Discovery of 2,3-Dihydro[1,4]dioxino[2,3-g]benzofuran Derivatives as Protease Activated Receptor 4 (PAR4) Antagonists with Potent Antiplatelet Aggregation Activity and Low Bleeding Tendency.

Patients with arterial embolic disease have benefited greatly from antiplatelet therapy. However, hemorrhage risk of antiplatelet agents cannot be ignored. Herein, we describe the discovery of 2,3-dihydro[1,4]dioxino[2,3-g]benzofuran compounds as novel PAR4 antagonists. Notably, the isomers 36 and 37 with the chemotype of phenoxyl methylene substituted on the 2,3-dihydro-1,4-dioxine ring exhibited potent in vitro antiplatelet activity (IC50 = 26.13 nM for 36 and 14.26 nM for 37) and significantly improved metabolic stability in human liver microsomes (T1/2 = 97.6 min for 36 and 11.1 min for BMS-986120). 36 also displayed good oral PK profiles (mice: T1/2 = 7.32 h and F = 45.11%). Both of them showed overall potent ex vivo antiplatelet activity at concentrations of 6 and 12 mg/kg, with no impact on the coagulation system and low bleeding liability. Our work will facilitate development of novel PAR4 antagonists as a safer therapeutic option for arterial embolism.

Ultra-high throughput-based screening for the discovery of antiplatelet drugs affecting receptor dependent calcium signaling dynamics.

Distinct platelet activation patterns are elicited by the tyrosine kinase-linked collagen receptor glycoprotein VI (GPVI) and the G-protein coupled protease-activated receptors (PAR1/4) for thrombin. This is reflected in the different platelet Ca2+ responses induced by the GPVI agonist collagen-related peptide (CRP) and the PAR1/4 agonist thrombin. Using a 96 well-plate assay with human Calcium-6-loaded platelets and a panel of 22 pharmacological inhibitors, we assessed the cytosolic Ca2+ signaling domains of these receptors and developed an automated Ca2+ curve algorithm. The algorithm was used to evaluate an ultra-high throughput (UHT) based screening of 16,635 chemically diverse small molecules with orally active physicochemical properties for effects on platelets stimulated with CRP or thrombin. Stringent agonist-specific selection criteria resulted in the identification of 151 drug-like molecules, of which three hit compounds were further characterized. The dibenzyl formamide derivative ANO61 selectively modulated thrombin-induced Ca2+ responses, whereas the aromatic sulfonyl imidazole AF299 and the phenothiazine ethopropazine affected CRP-induced responses. Platelet functional assays confirmed selectivity of these hits. Ethopropazine retained its inhibitory potential in the presence of plasma, and suppressed collagen-dependent thrombus buildup at arterial shear rate. In conclusion, targeting of platelet Ca2+ signaling dynamics in a screening campaign has the potential of identifying novel platelet-inhibiting molecules.

Amphetamine increases vascular permeability by modulating endothelial actin cytoskeleton and NO synthase via PAR-1 and VEGF-R.

Abuse of amphetamine-type stimulants is linked to cardiovascular adverse effects like arrhythmias, accelerated atherosclerosis, acute coronary syndromes and sudden cardiac death. Excessive catecholamine release following amphetamine use causes vasoconstriction and vasospasms, over time leading to hypertension, endothelial dysfunction or even cardiotoxicity. However, immediate vascular pathomechanisms related to amphetamine exposure, especially endothelial function, remain incompletely understood and were analyzed in this study. Pharmaco-pathological effects of acute d-amphetamine-sulfate (DAM) were investigated ex vivo using contraction-force measurements of rat carotid artery rings and in vitro using label-free, real-time electrochemical impedance spectroscopy (EIS) on endothelial and smooth muscle cells. Specific receptor and target blocking was used to identify molecular targets and to characterize intracellular signaling. DAM induced vasodilation represented by 29.3±2.5% decrease in vascular tone (p<0.001) involving vascular endothelial growth factor receptor (VEGF-R) and protease activated receptor 1 (PAR-1). EIS revealed that DAM induces endothelial barrier disruption (-75.9±1.1% of initial cellular impedance, p<0.001) also involving VEGF-R and PAR-1. Further, in response to DAM, Rho-associated protein kinase (ROCK) mediated reversible contraction of actin cytoskeleton resulting in endothelial barrier disruption. Dephosphorylation of Serine1177 (-50.8±3.7%, p<0.001) and Threonine495 (-44.8±6.5%, p=0.0103) of the endothelial NO synthase (eNOS) were also observed. Blocking of VEGF-R and PAR-1 restored baseline eNOS Threonine495 phosphorylation. DAM induced vasodilation, enhanced vascular permeability and actin cytoskeleton contraction and induced eNOS hypophosphorylation involving VEGF-R, PAR-1 and ROCK. These results may contribute to a better understanding of severe adverse cardiovascular effects in amphetamine abuse.

Neuroprotective Effects of Noncanonical PAR1 Agonists on Cultured Neurons in Excitotoxicity.

Serine proteases regulate cell functions through G protein-coupled protease-activated receptors (PARs). Cleavage of one peptide bond of the receptor amino terminus results in the formation of a new N-terminus ("tethered ligand") that can specifically interact with the second extracellular loop of the PAR receptor and activate it. Activation of PAR1 by thrombin (canonical agonist) and activated protein C (APC, noncanonical agonist) was described as a biased agonism. Here, we have supposed that synthetic peptide analogs to the PAR1 tethered ligand liberated by APC could have neuroprotective effects like APC. To verify this hypothesis, a model of the ischemic brain impairment based on glutamate (Glu) excitotoxicity in primary neuronal cultures of neonatal rats has been used. It was shown that the nanopeptide NPNDKYEPF-NH2 (AP9) effectively reduced the neuronal death induced by Glu. The influence of AP9 on cell survival was comparable to that of APC. Both APC and AP9 reduced the dysregulation of intracellular calcium homeostasis in cultured neurons induced by excitotoxic Glu (100 µM) or NMDA (200 µM) concentrations. PAR1 agonist synthetic peptides might be noncanonical PAR1 agonists and a basis for novel neuroprotective drugs for disorders related to Glu excitotoxicity such as brain ischemia, trauma and some neurodegenerative diseases.

Thrombomodulin Switches Signaling and Protease-Activated Receptor 1 Cleavage Specificity of Thrombin.

BACKGROUND: Cleavage of the extracellular domain of PAR1 (protease-activated receptor 1) by thrombin at Arg41 and by APC (activated protein C) at Arg46 initiates paradoxical cytopathic and cytoprotective signaling in endothelial cells. In the latter case, the ligand-dependent coreceptor signaling by EPCR (endothelial protein C receptor) is required for the protective PAR1 signaling by APC. Here, we investigated the role of thrombomodulin in determining the specificity of PAR1 signaling by thrombin. METHODS: We prepared a PAR1 knockout (PAR1-/-) EA.hy926 endothelial cell line by CRISPR/Cas9 and transduced PAR1-/- cells with lentivirus vectors expressing PAR1 mutants in which either Arg41 or Arg46 was replaced with an Ala. Furthermore, human embryonic kidney 293 cells were transfected with wild-type or mutant PAR1 cleavage reporter constructs carrying N-terminal Nluc (NanoLuc luciferase) and C-terminal enhanced yellow fluorescent protein tags. RESULTS: Characterization of transfected cells in signaling and receptor cleavage assays revealed that, upon interaction with thrombomodulin, thrombin cleaves Arg46 to elicit cytoprotective effects by a ß-arrestin-2 biased signaling mechanism. Analysis of functional data and cleavage rates indicated that thrombin-thrombomodulin cleaves Arg46>10-fold faster than APC. Upon interaction with thrombin, the cytoplasmic domain of thrombomodulin recruited both ß-arrestin-1 and -2 to the plasma membrane. Thus, the thrombin cleavage of Arg41 was also cytoprotective in thrombomodulin-expressing cells by ß-arrestin-1-biased signaling. APC in the absence of EPCR cleaved Arg41 to initiate disruptive signaling responses like thrombin. CONCLUSIONS: These results suggest that coreceptor signaling by thrombomodulin and EPCR determines the PAR1 cleavage and signaling specificity of thrombin and APC, respectively.

Endothelial Protein C Receptor and 3K3A-Activated Protein C Protect Mice from Allergic Contact Dermatitis in a Contact Hypersensitivity Model.

Endothelial protein C receptor (EPCR) is a receptor for the natural anti-coagulant activated protein C (aPC). It mediates the anti-inflammatory and barrier-protective functions of aPC through the cleavage of protease-activated receptor (PAR)1/2. Allergic contact dermatitis is a common skin disease characterized by inflammation and defective skin barrier. This study investigated the effect of EPCR and 3K3A-aPC on allergic contact dermatitis using a contact hypersensitivity (CHS) model. CHS was induced using 1-Fluoro-2,4-dinitrobenzene in EPCR-deficient (KO) and matched wild-type mice and mice treated with 3K3A-aPC, a mutant form of aPC with diminished anti-coagulant activity. Changes in clinical and histological features, cytokines, and immune cells were examined. EPCRKO mice displayed more severe CHS, with increased immune cell infiltration in the skin and higher levels of inflammatory cytokines and IgE than wild-type mice. EPCR, aPC, and PAR1/2 were expressed by the skin epidermis, with EPCR presenting almost exclusively in the basal layer. EPCRKO increased the epidermal expression of aPC and PAR1, whereas in CHS, their expression was reduced compared to wild-type mice. 3K3A-aPC reduced CHS severity in wild-type and EPCRKO mice by suppressing immune cell infiltration/activation and inflammatory cytokines. In summary, EPCRKO exacerbated CHS, whereas 3K3A-aPC could reduce the severity of CHS in both EPCRKO and wild-type mice.

The C-Type Lectin Receptor CD93 Regulates Platelet Activation and Surface Expression of the Protease Activated Receptor 4.

BACKGROUND: The C-type lectin receptor CD93 is a single pass type I transmembrane glycoprotein involved in inflammation, immunity, and angiogenesis. This study investigates the role of CD93 in platelet function. CD93 knockout (KO) mice and wild-type (WT) controls were compared in this study. METHODS: Platelet activation and aggregation were investigated by flow cytometry and light transmission aggregometry, respectively. Protein expression and phosphorylation were analyzed by immunoblotting. Subcellular localization of membrane receptors was investigated by wide-field and confocal microscopy. RESULTS: The lack of CD93 in mice was not associated to any evident bleeding defect and no alterations of platelet activation were observed upon stimulation with thromboxane A2 analogue and convulxin. Conversely, platelet aggregation induced by stimulation of the thrombin receptor PAR4 was significantly reduced in the absence of CD93. This defect was associated with a significant reduction of α-granule secretion, integrin αIIbß3 activation, and protein kinase C (PKC) stimulation. Resting WT and CD93-deficient platelets expressed comparable amounts of PAR4. However, upon stimulation with a PAR4 activating peptide, a more pronounced clearance of PAR4 from the platelet surface was observed in CD93-deficient platelets compared with WT controls. Confocal microscopy analysis revealed a massive movement of PAR4 in cytosolic compartments of activated platelets lacking CD93. Accordingly, platelet desensitization following PAR4 stimulation was more pronounced in CD93 KO platelets compared with WT controls. CONCLUSION: These results demonstrate that CD93 supports platelet activation triggered by PAR4 stimulation and is required to stabilize the expression of the thrombin receptor on the cell surface.

Genetic and nongenetic drivers of platelet reactivity in healthy Tanzanian individuals.

BACKGROUND: Platelets play a key role in hemostasis, inflammation, and cardiovascular diseases. Platelet reactivity is highly variable between individuals. The drivers of this variability in populations from Sub-Saharan Africa remain largely unknown. OBJECTIVES: We aimed to investigate the nongenetic and genetic determinants of platelet reactivity in healthy adults living in a rapidly urbanizing area in Northern Tanzania. METHODS: Platelet activation and reactivity were measured by platelet P-selectin expression and the binding of fibrinogen in unstimulated blood and after ex vivo stimulation with adenosine diphosphate and PAR-1 and PAR-4 ligands. We then analyzed the associations of platelet parameters with host genetic and nongenetic factors, environmental factors, plasma inflammatory markers, and plasma metabolites. RESULTS: Only a few associations were found between platelet reactivity parameters and plasma inflammatory markers and nongenetic host and environmental factors. In contrast, untargeted plasma metabolomics revealed a large number of associations with food-derived metabolites, including phytochemicals that were previously reported to inhibit platelet reactivity. Genome-wide single-nucleotide polymorphism genotyping identified 2 novel single-nucleotide polymorphisms (rs903650 and rs4789332) that were associated with platelet reactivity at the genome-wide level (P < 5 × 10-8) as well as a number of variants in the PAR4 gene (F2RL3) that were associated with PAR4-induced reactivity. CONCLUSION: Our study uncovered factors that determine variation in platelet reactivity in a population in East Africa that is rapidly transitioning to an urban lifestyle, including the importance of genetic ancestry and the gradual abandoning of the traditional East African diet.

KLK10 promotes the progression of KRAS mutant colorectal cancer via PAR1-PDK1-AKT signaling pathway.

Kirsten rat sarcoma virus (KRAS) gene mutation is common in colorectal cancer (CRC) and is often predictive of treatment failure and poor prognosis. To understand the mechanism, we compared the transcriptome of CRC patients with wild-type and mutant KRAS and found that KRAS mutation is associated with the overexpression of a secreted serine protease, kallikrein-related peptidase 10 (KLK10). Moreover, using in vitro and in vivo models, we found that KLK10 overexpression favors the rapid growth and liver metastasis of KRAS mutant CRC and can also impair the efficacy of KRAS inhibitors, leading to drug resistance and poor survival. Further functional assays revealed that the oncogenic role of KLK10 is mediated by protease-activated receptor 1 (PAR1). KLK10 cleaves and activates PAR1, which further activates 3-phosphoinositide-dependent kinase 1 (PDK1)-AKT oncogenic pathway. Notably, suppressing PAR1-PDK1-AKT cascade via KLK10 knockdown can effectively inhibit CRC progression and improve the sensitivity to KRAS inhibitor, providing a promising therapeutic strategy. Taken together, our study showed that KLK10 promotes the progression of KRAS mutant CRC via activating PAR1-PDK1-AKT signaling pathway. These findings expanded our knowledge of CRC development, especially in the setting of KRAS mutation, and also provided novel targets for clinical intervention.

Proteinase-activated Receptor 2: Springboard of Tumors.

Proteinase-activated receptors (PARs) were discovered more than 25 years ago and since then, their role in cancer has been under investigation. Research has primarily focused on the receptors located on the membrane of cancer cells and their impact on metabolism, intracellular signalling, and proliferation. Regarding the host response to cancer, studies have predominantly examined the relationship of thrombin receptors (PAR-1, PAR-3, and PAR-4) with blood clotting in distant metastatic spread. However, limited studies have examined the role of PARs, especially PAR-2, in the host anti-tumor immunity. This review article provides insights into the role of PAR-2 on cancer cells and immune competent cells involved in cancer development and progression. It also discussed the current knowledge of the importance of PAR-2 activation at various stages of cancer progression and its association with cancer-related pain.

Coagulation Factor Xa Has No Effects on the Expression of PAR1, PAR2, and PAR4 and No Proinflammatory Effects on HL-1 Cells.

Atrial fibrillation (AF), characterised by irregular high-frequency contractions of the atria of the heart, is of increasing clinical importance. The reasons are the increasing prevalence and thromboembolic complications caused by AF. So-called atrial remodelling is characterised, among other things, by atrial dilatation and fibrotic remodelling. As a result, AF is self-sustaining and forms a procoagulant state. But hypercoagulation not only appears to be the consequence of AF. Coagulation factors can exert influence on cells via protease-activated receptors (PAR) and thereby the procoagulation state could contribute to the development and maintenance of AF. In this work, the influence of FXa on Heart Like-1 (HL-1) cells, which are murine adult atrial cardiomyocytes (immortalized), was investigated. PAR1, PAR2, and PAR4 expression was detected. After incubations with FXa (5-50 nM; 4-24 h) or PAR1- and PAR2-agonists (20 µM; 4-24 h), no changes occurred in PAR expression or in the inflammatory signalling cascade. There were no time- or concentration-dependent changes in the phosphorylation of the MAP kinases ERK1/2 or the p65 subunit of NF-κB. In addition, there was no change in the mRNA expression of the cell adhesion molecules (ICAM-1, VCAM-1, fibronectin). Thus, FXa has no direct PAR-dependent effects on HL-1 cells. Future studies should investigate the influence of FXa on human cardiomyocytes or on other cardiac cell types like fibroblasts.

S. aureus drives itch and scratch-induced skin damage through a V8 protease-PAR1 axis.

Itch is an unpleasant sensation that evokes a desire to scratch. The skin barrier is constantly exposed to microbes and their products. However, the role of microbes in itch generation is unknown. Here, we show that Staphylococcus aureus, a bacterial pathogen associated with itchy skin diseases, directly activates pruriceptor sensory neurons to drive itch. Epicutaneous S. aureus exposure causes robust itch and scratch-induced damage. By testing multiple isogenic bacterial mutants for virulence factors, we identify the S. aureus serine protease V8 as a critical mediator in evoking spontaneous itch and alloknesis. V8 cleaves proteinase-activated receptor 1 (PAR1) on mouse and human sensory neurons. Targeting PAR1 through genetic deficiency, small interfering RNA (siRNA) knockdown, or pharmacological blockade decreases itch and skin damage caused by V8 and S. aureus exposure. Thus, we identify a mechanism of action for a pruritogenic bacterial factor and demonstrate the potential of inhibiting V8-PAR1 signaling to treat itch.

An siRNA library screen identifies CYLD and USP34 as deubiquitinases that regulate GPCR-p38 MAPK signaling and distinct inflammatory responses.

G protein-coupled receptors (GPCRs) are highly druggable and implicated in numerous diseases, including vascular inflammation. GPCR signals are transduced from the plasma membrane as well as from endosomes and controlled by posttranslational modifications. The thrombin-activated GPCR protease-activated receptor-1 is modified by ubiquitin. Ubiquitination of protease-activated receptor-1 drives recruitment of transforming growth factor-ß-activated kinase-1-binding protein 2 (TAB2) and coassociation of TAB1 on endosomes, which triggers p38 mitogen-activated protein kinase-dependent inflammatory responses in endothelial cells. Other endothelial GPCRs also induce p38 activation via a noncanonical TAB1-TAB2-dependent pathway. However, the regulatory processes that control GPCR ubiquitin-driven p38 inflammatory signaling remains poorly understood. We discovered mechanisms that turn on GPCR ubiquitin-dependent p38 signaling, however, the mechanisms that turn off the pathway are not known. We hypothesize that deubiquitination is an important step in regulating ubiquitin-driven p38 signaling. To identify specific deubiquitinating enzymes (DUBs) that control GPCR-p38 mitogen-activated protein kinase signaling, we conducted a siRNA library screen targeting 96 DUBs in endothelial cells and HeLa cells. We identified nine DUBs and validated the function two DUBs including cylindromatosis and ubiquitin-specific protease-34 that specifically regulate thrombin-induced p38 phosphorylation. Depletion of cylindromatosis expression by siRNA enhanced thrombin-stimulated p38 signaling, endothelial barrier permeability, and increased interleukin-6 cytokine expression. Conversely, siRNA knockdown of ubiquitin-specific protease-34 expression decreased thrombin-promoted interleukin-6 expression and had no effect on thrombin-induced endothelial barrier permeability. These studies suggest that specific DUBs distinctly regulate GPCR-induced p38-mediated inflammatory responses.

PAR1 regulates sepsis-induced vascular endothelial barrier dysfunction by mediating ERM phosphorylation via the RhoA/ROCK signaling pathway.

Sepsis begins with vascular endothelial barrier breakdown and causes widespread organ failure. Protease-activated receptor 1 (PAR1) is an important target for modulating vascular endothelial permeability; however, little research has been undertaken in sepsis, and its putative molecular mechanism remains unknown. The vascular endothelial permeability was examined by detecting FITC-dextran flux. F-actin was examined by immunofluorescence (IF). PAR1, ERM phosphorylation, and RhoA/ROCK signaling pathway expression in lipopolysaccharide (LPS)-stimulated human umbilical vein endothelial cells (HUVECs) line were examined by IF and Western blot. To develop the sepsis model, cecal ligation and puncture (CLP) were conducted. The PAR1 inhibitor SCH79797 was utilized to inhibit PAR1 expression in vivo. Vascular permeability in main organs weres measured by Evans blue dye extravasation. The pathological changes in main organs were examined by HE staining. The expression of PAR1, ERM phosphorylation, and the RhoA/ROCK signaling pathway was examined using IF, immunohistochemical and WB in CLP mice. In vitro, in response to LPS stimulation of HUVECs, PAR1 mediated the phosphorylation of ERM, promoted F-actin rearrangement, and increased endothelial hyperpermeability, all of which were prevented by inhibiting PAR1 or RhoA. Additionally, inhibiting PAR1 expression reduced RhoA and ROCK expression. In vivo, we showed that inhibiting PAR1 expression will reduce ezrin/radixin/moesin (ERM) phosphorylation to relieve vascular endothelial barrier dysfunction and thereby ameliorate multiorgan dysfunction syndrome (MODS) in CLP-induced septic mice. This study revealed that PAR1-mediated phosphorylation of ERM induced endothelial barrier dysfunction, which in turn led to MODS in sepsis, and that the RhoA/ROCK signaling pathway underlay these effects.

Thrombin receptor PAR1 silencing in endothelial colony-forming cells modifies stemness and vasculogenic properties.

BACKGROUND: The involvement of thrombin receptor PAR1 in blood vessel development has been largely demonstrated in knockout mice; however, its implication in adult mouse angiogenesis seems very moderate. OBJECTIVES: We aimed to explore the potential relationships between PAR1, stemness, and angiogenic properties of human endothelial colony-forming cells (ECFCs). METHODS AND RESULTS: PAR1 activation on ECFCs using the selective PAR1-activating peptide induced a significant decrease in CD133 expression (RTQ-PCR analysis). In line, silencing of PAR1 gene expression with siRNA increased CD133 mRNA as well as intracellular CD133 protein expression. To confirm the link between CD133 and PAR1, we explored the association between PAR1 and CD133 levels in fast and slow fibroblasts prone to reprogramming. An imbalance between PAR1 and CD133 levels was evidenced, with a decreased expression of PAR1 in fast reprogramming fibroblasts expressing a high CD133 level. Regarding in vitro ECFC angiogenic properties, PAR1 silencing with specific siRNA induced cell proliferation evidenced by the overexpression of Ki67. However, it did not impact migration properties nor ECFC adhesion on smooth muscle cells or human arterial endothelial cells. In a mouse model of hind-limb ischemia, PAR1 silencing in ECFCs significantly increased postischemic revascularization compared to siCtrl-ECFCs along with a significant increase in cutaneous blood flows (P < .0001), microvessel density (P = .02), myofiber regeneration (P < .0001), and human endothelial cell incorporation in muscle (P < .0001). CONCLUSION: In conclusion, our work describes for the first time a link between PAR1, stemness, and vasculogenesis in human ECFCs.

Protease-Activated Receptor 1-Mediated Damage of Podocytes in Diabetic Nephropathy.

There is clinical evidence that increased urinary serine proteases are associated with the disease severity in the setting of diabetic nephropathy (DN). Elevation of serine proteases may mediate [Ca2+]i dynamics in podocytes through the protease-activated receptors (PARs) pathway, including associated activation of nonspecific cation channels. Cultured human podocytes and freshly isolated glomeruli were used for fluorescence and immunohistochemistry stainings, calcium imaging, Western blot analysis, scanning ion conductance microscopy, and patch clamp analysis. Goto-Kakizaki, Wistar, type 2 DN (T2DN), and a novel PAR1 knockout on T2DN rat background rats were used to test the importance of PAR1-mediated signaling in DN settings. We found that PAR1 activation increases [Ca2+]i via TRPC6 channels. Both human cultured podocytes exposed to high glucose and podocytes from freshly isolated glomeruli of T2DN rats had increased PAR1-mediated [Ca2+]i compared with controls. Imaging experiments revealed that PAR1 activation plays a role in podocyte morphological changes. T2DN rats exhibited a significantly higher response to thrombin and urokinase. Moreover, the plasma concentration of thrombin in T2DN rats was significantly elevated compared with Wistar rats. T2DNPar1-/- rats were embryonically lethal. T2DNPar1+/- rats had a significant decrease in glomerular damage associated with DN lesions. Overall, these data provide evidence that, during the development of DN, elevated levels of serine proteases promote an excessive [Ca2+]i influx in podocytes through PAR1-TRPC6 signaling, ultimately leading to podocyte apoptosis, the development of albuminuria, and glomeruli damage. ARTICLE HIGHLIGHTS: Increased urinary serine proteases are associated with diabetic nephropathy. During the development of diabetic nephropathy in type 2 diabetes, the elevation of serine proteases could overstimulate protease-activated receptor 1 (PAR1). PAR1 signaling is involved in the development of DN via TRPC6-mediated intracellular calcium signaling. This study provides fundamental knowledge that can be used to develop efficient therapeutic approaches targeting serine proteases or corresponding PAR pathways to prevent or slow the progression of diabetes-associated kidney diseases.

Platelet Function Decreases with Increasing Severity of Liver Cirrhosis and Portal Hypertension-A Prospective Study.

BACKGROUND: Cirrhotic patients display an increased risk for both bleeding and thrombosis. We investigated platelet activation across Child-Pugh stages (CPSs) and portal hypertension (PH) severity. MATERIAL AND METHODS: A total of 110 cirrhotic patients were prospectively included. CPS and hepatic venous pressure gradient (HVPG) were determined. Platelet surface expression of P-selectin and activated glycoprotein (GP) IIb/IIIa were measured by flow cytometry before/after stimulation with protease-activated receptor (PAR)-1 (thrombin receptor activating peptide, TRAP) and PAR-4 (AYPGKF) agonists, epinephrine, and lipopolysaccharide (LPS). RESULTS: Platelet count was similar across CPS but lower with increasing PH severity. Expression of P-selectin and activated GPIIb/IIIa in response to TRAP and AYPGKF was significantly reduced in platelets of CPS-B/C versus CPS-A patients (all p < 0.05). Platelet P-selectin expression upon epinephrine and LPS stimulation was reduced in CPS-C patients, while activated GPIIb/IIIa in response to these agonists was lower in CPS-B/C (all p < 0.05). Regarding PH severity, P-selectin and activated GPIIb/IIIa in response to AYPGKF were lower in HVPG ≥20 mmHg patients (both p < 0.001 vs. HVPG < 10 mmHg). Similarly, activated GPIIb/IIIa was lower in HVPG ≥20 mmHg patients after TRAP stimulation (p < 0.01 vs. HVPG < 10 mmHg). The lower platelet surface expression of P-selectin and activated GPIIb/IIIa upon stimulation of thrombin receptors (PAR-1/PAR-4) in CPS-B/C and HVPG ≥20 mmHg patients was paralleled by reduced antithrombin-III levels in those patients (all p < 0.05). Overall, PAR-1- and PAR-4-mediated platelet activation correlated with antithrombin-III levels (p < 0.001). CONCLUSION: Platelet responsiveness decreases with increasing severity of liver cirrhosis and PH but is potentially counterbalanced by lower antithrombin-III levels.