Plasminogen-induced aggregation of PANC-1 cells requires conversion to plasmin and is inhibited by endogenous plasminogen activator inhibitor-1

PANC-1 cells express proteinase-activated receptors (PARs)-1, -2, and respond to their activation by transient elevation of cytosolic [Ca(2+)] and accelerated aggregation (Wei et al., 2006, J Cell Physiol 206:322-328). We studied the effect of plasminogen (PGN), an inactive precursor of the PAR-1-activating protease, plasmin (PN) on aggregation of pancreatic adenocarcinoma (PDAC) cells. A single dose of PGN time- and dose-dependently promoted PANC-1 cells aggregation in serum-free medium, while PN did not. PANC-1 cells express urokinase plasminogen activator (uPA), which continuously converted PGN to PN. This activity and PGN-induced aggregation were inhibited by the uPA inhibitor amiloride. PGN-induced aggregation was also inhibited by alpha-antiplasmin and by the PN inhibitor epsilon-aminocaproic acid (EACA). Direct assay of uPA activity revealed very low rate, markedly enhanced in the presence of PGN. Moreover, in PGN activator inhibitor 1-deficient PANC-1 cells, uPA activity and PGN-induced aggregation were markedly potentiated. Two additional human PDAC cell lines, MiaPaCa and Colo347, were assayed for PGN-induced aggregation. Both cell lines responded by aggregation and exhibited PGN-enhanced uPA activity. We hypothesized that the continuous conversion of PGN to PN by endogenous uPA is limited by PN's degradation and negatively controlled by endogenously produced PAI-1. Indeed, we found that PANC-1 cells inactivate PN with t1/2 of approximately 7 h, while the continuous addition of PN promoted aggregation. Our data suggest that PANC-1 cells possess intrinsic, PAI-1-sensitive mechanism for promotion of aggregation and differentiation by prolonged exposure to PGN and, possibly, additional precursors of PARs agonists.     

PAR-3 Knockdown Enhances Adhesion Rate of PANC-1 Cells via Increased Expression of Integrinαv and E-Cadherin

The balance between the adhesion of cancer cells to extracellular matrix and their migratory potential, as well as their proteolytic activity, are important parameters that determine cancer cells invasiveness and metastasis. Since thrombin has been implicated in cancer progression, we studied the role(s) of thrombin-activated receptors in the adhesion process. We stably knocked down proteinase-activated receptors (PARs) -1, or -3 in human pancreatic adenocarcinoma PANC-1 cells. PANC-1 cells exhibit rapid adhesion to cell culture treated plastic and much faster kinetics of adhesion to Matrigel coated surface. Knockdown of PAR-1 had no effect on cells'' adhesiveness, while PAR-3 knockdowns (KDs) exhibited much faster adhesion kinetics. PAR-3 KDs also exhibited slower in vitro wound closure than vector-control and PAR-1 KD cells. To study the molecular mechanism(s) of PAR-3 KD cells'' enhanced rate of adhesion, we assayed the expression of the molecules that mediate cell-surface and cell-cell adhesion. ITGαv, as well as ITGα6 and ITGα10 mRNAs, were greatly enriched (>40-fold) in a rapidly-adhering sub-population of PAR-3 KD cells. The whole population of both PAR-1 and -3 KDs exhibited enhanced expression of a number of integrins (ITGs) mRNAs. However, ITGαv mRNA and protein expression was increased in PAR-3 KD and markedly decreased in PAR-1 KD. PAR-3 KD cells also expressed more E-cadherin mRNA and protein. The enhanced adhesion kinetics of PAR-3 KDs was almost fully inhibited by calcium chelation, or by a HAV-motive decapeptide that affects E-cadherin intermolecular interactions. We propose that the enhanced rate of adhesion of PAR-3 KDs results from enhanced expression of E-cadherin, leading to a greater adhesion of free-floating cells to cells rapidly bound to the surface via their integrins, and particularly ITGαv.     

Reaction of mast cell proteases tryptase and chymase with protease activated receptors (PARs) on keratinocytes and fibroblasts

Protease activated receptors (PARs) compose a family of G protein signal transduction receptors activated by proteolysis. In this study, the susceptibility of PARs expressed on human keratinocytes and dermal fibroblasts to the human mast cell proteases tryptase and chymase was evaluated. PAR activation was measured by monitoring cytosolic [Ca²⁺] in cells loaded with the fluorescent Ca²⁺ probe Fura-2. Tryptase produced transient cytosolic Ca²⁺ mobilization in keratinocytes, but not in fibroblasts. Ca²⁺ mobilization in keratinocytes required enzymatically active tryptase, demonstrated desensitization, and was blocked by pretreatment of cells with the PAR-2 peptide agonist SLIGKV, trypsin, or the phospholipase inhibitor U73122. Heparin, a GAG that binds to tryptase, stabilizing its functional form, also inhibited tryptase-induced Ca²⁺ mobilization. The maximal response elicited by tryptase was smaller than that observed upon treatment of keratinocytes with trypsin, a known activator of PAR-2, and keratinocytes made refractory to tryptase by pretreatment with the protease remained responsive to trypsin. Pretreatment of keratinocytes with thrombin, an activator of PAR-1 and -3 (thrombin receptors), had no detectable effect on the tryptase or trypsin responses. These data suggest that in keratinocytes tryptase may be activating a subpopulation of PAR-2 receptors. Treatment of keratinocytes or fibroblasts with human chymase did not produce Ca²⁺ mobilization, nor did it affect Ca²⁺ mobilization produced by trypsin. However, chymase pretreatment of fibroblasts rapidly inhibited the ability of these cells to respond to thrombin. Inhibition was dependent on chymase enzymatic activity and was not significantly affected by the presence of heparin. This finding is consistent with studies indicating that PAR-1 may be susceptible to proteases with chymotrypsin-like specificity. These results suggest that the proteases tryptase and chymase secreted from mast cells in skin may affect the behavior of surrounding cells by the hydrolysis of PARs expressed by these cells. J. Cell. Physiol. 176:365–373, 1998. © 1998 Wiley-Liss, Inc.     

Plasmin-mediated activation of platelets occurs by cleavage of protease-activated receptor 4

The activation of plasmin from its circulating precursor plasminogen is the mechanism of several clot-busting drugs used to clinically treat patients who have suffered a stroke; however, plasmin thus generated has been shown to activate platelets directly. There has been speculation as to whether plasmin interacts with the protease-activated receptors (PARs) because of its similarity in amino acid specificity with the classic platelet activator thrombin. We have investigated whether plasmin activates platelets via PAR activation through multiple complementary approaches. At concentrations sufficient to induce human platelet aggregation, plasmin released very little calcium compared with that induced by thrombin, the PAR-1 agonist peptide SFLLRN, or the PAR-4 agonist peptide AYPGKF. Stimulation of platelets with plasmin initially failed to desensitize additional stimulation with SFLLRN or AYPGKF, but a prolonged incubation with plasmin desensitized platelets to further stimulation by thrombin. The desensitization of PAR-1 had no effect on plasmin-induced platelet aggregation and yielded an aggregation profile that was similar to plasmin in response to a low dose of thrombin. However, PAR-4 desensitization completely eliminated aggregation in response to plasmin. Inclusion of the PAR-1-specific antagonist BMS-200261 inhibited platelet aggregation induced by a low dose of thrombin but not by plasmin. Additionally, mouse platelets naturally devoid of PAR-1 showed a full aggregation response to plasmin in comparison to thrombin. Furthermore, human and mouse platelets treated with a PAR-4 antagonist, as well as platelets isolated from PAR-4 homozygous null mice, failed to aggregate in response to plasmin. Finally, a protease-resistant recombinant PAR-4 was refractory to activation by plasmin. We conclude that plasmin induces platelet aggregation primarily through slow cleavage of PAR-4.     

Protease-activated receptor-2 (PAR-2) in brain microvascular endothelium and its regulation by plasmin and elastase

Protease-activated receptors (PARs) mediate cell activation after proteolytic cleavage of their extracellular amino terminus. We have reported earlier that primary cultures of rat brain capillary endothelial (RBCE) cells express at least two receptors for thrombin: PAR-1 and PAR-3. In the present study we show that PAR-2 activation by trypsin or by the PAR-2 agonist peptide (SLIGRL) evokes [Ca(2+) ](i) signal in RBCE cells. Taking advantage of RBCE cells expressing PAR-1 and PAR-2, we show that trypsin activates both receptors. The relative agonist activity of trypsin and thrombin on PARs of RBCE cells compared with that of SLIGRL were 112% and 48%, respectively, whereas the potency of trypsin was 10(5) -fold higher than that of SLIGRL. Because under pathological conditions other proteases such as plasmin or leukocyte elastase may reach the cells of the blood-brain barrier, we investigated the effect of these proteases on RBCE cells. Elastase evoked a small increase in [Ca(2+) ](i) but preincubation of cells with elastase dose-dependently reduced the trypsin-induced [Ca(2+) ](i) signal. Plasmin had a 30% inhibitory effect on the trypsin-induced response, and reduced the SLIGRL signal by 20%. It is concluded that PAR-2 is functional in brain capillary endothelium, and that the main fibrinolytic proteases, plasmin and elastase, may regulate PAR-2 signalling under pathological conditions.     

Mesotrypsin, a brain trypsin, activates selectively proteinase-activated receptor-1, but not proteinase-activated receptor-2, in rat astrocytes

Proteinase-activated receptors (PARs), a subfamily of G protein-coupled receptors, which are activated by serine proteases, such as trypsin, play pivotal roles in the CNS. Mesotrypsin (trypsin IV) has been identified as a brain-specific trypsin isoform. However, its potential physiological role concerning PAR activation in the brain is largely unknown. Here, we show for the first time that mesotrypsin, encoded by the PRSS3 (proteinase, serine) gene, evokes a transient and pronounced Ca(2+) mobilization in both primary rat astrocytes and retinal ganglion RGC-5 cells, suggesting a physiological role of mesotrypsin in brain cells. Mesotrypsin mediates Ca(2+) responses in rat astrocytes in a concentration-dependent manner, with a 50% effective concentration (EC(50)) value of 25 nm. The maximal effect of mesotrypsin on Ca(2+) mobilization in rat astrocytes is much higher than that observed in 1321N1 human astrocytoma cells, indicating that the activity of mesotrypsin is species-specific. The pre-treatment of cells with thrombin or the PAR-1-specific peptide TRag (Ala-pFluoro-Phe-Arg-Cha-HomoArg-Tyr-NH(2), synthetic thrombin receptor agonist peptide), but not the PAR-2-specific peptide, reduces significantly the mesotrypsin-induced Ca(2+) response. Treatment with the PAR-1 antagonist SCH79797 confirms that mesotrypsin selectively activates PAR-1 in rat astrocytes. Unlike mesotrypsin, the two other trypsin isoforms, cationic and anionic trypsin, activate multiple PARs in rat astrocytes. Therefore, our data suggest that brain-specific mesotrypsin, via the regulation of PAR-1, is likely to be involved in multiple physiological/pathological processes in the brain.     

Macrophage migration inhibitory factor is induced by thrombin and factor Xa in endothelial cells

Macrophage migration inhibitory factor (MIF), a proinflammatory cytokine, has been shown to play a role in wound-healing processes. In this study, we investigated whether protease-activated receptor (PAR)-1 and PAR-2 mediated MIF expression in human endothelial cells. Thrombin, factor Xa (FXa), and trypsin induced MIF expression in human dermal microvascular endothelial cells and human umbilical vein endothelial cells, but other proteases, including kallikrein and urokinase, failed to do so. Thrombin-induced MIF mRNA expression was significantly reduced by the thrombin-specific inhibitor hirudin. Thrombin receptor activation peptide-6, a synthetic PAR-1 peptide, induced MIF mRNA expression, suggesting that PAR-1 mediates MIF expression in response to thrombin. The effects of FXa were blocked by antithrombin III, but not by hirudin, indicating that FXa might enhance MIF production directly rather than via thrombin stimulation. The synthetic PAR-2 peptide SLIGRL-NH(2) induced MIF mRNA expression, showing that PAR-2 mediated MIF expression in response to FXa. Concerning the signal transduction, a mitogen-activated protein kinase kinase inhibitor (PD98089) and a nuclear factor (NF)-kappaB inhibitor (SN50) suppressed the up-regulation of MIF mRNA in response to thrombin, FXa, and PAR-2 agonist stimulation, whereas a p38 inhibitor (SB203580) had little effect. These facts indicate that up-regulation of MIF by thrombin or FXa is regulated by p44/p42 mitogen-activated protein kinase-dependent pathways and NF-kappaB-dependent pathways. Moreover, we found that PAR-1 and PAR-2 mRNA expression in endothelial cells was enhanced by MIF. Furthermore, we examined the inflammatory response induced by PAR-1 and PAR-2 agonists injected into the mouse footpad. As shown by footpad thickness, an indicator of inflammation, MIF-deficient mice (C57BL/6) were much less sensitive to either PAR-1 or PAR-2 agonists than wild-type mice. Taken together, these results suggest that MIF contributes to the inflammatory phase of the wound healing process in concert with thrombin and FXa via PAR-1 and PAR-2.     

Phenotypic changes in mouse pancreatic stellate cell Ca2+ signaling events following activation in culture and in a disease model of pancreatitis

The specific characteristics of intracellular Ca 2+ signaling and the downstream consequences of these events were investigated in mouse pancreatic stellate cells (PSC) in culture and in situ using multiphoton microscopy in pancreatic lobules. PSC undergo a phenotypic transformation from a quiescent state to a myofibroblast-like phenotype in culture. This is believed to parallel the induction of an activated state observed in pancreatic disease such as chronic pancreatitis and pancreatic cancer. By day 7 in culture, the complement of cell surface receptors coupled to intracellular Ca 2+ signaling was shown to be markedly altered. Specifically, protease-activated receptors (PAR) 1 and 2, responsive to thrombin and trypsin, respectively, and platelet-derived growth factor (PDGF) receptors were expressed only in activated PSC (aPSC). PAR-1, ATP, and PDGF receptor activation resulted in prominent nuclear Ca 2+ signals. Nuclear Ca 2+ signals and aPSC proliferation were abolished by expression of parvalbumin targeted to the nucleus. In pancreatic lobules, PSC responded to agonists consistent with the presence of only quiescent PSC. aPSC were observed following induction of experimental pancreatitis. In contrast, in a mouse model of pancreatic disease harboring elevated K-Ras activity in acinar cells, aPSC were present under control conditions and their number greatly increased following induction of pancreatitis. These data are consistent with nuclear Ca 2+ signaling generated by agents such as trypsin and thrombin, likely present in the pancreas in disease states, resulting in proliferation of "primed" aPSC to contribute to the severity of pancreatic disease.     

Trypsin stimulates integrin alpha(5)beta(1)-dependent adhesion to fibronectin and proliferation of human gastric carcinoma cells through activation of proteinase-activated receptor-2

Trypsin is widely expressed in various non-pancreatic tissues at low levels and overexpressed in some types of human cancers. In the present study, we found that trypsin stimulates integrin-dependent adhesion and growth of MKN-1 human gastric carcinoma cells. MKN-1 cells expressed both proteinase-activated receptor-1 (PAR-1) and PAR-2, which are activated by thrombin and trypsin, respectively. Both trypsin and the PAR-2 ligand SLIGKV promoted integrin alpha(5)beta(1)-mediated adhesion of MKN-1 cells to fibronectin, and less effectively integrin alpha(v)beta(3)-mediated cell adhesion to vitronectin, but not that to type IV collagen or laminin-1 at all. Thrombin and the PAR-1 ligand SFLLRN promoted the cell adhesion to vitronectin more strongly than trypsin or the PAR-2 ligand, but not the cell adhesion to fibronectin at all. The cell adhesion-stimulating effect of the PAR-2 ligand was significantly reduced by the pre-treatment of cells with trypsin, indicating that the effect of trypsin is mediated by PAR-2 activation. The trypsin-stimulated cell adhesion to vitronectin, but not to fibronectin, was effectively inhibited by the G(i) protein blocker pertussis toxin, and both cell adhesions were completely inhibited by the Src kinase inhibitor herbimycin A. Furthermore, trypsin and the PAR-2 ligand stimulated growth of MKN-1 cells more strongly than thrombin or the PAR-1 ligand. These results show that trypsin regulates cellular adhesion and proliferation by inducing PAR-2/G protein signalings, and that the integrin alpha(5)beta(1)- and integrin alpha(v)beta(3)-dependent cell adhesions are regulated by different PAR/G protein signalings.     

Involvement of SNARE proteins in thrombin-induced platelet aggregation: evidence for the relevance of Ca2+ entry

Thrombin induces platelet activation through a variety of intracellular mechanisms, including Ca(2+) mobilization. The protein of the exocytotic machinery SNAP-25, but not VAMPs, is required for store-operated Ca(2+) entry, the main mechanism for Ca(2+) influx in platelets. Hence, we have investigated the role of the SNAP-25 and VAMPs in thrombin-induced platelet aggregation. Platelet stimulation with thrombin or selective activation of thrombin receptors PAR-1, PAR-4 or GPIb-IX-V results in platelet aggregation that, except for GPIb-IX-V receptor, requires Ca(2+) entry for full activation. Depletion of the intracellular Ca(2+) stores using pharmacological tools was unable to induce aggregation except when cytosolic Ca(2+) concentration reached a critical level (around 1.5 microM). Electrotransjection of cells with anti-SNAP-25 antibody reduced thrombin-evoked platelet aggregation, while electrotransjection of anti-VAMP-1, -2 and -3 antibody had no effect. These findings support a role for SNAP-25 but not VAMP-1, -2 and -3 in platelet aggregation, which is likely mediated by the regulation of Ca(2+) mobilization in human platelets.     

Streptococcal SpeB Cleaved PAR-1 Suppresses ERK Phosphorylation and Blunts Thrombin-Induced Platelet Aggregation

Background

The family of 4 related protease-activated receptors (PAR-1, 2, 3 & 4) expressed by mammalian cells allow to sense for and react to extracellular proteolytic activity. Since major human bacterial pathogens secret a wide array of protease(-s) we investigated whether they interfere with human PAR function.

Methodology/Principal Findings

Supernatants from cultures of major human bacterial pathogens were assayed for the presence of protease(-s) capable to cleave overexpressed human PAR-1, 2, 3 and 4 reporter constructs. Group A streptococcus (GAS) was found to secret a PAR-1-cleaving protease. Experiments involving genetical and pharmacological gain and loss of function identified streptococcal pyrogenic exotoxin B SpeB as the protease responsible. On the host’s side analysis of overexpressed PAR-1 carrying alanine substitutions and deletions showed the amino acid residue leucine₄₄ on PAR-1’s extracellular N-terminus to be the only cleavage site. Complementary studies on endogenously expressed PAR-1 using PAR-1 blocking antibodies further supported our conclusion. Through PAR-1 cleavage SpeB efficiently blunted thrombin-induced induction of the ERK-pathway in endothelial cells and prevented platelets aggregation in response to thrombin.

Conclusions/Significance

Our results identify a novel function of the streptococcal virulence factor SpeB. By cleaving human PAR-1 at the N-terminal amino acid residue leucine₄₄ SpeB rendered endothelial cells unresponsive to thrombin and prevented human platelets from thrombin-induced aggregation. These results suggest that by blunting PAR-1 signaling, SpeB modulates various innate host responses directed against invasive GAS potentially helping the invasive bacteria to escape. This may allow to tailor additional treatments in the future since upon invasion of the blood stream endothelial cells as well as platelets and mononuclear cells respond to PAR-1 agonists aiming to prevent further bacterial dissemination.     

Sustained stimulation of platelet thrombin receptor is associated with tyrosine dephosphorylation of a novel p67 peptide in a manner regulated by extracellular calcium

Signaling pathways elicited by protease-activated receptor-1 (PAR-1) agonists, thrombin receptor-activating peptide (TRAP) and thrombin, are markedly different. Here we show that TRAP-induced disaggregation of platelets is a function of extracellular calcium. Chelation of calcium with EGTA after the onset of aggregation precluded subsequent destabilization of the aggregates in TRAP-stimulated platelets, whereas disaggregation was not observed in the platelets stimulated with thrombin. TRAP-induced disaggregation was independent of the activity of the calcium-dependent thiol protease, calpain. Inhibition of phosphoinositide 3-kinase activity provoked further destabilization of the platelet aggregates in the presence of calcium; however, EGTA attenuated this effect. Activation of protein kinase C (PKC) by phorbol ester prevented disaggregation of the TRAP-stimulated platelets independent of the extracellular calcium. Two proteins of relative mobilities 67 and 75 kD were found to be significantly dephosphorylated on tyrosine in calcium-pretreated platelets as compared to the EGTA-treated platelets following continued stimulation with either TRAP or thrombin for 15 min. Inhibition of phosphoinositide 3-kinase by two pharmacologically independent inhibitors also caused dephosphorylation of p67, which was completely abrogated by chelation of extracellular calcium. Platelet activation by phorbol ester was not associated with disaggregation, although dephosphorylation of p67 was induced under this condition. SHP-1, an abundant tyrosine phosphatase in platelets, co-migrated with the p67 protein and co-localized to the actin-based cytoskeleton of aggregated platelets; however, its identity with p67 was ruled out from immunoprecipitation studies.     

Protease-activated receptor-2 (PAR-2) is a weak enhancer of mucin secretion by human bronchial epithelial cells in vitro

PAR-2, a member of a family of G-protein-coupled receptors, can be activated by serine proteases via proteolytic cleavage. PAR-2 expression is known to be upregulated in respiratory epithelium subsequent to inflammation in asthma and chronic obstructive pulmonary disease (COPD). Since these diseases also are characterized by excessive mucus production and secretion, we investigated whether PAR-2 could be linked to mucin hypersecretion by airway epithelium. Normal human bronchial epithelial (NHBE) cells in primary culture or the human bronchial epithelial cell lines, NCI-H292 and HBE-1, were used. NHBE, NCI-H292, and HBE-1 cells expressed prominent levels of PAR-2 protein. Short-term (30min) exposure of cells to the synthetic PAR-2 agonist peptide (SLIGKV-NH2) elicited a small but statistically significant increase in mucin secretion at high concentrations (100microM and 1000microM), compared to a control peptide with reversed amino acid sequence (VKGILS-NH2). Neither human lung tryptase nor bovine pancreatic trypsin, both PAR-2 agonists, affected NHBE cell mucin secretion when added over a range of concentrations. Knockdown of PAR-2 expression by siRNA blocked the stimulatory effect of the AP. The results suggest that, since PAR-2 activation only weakly increases mucin secretion by human airway epithelial cells in vitro, PAR-2 probably is not a significant contributor to mucin hypersecretion in inflamed airways.     

Thrombin-stimulated Pyk2 phosphorylation in human endothelium is dependent on intracellular calcium and independent of protein kinase C and Src kinases

G-protein-coupled receptor agonists (GPCAs) cause functional responses in endothelial cells including secretion, proliferation, and altering monolayer permeability. These events are mediated in part by activation of the p42/44 mitogen-activated protein kinase (MAPK) cascade. The cytosolic tyrosine kinase Pyk2 is postulated to link GPCA-induced changes in intracellular calcium to activation of the MAP kinase cascade. We have investigated the regulation of Pyk2 in human umbilical vein endothelial cells in response to GPCAs and show that (1) thrombin, a PAR-1 peptide, and histamine cause rapid concentration- and time-dependent phosphorylation on tyrosines 402 (Src kinase binding site), 881 (Grb2 binding site), and 580 (an autophosphorylation site), (2) thrombin-stimulated phosphorylation is dependent on intracellular calcium and independent of PKC and PI-3 kinase, and (3) inhibition of Src kinases has no significant effect on thrombin-stimulated phosphorylation, implying that tyrosine phosphorylation of Pyk2 is independent of Src binding.     

The correlation between rises in intracellular calcium and PGI2 production in cultured vascular endothelial cells.

Elevation of intracellular calcium in response to trypsin, bradykinin, thrombin or histamine is associated with a proportional increase in PGI2 production in cultured human umbilical vein endothelial cells (HUVEC), bovine pulmonary artery endothelial cells (CPAE), and bovine aortic endothelial cells (BAEC). The major agonists that induce increases in intracellular calcium and PGI2 production are thrombin and trypsin in HUVEC, bradykinin in CPAE, and bradykinin and trypsin in BAEC. These results suggest that endothelial cells derived from different species or sites require different agonists to induce increases in intracellular calcium and PGI2 production and that only agonists which increase intracellular calcium can stimulate PGI2 production.     

Characterization of thrombin-induced leukocyte rolling and adherence: a potential proinflammatory role for proteinase-activated receptor-4

It is commonly accepted that thrombin exerts its proinflammatory properties through the activation of proteinase-activated receptor (PAR)-1, although two other thrombin receptors have been discovered: PAR-3 and PAR-4. In this study, we have investigated the mechanisms and the receptors involved in thrombin-induced leukocyte/endothelial cell interactions by using selective agonists and antagonists of thrombin receptors in an in vivo intravital microscopy system. Topical addition of selective PAR-1 agonists to rat mesenteric venules failed to reproduce the increased leukocyte rolling and adhesion observed after thrombin topical addition. When added together with the selective PAR-1 antagonist RWJ-56110, thrombin was still able to provoke increased leukocyte rolling and adherence. The thrombin-induced leukocyte rolling and adherence was not affected by pretreatment of rats with an anti-platelet serum. Selective PAR-4-activating peptide was able to reproduce the effects of thrombin on leukocyte rolling and adhesion. Intraperitoneal injection of PAR-4-activating peptide also caused a significant increase in leukocyte migration into the peritoneal cavity. In rat tissues, PAR-4 expression was detected both on endothelium and isolated leukocytes. Taken together, these results showed that in rat mesenteric venules, thrombin exerts proinflammatory properties inducing leukocyte rolling and adherence, by a mechanism independent of PAR-1 activation or platelet activation. However, PAR-4 activation either on endothelial cells or on leukocytes might be responsible for the thrombin-induced effects. These findings suggest that PAR-4 activation could contribute to several early events in the inflammatory reaction, including leukocyte rolling, adherence and recruitment, and that in addition to PAR-1, PAR-4 could be involved in proinflammatory properties of thrombin.     

Early caspase-3 activation independent of apoptosis is required for cellular function

A number of pro-apoptotic stimuli induce the activation of caspase-9, an initiator protease that activates executioner caspases, such as caspase-3, leading to the development of programmed cell death. Here we demonstrate that cell (platelets and pancreatic acinar cells) stimulation with agonists induces a bimodal activation of caspase-3. The early caspase-3 activation occurs within 1 min of stimulation and is independent on caspase-9 or mitochondrial cytochrome c release suggesting that is a non-apoptotic event. The ability of agonists to induce early activation of caspase-3 is similar to that observed for other physiological processes. Activation of caspase-3 by physiological concentrations of cellular agonists, including thrombin or CCK-8, is independent of rises in cytosolic calcium concentration but requires PKC activation, and is necessary for agonist-induced activation of the tyrosine kinases Btk and pp60src and for several cellular functions, including store-operated calcium entry, platelet aggregation, or pancreatic secretion. Thus, early activation of caspase-3 seems to be a non-apoptotic event required for cellular function.     

Dissection of protease-activated receptor-1-dependent and -independent responses to thrombin in skeletal myoblasts

Thrombin exerts a number of effects on skeletal myoblasts in vitro. It stimulates proliferation and intracellular calcium mobilization and inhibits differentiation and apoptosis induced by serum deprivation in these cells. Many cellular responses to thrombin are mediated by protease-activated receptor-1 (PAR-1). Expression of PAR-1 is present in mononuclear myoblasts in vitro, but repressed when fusion occurs to form myotubes. In the current study, we used PAR-1-null mice to determine which of thrombin's effects on myoblasts are mediated by PAR-1. Thrombin inhibited fusion almost as effectively in cultures prepared from the muscle of PAR-1-null myoblasts as in cultures prepared from wild-type mice. Apoptosis was inhibited as effectively in PAR-1-null myoblasts as in wild-type myoblasts. These effects in PAR-1-null myoblasts were mediated by a secreted inhibitor of apoptosis and fusion, as demonstrated previously for normal rat myoblasts. Thrombin failed to induce an intracellular calcium response in PAR-1-null myoblast cultures, although these cells were able to mobilize intracellular calcium in response to activation of other receptors. PAR-1-null myoblasts also failed to proliferate in response to thrombin. These results demonstrate that thrombin's effects on myoblast apoptosis and fusion are not mediated by PAR-1 and that PAR-1 is the only thrombin receptor capable of inducing proliferation and calcium mobilization in neonatal mouse myoblasts.