Thrombin Receptor Expression Research Articles

Attenuation of type-1 diabetes-induced cardiovascular dysfunctions by direct thrombin inhibitor in rats: a mechanistic study.

Chronic diabetes is associated with ventricular dysfunctions in the absence of hypertension and coronary artery diseases. This condition is termed as diabetic cardiomyopathy (DCM). There is no favourable treatment available for the management of diabetic cardiomyopathy. Recent studies have reported increase in circulating thrombin level among diabetic patients which is responsible for hypercoagulability of blood. Thrombin induces inflammation and fibrosis, and enhances cardiac cell growth and contractility in vitro. In this study, we have investigated the effects of argatroban; a direct thrombin inhibitor against DCM in streptozotocin-induced type-1 diabetes. Diabetes was induced by single dose of streptozotocin (STZ; 50mg/kg, i.p.) in male Sprague-Dawley rats. After 4 weeks of diabetes induction, the animals were treated with argatroban (0.3 and 1mg/kg, i.p. daily) for the next 4 weeks. The effect of argatroban was evaluated against diabetes-associated cardiac dysfunction, structural alteration and protein expression. STZ-induced diabetic rats exhibited significant decline in left ventricular functions. Four weeks of treatments with argatroban significantly improved ventricular functions without affecting heart rate. Further, it also protected heart against structural changes induced by diabetes as shown by reduction in fibrosis, hypertrophy and apoptosis. The improvement in cardiac functions and structural changes was associated with significant reduction in left ventricular expression of thrombin receptor also termed as protease-activated receptor-1 or PAR1, p-AKT (ser-473), p-50 NFκB and caspase-3 proteins. This study demonstrates beneficial effects of argatroban via improvement in cardiac functions and structural changes in STZ-induced DCM. These effects may be attributed through reduction in cardiac inflammation, fibrosis and apoptosis.

Evidence for functional PAR-4 thrombin receptor expression in cardiac fibroblasts and its regulation by high glucose: PAR-4 in cardiac fibroblasts.

Thrombin promotes cardiac fibroblast proliferation and fibrosis via protease-activated receptor (PAR-1). PAR-4 is reportedly absent in cardiac fibroblasts. In smooth muscle cells, PAR-4 expression is also low but increases upon hyperglycemia and contributes to vascular remodelling in diabetic mice. We examined if PAR-4 is a glucose-responsive gene with remodelling-related functions in cardiac fibroblasts. Cardiac PAR-4 increased in mice with streptozotocin- or diabetogenic diet (DD)-induced diabetes. PAR-4 mRNA and protein were detectable in cardiac fibroblasts from chow-fed mice and increased in high (HG, 25mM) vs. low glucose (LG; 5.5mM) cultures. Conversely PAR-4 mRNA was higher in fibroblasts from DD-fed mice but reduced in LG cultures. Cardiac fibroblasts in HG culture responded more strongly to thrombin or PAR-4 activating peptide in terms of migration (wound-scratch assay), remodelling-associated gene expression (interleukin 6, alpha smooth muscle actin) and oxidative stress (dihydroethidium fluorescence). PAR-4 is expressed in mouse cardiac fibroblasts and is dynamically regulated by extracellular glucose in vitro and diabetes in vivo, thereby impacting on fibroblast functions relevant for cardiac remodelling. These findings add further evidence for the usefulness of the recently developed PAR-4 antagonists in clinical settings.

Modification of immunological features in human platelets during sepsis

ABSTRACTSepsis is an organic dysfunction that puts at risk the life of patients suffering this disorder due to an exacerbated immunological response to the infection mediated by the host. Platelets have been largely researched on sepsis owing to its role in Disseminated Intracellular Coagulation (DIC) and because thrombocytopenia is an important clinical feature of these patients. Nevertheless, a great number of evidence shows that platelets have also an important role in immunological response since they have pattern recognition receptors, chemokine receptors and granules with stored soluble mediators. In this work, the immunological features of platelets in individuals with sepsis are described. The results show that platelets of these individuals have high levels of surfaces expression of TLR4, CD62P, CD32 and thrombin receptor 1 (PAR-1), these platelets have also greater capability to join Escherichia coli, and show a different profile of soluble mediators (IL-1β, CD40L and TNF-α). Platelets from patients with sepsis form aggregates with neutrophils in circulation, but are unable to induce the production of reactive oxygen species. This research describes important features of platelets to help the understanding of the immunological role of these cells in sepsis.

P5364Dabigatran enhances platelet reactivity and platelet thrombin receptor expression in patients with atrial fibrillation

P5364Dabigatran enhances platelet reactivity and platelet thrombin receptor expression in patients with atrial fibrillation

Dabigatran reduces thrombin-induced platelet aggregation and activation in a dose-dependent manner.

Dabigatran is an oral anticoagulant and a reversible inhibitor of thrombin. Further, dabigatran might affect platelet function through a direct effect on platelet thrombin receptors. The aim was to investigate the effect of dabigatran on platelet activation and platelet aggregation. Healthy donor blood was incubated with dabigatran 0, 50, 500ng/mL, corresponding to the therapeutic range of dabigatran peak plasma concentrations, and 10,000ng/mL comprising a supra-therapeutic dabigatran plasma level. Platelet aggregation was tested with 96-well aggregometry. Flow cytometry was used to test platelet activation and platelet thrombin receptor expression (SPAN-12 and WEDE-15 expression). Agonists were thrombin, thrombin receptor-activating peptide, protease-activated receptor-4 agonist, collagen, collagen-related peptide, arachidonic acid, and adenosine diphosphate. All concentrations of dabigatran fully inhibited platelet aggregation for thrombin up to 2IU/mL, while dabigatran did not affect platelet aggregation by other agonists. Platelet activation (percentage of platelets positive for activated GPIIb/IIIa, CD63, P-selectin) was reduced after thrombin stimulation in samples with dabigatran levels ≥500ng/mL. After stimulation with thrombin, the percentage of activated GPIIb/IIIa-positive platelets was 99.8 ± 0.2% without dabigatran, 14.7 ± 4.7% with 500ng/mL dabigatran, and 4.2 ± 0.2% with 10,000ng/mL dabigatran, both p < 0.001 when compared to samples without dabigatran. Also, the receptor expression of GPIIb/IIIa, CD63, andP-selectin were reduced after dabigatran treatment. The expression of thrombin receptors was reduced at dabigatran on ≥ 500ng/mL. In conclusion, dabigatran exclusively inhibits thrombin-induced platelet activation and aggregation with a dose-dependent response. Platelet stimulation with other agonists was not affected by dabigatran.

Dabigatran enhances platelet reactivity and platelet thrombin receptor expression in patients with atrial fibrillation

Background The direct oral anticoagulant (DOAC) dabigatran is a direct thrombin inhibitor. Its landmark trial, the RE-LY study, observed a trend towards a higher incidence of myocardial infarctions (MIs) in dabigatran-treated patients. Since then, there have been discussions on whether dabigatran increases the risk of MI. Objective In this study, we aimed to assess platelet reactivity and platelet thrombin receptor expression in dabigatran-treated patients. Methods We conducted a cross-sectional study in 13 hospitalized patients with planned initiation of dabigatran medication. Platelet reactivity was measured by light-transmission aggregometry and platelet thrombin receptor expression was measured by flow cytometry analysis. Results Platelet reactivity was higher after initiation of dabigatran medication as compared with baseline (baseline 44±24% vs. dabigatran 70±25%). Accordingly, the density of both platelet thrombin receptors (protease activated receptor [PAR]-1 and PAR-4) on platelets increased during dabigatran treatment (PAR1, baseline 63±11% vs. dabigatran 70±10%; PAR4, baseline 1.1±0.5% vs. dabigatran 1.6±0.9%). Conclusions Dabigatran increases platelet reactivity by enhancing the thrombin receptor density on platelets. This finding should be considered while choosing the optimal DOAC in individualized medicine.

Disabled-2 is required for efficient hemostasis and platelet activation by thrombin in mice.

The essential role of platelet activation in hemostasis and thrombotic diseases focuses attention on unveiling the underlying intracellular signals of platelet activation. Disabled-2 (Dab2) has been implicated in platelet aggregation and in the control of clotting responses. However, there is not yet any in vivo study to provide direct evidence for the role of Dab2 in hemostasis and platelet activation. Megakaryocyte lineage-restricted Dab2 knockout (Dab2(-/-)) mice were generated to delineate in vivo functions of Dab2 in platelets. Dab2(-/-) mice appeared normal in size with prolonged bleeding time and impaired thrombus formation. Although normal in platelet production and granule biogenesis, Dab2(-/-) platelets elicited a selective defect in platelet aggregation and spreading on fibrinogen in response to low concentrations of thrombin, but not other soluble agonists. Investigation of the role of Dab2 in thrombin signaling revealed that Dab2 has no effect on the expression of thrombin receptors and the outside-in signaling. Dab2(-/-) platelets stimulated by low concentrations of thrombin were normal in Gαq-mediated calcium mobilization and protein kinase C activation, but were defective in Gα₁₂/₁₃-mediated RhoA-ROCKII activation. The attenuated Gα₁₂/₁₃ signaling led to impaired ADP release, Akt-mammalian target of rapamycin and integrin αIIbβ3 activation, fibrinogen binding, and clot retraction. The defective responses of Dab2(-/-) platelets to low concentrations of thrombin stimulation may contribute to the impaired hemostasis and thrombosis of Dab2(-/-) mice. This study sheds new insight in platelet biology and represents the first report demonstrating that Dab2 is a key regulator of hemostasis and thrombosis by functional interplay with Gα₁₂/₁₃-mediated thrombin signaling.

FcγRIIa Ligation Induces Platelet Hypersensitivity to Thrombotic Stimuli

Platelets are known for their important role in hemostasis, however their significance in other functions, including inflammation and infection, are becoming more apparent. Patients with systemic lupus erythematosus (SLE) are known to have circulating IgG complexes in their blood and are highly susceptible to thrombotic events. Because platelets express a single receptor for IgG, we tested the hypothesis that ligation of this receptor (FcγRIIa) induces platelet hypersensitivity to thrombotic stimuli. Platelets from SLE patients were considerably more sensitive to thrombin compared to healthy volunteers, and this correlated with elevated levels of surface IgG on SLE platelets. To test whether FcγRIIa ligation stimulated thrombin hypersensitivity, platelets from healthy volunteers were incubated with buffer or heat-aggregated IgG, then stimulated with increasing concentrations of thrombin. Interestingly, heat-aggregated IgG-stimulated platelets, but not buffer-treated platelets, were hypersensitive to thrombin, and hypersensitivity was blocked by an anti-FcγRIIa monoclonal antibody (mAb). Thrombin hypersensitivity was not due to changes in thrombin receptor expression (GPIbα or PAR1) but is dependent on activation of shared signaling molecules. These observations suggest that ligation of platelet FcγRIIa by IgG complexes induces a hypersensitive state whereby small changes in thrombotic stimuli may result in platelet activation and subsequent vascular complications such as transient ischemic attacks or stroke.

Effect of thrombin receptor expression on microvessel endothelial cells, and survival by coagulation state in oesophageal squamous cell carcinoma

PurposeThe authors aimed to evaluate the prognostic value of thrombin receptor (TR) expression in microvessel endothelial cells (VECs) by coagulation state in oesophageal squamous cell carcinoma (ESCC).MethodsIn 138 ESCC normal...

High Glucose Enhances Thrombin Responses via Protease-Activated Receptor-4 in Human Vascular Smooth Muscle Cells

Diabetes is associated with vascular remodeling and increased thrombin generation. Thrombin promotes vascular smooth muscle cell (SMC) mitogenesis and migration via protease-activated receptors (PAR)-1, PAR-3, and PAR-4. We investigated the effect of high glucose on expression and function of vascular thrombin receptors. In human vascular SMCs, high glucose (25 versus 5.5 mmol/L) induced a rapid and sustained increase in PAR-4 mRNA, protein, and cell surface expression. PAR-1 and PAR-3 expression were not changed. High glucose pretreatment (48 hours) enhanced thrombin or PAR-4-activating peptide but not PAR-1-activating peptide evoked intracellular calcium mobilization, migration, and tumor necrosis factor α gene expression. This enhancement of thrombin-stimulated migration and gene expression by high glucose was abolished by endogenous PAR-4 knockdown. PAR-4 regulation was prevented by inhibition of protein kinase (PK)C-β and -δ isoforms or nuclear factor (NF)κB. Nuclear translocation of NFκB in high glucose-stimulated SMCs led to PKC-dependent NFκB binding to the PAR-4 promoter in a chromatin immunoprecipitation assay. Furthermore, in situ hybridization and immunohistochemistry confirmed high abundance of PAR-4 in human diabetic vessels as compared with nondiabetic vessels. High glucose enhances SMC responsiveness to thrombin through transcriptional upregulation of PAR-4, mediated via PKC-β, -δ, and NFκB. This may play an important role in the vascular complications of diabetes.

Human cytomegalovirus increases HUVEC sensitivity to thrombin and modulates expression of thrombin receptors

Human cytomegalovirus (HCMV) establishes a life-long persistent infection. HCMV infection could be associated with chronic inflammatory diseases, such as cardiovascular disease and atherosclerosis. Here we observed that in HCMV (AD-169) pre-exposed human umbilical vein endothelial cells (HUVEC), thrombin-induced expression of IL-1alpha and M-CSF is markedly enhanced compared to the un-exposed cells. Study of the expression of thrombin receptor genes in HUVEC showed that HCMV triggered a time- and concentration-dependent expression of the thrombin receptors PAR1, PAR3 and PAR4 at the mRNA level. Induction of PAR1 and PAR3 mRNA expression is due to transcriptional activation of their promoters as shown by gene reporter assay. Furthermore, the virus induced expression of PAR1 and PAR3 but not PAR4 proteins, as analyzed by Western immunoblotting. However, flow cytometric analysis revealed that only PAR3, expressed at very low level in control HUVEC, is induced at the surface during the exposure to the virus. Our data suggest that although exposure to HCMV induces a minor increase of cell-surface receptors expression, it does make endothelial cells more responsive to additional thrombin stimulation.

Altered adhesive properties of cord blood endothelial outgrowth cells expressing IL‐1ra

The aim of this study was to examine the potential of endothelial outgrowth cells (EOCs) expanded from CD34(+) cord blood-derived cells (CB-EOCs) for overexpression of therapeutic transgenes. As proof of principle, we overexpressed icIL-1ra in CB-EOCs. Proinflammatory activation of CB-EOCs in response to cytokine stimulation (IL-1beta and tumor necrosis factor (TNF)) and during coculture with monocytes showed that icIL-1ra-expressing CB-EOCs express significantly reduced levels of ICAM-1, MCP-1 and thrombin receptor expression. Moreover, overexpression of icIL-1ra attenuated the IL-1beta-mediated proinflammatory activation by diminishing the expression of ICAM-1, SELE, MCP-1 and IL-1beta. Interestingly, overexpression of icIL-1ra also inhibited TNF-induced upregulation of ICAM-1. Expression of ICAM-1, VCAM-1, tissue factor and IL-1beta was also decreased on direct contact with monocytes. These changes in gene expression were accompanied by functional reduction in leukocyte rolling, adhesion of monocytes to CB-EOCs, as well as by a reduction in transendothelial migration of monocytes. Our findings show that CB-EOCs stably expressing transgenic icIL-1ra are protected against activation by not only IL-1beta but also TNFalpha-mediated proinflammatory stimuli and inhibit decisive pathomechanisms of inflammatory processes such as rolling, adhesion and transmigration of monocytes. Therefore, icIL-ra transgenic CB-EOCs may prove to be beneficial in the treatment of IL-1beta- and TNFalpha-mediated inflammatory vasculopathies.

Effects of Thrombin on RPE Cells Are Mediated by Transactivation of Growth Factor Receptors

To determine the expression of blood coagulation factors and thrombin receptors in retinal pigment epithelial (RPE) cells and whether the effects of thrombin on the chemotaxis and the secretion of VEGF are mediated by transactivation of growth factor receptors. Gene expression in acutely isolated and cultured human RPE cells was evaluated by RT-PCR. Alterations in gene expression and secretion of VEGF were determined by real-time RT-PCR and ELISA, respectively. Chemotaxis was examined with a Boyden chamber assay. RPE cells expressed the mRNA of the protease-activated receptors PAR1 and -3 and of various coagulation factors (III, V, VII, VIII, and X). Thrombin stimulated the expression and secretion of VEGF-A from RPE cells, decreased the expression of VEGFD, and increased the gene expression of VEGFR-1 (FLT1). The effects on the secretion of VEGF-A and the increase in FLT1 expression were mediated by stimulation of the secretion of TGF-beta1 and activation of the TGF-beta activin receptor-like kinase. Thrombin stimulated the chemotaxis of RPE cells, and this effect was mediated by transactivation of the PDGF receptor tyrosine kinase. The expression of different coagulation factors suggests that RPE cells provide a procoagulant surface for the formation of thrombin from prothrombin via the extrinsic coagulation pathway. Thrombin stimulates the secretion of VEGF-A, the expression of FLT1, and the chemotaxis of RPE cells via transactivation of TGF-beta and PDGF receptors, respectively.

Thrombin increases inflammatory cytokine and angiogenic growth factor secretion in human adipose cells in vitro

BackgroundAbdominal obesity is associated with pro-thrombotic and inflammatory states. Therefore, the purpose of this study was to examine the expression of thrombin receptors (PAR1 and PAR4) human adipose tissue and whether thrombin stimulates an inflammatory cytokine and growth factor profile in human adipose tissue.MethodsHuman adipose tissue, isolated preadipocytes and differentiated adipocytes were used in this study. PAR1 and PAR4 mRNA and protein were detected by RT-PCR and immunoblot analysis in both adipose tissue and adipose microvessels. In separate studies, IL-1β, IL-6, MCP-1, TNF-α, IL-10, FGF-2, VEGF, and PDGF production were measured from adipose tissue (n = 5), adipocytes (n = 5), and preadipocytes (n = 3) supernatants with and without thrombin (1 or 10 U/ml; 24 hrs) treatment.ResultsThrombin increased cytokine secretion of IL-1β, IL-6, MCP-1 and TNF-α and growth factor secretion of VEGF from adipocytes along with MCP-1 and VEGF from preadipocytes. The direct thrombin inhibitor lepirudin given in conjunction with thrombin prevented the thrombin-mediated increase in cytokine and growth factor secretion.ConclusionHere we show that thrombin PAR1 and PAR4 receptors are present and that thrombin stimulates inflammatory cytokine generation and growth factor release in human adipose tissue and cells in vitro. These data suggest that thrombin may represent a molecular link between obesity and associated inflammation.

Thrombin Receptor and Ventricular Arrhythmias after Acute Myocardial Infarction

The mechanism mediating the development of ventricular arrhythmia (VA) after acute myocardial infarction (AMI) is still uncertain. Thrombin receptor (TR) activation has been proven to be arrhythmogenic in many other situations, and we hypothesize that it may participate in the genesis of post-AMI VA. Using a left coronary artery ligation rat model of AMI, we found that a local injection of hirudin into the left ventricle (LV) significantly reduced the ratio of VA durations to infarction sizing, whereas injection of thrombin receptor-activating peptide (TRAP) increased the ratios of VA duration to infarction sizing. The effects of TR activation on whole-cell currents were investigated in isolated myocytes. TRAP increased a glibenclamide-sensitive outward current. Pretreatment of rats with glibenclamide (4 mg/kg intraperitoneally) eliminated the effects of a local injection of TRAP on the ratios of VA durations to infarction sizing. TR mRNA and protein expression in the ischemic left ventricle had reached its peak by 20 min postligation in the rat AMI model (P < 0.05). TR-immunoreactive myocytes were observed in infarcted LV but were seldom seen in the right ventricle or in the normal heart. By 60 min, TR transcript levels had returned to control levels. We conclude that increased TR activation and expression in the infarcted LV after AMI may contribute to VA through a mechanism involving glibenclamide-sensitive potassium channels.

Oncogene-Directed Therapies as Modulators of Cancer Coagulopathy, Angiogenesis and Tumor-Vascular Interface

Oncogenic lesions in the cancer cell genome not only alter the intrinsic properties of cancer cells themselves, but also trigger signaling events, the effects of which impact the relationship between tumors and their surrounding host tissues, including the vascular system. Manifestations of the latter include the onset and progression of tumor angiogenesis, and systemic activation of the hemostatic system, a process also known as cancer coagulopathy, or Trousseau syndrome. Indeed, tumor growth, invasion and metastasis are profoundly affected by the properties of the tumor - vascular interface. We postulated earlier that the link between the expression of cancercausing signaling alterations and cancer coagulopathy is at least twofold. First, cancer coagulopathy may emerge indirectly, as an unspecific consequence of vascular permeability and other features of tumor-associated vascular growth, including the pro- and anticoagulant actions of certain angiogenic mediators, some of which (e.g. VEGF) are direct regulatory targets of oncogenes and tumor suppressors. Second, activated oncogenes (K-ras, EGFR or PML-RARα, MET), and/or inactivation of tumor suppressors (e.g. p53 or PTEN) may deregulate some of the hemostatic proteins more directly, i.e. through changes in expression of tissue factor (TF), plasminogen activation inhibitor 1 (PAI-1) and cyclooxygenase 2 (COX-2) [1-4]. In addition to TF expression and release as plasma microvesieles, mutant K-ras may also upregulate expression of thrombin receptors (PAR-1) by cancer cells, thereby possibly rendering such cells hypersensitive to signals emanating from the hemostatic system (e.g. pericellular thrombin). Here, we present an argument that anti-cancer signal transduction inhibitors, also known as ‘targeted agents’ (e.g. Gleevec, Iressa, Erbitux, ATRA) could modulate various vascular processes in cancer, including angiogenesis and cancer coagulopathy, i.e. act as cancer - specific indirect antiangiogenic agents, possibly with a distinct element of anticoagulant activity.

Platelet inhibition with prasugrel (CS-747) compared with clopidogrel in patients undergoing coronary stenting: the subset from the JUMBO study

Background: Based on the preclinical and phase 1 studies, prasugrel, a novel platelet ADP P2Y12 receptor blocker, may be a more potent platelet inhibitor than clopidogrel. This study compared the...

Thrombin receptor expression is upregulated in prostate cancer

Aberrant expression of protease-activated receptors (PARs) has been associated with increased angiogenesis, tumor growth, and metastasis of various cancers. We assessed the status of PAR1 expression in prostate cancer. The study compared the abundance levels of PAR1 RNA and protein using real-time reverse-transcriptase polymerase chain reaction and immunoblotting in freshly resected prostate tissues from early localized-stage disease (n=9) to those from patients with advanced metastatic disease (n=7). PAR1 expression and localization was evaluated using immunohistochemical staining of prostate specimens with benign prostatic hyperplasia (n=27), early- (n=32) and advanced-stage (n=22) prostate cancer. Association analyses of PAR1 expression with expression of VEGF-family of growth factors, their receptors, and clinicopathological characteristics of the patients were also performed. PAR1 RNA expression in advanced-stage prostate was 2.39-fold higher (P=0.024) and its protein expression was 2.75-fold higher (P=5.89x10(-5)) when compared with early-stage prostate cancer. PAR1 expression was localized to endothelial cells in vascular network of prostate tumor areas. The expression of PAR1 correlated statistically significantly with advanced disease stage (P=0.0006) and pre-operative PSA levels (P=0.005) in these samples. These findings demonstrate that PAR1 expression is increased in prostate cancer. Its predominant expression in vascular network suggests that it may play a direct and crucial role in angiogenesis and could be a relevant target for therapeutic interventions to control or to prevent disease progression.

Characterization and Functional Activity of Thrombin Receptors in the Human Lens

To investigate the expression of thrombin receptors in the human lens, the activation of downstream signaling pathways, and the ability of thrombin to regulate lens cell growth. Thrombin receptor function in the human lens was determined first by measuring changes in intracellular calcium in response to thrombin and protease-activated receptor-activating peptides (PAR-APs). In the human capsular bag model, cell growth was assessed by phase microscope inspection of the cell coverage of the posterior capsular surface. In the human lens cell line FHL124, it was assessed by [(3)H]thymidine incorporation. Changes in p42/p44 ERK phosphorylation (p-ERK) and protein kinase B (PKB/Akt) phosphorylation (p-Akt) were monitored by Western blot. Reverse transcription-polymerase chain reaction (RT-PCR) applied to isolated lens epithelia and ex vivo capsular bag preparations as well as FHL124 cells determined expression of mRNA for the PARs. Brief exposures to thrombin (10 nM) and PAR1-AP (10 microM) induced an increase in cytosolic calcium in both anterior and equatorial lens cells, but activating peptides for PAR2, -3, and -4 failed to produce responses. Repeated exposure to thrombin produced a significant increase in cell coverage in the capsular bag model and increased [(3)H]thymidine incorporation into FHL124 cells. In the latter, exposure to thrombin (10 nM) and PAR1-AP (10 microM) induced biphasic increases in the phosphorylation of p42/p44 (p-ERK), with peak responses at 20 minutes and 12 hours. Thrombin also produced a 20-fold increase in p-Akt at 12 hours compared with the control, whereas PAR1-AP (10 microM) induced a much smaller response. PAR1-AP did not induce a significant increase in [(3)H]thymidine incorporation and PAR2-AP, PAR3-AP, and PAR4-AP failed to reproduce any of the thrombin-stimulated effects. mPAR1 and -3 were expressed in native lens cells, and this expression was conserved in ex vivo capsular bag preparations as well as in FHL124 cells. This study identifies thrombin receptors coupled to calcium, ERK, and Akt signaling that modulate growth in native lens tissue and cultured cells, and it appears that the PAR1 subtype is mainly responsible. PAR3 mRNA was also detected, but the receptor itself, if present, was not coupled to the above signaling elements.

Proliferation of dental pulp fibroblasts in response to thrombin involves mitogen-activated protein kinase signalling.

To examine the involvement of mitogen-activated protein kinases (MAPK) signalling on thrombin-stimulated human dental pulp fibroblasts (DPF). Dental pulp fibroblasts were isolated from dental pulp connective tissue of third molars and expanded in vitro. Expression of thrombin receptors was analysed by RT-PCR, and cell proliferation was measured by 3[H]-thymidine incorporation assay. Phosphorylation levels of MAPK were determined by Western blot analysis, and alkaline phosphatase activity was measured to serve as a marker for odontogenic differentiation. Statistical analysis was performed by Student's t-test. Dental pulp fibroblasts express the thrombin receptors protease-activated receptor-1 (PAR-1), PAR-3 and PAR-4. Measurement of 3[H]-thymidine incorporation revealed a dose-dependent increase of DNA synthesis in response to thrombin treatment. The thrombin-induced mitogenic activity was decreased by the extracellular signal-regulated protein kinase (ERK) signalling inhibitor PD98059 (P < 0.05), and by SB203580 (P < 0.05), a p38 MAPK inhibitor. Western blot analysis demonstrated increased phosphorylation of ERK in DPF following stimulation with thrombin, while p38 MAPK and c-Jun NH2-terminal kinase (JNK) were not activated. Alkaline phosphatase activity of DPF remained unchanged upon incubation with thrombin. These results suggest that signalling via MAPK mediates the mitogenic activity of thrombin on DPF and may thus play a role during the early stages of pulp repair.