RhoA GTPase switch controls Cx43-hemichannel activity through the contractile system

ATP-dependent paracrine signaling, mediated via the release of ATP through plasma membrane-embedded hemichannels of the connexin family, coordinates a synchronized response between neighboring cells. Connexin 43 (Cx43) hemichannels that are present in the plasma membrane need to be tightly regulated to ensure cell viability. In monolayers of bovine corneal endothelial cells (BCEC),Cx43-mediated ATP release is strongly inhibited when the cells are treated with inflammatory mediators, in particular thrombin and histamine. In this study we investigated the involvement of RhoA activation in the inhibition of hemichannel-mediated ATP release in BCEC. We found that RhoA activation occurs rapidly and transiently upon thrombin treatment of BCEC. The RhoA activity correlated with the onset of actomyosin contractility that is involved in the inhibition of Cx43 hemichannels. RhoA activation and inhibition of Cx43-hemichannel activity were both prevented by pre-treatment of the cells with C3-toxin as well as knock down of RhoA by siRNA. These findings provide evidence that RhoA activation is a key player in thrombin-induced inhibition of Cx43-hemichannel activity. This study demonstrates that RhoA GTPase activity is involved in the acute inhibition of ATP-dependent paracrine signaling, mediated by Cx43 hemichannels, in response to the inflammatory mediator thrombin. Therefore, RhoA appears to be an important molecular switch that controls Cx43 hemichannel openings and hemichannel-mediated ATP-dependent paracrine intercellular communication under (patho) physiological conditions of stress

Paradigm of biased PAR1 (protease-activated receptor-1) activation and inhibition in endothelial cells dissected by phosphoproteomics

Thrombin is the key serine protease of the coagulation cascade and mediates cellular responses by activation of PARs (protease-activated receptors). The predominant thrombin receptor is PAR1, and in endothelial cells (ECs), thrombin dynamically regulates a plethora of phosphorylation events. However, it has remained unclear whether thrombin signaling is exclusively mediated through PAR1. Furthermore, mechanistic insight into activation and inhibition of PAR1-mediated EC signaling is lacking. In addition, signaling networks of biased PAR1 activation after differential cleavage of the PAR1 N terminus have remained an unresolved issue. Here, we used a quantitative phosphoproteomics approach to show that classical and peptide activation of PAR1 induce highly similar signaling, that low thrombin concentrations initiate only limited phosphoregulation, and that the PAR1 inhibitors vorapaxar and parmodulin-2 demonstrate distinct antagonistic properties. Subsequent analysis of the thrombin-regulated phosphosites in the presence of PAR1 inhibitors revealed that biased activation of PAR1 is not solely linked to a specific G-protein downstream of PAR1. In addition, we showed that only the canonical thrombin PAR1 tethered ligand induces extensive early phosphoregulation in ECs. Our study provides detailed insight in the signaling mechanisms downstream of PAR1. Our data demonstrate that thrombin-induced EC phosphoregulation is mediated exclusively through PAR1, that thrombin and thrombin-tethered ligand peptide induce similar phosphoregulation, and that only canonical PAR1 cleavage by thrombin generates a tethered ligand that potently induces early signaling. Furthermore, platelet PAR1 inhibitors directly affect EC signaling, indicating that it will be a challenge to design a PAR1 antagonist that will target only those pathways responsible for tissue pathology

Allosteric Inhibition of Factor XIIIa. Non-Saccharide Glycosaminoglycan Mimetics, but Not Glycosaminoglycans, Exhibit Promising Inhibition Profile

Factor XIIIa (FXIIIa) is a transglutaminase that catalyzes the last step in the coagulation process. Orthostery is the only approach that has been exploited to design FXIIIa inhibitors. Yet, allosteric inhibition of FXIIIa is a paradigm that may offer a key advantage of controlled inhibition over orthosteric inhibition. Such an approach is likely to lead to novel FXIIIa inhibitors that do not carry bleeding risks. We reasoned that targeting a collection of basic amino acid residues distant from FXIIIa’s active site by using sulfated glycosaminoglycans (GAGs) or non-saccharide GAG mimetics (NSGMs) would lead to the discovery of the first allosteric FXIIIa inhibitors. We tested a library of 22 variably sulfated GAGs and NSGMs against human FXIIIa to discover promising hits. Interestingly, although some GAGs bound to FXIIIa better than NSGMs, no GAG displayed any inhibition. An undecasulfated quercetin analog was found to inhibit FXIIIa with reasonable potency (efficacy of 98%). Michaelis-Menten kinetic studies revealed an allosteric mechanism of inhibition. Fluorescence studies confirmed close correspondence between binding affinity and inhibition potency, as expected for an allosteric process. The inhibitor was reversible and at least 9-fold- and 26-fold selective over two GAG-binding proteins factor Xa (efficacy of 71%) and thrombin, respectively, and at least 27-fold selective over a cysteine protease papain. The inhibitor also inhibited the FXIIIa-mediated polymerization of fibrin in vitro. Overall, our work presents the proof-of-principle that FXIIIa can be allosterically modulated by sulfated non-saccharide agents much smaller than GAGs, which should enable the design of selective and safe anticoagulants

PAR1 Agonists Stimulate APC-Like Endothelial Cytoprotection and Confer Resistance to Thromboinflammatory Injury

Stimulation of protease-activated receptor 1 (PAR1) on endothelium by activated protein C (APC) is protective in several animal models of disease, and APC has been used clinically in severe sepsis and wound healing. Clinical use of APC, however, is limited by its immunogenicity and its anticoagulant activity. We show that a class of small molecules termed “parmodulins” that act at the cytosolic face of PAR1 stimulates APC-like cytoprotective signaling in endothelium. Parmodulins block thrombin generation in response to inflammatory mediators and inhibit platelet accumulation on endothelium cultured under flow. Evaluation of the antithrombotic mechanism showed that parmodulins induce cytoprotective signaling through Gβγ, activating a PI3K/Akt pathway and eliciting a genetic program that includes suppression of NF-κB–mediated transcriptional activation and up-regulation of select cytoprotective transcripts. STC1 is among the up-regulated transcripts, and knockdown of stanniocalin-1 blocks the protective effects of both parmodulins and APC. Induction of this signaling pathway in vivo protects against thromboinflammatory injury in blood vessels. Small-molecule activation of endothelial cytoprotection through PAR1 represents an approach for treatment of thromboinflammatory disease and provides proof-of-principle for the strategy of targeting the cytoplasmic surface of GPCRs to achieve pathway selective signaling

Potentiation of thrombus instability: a contributory mechanism to the effectiveness of antithrombotic medications

© The Author(s) 2018The stability of an arterial thrombus, determined by its structure and ability to resist endogenous fibrinolysis, is a major determinant of the extent of infarction that results from coronary or cerebrovascular thrombosis. There is ample evidence from both laboratory and clinical studies to suggest that in addition to inhibiting platelet aggregation, antithrombotic medications have shear-dependent effects, potentiating thrombus fragility and/or enhancing endogenous fibrinolysis. Such shear-dependent effects, potentiating the fragility of the growing thrombus and/or enhancing endogenous thrombolytic activity, likely contribute to the clinical effectiveness of such medications. It is not clear how much these effects relate to the measured inhibition of platelet aggregation in response to specific agonists. These effects are observable only with techniques that subject the growing thrombus to arterial flow and shear conditions. The effects of antithrombotic medications on thrombus stability and ways of assessing this are reviewed herein, and it is proposed that thrombus stability could become a new target for pharmacological intervention.Peer reviewedFinal Published versio

Small GTP-binding protein Rho-mediated signaling promotes proliferation of rheumatoid synovial fibroblasts

Rho is a major small GTP-binding protein that is involved in the regulation of various cell functions, including proliferation and cell migration, through activation of multiple signaling molecules in various types of cells. We studied its roles in synovial fibroblasts (SFs) in patients with rheumatoid arthritis (RA) and clarified its relevance to RA synovitis, with the following results. 1)We found that the thrombin receptor was overexpressed on RA synovial fibroblasts (RA SFs) and that thrombin induced a marked proliferation and progression of the cell cycle to the S phase in these cells. 2)We also found that thrombin efficiently activated Rho. 3)Rho activation and proliferation and the progression of the cell cycle to the S phase were completely blocked by p115RGS (an N-terminal regulator of the G-protein signaling domain of p115RhoGEF) and by the C-terminal fragments of Gα13 (an inhibitor of the interaction of receptors with G13). 4)Thrombin induced the secretion of IL-6 by RA SFs, but this action was blocked by p115RGS or Gα13. Our findings show that the actions of thrombin on the proliferation of RA SFs, cell-cycle progression to the S phase, and IL-6 secretion were mainly mediated by the G13 and RhoGEF pathways. These results suggest that p115RGS and Gα13 could be potent inhibitors of such functions. A rational design of future therapeutic strategies for RA synovitis could perhaps include the exploitation of the Rho pathway to directly reduce the growth of synovial cells

Myricetin, the main flavonoid in Syzygium cumini leaf, is a novel inhibitor of platelet thiol isomerases PDI and ERp5

Background: Flavonoids have been characterized as a prominent class of compounds to treat thrombotic diseases through the inhibition of thiol isomerases. Syzygium cumini is a flavonoid-rich medicinal plant that contains myricetin and gallic acid. Little is known about the potential anti-platelet properties of S. cumini and its constituent flavonoids. Objective To evaluate the anti-platelet effects and mechanism of action of a polyphenol-rich extract (PESc) from S. cumini leaf and its most prevalent polyphenols, myricetin and gallic acid. Methods PESc, myricetin and gallic acid were incubated with platelet-rich plasma and washed platelets to assess platelet aggregation and activation. In vitro platelet adhesion and thrombus formation as well as in vivo bleeding time were performed. Finally, myricetin was incubated with recombinant thiol isomerases to assess its potential to bind and inhibit these, whilst molecular docking studies predicted possible binding sites. Results: PESc decreased platelet activation and aggregation induced by different agonists. Myricetin exerted potent anti-platelet effects, whereas gallic acid did not. Myricetin reduced the ability of platelets to spread on collagen, form thrombi in vitro without affecting haemostasis in vivo. Fluorescence quenching studies suggested myricetin binds to different thiol isomerases with similar affinity, despite inhibiting only protein disulphide isomerase (PDI) and ERp5 reductase activities (IC50~3.5 μM). Finally, molecular docking studies suggested myricetin formed non-covalent bonds with PDI and ERp5. Conclusions: PESc and its most abundant flavonoid myricetin strongly inhibit platelet function. Additionally, myricetin is a novel inhibitor of ERp5 and PDI, unveiling a new therapeutic perspective for the treatment of thrombotic disorders

Mechanisms of modulation of brain microvascular endothelial cells function by thrombin.

Brain microvascular endothelial cells are a critical component of the blood-brain barrier. They form a tight monolayer which is essential for maintaining the brain homeostasis. Blood-derived proteases such as thrombin may enter the brain during pathological conditions like trauma, stroke, and inflammation and further disrupts the permeability of the blood-brain barrier, via incompletely characterized mechanisms. We examined the underlying mechanisms evoked by thrombin in rat brain microvascular endothelial cells (RBMVEC). Our results indicate that thrombin, acting on protease-activated receptor 1 (PAR1) increases cytosolic C

Adjunctive therapies to reduce thrombotic events in patients with a history of myocardial infarction : role of vorapaxar

© 2015 Farag et al. This work is published by Dove Medical Press Limited, and licensed under Creative Commons Attribution – Non Commercial (unported, v3.0) LicenseAcute myocardial infarction (AMI) is generally attributed to coronary atherothrombotic disease. Platelet activation is essential for thrombus formation and is thus an important target for pharmacological intervention to prevent and treat AMI. Despite contemporary treatment with dual antiplatelet therapy, including acetylsalicylic acid and adenosine diphosphate receptor antagonists, patients with prior AMI remain at increased risk of future thrombotic events. This has stimulated the search for more potent antithrombotic agents. Among these is the oral protease-activated receptor-1 antagonist vorapaxar, which represents a new oral antiplatelet agent to reduce thrombotic risk in patients with atherothrombotic disease. The TRACER and the TRA 2°P-TIMI 50 trials concluded that vorapaxar in addition to standard therapy reduced ischemic adverse cardiac events. A remarkable benefit was observed in patients with stable atherosclerotic disease, particularly those with a previous history of AMI. Although favorable effects were seen in reduction of adverse cardiac events, this was associated with excess major and intracranial bleeding, particularly in patients at high risk of bleeding and those with a history of stroke or transient ischemic attack. Currently, the lack of a reliable individualized risk stratification tool to assess patients for thrombotic and bleeding tendencies in order to identify those who might gain most net clinical benefit has led to limited use of vorapaxar in clinical practice. Vorapaxar may find a niche as an adjunct to standard care in patients at high risk of thrombotic events and who are at low risk of bleeding.Peer reviewe