Expression and Purification of Protease-Activated Receptor 4 (PAR4) and Analysis with Histidine Hydrogen-Deuterium Exchange.

Protease-activated receptors (PARs) are G-protein-coupled receptors that are activated by proteolysis of the N-terminus, which exposes a tethered ligand that interacts with the receptor. Numerous studies have focused on the signaling pathways mediated by PARs. However, the structural basis for initiation of these pathways is unknown. Here, we describe a strategy for the expression and purification of PAR4. This is the first PAR family member to be isolated without stabilizing modifications for biophysical studies. We monitored PAR4 activation with histidine hydrogen-deuterium exchange. PAR4 has nine histidines that are spaced throughout the protein, allowing a global view of solvent accessible and nonaccessible regions. Peptides containing each of the nine His residues were used to determine the t1/2 for each His residue in apo or thrombin-activated PAR4. The thrombin-cleaved PAR4 exhibited a 2-fold increase (p > 0.01) in t1/2 values observed for four histidine residues (His180, His229, His240, and His380), demonstrating that these regions have decreased solvent accessibility upon thrombin treatment. In agreement, thrombin-cleaved PAR4 also was resistant to thermolysin digestion. In contrast, the rate of thermolysin proteolysis following stimulation with the PAR4 activation peptide was the same as that of unstimulated PAR4. Further analysis showed the C-terminus is protected in thrombin-activated PAR4 compared to uncleaved or agonist peptide-treated PAR4. The studies described here are the first to examine the tethered ligand activation mechanism for a PAR family member biophysically and shed light on the overall conformational changes that follow activation of PARs by a protease.

Protease-activated receptors are potential regulators in the development of arterial endofibrosis in high-performance athletes.

OBJECTIVE: High-performance athletes can develop symptomatic arterial flow restriction during exercise caused by endofibrosis. The pathogenesis is poorly understood; however, coagulation enzymes, such as tissue factor (TF) and coagulation factor Xa, might contribute to the fibrotic process, which is mainly regulated through activation of protease-activated receptors (PARs). Therefore, the aim of this explorative study was to evaluate the presence of coagulation factors and PARs in endofibrotic tissue, which might be indicative of their potential role in the natural development of endofibrosis. METHODS: External iliac arterial specimens with endofibrosis (n = 19) were collected during surgical interventions. As control, arterial segments of the external iliac artery (n = 20) were collected post mortem from individuals with no medical history of cardiovascular disease who donated their body to medical science. Arteries were paraffinized and cut in tissue sections for immunohistochemical analysis. Positive staining within lesions was determined with ImageJ software (National Institutes of Health, Bethesda, Md). RESULTS: Endofibrotic segments contained a neointima, causing intraluminal stenosis, which was highly positive for collagen (+150%; P < .01) and elastin (+148%; P < .01) in comparison with controls. Intriguingly, endofibrosis was not limited to the intima because collagen (+213%) and elastin (+215%) were also significantly elevated in the media layer of endofibrotic segments. These findings were accompanied by significantly increased α-smooth muscle actin-positive cells, morphologically compatible with the presence of myofibroblasts. In addition, PAR1 and PAR4 and the membrane receptor TF were increased as well as coagulation factor X. CONCLUSIONS: We showed that myofibroblasts and the accompanying collagen and elastin synthesis might be key factors in the development of endofibrosis. The special association with increased presence of PARs, factor X, and TF suggests that protease-mediated cell signaling could be a contributing component in the mechanisms leading to endofibrosis.

The decreased expression of protease-activated receptor 4 in esophageal squamous carcinoma.

Protease-activated receptors (PARs) are a unique family of G-protein coupled receptors. PAR4, a member of PARs family, was reported to be related to the development of cancers. Whether PAR4 plays a role in the progress of esophageal squamous cancer is unknown. In this study, differential expression of PAR4 in esophageal squamous cancer was measured by real-time PCR (n = 28), western blot and tissue microarrays (n = 78). The results showed that PAR4 expression was remarkably decreased in esophageal squamous cancer tissues compared with the matched noncancerous tissues, especially in low differentiation and positive distant metastasis carcinoma tissues. Furthermore, the methylation level of PAR4 promoter in esophageal cancer cells and normal epithelial cells was determined. Human esophageal cancer cells TE-1 displayed significant hypermethylation of 19 CpG sites, but pronounced hypomethylation of the sites in esophageal epithelial cells HEEpiC. The results suggested that down-regulation expression of PAR4 occurs frequently in esophageal squamous cancers, and the loss of PAR4 expression may partly result from hypermethylation of the PAR4 promoter. That PAR4 expression difference in tumor progression possibly makes PAR4 become a molecular mark of tumor diagnosis.

Thrombin receptor levels in platelet concentrates during storage and their impact on platelet functionality.

BACKGROUND: Quality control of platelet (PLT) concentrates is challenging, due to PLT lesions, which are difficult to detect with routine methods. The search for reliable PLT lesion biomarkers is focused on the role of PLTs in primary hemostasis. PLT transfusions also have a significant impact on secondary hemostasis. In this phase, responsiveness of PLTs to small amounts of thrombin is crucial. PAR1 and PAR4 are protease-activated receptors and are responsible for thrombin reactivity of human PLTs. This study should elucidate if levels of those two receptors are changing in PLT concentrates during storage and if those changes have an impact on PLT aggregation and support of thrombin generation. STUDY DESIGN AND METHODS: PLT concentrates from buffy coat preparations were stored in SSP+ solution for 9 days at 22±2°C on a horizontal flatbed agitator, and samples were taken daily for analysis. PAR1 and PAR4 levels were evaluated using Western blot analysis. PLT aggregation was measured using Born aggregometry and specific PAR1 or PAR4 agonists. Thrombin generation was measured using calibrated automated thrombography. RESULTS: Levels of both receptors (PAR1 and PAR4) started to decrease after 5 days of storage. PAR1-mediated PLT aggregation remained constant, whereas PAR4-mediated PLT aggregation decreased with storage time. Rate of thrombin generation was accelerated after 5 days of storage. CONCLUSION: Decreasing levels of PARs in PLT concentrates after 5 days of storage influenced PAR4-mediated, but not PAR1-mediated, aggregation. Thrombin generation with senescent PLTs was increased, which may be attributed to other mechanisms promoting increased phosphatidylserine exposure.

Acute hypoxia and osteoclast activity: a balance between enhanced resorption and increased apoptosis.

Osteoclasts are the primary mediators of pathological bone resorption in many conditions in which micro-environmental hypoxia is associated with disease progression. However, effects of hypoxia on human osteoclast activity have not been reported. Mature human osteoclasts were differentiated from peripheral blood or obtained from giant cell tumour of bone. Osteoclasts were exposed to a constant hypoxic environment and then assessed for parameters including resorption (toluidine blue staining of dentine slices), membrane integrity (trypan blue exclusion), apoptosis (TUNEL, DAPI), and osteolysis-associated enzyme activity (TRAP, cathepsin K). 24 h exposure to 2% O(2) produced a 2.5-fold increase in resorption associated with increased TRAP and cathepsin K enzyme activity. Hypoxia-Inducible Factor-1alpha (HIF-1alpha) siRNA completely ablated the hypoxic increase in osteoclast resorption. 24 h at 2% O(2) also increased the number of osteoclasts with compromised membrane integrity from 6% to 21%, with no change in the total osteoclast number or the proportion of late-stage apoptotic cells. Transient reoxygenation returned the percentage of trypan blue-positive cells to normoxic levels, suggesting that osteoclasts can recover from the early stages of cell death. Repeated over an extended period, hypoxia/reoxygenation enhanced osteoclast differentiation at this pO(2). These data suggest that in diseased bone, where the pO(2) may fall to

Solid tumor cells express functional "tethered ligand" thrombin receptor.

Previous work demonstrated that alpha-thrombin promoted tumor cell adhesion to endothelium and extracellular matrix as well as enhanced the metastatic capacity of tumor cells. This study was initiated to investigate whether the thrombin effect on tumor cells is mediated through the "tethered ligand" thrombin receptor. RT-PCR analysis using primers based on the human thrombin receptors detected mRNA in human colon adenocarcinoma cells (clone A), whose authenticity was confirmed by Southern hybridization. The presence of thrombin receptor mRNA in rat (W256 carcinosarcoma) and mouse (melanoma) tumor cells was demonstrated by RT-PCR/Southern blotting using species-specific PCR primers. Sequencing of the PCR fragment of clone A cells revealed complete homology with the reported human cDNA sequence. Subsequently, tumor cells derived from three species, i.e., human, rat, and mouse, were found to express the thrombin receptor protein as revealed by immunoblotting using ligand peptide-derived mAb ATAP138, whose reactivity towards the M(r) approximately 66,000, potential thrombin receptor was blocked by preincubating the antibody with the immunogen peptide SFLLRNPNDKYEPF (TRP 14). Finally, peptides TRP 14 and TRP 7 (SFLLRNP), but not TRP 5 (FLLRN), were found to mimic alpha-thrombin in stimulating tumor cell adhesion to fibronectin, suggesting that the thrombin receptors expressed on solid tumor cells are biologically functional.

Analysis of the platelet-type thrombin receptor in 20 cases of large granular lymphocyte proliferations.

The platelet-type thrombin receptor was expressed by large granular lymphocytes (LGLs) in a variety of proliferative diseases. Twenty patients with LGL proliferative disease were examined, including five T cell clones and a variety of polyclonal proliferations, some secondary to rheumatoid arthritis and Felty's syndrome; 17/20 showed high number of CD3+, CD8+, and CD57+ lymphocytes and 9/20 also had high numbers of CD16+ or CD 56+ positive lymphocytes. The thrombin receptor was present on more than 20% of the LGLs in 13/20 patients. The clonal T cell expansions showed the highest receptor expression with greater than 75% cells positive. Regression analysis of all 20 cases showed striking and highly statistically significant positive Spearman rank correlation between the proportion of thrombin receptor and CD57-positive LGLs (r = 0.56, P = 0.009). A negative correlation with CD56 was also found (r = -0.46, P= 0.043). Dual antibody flow cytometry showed the receptor was more often co-expressed with CD57 (64%) than with CD16 (19%) or CD56 (11%). The expression of the platelet-type thrombin receptor by LGLs of this phenotype raises the possibility of a functional role for thrombin in the pathogenesis of LGL proliferative diseases.

Expression of and functional responses to protease-activated receptors on human eosinophils.

Eosinophil recruitment to airway tissue is a key feature of asthma, and release of a wide variety of toxic mediators from eosinophils leads to the tissue damage that is a hallmark of asthma pathology. Factors that control the release of these toxic mediators are targets for potential therapeutic intervention. Protease-activated receptors (PARs) are a novel class of receptors that are activated by cleavage of the N terminus of the receptor by proteases such as thrombin or trypsin-like enzymes. To date, PAR1-4 have been identified, and there are several studies that have demonstrated the expression of PARs in airway tissue, particularly the respiratory epithelium. We have investigated whether eosinophils express PARs and if activation of these receptors will then trigger a functional response. Using a combination of reverse transcriptase-polymerase chain reaction, Western blotting, and flow cytometry analysis, we have demonstrated that eosinophils express PAR1 and PAR2. FACS analysis showed that PAR1 could be clearly detected on the surface of the cells, whereas PAR2 appeared to be primarily intracellular. Trypsin and the PAR2 agonist peptide were seen in trigger shape change, release of cysteinyl leukotrienes, and most obviously, generation of reactive oxygen species. In contrast, thrombin had no effect on eosinophil function. The PAR1 agonist peptide did have a minor effect on eosinophil function, but this was most likely down to its ability to activate PAR1 and PAR2. These results demonstrate that PAR2 is the major PAR receptor that is capable of modulating eosinophil function.

Thrombomodulin and thrombin localization on the vascular endothelium; their internalization and transcytosis by plasmalemmal vesicles.

We have immunolocalized thrombomodulin (TM), (an endothelial cofactor protein for thrombin-mediated protein C activation), and its ligand thrombin (TH) by light- and electron microscopy on endothelial cells in culture and in situ. Our results show that: i) TM is expressed in small clusters on the basal and the apical surface of human umbilical vein endothelial cells (HUVEC), is absent from coated pits and coated vesicles, is internalized from the cell surface via plasmalemmal vesicles, and is associated with endosomes, including a tubulovesicular network within the cytoplasm; ii) exogenous TH binds at low surface density on HUVEC and colocalizes with TM in small TM clusters; iii) on the microvascular endothelium of heart, lung, and kidney, TM is expressed at high density on the luminal plasmalemma, is associated with a fraction of the plasmalemmal vesicle population; iv) and is found internalized in multivesicular bodies in TH perfused heart samples; v) in the same samples, perfused TH is detected at low surface density: on the luminal aspect of the endothelium within small TM clusters, in the introits and within plasmalemmal vesicles, in endosomal structures, on the abluminal plasmalemma, and within the pericapillary spaces. Our findings suggest that the high level of endothelial TM expression in situ provides a safety margin for random TH generation; they also demonstrate that bound TH is transcytosed across the endothelium by plasmalemmal vesicles which might represent an additional mechanism to remove TH from the circulation.

Protease-activated receptor 2, a receptor involved in melanosome transfer, is upregulated in human skin by ultraviolet irradiation.

Previous studies have shown that the protease-activated receptor 2 is involved in skin pigmentation through increased phagocytosis of melanosomes by keratinocytes. Ultraviolet irradiation is a potent stimulus for melanosome transfer. We show that protease-activated receptor 2 expression in human skin is upregulated by ultraviolet irradiation. Subjects with skin type I, II, or III were exposed to two or three minimal erythema doses of irradiation from a solar simulator. Biopsies were taken from nonexposed and irradiated skin 24 and 96 h after irradiation and protease-activated receptor 2 expression was detected using immunohistochemical staining. In nonirradiated skin, protease-activated receptor 2 expression was confined to keratinocytes in the lower one-third of the epidermis. After ultraviolet irradiation protease-activated receptor 2 expression was observed in keratinocytes in the upper two-thirds of the epidermis or the entire epidermis at both time points studied. Subjects with skin type I showed delayed upregulation of protease-activated receptor 2 expression, however, compared with subjects with skin types II and III. Irradiated cultured human keratinocytes showed upregulation in protease-activated receptor 2 expression as determined by immunofluorescence microscopy and Western blotting. Cell culture supernatants from irradiated keratinocytes also exhibited a dose-dependent increase in protease-activated receptor-2 cleavage activity. These results suggest an important role for protease-activated receptor-2 in pigmentation in vivo. Differences in protease-activated receptor 2 regulation in type I skin compared with skin types II and III suggest a potential mechanism for differences in tanning in subjects with different skin types.

Rat uterine stromal cells: thrombin receptor and growth stimulation by thrombin.

The estrogen-stimulated maturation of the immature rat uterus is mediated by peptide growth factors whose expression is regulated by estradiol. We present evidence that thrombin is a uterine growth factor. When an immature rat is given a single injection of estradiol, the uterus increases 50% in wet weight within 3 h through the imbibition of water and plasma proteins, including prothrombin. Tissue factor, the initiator of coagulation, is induced 3- to 4-fold over the same time period. Thrombin is generated in situ from prothrombin through the coagulation cascade. It acts as a growth factor through the proteolytically activated thrombin receptor. Thrombin's role as a growth factor in uterine stromal cells is proven by two lines of evidence: demonstrations that the proteolytically activated thrombin receptor is present and that cultured cells are stimulated to grow by thrombin. Thrombin receptor in the uterus is demonstrated by reverse transcription-PCR for receptor messenger RNA by specific [125I]peptide labeling of a membrane-bound binding protein of about 60 kDa and by Western blot with a thrombin receptor antipeptide antibody. Thrombin's effectiveness as a growth factor is shown by thrombin-stimulated growth of primary stromal cell cultures, with maximum stimulation at 100 nM. That the effect is mediated by the proteolytically activated thrombin receptor is shown by the inhibition of growth by hirudin, a highly specific inhibitor of thrombin; the absence of enhanced growth with Pro-Phe-Arg-chloromethyl ketone-thrombin, an active site-inhibited thrombin derivative; and the stimulation of growth by the thrombin receptor-activating peptide.

Effects of thrombin on steroid-modulated cultured endometrial stromal cell fibrinolytic potential.

By virtue of their unique chronic expression of tissue factor, the primary initiator of hemostasis, decidualized endometrial stromal cells are capable of significant thrombin generation after vascular disruption. In addition to its potent procoagulant effects, thrombin modifies endothelial and glomerular cell fibrinolytic activity. Therefore, we evaluated whether thrombin affected the expression of endometrial stromal cell urokinase-type (uPA) and tissue-type (tPA) plasminogen activators and their primary inhibitor, type 1 plasminogen activator inhibitor (PAI-1), and whether ovarian steroids modulated putative thrombin effects. Confluent stromal cell cultures were incubated in a defined medium containing vehicle control, 10(-8) mol/L estradiol (E2), 10(-7) mol/L medroxyprogesterone acetate (MPA), or E2 plus MPA for 4 days. The medium was then collected and exchanged for medium containing the corresponding steroids with or without thrombin and the specific thrombin inhibitor, D-phenyl-alanyl-propyl-arginine-chloromethyl ketone, for an additional 24 h. The conditioned medium was then collected and analyzed for immunoreactive (ir) uPA, tPA, and PAI-1 by enzyme-linked immunosorbent assay and for PA activity by chromogenic assay, whereas Northern analysis of the cells was employed to evaluate the expression of thrombin receptor, uPA, tPA, and PAI-1 messenger ribonucleic acid (mRNA) species. The latter studies revealed that confluent cultures incubated in defined medium expressed the 3.45-kilobase thrombin receptor message. Steady state levels of thrombin receptor mRNA were unaffected by exogenous steroids. Thrombin added in the absence of exogenous steroids elevated concentrations of ir tPA, uPA, and PAI-1 compared with control cultures. Conversely, in the absence of added thrombin, MPA added alone or together with E2 inhibited levels of ir tPA and uPA while stimulating PAI-1 levels despite the lack of a response to E2 alone. Interestingly, thrombin counteracted this progestin inhibition of tPA and uPA expression and augmented the progestin-enhanced expression of PAI-1. Northern analysis revealed that steady state levels of tPA and uPA mRNA were also enhanced by thrombin in both control and steroid-containing cultures. Net PA activity reflects the balance between PA and PAI-1. In the absence of thrombin, there is virtually no detectable tPA activity and minimal uPA activity in progestin-exposed cultures. However, thrombin elicited significant increases in tPA and uPA activity in control and E2-treated cultures. Despite the molar excess of PAI-1 in MPA-treated and E2- plus MPA-treated cultures, thrombin reversed progestin inhibition of PA activity. Predictably, the addition of D-phenyl-alanyl-propyl-arginine-chloromethyl ketone, blocked the effects of thrombin on PAI-1, tPA, and uPA protein and mRNA expression and PA activity. In summary, thrombin enhances endometrial stromal cell fibrinolytic and extracellular matrix-degrading protease activity in vitro. Such processes occurring in vivo would probably play a role in menstruation and abnormal uterine bleeding.

Evidence for functionally active protease-activated receptor-3 (PAR-3) in human vascular smooth muscle cells.

The present study investigates whether vascular smooth muscle cells of the human saphenous vein (SMC) express a functionally active protease-activated receptor-3 (PAR-3). PAR-3 mRNA was detected by RT-PCR. In the presence of thrombin, a rapid and transient increase in PAR-3 mRNA was observed. Stimulation of SMC with thrombin or the synthetic PAR-3-activating peptide, TFRGAP, resulted in transient mobilization of intracellular calcium. After a preceding challenge with thrombin, the calcium signal to TFRGAP was abolished, suggesting cleavage and subsequent desensitization of PAR-3 by thrombin. Activation of PAR-3 by TFRGAP elicited a time-dependent activation of the extracellular-signal-regulated kinase (ERK)-1/2 with a maximum response 10-20 min after stimulation. At 200 microM, TFRGAP increased [3H]-thymidine incorporation into cellular DNA about two-fold. These data indicate that PAR-3 is expressed in human SMC and triggers intracellular signaling. Thus, in the SMC PAR-3 might contribute to thrombin-induced responses.

Effect of protease-activated receptor (PAR)-1, -2 and -4-activating peptides, thrombin and trypsin in rat isolated airways.

Mechanisms of relaxation and contraction to protease-activated receptor- (PAR) tethered ligand peptides (SFLLRN/TFLLR, SLIGRL and GYPGKF (all C-terminally amidated) for PAR1, PAR2 and PAR4, respectively) and enzymes (thrombin and trypsin) were investigated in isolated segments of rat trachea, main and first order intrapulmonary bronchi. In airway segments previously exposed to SLIGRL, SFLLRN caused contractions that were potentiated by indomethacin, but were independent of mast cell degranulation. Contractions to TFLLR in the intrapulmonary bronchi were similarly potentiated by indomethacin. SLIGRL caused epithelium-dependent relaxations which were unaffected by N(G)-nitro-L-arginine, 1-H-oxodiazol-[1,2,4]-[4,3-a]quinoxaline-1-one or zinc-protoporphyrin-IX but were abolished by haemoglobin in all three regions of the airways. Relaxations to SLIGRL were markedly attenuated by indomethacin only in the main and intrapulmonary bronchi. GYPGKF caused epithelium-dependent relaxations in all three regions of the airway which were only significantly inhibited by indomethacin in the intrapulmonary bronchi. In general, thrombin and trypsin failed to cause any response in the airways tested. Intense PAR2-immunoreactivity was observed on airway epithelium. PAR1-immunoreactivity was faint on airway epithelium and smooth muscle, but was prevalent in mast cells. These findings indicate that PAR2 and possibly PAR4 present on rat airway epithelia mediate smooth muscle relaxation via cyclo-oxygenase-dependent and -independent mechanisms. PAR1-mediated contractions were most likely due to activation of smooth muscle receptors. The general failure of thrombin and trypsin to cause responses which may have been due to endogenous protease inhibitors, highlights the need for caution in assessing pathophysiological roles for PARs if only enzymes are used to activate PARs.

Tissue factor, plasminogen activator inhibitor-1, and thrombin receptor expression in human crescentic glomerulonephritis.

Glomerular fibrin deposition is a common histological feature of crescentic glomerulonephritis (CGN). Tissue factor (TF) is the most powerful activator of the coagulation system, whereas plasminogen activator inhibitor (PAI)-1 is a key modulator of the fibrinolytic pathway. Thrombin, released locally as the final step of the coagulation cascade and trapped within the fibrin clots, can induce the activation of glomerular cells, through the interaction with a specific receptor. To investigate the mechanisms underlying coagulation cascade activation and fibrin deposition and the role of this phenomenon in the pathogenesis of human CGN, TF, PAI-1, and thrombin receptor expression were studied in CGN biopsy specimens. Glomerular TF gene and protein expression were strikingly increased in CGN, in particular within the crescents and in the mesangial area, with the same distribution of fibrin deposits. Interestingly, very few infiltrating mononuclear cells were stained in TF immunohistochemistry. To better evaluate the involvement of monocytes in TF expression, TF mRNA and CD68 protein were studied by an in situ hybridization/immunohistochemistry combined technique. Only 16% of the cells expressing TF mRNA were CD68 positive. However, most of the TF signal was localized in the proximity of monocytes, suggesting that soluble mediator(s) released by these cells could induce TF expression. Indeed, interleukin-1 (IL-1), one of the main monocyte-derived cytokines, upregulated TF mRNA levels in cultured human mesangial cells in a time-dependent manner. Moreover, a striking increase in IL-1 expression was present within the cellular crescents in CGN biopsy specimens. Finally, we observed a marked upregulation of both PAI-1 and thrombin receptor mRNA levels in CGN with a pattern resembling TF and fibrin distribution. Surprisingly, thrombin receptor protein expression was strikingly downregulated in CGN, suggesting its continuous activation and degradation. In conclusion, we can hypothesize that TF and PAI-1, mainly expressed by resident cells, may play a pivotal role in the development and preservation of fibrin deposits in CGN. In addition, thrombin, released locally and accumulated within the fibrin clots, may represent a pathogenetic mediator of crescentic lesions.

Functional thrombin receptor PAR1 in primary cultures of human glioblastoma cells.

In this study we investigated primary cultures obtained from two glioblastomas surgically removed from a 64-year-old man and a 50-year-old woman, respectively. The presence of the tethered ligand thrombin receptor PAR1 (protease-activated receptor 1) in these cells was demonstrated at the level of receptor binding by using immunofluorescence studies with the monoclonal anti-PAR1 antibody Mab 31-2. Stimulation of human glioblastoma cells both with alpha-thrombin and the thrombin receptor activating peptide TRAP-6 resulted in a series of [Ca+]i spikes as shown by confocal laser fluorescence microscopy with fluo-3 as calcium sensitive fluorescence indicator. This effect was completely blocked with the thrombin receptor antagonist peptide T1. Our results demonstrate functional thrombin receptors (PAR1) in primary cultures of human glioblastomas for the first time.

Thrombin induced inhibition of neurite outgrowth from dorsal root ganglion neurons.

Thrombin is a multifunctional protease. Recent studies on cultured neuronal cells have suggested a function for thrombin in the development and maintenance of the nervous system. Thrombin has been found to induce neurite retraction and reverse stellation in neuroblastoma cell lines and rat astrocytes, respectively. The major focus of our study was to investigate the potential role of thrombin in peripheral nervous system development using the rat embryonic dorsal root ganglion model. We found a dose dependent inhibition of neurite outgrowth from explant dorsal root ganglion cultures upon exposure to 2 to 200 nM thrombin. This effect was reversed by the specific thrombin inhibitor, hirudin. A synthetic peptide that imitates the fully active receptor, thrombin receptor activating peptide, was also found to inhibit neurite outgrowth from dorsal root ganglia. bis-Benzimide stained neuronal cultures did not show any evidence of cell death after exposure to thrombin or thrombin receptor activating peptides. Immunohistochemical studies revealed specific staining of the thrombin receptor on neurons, with intense labeling along neurites. Enriched neuronal cultures exposed to thrombin and thrombin receptor activating peptides revealed rapid activation of phospholipase Cgamma-1, a second messenger associated with the thrombin receptor. These findings are the first to describe the localization of the thrombin receptor to dorsal root ganglion neurons. We propose that receptor activation is associated with thrombin induced inhibition of neurite outgrowth.

Thrombin receptor on rat primary hippocampal neurons: coupled calcium and cAMP responses.

We have tested the hypothesis that hippocampal neurons respond to thrombin via a neuronal thrombin receptor. A human neuroblastoma cell line, SK-N-SH, known to be thrombin responsive morphologically, responded both to thrombin and thrombin receptor agonist peptide (TRAP 42-55) with elevation of intracellular calcium. In Western blots of membranes from SK-N-SH cells and cultured rat hippocampal neurons using an antibody against the N-terminal peptide of the human thrombin receptor, putative receptor proteins of 66 and 47 kDa were detected in both cells. Neurons were treated with thrombin and TRAP 42-55 (TRAP-14) to determine their effects on intracellular levels of calcium and cAMP. Only 10% of the neurons showed a rapid response to thrombin, but most responded rapidly to agonist peptide with a prolonged elevation of intracellular free calcium. Neuronal cAMP levels were decreased by 40% after 24 h thrombin treatment. This decrease in cAMP level could be blocked by both the Gi-protein inhibitor, pertussis toxin, and the thrombin inhibitor, hirudin, suggesting a possible involvement of Gi-protein-coupled receptor activation. Furthermore, rapid calcium and cAMP responses were apparently induced by pre-treatment of neurons with thrombin for 24 h and subsequent washout. In summary, these data indicate that rat primary hippocampal neurons have thrombin receptors whose responses to thrombin apparently are up-regulated by 24 h thrombin pre-treatment. These results may have implications for synaptic remodeling, learning and memory.

Characterization of functional thrombin receptors in human pancreatic tumor cells (MIA PACA-2).

In this article, the "tethered ligand" thrombin receptor was identified on human pancreatic tumor cells, MIA PaCa-2, using immunofluorescence studies with a monoclonal anti-thrombin receptor antibody. Pharmacological characterization, using 3H-labeled thrombin receptor activating peptide-6 (TRAP-6) as radioligand, demonstrated a single class of high-affinity binding sites (KD = 9.1+/-1.8 x 10(-7) M) and a binding capacity of 13.9+/-0.7 fmol/mg protein. These binding sites represent functional thrombin receptors, as shown by alpha-thrombin- and TRAP-6-induced mobilization of free intracellular calcium, protein kinase C translocation from cytosol to the cell membrane, and stimulation of DNA synthesis in MIA PaCa-2 cells. These results provide the first identification of tethered ligand thrombin receptor in human pancreatic cancer cells and suggest thrombin receptor involvement in mechanisms of human pancreatic tumor progression.

Identification of G protein-coupled receptors potently stimulating migration of human transitional-cell carcinoma cells.

The expression of G protein-coupled receptors inducing calcium mobilization and stimulating cell migration was examined in human transitional-cell carcinoma (J82) cells. Measurements of cytoplasmic Ca2+ concentration ([Ca2+]i) and phospholipase C activity indicated that these cells express several calcium-mobilizing receptors, including those for lysophosphatidic acid (LPA), thrombin, bradykinin, bombesin and histamine, of which only the LPA response was sensitive (approximately 50%) to pertussis toxin (PTX). Migration of J82 cells was strongly stimulated by LPA and thrombin, by 5- to 20-fold, whereas bradykinin, bombesin and histamine were ineffective. Migration induced by either LPA or thrombin was inhibited by the actin cytoskeleton-disrupting agent, cytochalasin B, by the Rho protein-inactivating Clostridium difficile toxin B, by preventing [Ca2+]i transients with an intracellular calcium-chelating agent, and by the phorbol ester, phorbol 12-myristate 13-acetate, which also blocked the LPA- and thrombin-induced [Ca2+]i increases. On the other hand, ADP-ribosylation of Gi type G proteins by PTX abrogated the migratory response to LPA, without affecting the thrombin effect. Similarly, raising cAMP levels inhibited, by about 50%, the LPA- but not the thrombin-induced J82 cell migration. In conclusion, human transitional-cell carcinoma (J82) cells express various G protein-coupled, calcium-mobilizing receptors, out of which only those for LPA and thrombin stimulate cell migration, indicating that phospholipase C-derived second messengers per se are not sufficient for initiating this response. The complex signal transduction processes leading to LPA- and thrombin-stimulated motility of these human carcinoma cells apparently involve several common, essential factors, such as [Ca2+]i changes and Rho protein-regulated reorganization of the cytoskeleton, as well as some distinct components, most notably distinct subtypes of heterotrimeric G proteins and apparently also distinct cAMP-sensitive targets.