Different pathways for control of Na+/H+ exchange via activation of the thrombin receptor.
Ontology highlight
ABSTRACT: The aim of the present study was to clarify the control of Na+/H+ exchange in platelets activated via the thrombin receptor. When human BCECF-loaded platelets were stimulated with the thrombin-receptor-activating peptide (TRAP; amino acid sequence SFLLRN), which activates the receptor independently of proteolysis, the cytosolic pH (pHi) rose from 7.13 +/- 0.04 (n = 6) to 7.27 +/- 0.04 (n = 5), followed by a rapid decrease to resting values. Trypsin, which cleaves the receptor, induced a rapid and irreversible rise in pHi to 7.31 +/- 0.06 (n = 5). gamma-Thrombin, which cleaves the receptor but is unable to bind to the hirudin-like domain, induced a slow and irreversible rise in pHi to 7.31 +/- 0.04 (n = 14). alpha-Thrombin, which cleaves the receptor and binds to its hirudin-like domain, induced a rapid and irreversible rise in pHi to 7.31 +/- 0.04 (n = 22). Changes in pHi induced by TRAP, trypsin, gamma- and alpha-thrombin were accompanied by similar changes in cytosolic Ca2+ concentration ([Ca2+]i) and 32P-pleckstrin, a substrate of protein kinase C (PKC). The separate chelation of Ca2+i (30 microM BAPTA-AM) or inhibition of PKC (1 microM staurosporine) induced about 50% inhibition of the pHi responses triggered by TRAP, trypsin, gamma- and alpha-thrombin, but the combination induced complete inhibition. Thus the different types of activation of the thrombin receptor control Na+/H+ exchange via the same mechanism. Binding of thrombin to the hirudin-like domain accelerates exchange activation, whereas proteolysis of the receptor is essential for a sustained increase in pHi.
Project description:Spinal cord injury (SCI) will result in the significant elevation of thrombin production at lesion site via either breakage of blood-spinal cord barrier or upregulated expression within nerve cells. Thrombin-induced activation of the protease activated receptors (PARs) evokes various pathological effects that deteriorate the functional outcomes of the injured cord. The cellular consequences of thrombin action on the astrocytes, as well as the underlying mechanism are not fully elucidated by far. In the present study, SCI model of rats was established by contusion, and primary astrocytes were isolated for culture from newborn rats. The expression levels of thrombin and PAR1 receptor at lesion sites of the spinal cord were determined. The primary astrocytes cultured in vitro were stimulated with different concentration of thrombin, and the resultant morphological changes, inflammatory astrocytic responses, as well as PAR1-activated signal pathway of astrocytes were accordingly examined using various agonists or antagonists of the receptor. Thrombin was found to reverse astrocytic stellation, promote proliferation but inhibit migration of astrocytes. Furthermore, the serine protease was shown to facilitate inflammatory response of astrocytes through regulation of MAPKs/NFκB pathway. Our results have provided the morphological evidence of astrocytic reactivity in response to thrombin stimulation and its neuroinflammatory effects following SCI, which will be indicative for the fundamental insights of thrombin-induced neuropathology.
Project description:Thrombin is a dual action serine protease in the blood clotting cascade. Similar to other clotting factors, thrombin is mainly present in the blood in a zymogen form, prothrombin. Although the two cleavage events required to activate thrombin are well-known, little is known about why the thrombin precursors are inactive proteases. Although prothrombin is much larger than thrombin, prethrombin-2, which contains all of the same amino acids as thrombin, but has not yet been cleaved between Arg320 and Ile321, remains inactive. Crystal structures of both prethrombin-2 and thrombin are available and show almost no differences in the active site conformations. Slight differences were, however, seen in the loops surrounding the active site, which are larger in thrombin than in most other trypsin-like proteases, and have been shown to be important for substrate specificity. To explore whether the dynamics of the active site loops were different in the various zymogen forms of thrombin, we employed amide H/(2)H exchange experiments to compare the exchange rates of regions of thrombin with the same regions of prothrombin, prethrombin-2, and meizothrombin. Many of the surface loops showed less exchange in the zymogen forms, including the large loop corresponding to anion binding exosite 1. Conversely, the autolysis loop and sodium-binding site exchanged more readily in the zymogen forms. Prothrombin and prethrombin-2 gave nearly identical results while meizothrombin in some regions more closely resembled active thrombin. Thus, cleavage of the Arg320-Ile321 peptide bond is the key to formation of the active enzyme, which involves increased dynamics of the substrate-binding loops and decreased dynamics of the catalytic site.
Project description:We have investigated changes in cytoplasmic pH (pHi) in activated human platelets, using the fluorescent probe 2,7-biscarboxyethyl-5(6)-carboxyfluorescein. Stimulation of platelets by thrombin or 12-O-tetradecanoylphorbol 13-acetate increased pHi by about 0.11 pH unit above the resting value. This increase in pHi depended on the presence of external Na+ and was inhibited by ethylisopropylamiloride. The data suggest that protein kinase C mediates Na+/H+ exchange in human platelets.
Project description:Purpose:We analyzed the molecular mechanisms leading to glutamate release from rat primary cultures of RPE cells, under isosmotic conditions. Thrombin has been shown to stimulate glutamate release from astrocytes and retinal glia; however, the effect of thrombin on glutamate release from RPE cells has not been examined. Our previous work showed that upon the alteration of the blood-retina barrier, the serine protease thrombin could contribute to the transformation, proliferation, and migration of RPE cells. In this condition, elevated extracellular glutamate causes neuronal loss in many retinal disorders, including glaucoma, ischemia, diabetic retinopathy, and inherited photoreceptor degeneration. Methods:Primary cultures of rat RPE cells were preloaded with 1 µCi/ml 3H-glutamate in Krebs Ringer Bicarbonate (KRB) buffer for 30 min at 37 °C. Cells were rinsed and super-perfused with 1 ml/min KRB for 15 min. Stable release was reached at the 7th minute, and on the 8th minute, fresh KRB containing stimuli was added. Results:This study showed for the first time that thrombin promotes specific, dose-dependent glutamate release from RPE cells, induced by the activation of protease-activated receptor 1 (PAR-1). This effect was found to depend on the Ca2+ increase mediated by the phospholipase C-β (PLC-β) and protein kinase C (PKC) pathways, as well as by the reverse activity of the Na+/Ca2+ exchanger. Conclusions:Given the intimate contact of the RPE with the photoreceptor outer segments, diffusion of RPE-released glutamate could contribute to the excitotoxic death of retinal neurons, and the development of thrombin-induced eye pathologies.
Project description:Thrombin and thrombin receptor activation impact cardiomyocyte contraction and ventricular remodeling. However, there is some controversy regarding their effects in cardiac function, especially in cardiac dysfunction after acute myocardial infarction (AMI). A rat AMI model was created by left coronary artery ligation (LCA). Cardiac functional parameters, including the maximum left ventricular (LV) systolic pressure (LVSPmax), LV end-diastolic pressure (LVEDP), and the rise and fall rates in LV pressure (dp/dt max and dp/dt min, respectively), were measured. Hirudin decreased cardiac function within 120 minutes after AMI, whereas treatment with thrombin receptor-activating peptide (TRAP) reversed this hirudin-induced decrease in cardiac function. The mRNA and protein expression levels of inositol 1,4,5-trisphosphate receptor (IP3R) subtypes in infarct area tissues were analyzed by reverse transcription-polymerase chain reaction and immunoreaction. Hirudin decreased the expression levels of IP3R-1, -2, and -3 in the infarct area for up to 40 minutes after AMI, whereas TRAP treatment reversed these hirudin-induced effects. Treatment with the IP3R antagonist 2-aminoethoxydiphenyl borate (2.5 mg/kg) eliminated the effect of TRAP on the hirudin-induced decrease in cardiac function after AMI. Finally, TRAP increased the maximum binding capacity of the three IP3R subtypes, but only enhanced the affinity of IP3R-2. Thrombin and thrombin receptor activation improved cardiac function after AMI by an IP3R-mediated pathway, probably through the IP3R-2 subtype.
Project description:INTRODUCTION: Fibronectin (FN) is commonly used in the development of serum-free media for the expansion of mesenchymal stem cells (MSCs). This study was aimed to observe if thrombin could stimulate FN secretion by human bone marrow MSCs and investigate the potential underlying mechanisms. METHODS: PCR was performed to detect the expression of the protease-activated receptors (PARs) in MSCs. After thrombin treatment, the expression level and secretion of FN were observed by RT-PCR, immunofluorescence staining and ELISA, respectively, and the activation of ERK1/2 and NF kappa B pathways was revealed by Western blotting, with or without pre-treatment of small-molecule blockers specific for PAR-1 and -2. The phenotypic and functional activities of thrombin-treated MSCs were also observed. RESULTS: PCR analysis showed that human bone marrow MSCs expressed two subtypes of PARs, PAR-1 and PAR-2. Thrombin treatment enhanced MSCs to express FN at mRNA and protein levels and promoted FN secretion by MSCs, accompanied by potent adherence to the culture plastic. Thrombin induced prompt phosphorylation of ERK 1/2 and NF kappa B p65 and the stimulatory effects of thrombin on FN secretion were blunted by specific inhibitors of these signaling molecules. Blockage to PAR-1 and PAR-2 partially abrogated thrombin-elicited FN secretion by MSCs and ERK 1/2 phosphorylation, whereas that of NF kappa B p65 was unaffected. Moreover, thrombin-treated MSCs maintained the phenotypic features, in vitro osteogenesis and adipogenesis capacities, and inhibitory activity on Phytohemagglutinin-induced allogeneic lymphocyte proliferation. CONCLUSIONS: Thrombin could promote FN secretion by MSCs via PAR-mediated ERK 1/2 activation, while NF kappa B might be also involved in an undefined manner.
Project description:The molecular mechanism of thrombin activation by Na(+) remains elusive. Its kinetic formulation requires extension of the classical Botts-Morales theory for the action of a modifier on an enzyme to correctly account for the contribution of the E*, E, and E:Na(+) forms. The extended scheme establishes that analysis of k(cat) unequivocally identifies allosteric transduction of Na(+) binding into enhanced catalytic activity. The thrombin mutant N143P features no Na(+)-dependent enhancement of k(cat) yet binds Na(+) with an affinity comparable to that of wild type. Crystal structures of the mutant in the presence and absence of Na(+) confirm that Pro(143) abrogates the important H-bond between the backbone N atom of residue 143 and the carbonyl O atom of Glu(192), which in turn controls the orientation of the Glu(192)-Gly(193) peptide bond and the correct architecture of the oxyanion hole. We conclude that Na(+) activates thrombin by securing the correct orientation of the Glu(192)-Gly(193) peptide bond, which is likely flipped in the absence of cation. Absolute conservation of the 143-192 H-bond in trypsin-like proteases and the importance of the oxyanion hole in protease function suggest that this mechanism of Na(+) activation is present in all Na(+)-activated trypsin-like proteases.
Project description:The disease mechanisms underlying type 2 diabetes (T2D) remain poorly defined. Here we aimed to explore the pathophysiology of T2D by analyzing gene co-expression networks in human islets. Using partial correlation networks we identified a group of co-expressed genes ('module') including F2RL2 that was associated with glycated hemoglobin. F2Rl2 is a G-protein-coupled receptor (GPCR) that encodes protease-activated receptor-3 (PAR3). PAR3 is cleaved by thrombin, which exposes a 6-amino acid sequence that acts as a 'tethered ligand' to regulate cellular signaling. We have characterized the effect of PAR3 activation on insulin secretion by static insulin secretion measurements, capacitance measurements, studies of diabetic animal models and patient samples. We demonstrate that thrombin stimulates insulin secretion, an effect that was prevented by an antibody that blocks the thrombin cleavage site of PAR3. Treatment with a peptide corresponding to the PAR3 tethered ligand stimulated islet insulin secretion and single β-cell exocytosis by a mechanism that involves activation of phospholipase C and Ca(2+) release from intracellular stores. Moreover, we observed that the expression of tissue factor, which regulates thrombin generation, was increased in human islets from T2D donors and associated with enhanced β-cell exocytosis. Finally, we demonstrate that thrombin generation potential in patients with T2D was associated with increased fasting insulin and insulinogenic index. The findings provide a previously unrecognized link between hypercoagulability and hyperinsulinemia and suggest that reducing thrombin activity or blocking PAR3 cleavage could potentially counteract the exaggerated insulin secretion that drives insulin resistance and β-cell exhaustion in T2D.
Project description:Thrombin is a multifunctional serine protease that has a crucial role in blood coagulation. It is also a potent mesenchymal cell mitogen and chemoattractant and might therefore have an important role in the recruitment and local proliferation of mesenchymal cells at sites of tissue injury. We hypothesized that thrombin might also affect the deposition of connective tissue proteins at these sites by directly stimulating fibroblast procollagen production. To address this hypothesis, the effect of thrombin on procollagen production and gene expression by human foetal lung fibroblasts was assessed over 48 h. Thrombin stimulated procollagen production at concentrations of 1 nM and above, with maximal increases of between 60% and 117% at 10 nM thrombin. These effects of thrombin were, at least in part, due to increased steady-state levels of alpha1(I) procollagen mRNA. They could furthermore be reproduced with thrombin receptor-activating peptides for the protease-activated receptor 1 (PAR-1) and were completely abolished when thrombin was rendered proteolytically inactive with the specific inhibitors d-Phe-Pro-ArgCH2Cl and hirudin, indicating that thrombin is mediating these effects via the proteolytic activation of PAR-1. These results suggest that thrombin might influence the deposition of connective tissue proteins during normal wound healing and the development of tissue fibrosis by stimulating fibroblast procollagen production.
Project description:Thrombin-induced platelet activation via PAR1 and PAR4 is an important event in haemostasis. Although the underlying mechanisms responsible for ensuring efficient PAR1 activation by thrombin have been extensively studied, the potential involvement of recognitions sites outside the active site of the protease in thrombin-induced PAR4 activation is largely unknown. In this study, we developed a new assay to assess the importance of exosite I and II for PAR4 activation with α - and γ-thrombin. Surprisingly, we found that exosite II is critical for activation of PAR4. We also show that this dependency on exosite II likely represents a new mechanism, as it is unaffected by blockage of the previously known interaction between thrombin and glycoprotein Ibα.