Thrombin-induced Increase Research Articles

Long-term Stimulation of α7 Nicotinic Acetylcholine Receptor Rescues Hemorrhagic Neuron Loss via Apoptosis of M1 Microglia.

We previously revealed that long-term treatment with nicotine suppresses microglial activation, resulting in a protective effect against thrombin-induced shrinkage of the striatal tissue in organotypic slice cultures. Here, the effect of nicotine on impaired M1 and protective M2 microglial polarization was investigated using the BV-2 microglial cell line in the presence or absence of thrombin. Following nicotine treatment, α7 nicotinic acetylcholine receptor expression transiently increased and then gradually decreased until 14 days. Treatment with nicotine for 14 days slightly polarized M0 microglia to M2b and d subtypes. Co-exposure of thrombin and low concentration of interferon-γ recruited inducible NO synthase (iNOS)- and interleukin-1β-double-positive M1 microglia in a thrombin-concentration-dependent manner. Treatment with nicotine for 14 days significantly decreased the thrombin-induced increase of iNOS mRNA levels and conversely showed a tendency to increase arginase1 mRNA levels. Moreover, treatment with nicotine for 14 days suppressed thrombin-induced phosphorylation of p38 MAPK through the α7 receptor. Repeated intraperitoneal administration of α7 agonist PNU-282987 for 14 days selectively evoked the apoptosis of iNOS-positive M1 microglia at the perihematomal area and showed a neuroprotective effect in an in vivo intracerebral hemorrhage model. These findings revealed that long-term stimulation of α7 receptor causes suppression of thrombin-induced activation of p38 MAPK followed by apoptosis in neuropathic M1 microglia.

Augmentation by resveratrol of the inhibitory effect of ethanol on platelet aggregation

Ethanol and resveratrol have been shown to inhibit platelet aggregation. The aim of this study was to determine whether resveratrol has an additional effect on ethanol-induced inhibition of platelet aggregation. Ca2+ entry and subsequent aggregation of human platelets were measured by the fluorescence method and light transmittance method, respectively. Thromboxane B2 concentrations in media containing platelets were measured by using the enzyme-linked immunosorbent assay. Platelet aggregation induced by thrombin (0.025 U/ml) was significantly inhibited by preincubation of platelets with ethanol (0.5%). Preincubation with resveratrol (3.125 µM), which did not affect thrombin-induced platelet aggregation, significantly augmented the inhibitory effect of ethanol on platelet aggregation. Similar synergic effects of ethanol and resveratrol were found on aggregatory responses to collagen (2 µg/ml) and arachidonic acid (0.25 mM). On the other hand, the thrombin-induced increase in intracellular Ca2+ concentration ([Ca2+]i) was not affected by ethanol alone, resveratrol alone or both ethanol and resveratrol together. In nominally Ca2+-free medium, arachidonic acid (0.75 mM) caused a potent platelet aggregation, which was not affected by the presence of ethanol alone, resveratrol alone, or both of them together. Thromboxane B2 formation induced by thrombin was significantly inhibited by ethanol (0.5%) alone and resveratrol (3.125 µM) alone, and these inhibitory effects were significantly augmented in the presence of both ethanol and resveratrol together. Resveratrol shows an additive effect on ethanol-induced inhibition of platelet aggregation. This effect by resveratrol is partly explained by its inhibitory action on thromboxane A2 production in platelets. In addition, both ethanol and resveratrol attenuate platelet aggregation through acting on the Ca2+-dependent intra-platelet pathway after an increase in [Ca2+]i induced by thrombin.

Dabigatran reduces thrombin-induced neuroinflammation and AD markers in vitro: Therapeutic relevance for Alzheimer's disease.

Vascular risk factors such as atherosclerosis, diabetes, and elevated homocysteine levels are strongly correlated with onset of Alzheimer's disease (AD). Emerging evidence indicates that blood coagulation protein thrombin is associated with vascular and non-vascular risk factors of AD. Here, we examined the effect of thrombin and its direct inhibitor dabigatran on key mediators of neuro-inflammation and AD pathology in the retinoic acid (RA)-differentiated human neuroblastoma cell line SH-SY5Y. SH-SY5Y cells exposed to thrombin concentrations (10-100nM) +/- 250nM dabigatran for 24h were analyzed for protein and gene expression. Electrophoretic mobility shift assay (EMSA) was used to determine DNA binding of NFkB. Western blotting, qRT-PCR and ELISA were used to measure the protein, mRNA, and activity levels of known AD hallmarks and signaling molecules. Dabigatran treatment attenuated thrombin-induced increase in DNA binding of NFκB by 175% at 50nM and by 77% at 100nM thrombin concentration. Thrombin also augmented accumulation of Aβ protein expression and phosphorylation of p38 MAPK, a downstream molecule in the signaling cascade, expression of pro-apoptotic mediator caspase 3, APP, tTau and pTau. Additionally, thrombin increased BACE1 activity, GSK3β expression, and APP, BACE1, Tau and GSK3β mRNA levels. Co-incubation with dabigatran attenuated thrombin-induced increases in the protein, mRNA, and activities of the aforesaid molecules to various extents (between -31% and -283%). Our data demonstrates that thrombin promotes AD-related pathological changes in neuronal cultures and suggests that use of direct oral anticoagulants may provide a therapeutic benefit against thrombin-driven neuroinflammation and downstream pathology in AD.

Thrombin-activated PAR1 membrane expression is regulated by Rab11a-RCP complex dissociation

PAR1 activation by thrombin promotes intracellular signaling leading to RPE cell transformation, proliferation, and migration, characteristic of fibroproliferative eye diseases. Due to the cleavage of PAR1 N-terminal domain, carried by thrombin, the arrest of PAR1 signaling is achieved by transport into lysosomes and degradation. Recent findings suggest that the GTPase Rab11a in conjunction with its effector RCP may direct PAR1 to lysosomes. Hereby we demonstrate that thrombin-induced PAR1 internalization and lysosomal targeting requires the disassembly of the Rab11a/RCP complex, and that this process depends on thrombin-induced intracellular calcium increase and calpain activation. These findings unveil a novel mechanism that regulates thrombin activated PAR1 internalization and degradation.

Mechanisms of thrombin-Induced myometrial contractions: Potential targets of progesterone.

Intrauterine bleeding during pregnancy is a major risk factor for preterm birth. Thrombin, the most abundant coagulation factor in blood, is associated with uterine myometrial contraction. Here, we investigated the molecular mechanism and signaling of thrombin-induced myometrial contraction. First, histologic studies of placental abruption, as a representative intrauterine bleeding, revealed that thrombin was expressed within the infiltrating hemorrhage and that thrombin receptor (protease-activated receptor 1, PAR1) was highly expressed in myometrial cells surrounding the hemorrhage. Treatment of human myometrial cells with thrombin resulted in augmented contraction via PAR1. Thrombin-induced signaling to myosin was then mediated by activation of myosin light chain kinase- and Rho-induced phosphorylation of myosin light chain-2. In addition, thrombin increased prostaglandin-endoperoxidase synthase-2 (PTGS2 or COX2) mRNA and prostaglandin E2 and F2α synthesis in human myometrial cells. Thrombin significantly increased the mRNA level of interleukine-1β, whereas it decreased the expressions of prostaglandin EP3 and F2α receptors. Progesterone partially blocked thrombin-induced myometrial contractions, which was accompanied by suppression of the thrombin-induced increase of PTGS2 and IL1B mRNA expressions as well as suppression of PAR1 expression. Collectively, thrombin induces myometrial contractions by two mechanisms, including direct activation of myosin and indirect increases in prostaglandin synthesis. The results suggest a therapeutic potential of progesterone for preterm labor complicated by intrauterine bleeding.

Mammalian target of rapamycin and p70S6K mediate thrombin-induced nuclear factor-κB activation and IL-8/CXCL8 release in human lung epithelial cells

Thrombin plays a crucial role in lung inflammatory diseases such as asthma and chronic obstructive pulmonary disease (COPD). Thrombin induces the release of interleukin-8 (IL-8)/CXCL8 by lung epithelial cells, and this phenomenon plays a vital role in lung inflammation. Our previous studies have indicated that thrombin stimulates IL-8/CXCL8 expression through PI3K/Akt/IκB kinase (IKK)α/β/nuclear factor-κB (NF-κB) and p300 pathways in human lung epithelial cells. In the present study, we explored the roles of mammalian target of rapamycin (mTOR) and p70S6 kinase (p70S6K) in thrombin-induced NF-κB activation and IL-8/CXCL8 release in human lung epithelial cells. In this study, we found that rapamycin (an mTOR inhibitor) and p70S6K siRNA diminished thrombin-induced IL-8/CXCL8 release. Thrombin induced mTOR Ser2448 phosphorylation and p70S6K Thr389 phosphorylation in a time-dependent manner. Moreover, rapamycin attenuated thrombin-stimulated p70S6K phosphorylation. We also found that transfection of cells with the dominant negative mutant of Akt (Akt DN) reduced the thrombin-induced increase in mTOR phosphorylation and p70S6K phosphorylation. Moreover, thrombin-stimulated p300 phosphorylation was attenuated by Akt DN, rapamycin, and p70S6K siRNA. Thrombin triggered p70S6K translocation from the cytosol to the nucleus in a time-dependent manner. Thrombin induced the complex formation of p70S6K, p300, and p65; acetylation of p65 Lys310, and recruitment of p70S6K, p300, and p65 to the κB-binding site of the IL-8/CXCL8 promoter region. In conclusion, these results indicate that thrombin initiates the Akt-dependent mTOR/p70S6K signaling pathway to promote p300 phosphorylation and NF-κB activation and finally induces IL-8/CXCL8 release in human lung epithelial cells.

Validation of a Miniaturized Permeability Assay Compatible with CRISPR-Mediated Genome-Wide Screen

The impermeability of the luminal endothelial cell monolayer is crucial for the normal performance of the vascular and lymphatic systems. A key to this function is the integrity of the monolayer’s intercellular junctions. The known repertoire of junction-regulating genes is incomplete. Current permeability assays are incompatible with high-throughput genome-wide screens that could identify these genes. To overcome these limitations, we designed a new permeability assay that consists of cell monolayers grown on ~150 μm microcarriers (MCs). Each MC functions as a miniature individual assay of permeability (MAP). We demonstrate that false-positive results can be minimized, and that MAP sensitivity to thrombin-induced increase in monolayer permeability is similar to the sensitivity of impedance measurement. We validated the assay by showing that the expression of single guide RNAs (sgRNAs) that target genes encoding known thrombin signaling proteins blocks effectively thrombin-induced junction disassembly, and that MAPs carrying such cells can be separated effectively by fluorescence-assisted sorting from those that carry cells expressing non-targeting sgRNAs. These results indicate that MAPs are suitable for high-throughput experimentation and for genome-wide screens for genes that mediate the disruptive effect of thrombin on endothelial cell junctions.

In Models of Intracerebral Hemorrhage, Rivaroxaban is Superior to Warfarin to Limit Blood Brain Barrier Disruption and Hematoma Expansion.

Intracerebral hemorrhage (ICH) during oral anticoagulation therapy with an oral vitamin K epoxidase reductase such as warfarin is a life-threatening complication. However, whether direct oral anticoagulants (DOACs) are associated with larger hematoma volume and higher mortality rates remains controversial. We evaluated the hematoma volume and pathophysiology of ICH during anticoagulation with warfarin or rivaroxaban, an orally active direct factor Xa inhibitor. Mice were orally pretreated with rivaroxaban (10 or 30 mg/kg), warfarin (4 mg/kg), or vehicle. ICH was induced by intrastriatal collagenase-injection. Hematoma volume and neurological deficits 24 h after ICH induction were significantly decreased in the rivaroxaban-pretreated group in comparison with the warfarin-pretreated group. Rivaroxaban did not increase the hematoma volume relative to that observed for vehicle, and improved survival rate 7 days after ICH induction compared with warfarin. We evaluated blood-brain barrier (BBB) permeability 6 h after ICH induction using Evans blue spectrophotometry. Evans blue extravasation was significantly reduced in the rivaroxaban group compared with the warfarin group. To investigate the mechanism underlying hematoma expansion and BBB permeability, we focused on thrombin, a clot-derived factor and one of the major contributors to ICH-induced brain injury. To investigate the effects of anticoagulant agents on thrombin-induced injuries, human brain endothelial cells were used in membrane permeability assays. Rivaroxaban, but not warfarin, significantly mitigated the thrombin-induced increase in membrane permeability. These findings indicate that rivaroxaban decreases BBB disruption after ICH, and limits early hematoma expansion in these experimental models compared with warfarin. Our study suggests that rivaroxaban has advantages over warfarin with respect to ICH, an important complication during long-term anticoagulation therapy.

P300 and C/EBPβ-regulated IKKβ expression are involved in thrombin-induced IL-8/CXCL8 expression in human lung epithelial cells

Asthma and chronic obstructive pulmonary disease (COPD) are common chronic lung inflammatory diseases. Thrombin and interleukin (IL)-8/C-X-C chemokine ligand 8 (CXCL8) play critical roles in lung inflammation. Our previous study showed that c-Src-dependent IκB kinase (IKK)/IκBα/nuclear factor (NF)-κB and mitogen-activated protein kinase kinase kinase 1 (MEKK1)/extracellular signal-regulated kinase (ERK)/ribosomal S6 protein kinase (RSK)-dependent CAAT/enhancer-binding protein β (C/EBPβ) activation are involved in thrombin-induced IL-8/CXCL8 expression in human lung epithelial cells. In this study, we aimed to investigate the roles of p300 and C/EBPβ-reliant IKKβ expression in thrombin-induced IL-8/CXCL8 expression. Thrombin-induced increases in IL-8/CXCL8-luciferase activity and IL-8/CXCL8 release were inhibited by p300 small interfering (siRNA). Thrombin-caused histone H3 acetylation was attenuated by p300 siRNA. Stimulation of cells with thrombin for 12h resulted in increases in IKKβ expression and phosphorylation in human lung epithelial cells. However, thrombin did not affect p65 expression. Moreover, 12h of thrombin stimulation produced increases in IKKβ expression and phosphorylation, and IκBα phosphorylation, which were inhibited by C/EBPβ siRNA. Finally, treatment of cells with thrombin caused increases in p300 and C/EBPβ complex formation, p65 and C/EBPβ complex formation, and recruitment of p300, p65, and C/EBPβ to the IL-8/CXCL8 promoter. These results imply that p300-dependent histone H3 acetylation and C/EBPβ-regulated IKKβ expression contribute to thrombin-induced IL-8/CXCL8 expression in human lung epithelial cells. Results of this study will help clarify C/EBPβ signaling pathways involved in thrombin-induced IL-8/CXCL8 expression in human lung epithelial cells.

Thrombin Induces Inositol Trisphosphate–Mediated Spatially Extensive Responses in Lung Microvessels

Activation of plasma membrane receptors initiates compartmentalized second messenger signaling. Whether this compartmentalization facilitates the preferential intercellular diffusion of specific second messengers is unclear. Toward this, the receptor-mediated agonist, thrombin, was instilled into microvessels in a restricted region of isolated blood-perfused mouse lungs. Subsequently, the thrombin-induced increase in endothelial F-actin was determined using confocal fluorescence microscopy. Increased F-actin was evident in microvessels directly treated with thrombin and in those located in adjoining thrombin-free regions. This increase was abrogated by inhibiting inositol trisphosphate-mediated calcium release with Xestospongin C (XeC). XeC also inhibited the thrombin-induced increase in the amplitude of endothelial cytosolic Ca2+ oscillations. Instillation of thrombin and XeC into adjacent restricted regions increased F-actin in microvessels in the thrombin-treated and adjacent regions but not in those in the XeC-treated region. Thus, inositol trisphosphate, and not calcium, diffused interendothelially to the spatially remote thrombin-free microvessels. Thus, activation of plasma membrane receptors increased the ambit of inflammatory responses via a second messengerdifferent from that used by stimuli that induce cell-wide increases in second messengers. Thrombin however failed to induce the spatially extensive response in microvessels of mice lacking endothelialconnexin43, suggesting a role for connexin43 gap junctions. Compartmental second messengersignaling and interendothelial communication define the specific second messenger involvedin exacerbating proinflammatory responses to receptor-mediated agonists.

Evaluation of effects of various drugs on platelet functions using phorbol 12-myristate 13-acetate-induced megakaryocytic human erythroid leukemia cells.

BackgroundThe hyperfunction and activation of platelets have been strongly implicated in the development and recurrence of arterial occlusive disease, and various antiplatelet drugs are used to treat and prevent such diseases. New antiplatelet drugs and many other drugs have been developed, but some drugs may have adverse effects on platelet functions.ObjectiveThe aim of this study was to establish an evaluation method for evaluating the effect and adverse effect of various drugs on platelet functions.Materials and methodsHuman erythroid leukemia (HEL) cells were used after megakaryocytic differentiation with phorbol 12-myristate 13-acetate as an alternative to platelets. Drugs were evaluated by changes in intracellular Ca2+ concentration ([Ca2+]i) mobilization in Fura2-loaded phorbol 12-myristate 13-acetate-induced HEL cells. Aspirin and cilostazol were selected as antiplatelet drugs and ibuprofen and sodium valproate as other drugs.ResultsThere was a positive correlation between [Ca2+]i and platelet aggregation induced by thrombin. Aspirin (5.6–560 µM) and cilostazol (5–10 µM) significantly inhibited thrombin-induced increases in [Ca2+]i in a concentration-dependent manner. On the other hand, ibuprofen (8–200 µM) and sodium valproate (50–1,000 µg/mL) also significantly inhibited thrombin-induced increases in [Ca2+]i in a concentration-dependent manner. Furthermore, the interaction effects of the simultaneous combined use of aspirin and ibuprofen or sodium valproate were evaluated. When the inhibitory effect of aspirin was higher than that of ibuprofen, the effect of aspirin was reduced, whereas when the inhibitory effect of aspirin was lower than that of ibuprofen, the effect of ibuprofen was reduced. The combination of aspirin and sodium valproate synergistically inhibited thrombin-induced [Ca2+]i.ConclusionIt is possible to induce HEL cells to differentiate into megakaryocytes, which are a useful model for the study of platelet functions, and the quantification of the inhibition of thrombin-induced increases in [Ca2+]i is applicable to the evaluation of the effects of various drugs on platelets.

Dual role of vinculin in barrier-disruptive and barrier-enhancing endothelial cell responses

Endothelial cell (EC) barrier disruption induced by edemagenic agonists such as thrombin is a result of increased actomyosin contraction and enforcement of focal adhesions (FA) anchoring contracting stress fibers, which leads to cell retraction and force-induced disruption of cell junctions. In turn, EC barrier enhancement by oxidized phospholipids (OxPAPC) and other agonists is a result of increased tethering forces due to enforcement of the peripheral actin rim and enhancement of cell–cell adherens junction (AJ) complexes promoting EC barrier integrity. This study tested participation of the mechanosensitive adaptor, vinculin, which couples FA and AJ to actin cytoskeleton, in control of the EC permeability response to barrier disruptive (thrombin) and barrier enhancing (OxPAPC) stimulation. OxPAPC and thrombin induced different patterns of FA remodeling. Knockdown of vinculin attenuated both, OxPAPC-induced decrease and thrombin-induced increase in EC permeability. Thrombin stimulated the vinculin association with FA protein talin and suppressed the interaction with AJ protein, VE-cadherin. In contrast, OxPAPC stimulated the vinculin association with VE-cadherin. Thrombin and OxPAPC induced different levels of myosin light chain (MLC) phosphorylation and caused different patterns of intracellular phospho-MLC distribution. Thrombin-induced talin-vinculin and OxPAPC-induced VE-cadherin–vinculin association were abolished by myosin inhibitor blebbistatin. Expression of the vinculin mutant unable to interact with actin attenuated EC permeability changes and MLC phosphorylation caused by both, thrombin and OxPAPC. These data suggest that the specific vinculin interaction with FA or AJ in different contexts of agonist stimulation is defined by development of regional actyomyosin-based tension and participates in both, the barrier-disruptive and barrier-enhancing endothelial responses.

Thrombin-induced IL-8/CXCL8 release is mediated by CK2, MSK1, and NF-κB pathways in human lung epithelial cells

Airway inflammation plays a major role in the pathophysiology of lung inflammatory diseases such as asthma. Thrombin, a serine protease, is known to mediate central functions in thrombosis and hemostasis and also plays a critical role in lung inflammation via producing chemokine release including interleukin (IL)-8/CXCL8. Our previous studies showed that c-Src- and Rac-dependent nuclear factor (NF)-κB signaling pathways participate in thrombin-induced IL-8/CXCL8 release in human lung epithelial cells. In this study, we further investigated the role of casein kinase 2 (CK2)/mitogen stress-activated protein kinase 1 (MSK1)-dependent p65 phosphorylation in thrombin-induced NF-κB activation and IL-8/CXCL8 release. Thrombin-induced IL-8/CXCL8 release was inhibited by CK2 inhibitors (apigenin and tetrabromobenzotriazole, TBB), small interfering RNA of CK2β (CK2β siRNA), and MSK1 siRNA. Treatment of cells with thrombin caused increases in CK2β phosphorylation at Ser209, which was inhibited by a protein kinase C α (PKCα) inhibitor (Ro-32-0432). Thrombin-induced MSK1 phosphorylation at Ser581 and Akt phosphorylation at Ser473 were inhibited by apigenin. Moreover, the thrombin-induced increase in IL-8/CXCL8 release was attenuated by p65 siRNA. Stimulation of cells with thrombin resulted in an increase in p65 phosphorylation at Ser276, which was inhibited by apigenin and MSK1 siRNA. Thrombin-induced κB-luciferase activity was also inhibited by apigenin and MSK1 siRNA. Taken together, these results show that thrombin activates the PKCα/CK2/MSK1 signaling pathways, which in turn initiates p65 phosphorylation and NF-κB activation, and ultimately induces IL-8/CXCL8 release in human lung epithelial cells.

TAK1 Signaling Downstream of PAR‐1 in Endothelial Cells Restores Lung Vascular Barrier Integrity

The proinflammatory mediator thrombin increases vascular permeability by disassembling vascular endothelial adherens junctions (AJs). However, the intrinsic signaling mechanisms that mediate the reassembly of endothelial AJs are poorly understood. Here we show that transforming growth factor‐β‐activated kinase (TAK1) activation downstream of protease‐activated receptor‐1 (PAR‐1) signals the reassembly of endothelial AJs. PAR‐1 agonist, thrombin induced a time‐dependent phosphorylation of TAK1 in lung endothelial cells (ECs). Silencing of stromal interaction molecule 1 (STIM1) suppressed thrombin‐induced TAK1 phosphorylation in ECs, indicating that store‐operated calcium entry (SOCE) is essential for PAR‐1‐mediated TAK1 activation. Inhibition of TAK1 augmented thrombin‐induced permeability increase both in vitro and in intact lung microvessels. Consistent with these findings, inhibition of TAK1 impaired reassembly VE‐cadherin at AJs after thrombin treatment. We also observed that thrombin‐induced SOCE was markedly increased in TAK1 deficient ECs. Since global as well as EC‐restricted TAK1 knockout mice are embryonically lethal, to study the in vivo role of EC‐expressed TAK1, we generated tamoxifen‐inducible EC‐restricted TAK1 knockout (TAK1iΔEC) mice. Surprisingly, we observed markedly reduced expression of VE‐cadherin and its associated protein β‐catenin in lungs of TAK1iΔEC mice compared with their wild type littermates. We also observed enhanced lung vascular permeability as assessed by Evens blue dye‐conjugated albumin uptake in TAK1iΔEC mice. These findings support the notion that TAK1 signaling in endothelial cells is required to maintain lung vascular barrier integrity.

CLL2-1, a chemical derivative of orchid 1,4-phenanthrenequinones, inhibits human platelet aggregation through thiol modification of calcium-diacylglycerol guanine nucleotide exchange factor-I (CalDAG-GEFI)

CalDAG-GEFI is a guanine nucleotide exchange factor, which actives small GTPase Rap1 and plays an important role in platelet aggregation. Our previous study has shown that CalDAG-GEFI contains redox-sensitive thiols, and its function can be inhibited by thiol modification. In the present study, the effect of CLL2-1, a 1,4-phenanthrenequinone, on CalDAG-GEFI and platelet functions was investigated. In human platelets, CLL2-1 prevented platelet aggregation caused by various stimulators. Flow cytometric analysis revealed that CLL2-1 inhibited GPIIb/IIIa activation and P-selectin secretion. Moreover, CLL2-1 prevented Rap1 activation caused by thrombin, the Ca2+ ionophore A23187, and the diacylglycerol mimetic phorbol 12-myristate 13-acetate, while only slightly inhibited thrombin-induced increases in [Ca2+]i and did not inhibit protein kinase C activation. Western blots after reducing SDS-PAGE showed that treatment of either platelets or platelet lysates with CLL2-1 led to a decrease of monomeric CalDAG-GEFI and appearance of cross-linked oligomers of CalDAG-GEFI, and these effects were inhibited by pretreatment of platelets or lysates with thiol reducing agents prior to the addition of CLL2-1, indicating thiol modification of CalDAG-GEFI by CLL2-1. Furthermore, the thiol reducing agents also prevented the inhibitory effect of CLL2-1 on Rap1 activation, GPIIb/IIIa activation, and platelet aggregation. In CalDAG-GEFI-overexpressing human embryonic kidney 293T cells, CLL2-1 also inhibited CalDAG-GEFI-mediated Rap1 activation. Taken together, our results suggest that the antiplatelet effect of CLL2-1 is due to, at least in part, inhibition of CalDAG-GEFI-mediated Rap1 activation, and provide the basis for development of novel antiplatelet drugs.

Proteomic Effects of the Coagulation Proteinase Thrombin on LX-2 Hepatic Stellate Cells

Summary Background: The aim of this study was to characterize the effects of the coagulation proteinase thrombin on proteomic level in human hepatic stellate LX-2 cells. Methods: Proteomic analyses were performed using surface-enhanced laser desorption/ionization-time of flight mass spectrometry (SELDI-TOF-MS). The protein profiles obtained from LX-2 cell lysates using strong anion exchanger Q10 ProteinChip arrays were statistically analyzed. Results: The peak intensities of 50 protein/peptide clusters were identified as being different between nonstimulated and LX-2 cells treated with thrombin for 6 h and 24 h, respectively. As the most significantly enhanced single signal in LX-2 cells stimulated with thrombin, a protein with a molecular mass of 13.560 kDa has been identified that corresponds exactly to calcium dependent phospholipase 2 (cPLA2). Thrombin-induced increase in the cPLA2 protein expression in LX-2 cells was confirmed by using the Western blotting technique. Conclusions: Together with the finding that thrombin induced phosphorylating activation of cPLA2 in LX-2 cells, our data point to an important function of the thrombin-mediated modulation of cytosolic phospholipase A2 in hepatic stellate cells.

Low anticoagulant heparin blocks thrombin-induced endothelial permeability in a PAR-dependent manner

Acute lung injury and acute respiratory distress syndrome are accompanied by thrombin activation and fibrin deposition that enhance lung inflammation, activate endothelial cells and disrupt lung paracellular permeability. Heparin possesses anti-inflammatory properties but its clinical use is limited by hemorrhage and heparin induced thrombocytopenia. We studied the effects of heparin and low anticoagulant 2-O, 3-O desulfated heparin (ODSH) on thrombin-induced increases in paracellular permeability of cultured human pulmonary endothelial cells (ECs). Pretreatment with heparin or ODSH blocked thrombin-induced decrease in the EC transendothelial electrical resistance (TER), attenuated thrombin-stimulated paracellular gap formation and actin cytoskeletal rearrangement. Our data demonstrated that heparin and ODSH had inhibitory effects on thrombin-induced RhoA activation and intracellular calcium elevation. Thrombin-stimulated phosphorylation of the cytoskeletal regulatory proteins, myosin light chain and ezrin/radixin/moesin was also reduced. In these effects, low anticoagulant ODSH was more potent than heparin. Heparin or ODSH alone produced decreases in the EC TER that were abolished by siRNA-mediated depletion of the thrombin receptor, PAR-1. We also demonstrated that, in contrast to heparin, ODSH did not possess thrombin-binding activity. Results suggest that heparin and low anticoagulant ODSH can interfere with thrombin-activated signaling.

Thrombin-Induced CCAAT/Enhancer-Binding Protein β Activation and IL-8/CXCL8 Expression via MEKK1, ERK, and p90 Ribosomal S6 Kinase 1 in Lung Epithelial Cells

Thrombin, a serine protease, is a well-known coagulation factor generated during vascular injury and plays an important role in lung inflammation. We previously showed that the c-Src- and Rac/PI3K/Akt-dependent NF-κB pathways are involved in thrombin-induced IL-8/CXCL8 expression in human lung epithelial cells (A549). In this study, we investigated the role of the MEK kinase (MEKK)1/ERK/p90 ribosomal S6 kinase (RSK)1-dependent C/EBPβ signaling pathway in thrombin-induced IL-8/CXCL8 expression. Thrombin-induced IL-8/CXCL8 release and IL-8/CXCL8-luciferase activity were attenuated by small interfering RNA (siRNA) of C/EBPβ and by cells transfected with the C/EBPβ site mutation of the IL-8/CXCL8 construct. Moreover, thrombin-induced κB-luciferase activity was also inhibited by C/EBPβ siRNA. The thrombin-induced increases in IL-8/CXCL8 release and IL-8/CXCL8-luciferase were also inhibited by MEKK1 siRNA, PD98059 (an MEK inhibitor), U0126 (an ERK inhibitor), and RSK1 siRNA. Treatment of cells with thrombin caused an increase in C/EBPβ phosphorylation at Thr(235), C/EBPβ-luciferase activity, recruitment of C/EBPβ to the IL-8/CXCL8 promoter, and C/EBPβ-specific DNA complex formation. Furthermore, thrombin-mediated C/EBPβ phosphorylation and C/EBPβ-luciferase activity were inhibited by MEKK1 siRNA, PD98059, and RSK1 siRNA. Stimulation of cells with thrombin resulted in an increase in RSK1 phosphorylation at Thr(359)/Ser(363), and this effect was inhibited by MEKK1 siRNA and PD98059. The thrombin-induced increase in ERK activation was inhibited by MEKK1 siRNA. These results imply that thrombin activates the MEKK1/ERK/RSK1 signaling pathway, which in turn initiates C/EBPβ activation, recruitment of C/EBPβ to the IL-8/CXCL8 promoter, and C/EBPβ-specific DNA complex formation, and ultimately induces IL-8/CXCL8 expression and release in lung epithelial cells.

Endothelial permeability in vitro and in vivo: Protective actions of ANP and omapatrilat in experimental atherosclerosis

Increased arterial endothelial cell permeability (ECP) is considered an initial step in atherosclerosis. Atrial natriuretic peptide (ANP) which is rapidly degraded by neprilysin (NEP) may reduce injury-induced endothelial cell leakiness. Omapatrilat represents a first in class of pharmacological agents which inhibits both NEP and angiotensin converting enzyme (ACE). We hypothesized that ANP prevents thrombin-induced increases of ECP in human aortic ECs (HAECs) and that omapatrilat would reduce aortic leakiness and atherogenesis and enhance ANP mediated vasorelaxation of isolated aortas. Thrombin induced ECP determined by I125 albumin flux was assessed in HAECs with and without ANP pretreatment. Next we examined the effects of chronic oral administration of omapatrilat (12mg/kg/day, n=13) or placebo (n=13) for 8 weeks on aortic leakiness, atherogenesis and ANP-mediated vasorelaxation in isolated aortas in a rabbit model of atherosclerosis produced by high cholesterol diet. In HAECs, thrombin-induced increases in ECP were prevented by ANP. Omapatrilat reduced the area of increased aortic leakiness determined by Evans-blue dye and area of atheroma formation assessed by Oil-Red staining compared to placebo. In isolated arterial rings, omapatrilat enhanced vasorelaxation to ANP compared to placebo with and without the endothelium. ANP prevents thrombin-induced increases in ECP in HAECs. Chronic oral administration of omapatrilat reduces aortic leakiness and atheroma formation with enhanced endothelial independent vasorelaxation to ANP. These studies support the therapeutic potential of dual inhibition of NEP and ACE in the prevention of increased arterial ECP and atherogenesis which may be linked to the ANP/cGMP system.

Ezrin/radixin/moesin proteins differentially regulate endothelial hyperpermeability after thrombin

Endothelial cell (EC) barrier disruption induced by inflammatory agonists such as thrombin leads to potentially lethal physiological dysfunction such as alveolar flooding, hypoxemia, and pulmonary edema. Thrombin stimulates paracellular gap and F-actin stress fiber formation, triggers actomyosin contraction, and alters EC permeability through multiple mechanisms that include protein kinase C (PKC) activation. We previously have shown that the ezrin, radixin, and moesin (ERM) actin-binding proteins differentially participate in sphingosine-1 phosphate-induced EC barrier enhancement. Phosphorylation of a conserved threonine residue in the COOH-terminus of ERM proteins causes conformational changes in ERM to unmask binding sites and is considered a hallmark of ERM activation. In the present study we test the hypothesis that ERM proteins are phosphorylated on this critical threonine residue by thrombin-induced signaling events and explore the role of the ERM family in modulating thrombin-induced cytoskeletal rearrangement and EC barrier function. Thrombin promotes ERM phosphorylation at this threonine residue (ezrin Thr567, radixin Thr564, moesin Thr558) in a PKC-dependent fashion and induces translocation of phosphorylated ERM to the EC periphery. Thrombin-induced ERM threonine phosphorylation is likely synergistically mediated by protease-activated receptors PAR1 and PAR2. Using the siRNA approach, depletion of either moesin alone or of all three ERM proteins significantly attenuates thrombin-induced increase in EC barrier permeability (transendothelial electrical resistance), cytoskeletal rearrangements, paracellular gap formation, and accumulation of phospho-myosin light chain. In contrast, radixin depletion exerts opposing effects on these indexes. These data suggest that ERM proteins play important differential roles in the thrombin-induced modulation of EC permeability, with moesin promoting barrier dysfunction and radixin opposing it.