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Exposure of platelets to collagen triggers the formation of a platelet clot. Pharmacological agents capable of inhibiting platelet activation by collagen are thus of potential therapeutic interest. Thrombus formation is initiated by the interaction of the GPIb-V-IX complex with collagen-bound vWF, while GPVI interaction with collagen triggers platelet activation that is reinforced by ADP and thromboxane A2. Losartan is an angiotensin II (Ang II) type I receptor (AT1R) antagonist proposed to have an antiplatelet activity via the inhibition of both the thromboxane A2 (TXA2) receptor (TP) and the glycoprotein VI (GPVI). Here, we characterized in vitro the effects of losartan at different doses on platelet responses: losartan inhibited platelet aggregation and secretion induced by 1 μg . mL(-1) and 10 μg . mL(-1) of collagen with an IC50 of ~ 6 μM. Losartan inhibited platelet responses induced by the GPVI specific collagen related peptide but not by the α2β1 specific peptide. However, losartan did not inhibit the binding of recombinant GPVI to collagen, which is not in favor of a simple competition. Indeed, the clustering of GPVI observed in flow cytometry and using the Duolink methodology, was inhibited by losartan. The impact of a therapeutic dose of losartan (100 mg/day) on platelet responses was analyzed ex vivo in a double blind study. No statistically significant differences were observed between losartan-treated (n=25) and non-treated (n=30) patients in terms of collagen and U46619-induced platelet activation. These data indicate that in treated patients, losartan does not achieve a measurable antiplatelet effect but provide the proof of concept that inhibiting collagen-induced GPVI clustering is of pharmacological interest to obtain an antithrombotic efficacy. ClinicalTrials.gov NCT00763893.

The deleterious effects of diabetes in the heart are increasingly attributed to inflammatory signaling through the NLRP3 (NOD, LRR and PYD domains-containing protein 3) inflammasome. Thrombin antagonists reduce cardiac remodeling and dysfunction in diabetic mice, in part by suppressing fibrin-driven inflammation. The role of cellular thrombin receptor subtypes in this context is not known. We sought to determine the causal involvement of protease-activated receptors (PAR) in inflammatory signaling of the diabetic heart. Mice with diet-induced diabetes showed increased abundance of pro-caspase-1 and pro-interleukin (IL)-1β in the left ventricle (LV), indicating transcriptional NLRP3 inflammasome priming, and augmented cleavage of active caspase-1 and IL-1β, pointing to canonical NLRP3 inflammasome activation. Caspase-11 activation, which mediates non-canonical NLRP3 inflammasome signaling, was not augmented. Formation of the plasma membrane pore-forming protein N-terminal gasdermin D (GDSMD), a prerequisite for IL-1β secretion, was also higher in diabetic vs. control mouse LV. NLRP3, ASC and IL-18 expression did not differ between the groups, nor did expression of PAR1 or PAR2. PAR3 was nearly undetectable. LV abundance of PAR4 by contrast increased with diabetes and correlated positively with active caspase-1. Genetic deletion of PAR4 in mice prevented the diet-induced cleavage of caspase-1, IL-1β and GDSMD. Right atrial appendages from patients with type 2 diabetes also showed higher levels of PAR4, but not of PAR1 or PAR2, than non-diabetic atrial tissue, along with increased abundance of cleaved caspase-1, IL-1β and GSDMD. Human cardiac fibroblasts maintained in high glucose conditions to mimic diabetes also upregulated PAR4 mRNA and protein, and increased PAR4-dependent IL-1β transcription and secretion in response to thrombin, while PAR1 and PAR2 expressions were unaltered. In conclusion, PAR4 drives caspase-1-dependent IL-1β production through the canonical NLRP3 inflammasome pathway in the diabetic heart, providing mechanistic insights into diabetes-associated cardiac thromboinflammation. The emerging PAR4-selective antagonists may provide a feasible approach to prevent cardiac inflammation in patients with diabetes.

Background: The large and increasing number of chemicals released into the environment demands more efficient and cost-effective approaches for assessing environmental chemical toxicity. The U.S. Tox21 program has responded to this challenge by proposing alternative strategies for toxicity testing, among which the quantitative high-throughput screening (qHTS) paradigm has been adopted as the primary tool for generating data from screening large chemical libraries using a wide spectrum of assays. Objectives: The goal of this study was to develop methods to evaluate the data generated from these assays to guide future assay selection and prioritization for the Tox21 program. Methods: We examined the data from the Tox21 pilot-phase collection of approximately 3,000 environmental chemicals profiled in qHTS format against a panel of 10 human nuclear receptors (AR, ERα, FXR, GR, LXRβ, PPARγ, PPARδ, RXRα, TRβ, and VDR) for reproducibility, concordance of biological activity profiles with sequence homology of the receptor ligand binding domains, and structure-activity relationships. Results: We determined the assays to be appropriate in terms of biological relevance. We found better concordance for replicate compounds for the agonist-mode than for the antagonist-mode assays, likely due to interference of cytotoxicity in the latter assays. This exercise also enabled us to formulate data-driven strategies for discriminating true signals from artifacts, and to prioritize assays based on data quality. Conclusions: The results demonstrate the feasibility of qHTS to identify the potential for environmentally relevant chemicals to interact with key toxicity pathways related to human disease induction.

Platelet activation may be a central mediator of a chain of events leading to microinfarcts, leakage of thrombin and fibrin through the blood-brain barrier and chronic neuroinflammation that typify Alzheimer's Disease (AD). The platelet thrombin receptor Protease Activated Receptor 4 (PAR4) is responsible for platelet activation and amplification of thrombin generation. Thrombin cleaves fibrinogen to fibrin, and pathologic fibrin deposition in the cerebral microvasculature is itself a risk factor for Alzheimer's disease. Fibrin interaction with vascular amyloid β (Aβ) leads to degradation-resistant blood clots. This process initiates inflammation both in the vessel and adjacent parenchyma. Inflammation in general has been reported to turn on expression of PAR4 in endothelial cells not typically expressing PAR4. 1) Data were acquired from the Religious Orders Study (ROS) and the Rush Memory and Aging Project (MAP) to study the correlation of the PAR4 gene expression or methylation with AD diagnosis and longitudinal cognitive decline. 2) 5xFAD amyloid model mice were crossed with PAR4KO mice to test for pathology and markers of inflammation. 1) PAR4 gene F2RL3 mRNA was elevated in AD cases and was associated with worse retrospective longitudinal cognitive performance. We also report a significant association of F2RL3 epigenetic demethylation with cognitive decline. 2) 5xFAD mice exhibit increased PAR4 protein expression on vascular endothelial cells. 5xFAD mice exhibit increased vascular fibrin deposits compared to WT mice, and this fibrin deposition is reduced in 5xFAD/PAR4KO compared to 5xFAD. In a human study the PAR4 gene F2RL3 mRNA expression is associated with multiple AD-relevant outcomes and its encoded product, PAR4, may play a role in disease pathogenesis. PAR4 is a platelet receptor that functions upstream of fibrin deposition in 5xFAD amyloid mice, leading to misexpression in the microvasculature. Fibrin deposits have been reported to be inflammatory in 5xFAD and future work will focus on teasing apart the contribution of PAR4 mediated fibrin deposits in inflammation and microglial activation.

Pulmonary hypertension (PH) is an incurable disease characterized by pulmonary vascular remodeling. Endothelial injury and inflammation are the key triggers of disease initiation. Recent findings suggest that STING (stimulator of IFN genes) activation plays a critical role in endothelial dysfunction and IFN signaling. Here, we investigated the involvement of STING in the pathogenesis of PH. Patients with PH and rodent PH model samples, a Sugen 5416/hypoxia PH model, and pulmonary artery endothelial cells (PAECs) were used to evaluate the hypothesis. We found that the cyclic guanosine monophosphate–AMP synthase–STING signaling pathway was activated in lung tissues from rodent PH models and patients with PH and in TNF-α–induced PAECs in vitro. Specifically, STING expression was significantly elevated in the endothelial cells in PH disease settings. In the Sugen 5416/hypoxia mouse model, genetic knockout or pharmacological inhibition of STING prevented the progression of PH. Functionally, knockdown of STING reduced the proliferation and migration of PAECs. Mechanistically, STING transcriptionally regulates its binding partner F2RL3 (F2R-like thrombin or trypsin receptor 3) through the STING-NF-κB axis, which activated IFN signaling and repressed BMPR2 (bone morphogenetic protein receptor 2) signaling both in vitro and in vivo. Further analysis revealed that F2RL3 expression was increased in PH settings and identified negative feedback regulation of F2RL3/BMPR2 signaling. Accordingly, a positive correlation of expression amounts between STING and F2RL3/IFN-stimulated genes was observed in vivo. Our findings suggest that STING activation in PAECs plays a critical role in the pathobiology of PH. Targeting STING may be a promising therapeutic strategy for preventing the development of PH.

Background: Selective platelet release of pro- or anti-angiogenic factors distinctly regulated angiogenesis. We hypothesised that selective release of platelet angiogenic factors could differently regulate tumour growth. Methods: Breast cancer cell proliferation, cancer cell-induced endothelial tube formation in vitro , and tumour growth in vivo were studied in the presence of protease-activated receptor 1-stimulated platelet releasate (PAR1-PR; rich in pro-angiogenic factors) or PAR4-PR (rich in anti-angiogenic factors). Results: The PAR1-PR and PAR4-PR supplementation (10%) similarly enhanced cell proliferation of MCF-7 and MDA-MB-231 breast cancer cells. The cancer cells triggered capillary-like tube formation of endothelial cells that was further enhanced by pro-angiogenic factor-rich PAR1-PR. The VEGF, but not SDF-1 α , receptor blockade abolished PAR1-PR/PAR4-PR-enhanced cancer cell proliferation. Integrin blockade by RGDS had identical effects as VEGF inhibition. The Src and ERK inhibition diminished, whereas PI3K and PKC blockade abolished platelet releasate-enhanced cancer cell proliferation. Using a model of subcutaneous implantation of MDA-MB-231 cells in nude mice, PAR1-PR enhanced tumour growth more markedly than PAR4-PR, and seemed to achieve the exaggeration by promoting more profound tumour angiogenesis. Conclusions: Platelet releasate increases breast cancer cell proliferation through VEGF–integrin cooperative signalling. Pro-angiogenic factor-rich platelet releasate enhances cancer cell-induced angiogenesis more markedly, and thus exaggerates tumour growth in vivo .

Protease-activated receptors (PARs) are a class of integral membrane proteins that are cleaved by a variety of proteases, most notably thrombin, to reveal a tethered ligand and promote activation. PARs are critical mediators of platelet function in hemostasis and thrombosis, and therefore are attractive targets for anti-platelet therapies. Animal models studying platelet PAR physiology have relied heavily on genetically modified mouse strains, which have provided ample insight but have some inherent limitations. The current review aims to summarize the notable PAR expression and functional differences between the mouse and human, in addition to highlighting some recently developed tools to further study human physiology in mouse models.

Graft-vs.-host disease (GvHD) is a major complication of allogenic hematopoietic stem-cell(HSC) transplantation. GvHD is associated with loss of endothelial thrombomodulin, but the relevance of this for the adaptive immune response to transplanted HSCs remains unknown. Here we show that the protease-activated protein C (aPC), which is generated by thrombomodulin, ameliorates GvHD aPC restricts allogenic T-cell activation via the protease activated receptor (PAR)2/PAR3 heterodimer on regulatory T-cells (T regs , CD4 + FOXP3 + ). Preincubation of pan T-cells with aPC prior to transplantation increases the frequency of T regs and protects from GvHD. Preincubation of human T-cells (HLA-DR4 − CD4 + ) with aPC prior to transplantation into humanized (NSG-AB°DR4) mice ameliorates graft-vs.-host disease. The protective effect of aPC on GvHD does not compromise the graft vs. leukaemia effect in two independent tumor cell models. Ex vivo preincubation of T-cells with aPC, aPC-based therapies, or targeting PAR2/PAR3 on T-cells may provide a safe and effective approach to mitigate GvHD. Graft-vs.-host disease is a complication of allogenic hematopoietic stem cell transplantation, and is associated with endothelial dysfunction. Here the authors show that activated protein C signals via PAR2/PAR3 to expand T reg cells, mitigating the disease in mice.

Activation of intravesical protease activated receptor 4 (PAR4) leads to release of urothelial macrophage migration inhibitory factor (MIF). MIF then binds to urothelial MIF receptors to release urothelial high mobility group box-1 (HMGB1) and elicit bladder hyperalgesia. Since MIF binds to multiple receptors, we investigated the contribution of individual urothelial MIF receptors to PAR4-induced HMGB1 release in vivo and in vitro and bladder pain in vivo. We tested the effect of intravesical pre-treatment with individual MIF or MIF receptor (CD74, CXCR4, CXCR2) antagonists on PAR4-induced HMGB1 release in vivo (female C57/BL6 mice) and in vitro (primary human urothelial cells) and on PAR4-induced bladder hyperalgesia in vivo (mice). In mice, PAR4 induced HMGB1 release and bladder hyperalgesia through activation of intravesical MIF receptors, CD74 and CXCR4. CXCR2 was not involved in these effects. In primary urothelial cells, PAR4-induced HMGB1 release through activation of CD74 receptors. Micturition parameters in mice were not changed by any of the treatments. Urothelial MIF receptors CD74 and CXCR4 mediate bladder pain through release of urothelial HMGB1. This mechanism may set up persistent pain loops in the bladder and warrants further investigation. Urothelial CD74 and CXCR4 may provide novel targets for interrupting bladder pain.

In exploring the possibility that racial differences in platelet function might exist, Paul Bray and his colleagues report that platelets from blacks have a greater propensity to aggregate than those from whites in response to activation of the PAR4 thrombin receptor. Mechanistically, this difference in platelet function seems to reflect differences in the expression of the microRNA miR-376c and its target, the enzyme phosphatidylcholine transfer protein. Racial differences in the pathophysiology of atherothrombosis are poorly understood. We explored the function and transcriptome of platelets in healthy black ( n = 70) and white ( n = 84) subjects. Platelet aggregation and calcium mobilization induced by the PAR4 thrombin receptor were significantly greater in black subjects. Numerous differentially expressed RNAs were associated with both race and PAR4 reactivity, including PCTP (encoding phosphatidylcholine transfer protein), and platelets from black subjects expressed higher levels of PC-TP protein. PC-TP inhibition or depletion blocked PAR4- but not PAR1-mediated activation of platelets and megakaryocytic cell lines. miR-376c levels were differentially expressed by race and PAR4 reactivity and were inversely correlated with PCTP mRNA levels, PC-TP protein levels and PAR4 reactivity. miR-376c regulated the expression of PC-TP in human megakaryocytes. A disproportionately high number of microRNAs that were differentially expressed by race and PAR4 reactivity, including miR-376c , are encoded in the DLK1-DIO3 locus and were expressed at lower levels in platelets from black subjects. These results suggest that PC-TP contributes to the racial difference in PAR4-mediated platelet activation, indicate a genomic contribution to platelet function that differs by race and emphasize a need to consider the effects of race when developing anti-thrombotic drugs.