which the protease was removed. The data show the recovery of binding sites for antibodies WEDE15 and SPAN12 over the next 5 h expressed as a percentage of antibody binding to cells not exposed to thrombin. The results are the mean ± S.E. of four ( A) and three ( B) studies. Part C shows the number of cleaved receptors remaining on the cell surface calculated as the difference between the results obtained with antibodies WEDE15 (total receptors) and SPAN12 (intact receptors).



Interestingly, when these studies were repeated with subconfluent endothelial cells (70-80% coverage), two notable differences were observed. First, although the kinetics of cleavage and initial clearance of receptors from the cell surface appeared unaffected by cell density, the reappearance of intact receptors on the cell surface, as determined by antibody SPAN12 binding, occurred more slowly on the subconfluent cells (Fig. 5 B). Also, in contrast to the confluent cells, the number of cleaved receptors detected on the cell surface after the 10-min thrombin incubation was constant over the 5-h period of observation (Fig. 5 C). This suggests that subconfluent cells are either less capable of clearing the residual cleaved receptors remaining on the cell surface or can recycle cleaved receptors at a rate equivalent to receptor internalization during the second phase of receptor clearance. This also suggests that either the proliferative status of the endothelial cells or the presence of uniform cell contacts within an intact monolayer can affect the movement of cleaved receptors to and from the cell surface after thrombin treatment.

Finally, to determine whether the newly restored receptors were functional, endothelial cells that had been transiently exposed to thrombin were re-stimulated with thrombin and changes in the cytosolic free Caconcentration were measured. The results are shown in Fig. 6, expressed as a percentage of the initial response to thrombin. The recovery of a thrombin response could be detected as early as 30 min after exposure to thrombin. By 2 h the magnitude of the Caresponse reached approximately 60% of control.


Figure 6: Recovery of Caresponse after exposure to thrombin. Endothelial cells loaded with Fura-2/AM were exposed to thrombin (1 unit/ml) for 10 min, hirudin (5 units/ml) was added, and the incubation was continued. The cells were re-stimulated with thrombin (6 units/ml) at the time points indicated. The results shown are the mean ± S.E. from three studies expressed as a fraction of the Caresponse obtained in cells not exposed to thrombin.



Receptor Localization

Fluorescence microscopy was used to complement the results obtained by flow cytometry and examine the distribution of thrombin receptors in resting and activated endothelial cells. Intact unstimulated cells exhibited a uniform punctate staining with antibodies SPAN12 and ATAP2 (Fig. 7, A and D). Exposure to thrombin for 10 min abolished the binding of SPAN12 (Fig. 7 E). Exposure to thrombin also caused a decrease in the binding of antibody ATAP2 (Fig. 7 B). Note that this figure tends to underrepresent the amount of cleaved receptor remaining on the cell surface following exposure to thrombin. A more quantitative measure is provided by the flow cytometry data presented in Fig. 3. Two h after the thrombin was removed there was substantial recovery in the binding of both antibodies (Fig. 7, C and F). Binding of a control antibody SSA6, directed against the integrin, glycoprotein IIIa, was unchanged following exposure of the endothelial cells to thrombin (Fig. 7, G and H) and there was negligible staining by secondary antibody alone (Fig. 7 I). In preliminary studies with permeabilized cells, staining of small intracellular vesicles with antibody ATAP2, but not with antibody SPAN12, was seen 10 min after the addition of thrombin (not shown). The presence of cleaved thrombin receptors in vesicles has been demonstrated in thrombin-treated HEL and CHRF-288 cells as well as in transfected Rat-1 cells and an endothelial cell line (10, 13, 18) . Thrombin and transferrin receptor colocalization studies performed with the megakaryoblastic cell lines have shown the intracellular structures to be endosomes, and this is presumably also the case in endothelial cells (13) .


Figure 7: Immunofluorescent detection of the thrombin receptor on intact, naive, or thrombin-treated HUVEC. Panels A-C show HUVEC that were stained with antibody ATAP2: control ( A), after 10 min incubation with thrombin ( B) or 2 h after a 10-min exposure to thrombin ( C). Panels D-F show cells prepared the same way, but stained with antibody SPAN12. Panels G and H show control and thrombin-treated endothelial cells, respectively, that were stained with antibody SSA6 directed against the chain of the endothelial cell vitronectin receptor (). Panel I shows the result obtained in the absence of the primary antibody. The results shown are representative of seven such studies.




DISCUSSION

Unlike other G protein-coupled receptors, the activation of thrombin receptors by thrombin is essentially a ``one time'' event since cleaved thrombin receptors are normally unable to respond to thrombin a second time (1, 9) . For a cell to recover responsiveness to thrombin, new receptors must be expressed on the cell surface. This process is presumably initiated by receptor activation. In the present studies, we have examined the clearance and replacement of activated thrombin receptors on human umbilical vein endothelial cells. Endothelial cells were of particular interest for several reasons. As with platelets, thrombin is a biologically important agonist for endothelial cells, stimulating the synthesis and release of vasoactive compounds such as prostaglandin Iand NO (4, 19, 20) . However, in contrast to platelets, endothelial cells may be exposed to thrombin repeatedly during their life cycle. Also, until recently most of the information available about the trafficking of thrombin receptors has been obtained with megakaryoblastic cell lines or with epitope-tagged thrombin receptors expressed in cell lines. HUVEC provided an opportunity to examine the behavior of endogenous thrombin receptors in early passage, non-transformed cells.

The results that were obtained demonstrate some notable differences between endothelial cells, the megakaryoblastic cell lines, and platelets. In HEL and CHRF-288 cells, receptor cleavage is followed by rapid internalization of essentially all of the receptors and recycling of up to 40%, none of which can be re-activated by thrombin. Recovery of thrombin-responsive receptors is dependent upon protein synthesis and requires up to 16 h for completion. On platelets, thrombin receptors are cleaved and internalized, but not replaced.() In contrast, in this study we observed that cleaved thrombin receptors on endothelial cells are cleared in a biphasic manner, the first phase involving the rapid clearance of 50-60% of the receptors within 10 min, the second resulting in the removal of the remaining cleaved receptors over a 5-h period. The reappearance of intact receptors on the cell surface was detectable within 30 min, reached 60% by 2 h and 90% by 5 h, and was accompanied by a recovery of thrombin responsiveness. This process was independent of protein synthesis and appears to be due to an intracellular pool of intact receptors that can be mobilized to the cell surface (Fig. 8).


Figure 8: A model for thrombin receptor clearance and replacement on human umbilical vein endothelial cells. In endothelial cells, cleaved thrombin receptors are cleared from the cell surface in a two-phase process that is complete within a few hours. Replacement of the cleaved receptors with intact receptors initially occurs from an intracellular reserve of receptors. Recycling of cleaved receptors has not been resolved, but some of the cleaved receptors that are not initially internalized appear to retain or recover the ability to be activated a second time by SFLLRN.



These observations raise several issues. First, despite the marked differences in events following thrombin receptor activation in endothelial cells, platelets, and HEL cells, the primary structure and activation mechanism of the thrombin receptor is apparently the same in all the cells (1, 13, 21) . This implies that cell-specific factors unrelated to the thrombin receptor sequence dictate the manner in which cleaved thrombin receptors are cleared and replaced, although what these factors are remains to be determined. Phosphorylation by members of the G protein-coupled receptor kinase family, which plays an important role in receptor desensitization, does not appear to be required for G protein-coupled receptor internalization (22) . Recent studies have implicated a conserved P XXY sequence near the beginning of the cytoplasmic tail of many G protein-coupled receptors in receptor internalization and have shown that swapping a proline-rich domain in the third cytoplasmic loop of the - and -adrenergic receptors results in a switch in their patterns of internalization (23, 24) . At present, little is known about the structural features involved in thrombin receptor internalization beyond the presence of a PLIY sequence at the end of the last transmembrane domain, and nothing is known about proteins that might directly interact with the receptor causing it to be internalized.

In endothelial cells, two mechanisms of internalization appear to coexist, one of which is rapid and becomes saturated when only 60% of the receptors have been internalized, while the second is responsible for the much slower clearance of the remaining receptors. Rapid partial receptor clearance has also been observed in Rat-1 cells transfected with epitope-tagged thrombin receptors (11, 14, 18) . In HEL and CHRF-288 cells, a substantial population of cleaved receptors recycles back to the cell surface. The extremely rapid and essentially complete internalization of activated thrombin receptors in these cells may be responsible in part for the recycling of cleaved receptors because the very large receptor load may overwhelm the sorting mechanisms in the endosomes, a hypothesis that remains to be tested. A biological role for the recycled cleaved receptors has not been identified. Since we never observed an increase in the number of cleaved receptors on the endothelial cell surface, we could not establish that thrombin receptor recycling occurs in endothelial cells. However, it is theoretically possible that the slower phase of receptor removal reflects the difference in rate between continued removal and receptor recycling, which would not be detected by the methods that were used.

One factor that appears to affect the rate of thrombin receptor clearance and recovery on HUVEC was cell density. The rate of recovery of intact receptors on the cell surface was faster in confluent cultures when compared to subconfluent cultures, as was the rate of clearance of the cleaved receptors during the second, slower phase of receptor internalization (Fig. 5). Endothelial cells normally exist in vivo as a confluent, quiescent monolayer unless there is damage to the vascular wall, caused either by local pathology or a therapeutic intervention such as angioplasty. Based on our data obtained in vitro, there may be a slower recovery of thrombin responsiveness under conditions in which the endothelial lining of the vascular wall is disrupted. Whether these differences actually occur in vivo and whether they apply to arterial, as well as venous, endothelial cells will have to be determined.

A third issue is the extent to which activated thrombin receptors on endothelial cells become refractory to further stimulation. The refractoriness of activated thrombin receptors to reactivation by thrombin is thought to be due to two events: phosphorylation of sites in the cytoplasmic domains of the receptor (1, 11) and cleavage of the receptor N terminus. Cleavage prevents thrombin from activating the receptor a second time, but does not inhibit responses to SFLLRN. Phosphorylation would be expected to prevent responses to both. When endothelial cells were incubated with thrombin at concentrations sufficient to cleave all of the cell surface receptors, they were unable to respond a second time to thrombin, but showed a continued, although diminished, responsiveness to SFLLRN. Conversely, when the cells were initially activated by SFLLRN they showed a diminished response to both thrombin and SFLLRN, but in order to see this response it was necessary to wash out the peptide. Since SFLLRN does not inhibit receptor cleavage by thrombin, several possible mechanisms might account for these observations. The first possibility is that although thrombin may cleave and activate all of its receptors, it may not desensitize all of them, in which case those remaining on the cell surface can respond to SFLLRN even when a cleaved N terminus precludes a second response to thrombin. The second possibility is that all of the receptors have, in fact, initially been desensitized, but some of the receptors remaining on the cell surface have become resensitized, perhaps after dephosphorylation. A third possibility, that the second response is due to expression of new receptors from the internal pool, seems less likely in view of the short time between stimuli and the failure of the cells to respond a second time to thrombin after initially being exposed to thrombin. What is less clear is the biological relevance of the continued response since it isn't seen with further additions of thrombin. Theoretically, either the tethered ligand itself or a locally generated homologous peptide could cause receptor reactivation, but whether this occurs in vivo remains to be demonstrated.

Finally, the endothelial cells were able to restore intact thrombin receptors to the cell surface at a rate far faster than HEL or CHRF-288 cells. Following a transient exposure to thrombin, new receptors appeared almost immediately and a nearly full complement of receptors was restored within a few hours. Since this process occurred independently of protein synthesis, it implies that endothelial cells possess an intracellular pool of intact receptors that can rapidly replace those cleaved by thrombin (Fig. 8). This pool appears to contain at least as many receptors as are initially present on the cell surface, allowing endothelial cells to quickly recover following exposure to thrombin. In addition to our data, two sets of observations from other investigators appear to support this conclusion. In the study by Hein and co-workers (18) , fibroblasts expressing epitope-tagged human thrombin receptors were found to contain an intracellular receptor pool, visible as immunoreactivity in the perinuclear space that appears similar to observations we have made. Corroborative studies with an endothelial cell line suggested that a similar pool was also present in those cells as well. Functional studies with the endothelial cell line were also presented in that study and are consistent with our findings with human umbilical vein endothelial cells. In the second set of studies, Horvat and Palade (25) stained permeabilized endothelial cells with receptor-directed antibodies and noted a large number of intracellular thrombin receptors associated with a branching tubulovesicular network. They concluded that most of the thrombin receptors in resting endothelial cells are intracellular, rather than on the cell surface. Of note, their localization of thrombin receptors on the branching tubulovesicular network appears different from the perinuclear staining observed by either Hein et al. (18) or by us. The reason for this difference is currently unknown.

In conclusion, these results demonstrate that there are substantial differences in the regulation of thrombin receptors in different cells. Biologically, this may make a great deal of sense. Platelets are activated by thrombin, participate in the formation of the hemostatic plug, and then have no further need (that has been identified) to be activated by thrombin. Endothelial cells, on the other hand, do not typically undergo irreversible changes in response to local thrombin generation. Given their need to be able to synthesize and release vasoactive products, it is reasonable that they would have a mechanism for rapidly restoring thrombin responsiveness once the initial thrombin-generating event has passed. Replacement of cleaved receptors with intact receptors from an existing pool provides one mechanism for accomplishing this.


FOOTNOTES

*
This work was supported in part by National Institutes of Health Grants HL40387 and HL49987. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked `` advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Dept. of Physiology, Thomas Jefferson University, 1020 Locust St., Philadelphia, PA 19107. Tel.: 215-955-6715; Fax: 215-955-2073.

M. J. Woolkalis and L. F. Brass, manuscript in preparation.

The abbreviation used is: HUVEC, human umbilical vein endothelial cells.

M. Molino and L. F. Brass, manuscript in preparation.


ACKNOWLEDGEMENTS

We thank Dr. A. Sue Menko for the use of the Nikon microscope for the indirect immunofluorescence studies.


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