An orthosteric/allosteric bivalent peptide agonist comprising covalently linked protease-activated receptor-derived peptides mimics in vitro and in vivo activities of activated protein C.

BACKGROUND: Activated protein C (APC) has anticoagulant and cytoprotective cell-signaling activities, which often require protease-activated receptor (PAR) 1 and PAR3 and PAR cleavages at noncanonical sites (R46-N47 and R41-G42, respectively). Some PAR1-derived (P1) peptides and PAR3-derived (P3) peptides, eg, P1-47-66 and P3-42-65, mimic APC's cell signaling. In anti-inflammatory assays, these 2 peptides at low concentrations synergistically attenuate cellular inflammation. OBJECTIVES: To determine whether a P1 peptide covalently linked to a P3 peptide mimics APC's anti-inflammatory and endothelial barrier stabilization activities. METHODS: Anti-inflammatory assays employed stimulated THP-1 cells and caspase-1 measurements. Cultured human EA.hy926 or murine aortic endothelial cells (ECs) exposed to thrombin were monitored for transendothelial electrical resistance. Bivalent covalently linked P1:P3 peptides were studied for APC-like activities. RESULTS: In anti-inflammatory assays, P1-47-55 was as active as P1-47-66 and some P3 peptides (eg, P3-44-54 and P3-51-65) were as active as P3-42-65. The bivalent P1:P3 peptide comprising P1-47-55-(Gly[10 residues])-P3-51-65 (designated "G10 peptide") was more potently anti-inflammatory than the P1 or P3 peptide alone. In transendothelial electrical resistance studies of thrombin-challenged ECs, P1-47-55 and the G10 peptide mimicked APC's protective actions. In dose-response studies, the G10 peptide was more potent than the P1-47-55 peptide. In murine EC studies, the murine PAR-sequence-derived G10 peptide mimicked murine APC's activity. Anti-PAR1 and anti-PAR3 antibodies, but not anti-endothelial protein C receptor antibodies, abated G10's cytoprotection, showing that G10's actions involve PAR1:PAR3. G10 significantly increased survival in murine endotoxemia. CONCLUSION: The PAR-sequence-derived G10 peptide is a bivalent agonist that mimics APC's cytoprotective, anti-inflammatory, and endothelial barrier-stabilizing actions and APC's protection against endotoxemic mortality.

Redox regulation of platelet function and thrombosis.

Platelets are well-known players in several cardiovascular diseases such as atherosclerosis and venous thrombosis. There is increasing evidence demonstrating that reactive oxygen species (ROS) are generated within activated platelets. Nicotinamide adenine dinucleotide phosphate oxidase (NOX) is a major source of ROS generation in platelets. Ligand binding to platelet receptor glycoprotein (GP) VI stimulates intracellular ROS generation consisting of a spleen tyrosine kinase-independent production involving NOX activation and a following spleen tyrosine kinase-dependent generation. In addition to GPVI, stimulation of platelet thrombin receptors (protease-activated receptors [PARs]) can also trigger NOX-derived ROS production. Our recent study found that mitochondria-derived ROS production can be induced by engagement of thrombin receptors but not by GPVI, indicating that mitochondria are another source of PAR-dependent ROS generation apart from NOX. However, mitochondria are not involved in GPVI-dependent ROS generation. Once generated, the intracellular ROS are also involved in modulating platelet function and thrombus formation; therefore, the site-specific targeting of ROS production or clearance of excess ROS within platelets is a potential intervention and treatment option for thrombotic events. In this review, we will summarize the signaling pathways involving regulation of platelet ROS production and their role in platelet function and thrombosis, with a focus on GPVI- and PAR-dependent platelet responses.

A mouse model of the protease-activated receptor 4 Pro310Leu variant has reduced platelet reactivity.

BACKGROUND: Protease-activated receptor 4 (PAR4) mediates thrombin signaling on platelets and other cells. Our recent structural studies demonstrated that a single nucleotide polymorphism in extracellular loop 3 and PAR4-P310L (rs2227376) leads to a hyporeactive receptor. OBJECTIVES: The goal of this study was to determine how the hyporeactive PAR4 variant in extracellular loop 3 impacts platelet function in vivo using a novel knock-in mouse model (PAR4-322L). METHODS: A point mutation was introduced into the PAR4 gene F2rl3 via CRISPR/Cas9 to create PAR4-P322L, the mouse homolog to human PAR4-P310L. Platelet response to PAR4 activation peptide (AYPGKF), thrombin, ADP, and convulxin was monitored by αIIbß3 integrin activation and P-selectin translocation using flow cytometry or platelet aggregation. In vivo responses were determined by the tail bleeding assay and the ferric chloride-induced carotid artery injury model. RESULTS: PAR4-P/L and PAR4-L/L platelets had a reduced response to AYPGKF and thrombin measured by P-selectin translocation or αIIbß3 activation. The response to ADP and convulxin was unchanged among genotypes. In addition, both PAR4-P/L and PAR4-L/L platelets showed a reduced response to thrombin in aggregation studies. There was an increase in the tail bleeding time for PAR4-L/L mice. The PAR4-P/L and PAR4-L/L mice both showed an extended time to arterial thrombosis. CONCLUSION: PAR4-322L significantly reduced platelet responsiveness to AYPGKF and thrombin, which is in agreement with our previous structural and cell signaling studies. In addition, PAR4-322L had prolonged arterial thrombosis time. Our mouse model provides a foundation to further evaluate the role of PAR4 in other pathophysiological contexts.

Staphylococcus aureus: The Bug Behind the Itch in Atopic Dermatitis.

Pruritus or itch is a defining symptom of atopic dermatitis (AD). The origins of itch are complex, and it is considered both a defense mechanism and a cause of disease that leads to inflammation and psychological stress. Considerable progress has been made in understanding the processes that trigger itch, particularly the pruritoceptive origins that are generated in the skin. This perspective review discusses the implications of a recent observation that the V8 protease expressed by Staphylococcus aureus can directly trigger sensory neurons in the skin through activation of protease-activated receptor 1. This may be a key to understanding why itch is so common in AD because S. aureus commonly overgrows in this disease owing to deficient antimicrobial defense from both the epidermis and the cutaneous microbiome. Increased understanding of the role of microbes in AD provides increased opportunities for safely improving the treatment of this disorder.

Microscopic in-situ analysis of the mucosal healing around implants treated by protease activated receptor 4-agonist peptide or perpendicularly protruded type I collagen in rats.

Enhanced mucosal sealing around titanium implants can reduce complications such as peri-implantitis. The present study aims to investigate the mucosal healing at the early stage around the protease activated receptor 4-agonist peptide (PAR4-AP)- or perpendicularly protruded type I collagen (pCol)-treated titanium implants. A total of 72 implants were placed in 36 rats in the study. Following extractions, two tissue-level implants among the following three different surfaces, PAR4-AP-coated (PAR4 group, n = 24), pCol-treated (pCol group, n = 24) and non-treated (control group, n = 24) ones, were placed in the maxillae of each rat based on a split-mouth design. The specimens retrieved at 8 h (n = 8 per group), 3 days (n = 8 per group), and 2 weeks (n = 8 per group), were immunostained and tissue-cleared, and the signals of laminin-5 and collagen fibers were observed under multiphoton microscopy. Statistical analyses were performed using linear mixed model with post hoc tests to compare differences between the groups. While there was no intergroup difference at 8 h, the laminin-5 at 3 days was more abundant near the PAR4-group-surface, and its area was significantly larger in the PAR4 group (0.0204 ± 0.0194 mm2 ) than the control (0.0019 ± 0.0025 mm2 , p = .001) and pCol (0.0023 ± 0.0022 mm2 , p < .001) groups. The pCol group showed a significantly larger area of collagen fibers (0.0230 ± 0.0148 mm2 ) compared to the control (0.0035 ± 0.0051 mm2 , p = .002) and PAR4 (0.0031 ± 0.0057 mm2 , p < .001) groups at 3 days. At 3 days and 2 weeks, the collagen fiber orientation of the pCol group showed a more perpendicular manner compared to the control and PAR4 groups. The signal of basal lamina and collagen fibers were stronger around the PAR4-AP- and pCol-treated titanium surfaces, respectively during the early healing stage. This could have implications for improved mucosal sealing around dental implants, potentially reducing complications such as peri-implantitis.

New insight into the agonism of protease-activated receptors as an immunotherapeutic strategy.

The activation and mobilization of immune cells play a crucial role in immunotherapy. Existing therapeutic interventions, such as cytokines administration, aim to enhance immune cell activity. However, these approaches usually result in modest effectiveness and toxic side effects, thereby restricting their clinical application. Protease-activated receptors (PARs), a subfamily of G protein-coupled receptors, actively participate in the immune system by directly activating immune cells. The activation of PARs by proteases or synthetic ligands can modulate immune cell behavior, signaling, and responses to treat immune-related diseases, suggesting the significance of PARs agonism in immunotherapy. However, the agonism of PARs in therapeutical applications remains rarely discussed, since it has been traditionally considered that PARs activation facilitates disease progressions. This review aims to comprehensively summarize the activation, rather than inhibition, of PARs in immune-related physiological responses and diseases. Additionally, we will discuss the emerging immunotherapeutic potential of PARs agonism, providing a new strategic direction for PARs-mediated immunotherapy.

Rivaroxaban, a direct inhibitor of coagulation factor Xa, attenuates adverse cardiac remodeling in rats by regulating the PAR-2 and TGF-ß1 signaling pathways.

Background: Factor Xa (FXa) not only plays an active role in the coagulation cascade but also exerts non-hemostatic signaling through the protease-activated receptors (PARs). This study aimed to investigate whether the FXa inhibitor, Rivaroxaban (RIV), attenuates adverse cardiac remodeling in rats with myocardial infarction (MI) and to identify the underlying molecular mechanisms it uses. Methods: An MI model was induced in eight-week-old, male Wistar rats, by permanent ligation of the left anterior descending coronary artery. MI rats were randomly assigned to receive RIV or protease-activated receptors 2-antagonist (PAR-2 antagonist, FSLLRY) treatment for four weeks. Histological staining, echocardiography and hemodynamics were used to assess the cardioprotective effects of RIV. Meanwhile, pharmacological approaches of agonist and inhibitor were used to observe the potential pathways in which RIV exerts antifibrotic effects in neonatal rat cardiac fibroblasts (CFs). In addition, real-time PCR and western blot analysis were performed to examine the associated signaling pathways. Results: RIV presented favorable protection of left ventricular (LV) cardiac function in MI rats by significantly reducing myocardial infarct size, ameliorating myocardial pathological damage and improving left ventricular (LV) remodeling. Similar improvements in the PAR-2 antagonist FSLLRY and RIV groups suggested that RIV protects against cardiac dysfunction in MI rats by ameliorating PAR-2 activation. Furthermore, an in vitro model of fibrosis was then generated by applying angiotensin II (Ang II) to neonatal rat cardiac fibroblasts (CFs). Consistent with the findings of the animal experiments, RIV and FSLLRY inhibited the expression of fibrosis markers and suppressed the intracellular upregulation of transforming growth factor ß1 (TGFß1), as well as its downstream Smad2/3 phosphorylation effectors in Ang II-induced fibrosis, and PAR-2 agonist peptide (PAR-2 AP) reversed the inhibition effect of RIV. Conclusions: Our findings demonstrate that RIV attenuates MI-induced cardiac remodeling and improves heart function, partly by inhibiting the activation of the PAR-2 and TGF-ß1 signaling pathways.

Unraveling coagulation factor-mediated cellular signaling.

Blood coagulation is initiated in response to blood vessel injury or proinflammatory stimuli, which activate coagulation factors to coordinate complex biochemical and cellular responses necessary for clot formation. In addition to these critical physiologic functions, plasma protein factors activated during coagulation mediate a spectrum of signaling responses via receptor-binding interactions on different cell types. In this review, we describe examples and mechanisms of coagulation factor signaling. We detail the molecular basis for cell signaling mediated by coagulation factor proteases via the protease-activated receptor family, considering new insights into the role of protease-specific cleavage sites, cofactor and coreceptor interactions, and distinct signaling intermediate interactions in shaping protease-activated receptor signaling diversity. Moreover, we discuss examples of how injury-dependent conformational activation of other coagulation proteins, such as fibrin(ogen) and von Willebrand factor, decrypts their signaling potential, unlocking their capacity to contribute to aberrant proinflammatory signaling. Finally, we consider the role of coagulation factor signaling in disease development and the status of pharmacologic approaches to either attenuate or enhance coagulation factor signaling for therapeutic benefit, emphasizing new approaches to inhibit deleterious coagulation factor signaling without impacting hemostatic activity.

Species Differences in Platelet Protease-Activated Receptors.

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.

Protease-activated receptors and glycoprotein VI cooperatively drive the platelet component in thromboelastography.

BACKGROUND: Thromboelastography (TEG) is used for real-time determination of hemostatic status in patients with acute risk of bleeding. Thrombin is thought to drive clotting in TEG through generation of polymerized fibrin and activation of platelets through protease-activated receptors (PARs). However, the specific role of platelet agonist receptors and signaling in TEG has not been reported. OBJECTIVES: Here, we investigated the specific receptors and signaling pathways required for platelet function in TEG using genetic and pharmacologic inhibition of platelet proteins in mouse and human blood samples. METHODS: Clotting parameters (R time, α-angle [α], and maximum amplitude [MA]), were determined in recalcified, kaolin-triggered citrated blood samples using a TEG 5000 analyzer. RESULTS: We confirmed the requirement of platelets, platelet contraction, and αIIbß3 integrin function for normal α and MA. Loss of the integrin adaptor Talin1 in megakaryocytes/platelets (Talin1mKO) also reduced α and MA, but only minimal defects were observed in samples from mice lacking Rap1 GTPase signaling. PAR4mKO samples showed impaired α but normal MA. However, impaired TEG traces similar to those in platelet-depleted samples were observed with samples from PAR4mKO mice depleted of glycoprotein VI on platelets or with addition of a Syk inhibitor. We reproduced these results in human blood with combined inhibition of PAR1, PAR4, and Syk. CONCLUSION: Our results demonstrate that standard TEG is not sensitive to platelet signaling pathways critical for integrin inside-out activation and platelet hemostatic function. Furthermore, we provide the first evidence that PARs and glycoprotein VI play redundant roles in platelet-mediated clot contraction in TEG.

PARs in the inflammation-cancer transformation of CRC.

Colorectal cancer (CRC) is one of the common malignancies with a global trend of increasing incidence and mortality. There is an urgent need to identify new predictive markers and therapeutic targets for the treatment of CRC. Protease-activated receptors (PARs) are a class of G-protein-coupled receptors, with currently identified subtypes including PAR1, PAR2, PAR3 and PAR4. Increasingly, studies suggest that PARs play an important role in the growth and metastasis of CRC. By targeting multiple signaling pathways may contribute to the pathogenesis of CRC. In this review, we first describe recent studies on the role of PARs in CRC inflammation-cancer transformation, focusing on the important role of PARs in signaling pathways associated with inflammation-cancer transformation, and summarize the progress of research on PARs-targeted drugs.

Protease-activated receptors in health and disease.

Proteases are signaling molecules that specifically control cellular functions by cleaving protease-activated receptors (PARs). The four known PARs are members of the large family of G protein-coupled receptors. These transmembrane receptors control most physiological and pathological processes and are the target of a large proportion of therapeutic drugs. Signaling proteases include enzymes from the circulation; from immune, inflammatory epithelial, and cancer cells; as well as from commensal and pathogenic bacteria. Advances in our understanding of the structure and function of PARs provide insights into how diverse proteases activate these receptors to regulate physiological and pathological processes in most tissues and organ systems. The realization that proteases and PARs are key mediators of disease, coupled with advances in understanding the atomic level structure of PARs and their mechanisms of signaling in subcellular microdomains, has spurred the development of antagonists, some of which have advanced to the clinic. Herein we review the discovery, structure, and function of this receptor system, highlight the contribution of PARs to homeostatic control, and discuss the potential of PAR antagonists for the treatment of major diseases.

Protease inhibitor from Libidibia ferrea seeds attenuates inflammatory and nociceptive responses in mice.

ETHNOPHARMACOLOGICAL RELEVANCE: Libidibia ferrea (Mart. ex. Tul.) L.P. Queiroz is a Brazilian native tree locally known as jucá and pau-ferro, and it has been used in folk medicine for relieving, asthma, bronchitis, sore throat, rheumatism, enterocolitis and fever. The anti-inflammatory properties of L. ferrea were confirmed for its stem, fruit, leaves, bark and seeds extracts, however little is known about the natural compounds that may be associated with that response. AIM OF THIS STUDY: In a normal physiological condition, many enzymes play an important role in catalyzing biological functions. Among them, proteases are of great interest. Although they take part of many biological systems, as the inflammatory process, when deregulated, proteases may cause system malfunctions, such as under- or overproduction of cytokines, or immune cells activation. Thus, protease inhibitors prevent these immune responses by regulating proteases. The objective of this study was to evaluate the anti-inflammatory and anti-nociceptive response of a protease inhibitor purified from L. ferrea seeds (LfTI). MATERIALS AND METHODS: In vitro (5, 50 and 250 µg/mL of LfTI) and in vivo (0.6, 3 e 15 mg/kg of LfTI) assays were performed. Male Swiss mice weighing 18-25 g were used for cell harvesting and for the in vivo assays. The anti-inflammatory activity was analyzed in vitro by macrophage cytotoxicity, hydrogen peroxide (H2O2) production, and cell adhesion assays; and in vivo by leukocyte recruitment, nitric oxide (NO) production, vascular permeability, paw edema and mast cell degranulation assays. The anti-nociceptive activity was evaluated through abdominal writhing test induced by acetic acid and formalin sensitization. RESULTS: Our results showed that, in vitro, LfTI is not cytotoxic. Also, LfTI (50 µg/mL) inhibited macrophage H2O2 production (48.2%), and adhesion (48.4%). LfTI (0.6, 3 e 15 mg/kg) decreased polymorphonuclear cell recruitment dose-dependently, and it inhibited NO production (53%), vascular permeability (40.7%) and paw edema at 3 mg/kg at different time, but it did not inhibit mast cell degranulation. Besides, LfTI did not inhibit either the number of writhing or the licking time in the formalin test in the second phase (inflammatory). However, LfTI (3 mg/kg) inhibited licking time at the first phase (neurogenic) in the formalin sensitization (46.1%). CONCLUSIONS: Our results show that LfTI has anti-inflammatory and antinociceptive (neurogenic pain) effects, and these effects might be associated with the inhibition of inflammatory proteases and/or protease-activated receptors activation hindering.

Protease-Activated Receptor Antagonist for Reducing Cardiovascular Events - A Review on Vorapaxar.

Acute Coronary Syndrome (ACS) is a term that describes pathologies related to myocardial ischemia, and is comprised of unstable angina, non-ST elevation myocardial infarction, and ST elevation myocardial infarction. Urgent management of ACS is typically necessary to prevent future morbidity and mortality. Current medical recommendations of ACS management involve use of dual antiplatelet therapy, typically with aspirin and clopidogrel. However, newer therapies are being designed and researched to improve outcomes for patients with ACS. Vorapaxar is a novel antiplatelet therapy that inhibits thrombin-mediated platelet aggregation to prevent recurrence of ischemic events. It has been Food and Drug Administration approved for reduction of thrombotic cardiovascular events in patients with a history of MI or peripheral arterial disease with concomitant use of clopidogrel and/or aspirin, based upon the findings of the TRA 2°P-TIMI 50 trial. However, Vorapaxar was also found to have a significantly increased risk of bleeding, which must be considered when administering this drug. Based upon further subgroup analysis of both the TRA 2°P-TIMI 50 trial and TRACER trial, Vorapaxar was found to be potentially beneficial in patients with peripheral artery disease, coronary artery bypass grafting, and ischemic stroke. There are current trials in progress that are further evaluating the use of Vorapaxar in those conditions, and future research and trials are necessary to fully determine the utility of this drug.

Downregulation of Matriptase Inhibits Porphyromonas gingivalis Lipopolysaccharide-Induced Matrix Metalloproteinase-1 and Proinflammatory Cytokines by Suppressing the TLR4/NF-κB Signaling Pathways in Human Gingival Fibroblasts.

Matriptases are cell surface proteolytic enzymes belonging to the type II transmembrane serine protease family that mediate inflammatory skin disorders and cancer progression. Matriptases may affect the development of periodontitis via protease-activated receptor-2 activity. However, the cellular mechanism by which matriptases are involved in periodontitis is unknown. In this study, we examined the antiperiodontitis effects of matriptase on Porphyromonas gingivalis-derived lipopolysaccharide (PG-LPS)-stimulated human gingival fibroblasts (HGFs). Matriptase small interfering RNA-transfected HGFs were treated with PG-LPS. The mRNA and protein levels of proinflammatory cytokines and matrix metalloproteinase 1 (MMP-1) were evaluated using the quantitative real-time polymerase chain reaction (qRT-PCR) and an enzyme-linked immunosorbent assay (ELISA), respectively. Western blot analyses were performed to measure the levels of Toll-like receptor 4 (TLR4)/interleukin-1 (IL-1) receptor-associated kinase (IRAK)/transforming growth factor ß-activated kinase 1 (TAK1), p65, and p50 in PG-LPS-stimulated HGFs. Matriptase downregulation inhibited LPS-induced proinflammatory cytokine expression, including the expression of IL-6, IL-8, tumor necrosis factor-α (TNF-α), and IL-Iß. Moreover, matriptase downregulation inhibited PG-LPS-stimulated MMP-1 expression. Additionally, we confirmed that the mechanism underlying the effects of matriptase downregulation involves the suppression of PG-LPS-induced IRAK1/TAK1 and NF-κB. These results suggest that downregulation of matriptase PG-LPS-induced MMP-1 and proinflammatory cytokine expression via TLR4-mediated IRAK1/TAK1 and NF-κB signaling pathways in HGFs.

Diversification of PAR signaling through receptor crosstalk.

Protease activated receptors (PARs) are among the first receptors shown to transactivate other receptors: noticeably, these interactions are not limited to members of the same family, but involve receptors as diverse as receptor kinases, prostanoid receptors, purinergic receptors and ionic channels among others. In this review, we will focus on the evidence for PAR interactions with members of their own family, as well as with other types of receptors. We will discuss recent evidence as well as what we consider as emerging areas to explore; from the signalling pathways triggered, to the physiological and pathological relevance of these interactions, since this additional level of molecular cross-talk between receptors and signaling pathways is only beginning to be explored and represents a novel mechanism providing diversity to receptor function and play important roles in physiology and disease.

Factor VII activating protease (FSAP) inhibits the outcome of ischemic stroke in mouse models.

The outcome of ischemic stroke can be improved by further refinements of thrombolysis and reperfusion strategies. Factor VII activating protease (FSAP) is a circulating serine protease that could be important in this context. Its levels are raised in patients as well as mice after stroke and a single nucleotide polymorphism (SNP) in the coding sequence, which results in an inactive enzyme, is linked to an increased risk of stroke. In vitro, FSAP cleaves fibrinogen to promote fibrinolysis, activates protease-activated receptors, and decreases the cellular cytotoxicity of histones. Based on these facts, we hypothesized that FSAP can be used as a treatment for ischemic stroke. A combination of tissue plasminogen activator (tPA), a thrombolytic drug, and recombinant serine protease domain of FSAP (FSAP-SPD) improved regional cerebral perfusion and neurological outcome and reduced infarct size in a mouse model of thromboembolic stroke. FSAP-SPD also improved stroke outcomes and diminished the negative consequences of co-treatment with tPA in the transient middle cerebral artery occlusion model of stroke without altering cerebral perfusion. The inactive MI-isoform of FSAP had no impact in either model. FSAP enhanced the lysis of blood clots in vitro, but in the tail transection model of hemostasis, FSAP-SPD treatment provoked a faster clotting time indicating that it also has pro-coagulant actions. Thus, apart from enhancing thrombolysis, FSAP has multiple effects on stroke progression and represents a promising novel therapeutic strategy in the treatment of ischemic stroke.

Candida albicans Sap6 Initiates Oral Mucosal Inflammation via the Protease Activated Receptor PAR2.

Candida albicans Sap6, a secreted aspartyl protease (Sap), contributes to fungal virulence in oral candidiasis. Beside its protease activity, Sap6 contains RGD (RGDRGD) motif required for its binding to host integrins. Sap6 activates immune cells to induce proinflammatory cytokines, although its ability to interact and activate human oral epithelial cells (OECs) remain unknown. Addition of purified recombinant Sap6 (rSap6) to OECs resulted in production of IL-1ß and IL-8 cytokines similar to live hyphal C. albicans. OECs exposed to rSap6 showed phosphorylation of p38 and MKP1 and expression of c-Fos not found with C. albicans Δsap6, heat-inactivated Sap6, or rSap6ΔRGD . Heat inactivated rSap6 was able to induce IL-1ß but not IL-8 in OECs, while rSap6ΔRGD induced IL-8 but not IL-1ß suggesting parallel signaling pathways. C. albicans hyphae increased surface expression of Protease Activated Receptors PAR1, PAR2 and PAR3, while rSap6 increased PAR2 expression exclusively. Pretreatment of OECs with a PAR2 antagonist blocked rSap6-induced p38 MAPK signaling and IL-8 release, while rSap6ΔRGD had reduced MKP1 signaling and IL-1ß release independent from PAR2. OECs exposed to rSap6 exhibited loss of barrier function as measured by TEER and reduction in levels of E-cadherin and occludin junctional proteins that was prevented by pretreating OECs with a PAR2 antagonist. OECs treated with PAR2 antagonist also showed reduced rSap6-mediated invasion by C. albicans cells. Thus, Sap6 may initiate OEC responses mediated both through protease activation of PAR2 and by its RGD domain. This novel role of PAR2 suggests new drug targets to block C. albicans oral infection.

Protease-activated receptor-mediated platelet aggregation in patients with type 2 diabetes on potent P2Y12 inhibitors.

BACKGROUND: Antiplatelet therapy is a cornerstone in the secondary prevention of ischemic events following percutaneous coronary intervention (PCI). The new P2Y12 receptor inhibitors prasugrel and ticagrelor have been shown to improve patients' outcomes. Whether or not these drugs have equal efficacy in individuals with or without diabetes is disputed. Furthermore, platelets can be activated by thrombin, which is, at least in part, independent of P2Y12 -mediated platelet activation. Protease-activated receptor (PAR)-1 and -4 are thrombin receptors on human platelets. We sought to compare the in vitro efficacy of prasugrel (n = 121) and ticagrelor (n = 99) to inhibit PAR-mediated platelet aggregation in individuals with type 2 diabetes (prasugrel n = 26, ticagrelor n = 29). MATERIALS AND METHODS: We compared P2Y12 -, PAR-1- and PAR-4-mediated platelet aggregation as assessed by multiple electrode platelet aggregometry between prasugrel- and ticagrelor-treated patients without and with type 2 diabetes who underwent acute PCI. RESULTS: Overall, there were no differences of P2Y12 -, PAR-1- and PAR-4-mediated platelet aggregation between prasugrel- and ticagrelor-treated patients. However, both drugs inhibited P2Y12 -mediated platelet aggregation stronger, and thereby to a similar extent in patients with type 2 diabetes than in those without diabetes. There was no correlation between either P2Y12 -, or PAR-1- or PAR-4-mediated platelet aggregation and levels of HbA1c or the body mass index (BMI). However, we observed patients with high residual platelet reactivity in response to PAR-1 and PAR-4 stimulation in all cohorts. CONCLUSION: Prasugrel and ticagrelor inhibit P2Y12 - and PAR-mediated platelet aggregation in individuals with diabetes to a similar extent, irrespective of HbA1c levels and BMI.