Thrombin Receptor mRNA Expression Research Articles

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.

Effects of puerarin on proliferation of vascular smooth muscle cells induced by thrombin

To investigate the effects of puerarin on the proliferation of vascular smooth muscle cells (VSMC) induced by thrombin and the mechanism thereof. VSMCs were isolated from the thoracic aorta of a SD rat and cultured, then co-cultured with thrombin of the concentration 0.1, 0.3, 1.0, 3.0, and 10 U/L for 24 h, thrombin of the concentration of 1 U/L for 0, 6, 12, 24, 36, and 48 h respectively, or thrombin of the concentration of 1 U/L combined with puerarin of the concentrations of 1.5 x 10(-5), 1.5 x 10(-4), or 1.5 x 10(-3) mol/L for 24 h. Flow cytometry was used to detect the cell number and cell cycle. Western blotting was used to indicate the protein expression of the oncogenes c-fos and bcl-2 RT-PCR was used to evaluate the thrombin receptor (TR) mRNA expression. he numbers of the groups of VSMCs stimulated by 0.1, 0.3, 1.0, 3.0, and 10 U/L thrombin for 24 hours were 4.82 x 10(4)/ml +/- 0.11 x 10(4)/ml, 6.37 x 10(4)/ml +/- 0.09 x 10(4)/ml, 8.78 x 10(4)/ml +/- 0.08 x 10(4)/ml, 7.37 x 10(4)/ml +/- 0.07 x 10(4)/ml, and 5.28 x 10(4)/ml +/- 0.12 x 10(4)/ml respectively, all significantly higher than that of the control group (4.08 +/- 0.054 x 10(4)/ml, all P < 0.05). The effect of thrombin was in a dose-dependent manner within a concentration range of 0.1 - 1.0 U/L. The suppression rates of VSMC proliferation in the combination groups with puerarin of the concentrations of 1.5 x 10(-5), 1.5 x 10(-4), and 1.5 x 10(-3) mol/L were 10.9% +/- 1.6%, 32.1% +/- 3.3%, and 42.6% +/- 5.2% respectively in comparison with the thrombin group (all P < 0.05). The c-fos protein expression of the VSMCs after thrombin stimulation for 24 h increased by 156.0% +/- 11.3% (P < 0.05), and the bcl-2 protein expression of the VSMCs pretreated with puerarin of the concentrations of 1.5 x 10(-5), 1.5 x 10(-4), and 1.5 x 10(-3) mol/L, and then stimulated by thrombin was significantly lower than that of the VSMCs only stimulated by thrombin with the suppression rates of 20.7% +/- 2.1%, 31.6% +/- 5.2%, and 44.5% +/- 7.5% respectively (all P < 0.05). The bcl-2 protein expression of the VSMCs after thrombin stimulation for 24 h increased by 96.7% +/- 8.3% (P < 0.05), and the bcl-2 protein expression of the VSMCs pretreated with puerarin of the concentrations of 1.5 x 10(-5), 1.5 x 10(-4), and 1.5 x 10(-3) mol/L, and then stimulated by thrombin was significantly lower than that of the VSMCs only stimulated by thrombin with the suppression rates of 7.1% +/- 0.8%, 18.8% +/- 1.2%, and 39.6% +/- 6.4% respectively (all P < 0.05). The stimulation of thrombin increased the TR mRNA expression by 183.9% +/- 9.4%. The puerarin of the concentrations of 1.5 x 10(-5) mol/L and 1.5 x 10(-4) mol/L decreased the increase of TR mRNA expression induced by thrombin, however, without significant differences (both P > 0.05), and puerarin of the concentration of 1.5 x 10(-3) mol/L significantly suppressed the increase of TR mRNA expression induced by thrombin by 17.6% +/- 1.7% (P < 0.05). Puerarin suppresses the proliferation and DNA synthesis of VSMC induced by thrombin. The inhibitory effect of puerarin is closely related with the suppression of the protein expression of c-fos and bcl-2n, and partly related with the suppression of the TR mRNA expression.

Calorie restriction decreases platelet-derived growth factor (PDGF)-A and thrombin receptor mRNA expression in autoimmune murine lupus nephritis.

Calorie restriction (CR) and supplementation with fish oil (FO) are known to increase the life span and diminish histological evidence of glomerulonephritis in lupus prone (NZB x NZW)F1 (B/W) mice. Cellular proliferation is an important pathological element in the development of lupus nephritis, and we have examined the expression of thrombin receptor (TR) and the mitogenic agents PDGF-A and -B. Weanling B/W mice were fed either ad libitum or a calorie restricted (CR; 40% less calories than ad libitum) diet supplemented with either 5% (w/w) corn oil (CO) or FO. CR animals consumed 2.7-3.0 g of wet food per day versus 4.5-5.0 g for the ad libitum animals. Renal RNA was extracted from young (3.5-4.0 months of age) and old (8-10 months of age) mice. Densitometric analysis (reference gene GAPDH) of blots from Northern (PDGF-A and -B) and ribonuclease protection assays (TR) produced the following data: (i) in young mice no signal was detected for PDGF-A, -B and TR in all four groups, while the signals were readily detectable in old mice; (ii) in old mice low and similar levels of PDGF-B were detected, and neither CR nor the source of lipid altered its expression; (iii) CR significantly inhibited PDGF-A and TR expression in both CO (ad libitum versus CR; PDGF-A, 3.25-fold, P < 0.025; TR, 3.7-fold, P < 0.01) and FO (ad libitum versus CR; PDGF-A, 4.56-fold, P < 0.01; TR, 3.6-fold, P < 0.025) groups; (iv) although FO (versus CO) produced a trend towards decreased expression, results were not statistically significant. We conclude that suppression of renal disease in lupus-prone mice by CR is accompanied by decreased expression of PDGF-A and the thrombin receptor.

Enhanced contractile response to thrombin in the pregnant rat myometrium.

Thrombin causes various cellular events by activating protease-activated receptors (PARs). Here, we showed, for the first time, that thrombin induced myometrial contraction. To determine the mechanism of thrombin-induced myometrial contraction, we simultaneously measured intracellular Ca(2+) concentration ([Ca(2+)](i)) and tension of fura-PE3-loaded rat myometrium using front-surface fluorimetry. The expression of thrombin receptor mRNA in the rat myometrium were determined by reverse transcription-polymerase chain reaction analysis (RT - PCR analysis). Thrombin (0.01 - 3 u ml(-1)) caused dose-dependent increase in [Ca(2+)](i) and tension in the rat myometrium, and this effect was greatly enhanced in the pregnant myometrium. PAR1-activating peptide mimicked the effects of thrombin. In Ca(2+)-free PSS, thrombin induced no increase in [Ca(2+)](i) and tension in the pregnant myometrium. Both diltiazem (10 microM) and SK-F 96365 (10 microM) significantly inhibited the thrombin-induced elevations of [Ca(2+)](i) and tension, and their effects were additive. RT - PCR analysis revealed an approximately 10 fold increase in the level of thrombin receptor mRNA in the pregnant myometrium compared to that obtained in the non-pregnant myometrium. In conclusion, the contractile response to thrombin was greatly enhanced in the pregnant myometrium, mainly due to the up-regulation of thrombin receptor. We propose that initiation of a post-parturitional myometrial contraction is one of the most important physiological roles of thrombin receptor.

Thrombin-induced DNA synthesis of cultured human dental pulp cells is dependent on its proteolytic activity and modulated by prostaglandin E 2

To clarify the roles of alpha-thrombin and prostaglandin E2 (PGE2) in the healing and inflammatory processes of dental pulp, their effects on the DNA synthesis of human pulp cells were investigated by measurement of [3H]thymidine incorporation. At a concentration range of 1 to 25 units/ml, alpha-thrombin stimulated DNA synthesis of the pulp cells by 1.5 to 2.6-fold. On the contrary, PGE2 (> 0.05 microgram/ml) suppressed DNA synthesis by 24 to 39%. Using reverse transcriptase-polymerase chain reaction, thrombin receptor mRNA expression was identified in the pulp cells. Furthermore, alpha-thrombin-induced DNA synthesis could be inhibited by antithrombin III (2 units/ml) with heparin (2 units/ml) or D-Phe-Pro-ArgCH2Cl (50 micrograms/ml). PGE2 (0.1 to 0.5 microgram/ml) also inhibited the thrombin-induced DNA synthesis by 39 to 64%. These results imply that pulp cells express the thrombin receptor that is activated by the serine protease activity of thrombin. Interactions of thrombin and PGE2 are important in modulating the inflammatory and healing processes of the pulp.

Antibody to thrombin receptor inhibits neointimal smooth muscle cell accumulation without causing inhibition of platelet aggregation or altering hemostatic parameters after angioplasty in rat.

An antibody was raised in rabbits against SFFLRNPSEDTFEQF peptide, which is an NH2-terminal peptide of the thrombin-cleaved rat thrombin receptor. In vitro, the antibody inhibited rat smooth muscle cell proliferation but had no effect on rat platelet aggregation or clotting time. These data indicate that the antibody is a specific blocker of the thrombin receptor-signaling pathway in rat smooth muscle cells but does not work as a blocker in rat platelets, suggesting the existence of a second thrombin receptor in the platelets. Using an in vivo balloon catheter-induced injury model in rats, we examined the effect of the anti-rat thrombin receptor IgG on intimal smooth muscle cell accumulation 2 weeks after angioplasty. Analysis of the ratio of intimal to medial cross-sectional areas showed that injection of immune IgG resulted in 43.7% and 53.1% reduction (P<0.01) of neointimal smooth muscle cell accumulation compared with saline and nonimmune IgG treatment, respectively. Moreover, the injection of immune IgG caused a significant decrease of thrombin receptor mRNA expression and also 40.5% and 43.0% decreases (P<0.01) of the proliferating cell nuclear antigen (PCNA) index in the intima compared with the PCNA index after saline and nonimmune IgG treatment, respectively. The suppression of the PCNA index was also observed in the immune IgG-treated group at an early stage after angioplasty. These results suggest that thrombin receptor activation is involved in the proliferation and accumulation of neointimal smooth muscle cells induced by balloon injury.

Thrombin receptor expression is increased by angiotensin II in cultured and native vascular smooth muscle cells

The factors involved in restenosis after balloon angioplasty are poorly characterized but the local concentration of the potent mitogens angiotensin II (AII) and thrombin is known to be increased at sites of vascular injury. We investigated the possibility of a synergistic interaction between AII and thrombin by studying the effects of AII on the expression of the thrombin receptor in rat aortic smooth muscle cells (VMSC). Thrombin receptor mRNA expression was studied by Northern blot analysis and RT-PCR using total RNA extracted from VMSC or from endothelium-denuded rat aortae. As a measure of thrombin receptor protein expression, we assessed either the thrombin-stimulated release of 6-keto-prostaglandin F1 alpha from VMSC or the contraction of endothelium-denuded rat aortic rings. The thrombin receptor mRNA was expressed at a low level in both cultured and native VMSC. AII concentration- and time-dependently increased expression of thrombin receptor mRNA in VMSC and augmented the thrombin-induced release of 6-keto-prostaglandin F1 alpha as well as the thrombin induced contraction. Blockade of the angiotensin subtype 1 (AT1) receptor by EXP3174 or D8731 prevented the AII-mediated increase in thrombin receptor expression. The effect of AII on the increase in thrombin receptor mRNA expression was enhanced by the protein kinase C inhibitor Ro 31-8220, but was unaffected by prolonged incubation with phorbol myristate acetate or the tyrosine kinase inhibitors genistein and erbstatin A. These results demonstrate that AII enhances the expression of thrombin receptor in cultured and native VMSC. In cultured cells, this effect is mediated by the activation of the AT1 receptor subtype. This synergistic effect between AII and the thrombin receptor may promote the extensive proliferation of smooth muscle cells in response to vascular injury.

Vascular thrombin receptor regulation in hypertensive rats.

Thrombin has been implicated as an important mediator of vascular lesion formation in atherosclerosis and restenosis. To investigate a potential role for thrombin signaling in the vascular response to hypertension, we have studied thrombin receptor (TR) expression and regulation in hypertensive rats. Aortic TR mRNA was upregulated by angiotensin II (Ang II)-induced hypertension (10.7 +/- 2.5 times control, P < .02), which correlated with a 4-fold increase in thrombin-induced constriction in isolated endothelium-denuded aortic rings. The AT1 receptor antagonist losartan normalized blood pressure and TR mRNA. Conversely, lowering blood pressure to the same degree with hydralazine did not abolish the upregulation of TR mRNA expression. When low-renin low-Ang II hypertension was induced in Dahl salt-sensitive rats, there was no detectable increase in the expression of aortic thrombin receptor mRNA. Finally, treatment with a chimeric heparin-binding form of the recombinant human Cu/Zn superoxide dismutase caused complete inhibition of TR mRNA upregulation, suggesting that an increased rate of superoxide anion production is an important signaling mechanism. Thus, increased TR expression via a redox-sensitive mechanism in the aortic smooth muscle of rats treated with Ang II represents a novel in vivo mechanism through which the hypertensive effects of Ang II are mediated.

Failure of heparin to inhibit the expression of the thrombin receptor following endothelial injury of the rabbit carotid artery

The effect of heparin on thrombin receptor expression was evaluated in an experimental model of myointimal smooth muscle cell proliferation in rabbits. Myointimal hyperplasia was induced by an air-drying injury of the carotid artery and thrombin receptor expression following endothelial injury was measured by in situ hybridisation and immunohistochemistry. In healthy arteries, thrombin receptor mRNA and protein were detected in the endothelial cells only. In contrast, 14 days after endothelial injury, thrombin receptor mRNA expression increased in the smooth muscle cells present in the neointima, predominantly in areas of active cell proliferation. A 2-week subcutaneous treatment with heparin (10 mg/kg per day, s.c.) inhibited smooth muscle cell hyperplasia occurring in the intima following deendothelialization (80±7.8% inhibition, P<0.001). The 14-day heparin treatment strongly reduced thrombin receptor gene and protein expression observed in the endothelial cells in healthy arteries but did not affect thrombin receptor expression which occurred in smooth muscle cells which have proliferated in the neointima as a consequence of endothelial injury. These results therefore establish that thrombin receptor expression during intimal hyperplasia is an heparin-insensitive event.

The Thrombin Receptor Is Present in Myoblasts and Its Expression Is Repressed upon Fusion

Cultured myoblasts derived from limb muscle of newborn rats express thrombin receptor immunoreactivity on their surface. Receptor expression is repressed upon myoblast fusion. This is due at least in part to a decrease in the amount of the thrombin receptor mRNA. Addition of thrombin triggers calcium transients only in mono- but not multinucleated muscle cells. Furthermore, thrombin increases the rate of myoblast proliferation that coincides with an activation of mitogen-activated protein kinase. Northern analysis of thrombin receptor mRNA expression in skeletal muscle showed that the transcript is present at a relatively high level at birth, but is almost undetectable in the adult. By in situ hybridization, the mRNA at birth appeared to be present mostly in mononucleated cells grouped in clusters, but not in muscle fibers. Very few nuclei surrounded by a mRNA signal were present on muscle sections of rats 24 days postnatally. These results suggest that the thrombin receptor plays a role in muscle development.

Thrombin activates the sarcolemmal Na(+)-H+ exchanger. Evidence for a receptor-mediated mechanism involving protein kinase C.

Thrombin can activate the plasma membrane Na(+)-H+ exchanger in a variety of noncardiac cells. We have studied (1) the effect of thrombin on the activity of the sarcolemmal Na(+)-H+ exchanger in freshly isolated quiescent ventricular myocytes from the adult rat heart and (2) the signaling mechanism(s) underlying any effect. Reverse-transcription polymerase chain reaction analysis revealed thrombin receptor mRNA expression in a myocyte-enriched cell preparation. As an index of Na(+)-H+ exchanger activity, acid efflux rates (JHS) were determined in single myocytes (n = 4 to 11 per group) loaded with the pH-sensitive fluoroprobe carboxy-seminaphthorhodafluor-1 after two consecutive intracellular acid pulses (induced by transient exposure to 20 mmol/L NH4Cl) in bicarbonate-free medium. At a pHi of 6.9, JH did not change significantly during the second pulse relative to the first in control cells. However, when the second pulse occurred in the presence of 0.2, 1, or 5 U/mL thrombin, JH increased by 30%, 62% (P < .05), and 87% (P < .05), respectively. A hexameric thrombin receptor-activating peptide (SFLLRN) mimicked the effect of thrombin and increased JH by 73% (P < .05) at 25 mumol/L. In contrast, an inactive control peptide (FLLRN) was without effect at 25 mumol/L. In cells pretreated with 100 nmol/L GF109203X or 5 mumol/L chelerythrine (protein kinase C inhibitors), neither 5 U/mL thrombin nor 25 mumol/L SFLLRN produced a significant increase in JH. In the presence of 10 mumol/L HOE-694 (a Na(+)-H+ exchanger inhibitor), pHi did not recover after an acid load, even during exposure to 5 U/mL thrombin or 25 mumol/L SFLLRN, confirming that the Na(+)-H+ exchanger was the primary acid efflux mechanism under the conditions used. Neither 5 U/mL thrombin nor 25 mumol/L SFLLRN affected resting pHi and Ca2+ or background acid loading. We conclude that (1) adult rat ventricular myocytes express a functional thrombin receptor, whose stimulation results in increased activity of the sarcolemmal Na(+)-H+ exchanger, and (2) this effect appears to occur through a protein kinase C-mediated mechanism.

Thrombin Receptor-Mediated Synovial Proliferation in Patients with Rheumatoid Arthritis

Synovial cell proliferation is one of the pathological bases of rheumatoid arthritis (RA). Several cytokines including IL-1 and IL-6 and growth factors have been shown to be involved in the synovial cell proliferation in RA. Thrombin is a multifunctional protease and acts as a mitogen for several cell types through its specific receptor. To assess whether thrombin is involved in overproliferation of rheumatoid synovial cells, we measured the concentration of thrombin-antithrombin III (ATIII) complex (TAT) in synovial fluid obtained from patients with RA or osteoarthritis (OA). We also examined the effect of thrombin or thrombin receptor agonist peptide (TRAP) on cell growth of synovial cell clones (SCCs) established from an RA patient. The concentrations of TAT in the synovial fluid from patients with RA were significantly higher than in those with OA. Moreover, both thrombin and TRAP enhanced proliferation of synovial cells in vitro. We also characterized the expression of thrombin receptor mRNA by reverse transcription-PCR. The expression of mRNA for thrombin receptor was up-regulated by thrombin or TRAP stimulation. Thrombin receptor antigen was also detected on both SCCs and synovial tissue from RA patients by immunostaining using a monoclonal antibody against thrombin receptor. These findings indicate that thrombin may act as a mitogen for synovial cells through thrombin receptor and may play some role in synovial overproliferation and remodeling in RA.

Thrombin-induced mitogenesis in cultured aortic smooth muscle cells requires prolonged thrombin exposure

Thrombin has been implicated in vascular smooth muscle cell (VSMC) proliferation after vessel injury. Its proliferative effects, which are mediated via proteolytic activation of a receptor similar or identical to the cloned thrombin receptor (TR), have markedly delayed kinetics. The present study demonstrates that, despite rapid thrombin receptor activation and similar time to S phase entry compared with classic polypeptide growth factors, prolonged thrombin exposure is required to promote maximal VSMC mitogenesis. Flow cytometric analysis of thrombin-stimulated cells revealed that thrombin induced a progressive increase in the growth fraction over 3 days in culture, an effect that was blocked by hirudin even late after thrombin addition. Northern blot hybridization after thrombin stimulation demonstrated that thrombin upregulates TR mRNA expression within 6 h. These findings indicate that VSMC proliferate in response to prolonged thrombin exposure and suggest that the mitogenic delay may involve not only the thrombin-dependent synthesis and activation of newly made TR but also the progressive thrombin-dependent recruitment of cells into the growth fraction.

Cellular localization of thrombin receptor mRNA in rat brain: expression by mesencephalic dopaminergic neurons and codistribution with prothrombin mRNA

Cell culture studies demonstrating that the serine protease thrombin can induce neuronal and glial process retraction, glial proliferation, and changes in gene expression suggest a role for thrombin in CNS development, plasticity, and response to injury. Most cellular responses to thrombin are mediated by proteolytic activation of the cloned thrombin receptor (TR), a member of the seven transmembrane domain, G-protein-coupled receptor superfamily. As a step toward understanding the role of thrombin and its receptor in the CNS, Northern blot, in situ hybridization, and immunohistochemical techniques were used to analyze the cellular localization of TR mRNA in weanling-age rat brain. TR mRNA was broadly distributed across the neuraxis, although expression was very focal and often anatomically limited within specific neural structures. The greatest hybridization was associated with individual neurons in neocortex, cingulate/retrosplenial cortex, and subiculum, subsets of nuclei in hypothalamus, thalamus, pretectum, and ventral mesencephalon, and discrete cell layers in the hippocampus, cerebellum, and olfactory bulb. Patterns of hybridization included neuronal, glial, and ependymal cells, although white matter was uniformly negative, as were most cerebrovascular endothelial cells. Expression of TR mRNA by astroglia and dopaminergic neurons was confirmed by colocalization with immunoreactivity for glial fibrillary acidic protein (GFAP) in hippocampus and tyrosine hydroxylase in the substantia nigra. Comparison between TR and prothrombin (thrombin's precursor) cRNA hybridization demonstrated distinct but overlapping brain distributions of these transcripts, most clearly evident in postnatally developing, laminated structures. These results suggest widespread utilization of, and multiple physiologic, and possibly pathophysiologic, functions for, the thrombin/TR cell signaling system in the CNS.

Thrombin, Phorbol Ester, and cAMP Regulate Thrombin Receptor Protein and mRNA Expression by Different Pathways

Human mesangial cells have been used to study the regulation of thrombin receptor protein and mRNA expression during cross-talk between different signal transduction pathways. Persistent activation of thrombin receptor by thrombin led to homologous down-regulation of thrombin receptor protein. However, thrombin receptor mRNA expression was not affected, suggesting that increased receptor degradation is responsible for homologous down-regulation. Chronic activation of protein kinase C by phorbol 12-myristate 13-acetate (PMA) and of adenylylcyclase by prostaglandin E1 (PGE1) resulted in heterologous down-regulation of thrombin receptor protein. In contrast to thrombin, PMA and PGE1 reduced in parallel thrombin receptor mRNA levels to 51% and 24% of control, respectively, indicating that heterologous down-regulation of thrombin receptor protein is, at least in part, due to inhibition of receptor mRNA expression. The mechanisms of heterologous down-regulation of thrombin receptor protein have been studied in detail and compared to homologous down-regulation. PMA-induced down-regulation was completely blocked by GF 109 203 X, an inhibitor of protein kinase C. However, the loss of thrombin receptor induced by thrombin was not prevented by GF 109 203 X, indicating that homologous regulation is not dependent on protein kinase C activation. The heterologous effect of PGE1 was mimicked by 8-bromo-cAMP, isobutylmethylxanthine, and forskolin, suggesting that an increase in intracellular cAMP level is involved in heterologous regulation. Interestingly, heterologous down-regulation induced by PGE1 seems not to require previous internalization of thrombin receptor. These data indicate that thrombin receptor protein and mRNA expression can be regulated in homologous and heterologous ways by different mechanisms.

Characterization of thrombin receptor expression during vascular lesion formation.

Blood vessels respond to injury by initiating cell proliferation and migration that result in vascular lesion formation. To determine the roles of thrombin and the thrombin receptor in this process, we characterized thrombin receptor expression in normal and injured arteries, thrombin receptor-mediated smooth muscle cell mitogenesis, and the regulation of thrombin receptor mRNA expression in vitro. Thrombin receptor mRNA was not detected in normal rat or baboon arteries by in situ hybridization. Immunohistochemistry using an antithrombin receptor antibody (TR-R9), directed against the thrombin cleavage site of the rat aortic smooth muscle cell thrombin receptor, revealed low-level staining for thrombin receptor protein in endothelial cells and smooth muscle cells of normal arteries. In contrast, balloon catheter injury increased thrombin mRNA expression in medial smooth muscle cells within 6 hours. This increased thrombin receptor expression continued within the media and in neointimal cells throughout vascular lesion formation, predominantly in areas of active cell proliferation. In vitro, alpha-thrombin stimulates rat aortic smooth muscle cell proliferation in a concentration-dependent manner. That thrombin receptor activation is required for the mitogenic response was confirmed by demonstrating that the polyclonal antibody TR-R9 inhibits thrombin-induced cell proliferation. Thrombin receptor mRNA synthesis was induced by both basic fibroblast growth factor (maximal stimulation of 1.8-fold at 1 hour) and platelet-derived growth factor (maximal stimulation of 2.4-fold at 8 and 24 hours) in quiesced cultured rat aortic smooth muscle cells. In summary, upregulation of smooth muscle cell thrombin receptor expression occurs very early after vascular injury and continues throughout neointimal development.(ABSTRACT TRUNCATED AT 250 WORDS)

Thrombin and thrombin receptor agonist peptide induce early events of T cell activation and synergize with TCR cross-linking for CD69 expression and interleukin 2 production.

Thrombin stimulation of the T leukemic cell line Jurkat induced a transient increase in [Ca2+]i. Proteolytic activity of the enzyme was required for this effect since diisopropyl fluorophosphate-thrombin failed to increase [Ca2+]i. Furthermore, hirudin and anti-thrombin III inhibited the thrombin-induced [Ca2+]i rise in Jurkat T cells. A synthetic thrombin receptor agonist peptide (TRP) of 7 residues (SFLLRNP) was found to be as effective as thrombin for [Ca2+]i mobilization, and both agonists induced Ca2+ release exclusively from internal stores. Thrombin stimulated tyrosine phosphorylation of several proteins of molecular mass 40, 42, 70, 120, and 130 kDa. There was a good correlation between thrombin-induced tyrosine phosphorylation of the latter three proteins and Ca2+ mobilization. Thrombin and TRP also caused translocation of protein kinase C from the cytosol to the plasma membrane. As a likely consequence of these events, thrombin activated the nuclear factor NF-kB. Several cell lines of hematopoietic origin including the leukemic T cell line HPB.ALL and the erythroleukemic cell line K562 were responsive to thrombin, whereas others such as THP1, a myelomonocytic cell line, and BL2, a Burkitt lymphoma were refractory to thrombin or TRP stimulation. The magnitude of the thrombin response in the different cell types paralleled the expression of the thrombin receptor mRNA. We found that activation of Jurkat T cells by a combination of phytohemagglutinin and phorbol 12-myristate 13-acetate led to a dramatic inhibition of thrombin receptor mRNA expression and to a concomitant loss of the thrombin response. Finally, we demonstrate that thrombin and TRP enhanced CD69 expression and interleukin 2 production induced by T cell receptor cross-linking in both Jurkat T cells and peripheral blood lymphocytes. These findings highlight the role of thrombin as a potential regulator of T lymphocyte activation.