Cleavage Of Protease-activated Receptors Research Articles

Biased agonism of protease-activated receptor-1 regulates thromboinflammation in murine sickle cell disease

AbstractSickle cell disease (SCD) is a hereditary hemoglobinopathy marked by hemolytic anemia and vaso-occlusive events (VOEs). Chronic endothelial activation, inflammation, and coagulation activation contribute to vascular congestion, VOEs, and end-organ damage. Coagulation proteases such as thrombin and activated protein C (APC) modulate inflammation and endothelial dysfunction by activating protease-activated receptor 1 (PAR1), a G-protein–coupled receptor. Thrombin cleaves PAR1 at Arg41, while APC cleaves PAR1 at Arg46, initiating either proinflammatory or cytoprotective signaling, respectively, a signaling conundrum known as biased agonism. Our prior research established the role of thrombin and PAR1 in vascular stasis in an SCD mouse model. However, the role of APC and APC-biased PAR1 signaling in thrombin generation, inflammation, and endothelial activation in SCD remains unexplored. Inhibition of APC in SCD mice increased thrombin generation, inflammation, and endothelial activation during both steady state and tumor necrosis factor α challenge. To dissect the individual contributions of thrombin-PAR1 and APC-PAR1 signaling, we used transgenic mice with point mutations at 2 PAR1 cleavage sites, ArgR41Gln (R41Q) imparting insensitivity to thrombin and Arg46Gln (R46Q) imparting insensitivity to APC. Sickle bone marrow chimeras expressing PAR1-R41Q exhibited reduced thrombo-inflammatory responses compared with wild type PAR1 or PAR1-R46Q mice. These findings highlight the potential benefit of reducing thrombin-dependent PAR1 activation while preserving APC-PAR1 signaling in SCD thromboinflammation. These results also suggest that pharmacological strategies promoting biased PAR1 signaling could effectively mitigate vascular complications associated with SCD.

Therapeutic Effects of a Novel Aptamer on Coronaviral Infection-Induced Lung Injury and Systemic Inflammatory Responses.

Coronaviral infection-induced acute lung injury has become a major threat to public health, especially through the ongoing pandemic of COVID-19. Apta-1 is a newly discovered Aptamer that has anti-inflammatory effects on systemic septic responses. The therapeutic effects of Apta-1 on coronaviral infection-induced acute lung injury and systemic responses were evaluated in the present study. Female A/J mice (at 12-14 weeks of age) were challenged with murine hepatitis virus 1 (MHV-1), a coronavirus, at 5000 PFU intranasally, followed by Apta-1 intravenously administered (100 mg/kg, twice) 1.5 h or 2 days after viral delivery. Animals were sacrificed at Day 2 or Day 4. Lung tissues were examined with H&E, immunohistochemistry staining, and western blotting. RT-qPCR was used for cytokine gene expression. Serum and plasma were collected for laboratory assessments. Apta-1 treatment reduced viral titers, prevented MHV-1-induced reduction of circulating blood volume and hemolysis, reduced alveolar space hemorrhage, and protease-activated receptor 1 (PAR-1) cleavage. Apta-1 treatment also significantly reduced chemokine (MKC, MCP-1, and RANTES) levels, as well as AST, ALT, total bilirubin, and reduced unconjugated bilirubin levels in the serum. Apta-1 showed therapeutic benefits in coronaviral infection-induced hemorrhage and PAR-1 cleavage in the lung. It also has anti-inflammatory effects systemically.

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.

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

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. 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. 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 β-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 β-arrestin-1 and -2 to the plasma membrane. Thus, the thrombin cleavage of Arg41 was also cytoprotective in thrombomodulin-expressing cells by β-arrestin-1-biased signaling. APC in the absence of EPCR cleaved Arg41 to initiate disruptive signaling responses like thrombin. These results suggest that coreceptor signaling by thrombomodulin and EPCR determines the PAR1 cleavage and signaling specificity of thrombin and APC, respectively.

Selective modulation of activated protein C activities by a nonactive site-targeting nanobody library.

Activated protein C (APC) is a pleiotropic coagulation protease with anticoagulant, anti-inflammatory, and cytoprotective activities. Selective modulation of these APC activities contributes to our understanding of the regulation of these physiological mechanisms and permits the development of therapeutics for the pathologies associated with these pathways. An antibody library targeting the nonactive site of APC was generated using llama antibodies (nanobodies). Twenty-one nanobodies were identified that selectively recognize APC compared with the protein C zymogen. Overall, 3 clusters of nanobodies were identified based on the competition for APC in biolayer interferometry studies. APC functional assays for anticoagulant activity, histone H3 cleavage, and protease-activated receptor 1 (PAR1) cleavage were used to understand their diversity. These functional assays revealed 13 novel nanobody-induced APC activity profiles via the selective modulation of APC pleiotropic activities, with the potential to regulate specific mechanisms for therapeutic purposes. Within these, 3 nanobodies (LP2, LP8, and LP17) inhibited all 3 APC functions. Four nanobodies (LP1, LP5, LP16, and LP20) inhibited only 2 of the 3 functions. Monofunction inhibition specific to APC anticoagulation activity was observed only by 2 nanobodies (LP9 and LP11). LP11 was also found to shift the ratio of APC cleavage of PAR1 at R46 relative to R41, which results in APC-mediated biased PAR1 signaling and APC cytoprotective effects. Thus, LP11 has an activity profile that could potentially promote hemostasis and cytoprotection in bleedings associated with hemophilia or coagulopathy by selectively modulating APC anticoagulation and PAR1 cleavage profile.

Exogenous human α-Synuclein acts in vitro as a mild platelet antiaggregant inhibiting α-thrombin-induced platelet activation

α-Synuclein (αSyn) is a small disordered protein, highly conserved in vertebrates and involved in the pathogenesis of Parkinson’s disease (PD). Indeed, αSyn amyloid aggregates are present in the brain of patients with PD. Although the pathogenic role of αSyn is widely accepted, the physiological function of this protein remains elusive. Beyond the central nervous system, αSyn is expressed in hematopoietic tissue and blood, where platelets are a major cellular host of αSyn. Platelets play a key role in hemostasis and are potently activated by thrombin (αT) through the cleavage of protease-activated receptors. Furthermore, both αT and αSyn could be found in the same spatial environment, i.e. the platelet membrane, as αT binds to and activates platelets that can release αSyn from α-granules and microvesicles. Here, we investigated the possibility that exogenous αSyn could interfere with platelet activation induced by different agonists in vitro. Data obtained from distinct experimental techniques (i.e. multiple electrode aggregometry, rotational thromboelastometry, immunofluorescence microscopy, surface plasmon resonance, and steady-state fluorescence spectroscopy) on whole blood and platelet-rich plasma indicate that exogenous αSyn has mild platelet antiaggregating properties in vitro, acting as a negative regulator of αT-mediated platelet activation by preferentially inhibiting P-selectin expression on platelet surface. We have also shown that both exogenous and endogenous (i.e. cytoplasmic) αSyn preferentially bind to the outer surface of activated platelets. Starting from these findings, a coherent model of the antiplatelet function of αSyn is proposed.

Thrombin acts as inducer of proinflammatory macrophage migration inhibitory factor in astrocytes following rat spinal cord injury

BackgroundThe danger-associated molecular patterns (DAMPs) are critical contributors to the progressive neuropathology and thereafter affect the functional outcomes following spinal cord injury (SCI). Up to now, the regulatory mechanisms on their inducible production from the living cells remain elusive, aside from their passive release from the necrotic cells. Thrombin is immediately activated by the damaged or stressed central nervous system (CNS), which potently mediates inflammatory astrocytic responses through proteolytic cleavage of protease-activated receptors (PARs). Therefore, SCI-activated thrombin is conceived to induce the production of DAMPs from astrocytes at lesion site.MethodsRat SCI model was established by the cord contusion at T8–T10. The expression of thrombin and macrophage migration inhibitory factor (MIF) was determined by ELISA and Western blot. The PAR1, PAR3, and PAR4 receptors of thrombin were examined by PCR and immunohistochemistry. Primary astrocytes were isolated and purified from the spinal cord, followed by stimulation with different concentrations of thrombin either for transcriptome sequencing or for analysis of thrombin-mediated expression of MIF and related signal pathways in the presence or absence of various inhibitors. The post-injury locomotor functions were assessed using the Basso, Beattie, and Bresnahan (BBB) locomotor scale.ResultsMIF protein levels were significantly elevated in parallel with those of thrombin induced by SCI. Immunostaining demonstrated that PAR1 receptor, together with MIF, was abundantly expressed in astrocytes. By transcriptome sequencing and bioinformatical analysis of thrombin-stimulated primary astrocytes, MIF was identified to be dynamically regulated by the serine protease. Investigation of the underlying mechanism using various inhibitors revealed that thrombin-activated PAR1 was responsible for the MIF production of astrocytes through modulation of JNK/NFκB pathway. Administration of PAR1 inhibitor at lesion sites following SCI significantly reduced the protein levels of MIF and ameliorated functional deficits of rat locomotion.ConclusionSCI-activated thrombin is a robust inducer of MIF production from astrocytes. Exploring the roles of thrombin in promoting the production of DAMPs from astrocytes at lesion site will provide an alternative strategy for the clinical therapy of CNS inflammation.

Regulation of activated Protein C/PAR1 endothelial cytoprotective signaling by G protein‐coupled receptor kinases

Endothelial dysfunction is a hallmark of inflammation and associated with vascular diseases. Our goal is to define pathways by which the endothelium can resist injury and disruption to facilitate the advancement of new targets for therapeutic development. Activated Protein C (APC) has been shown in preclinical studies to increase endothelial barrier protection and reduce inflammatory responses and signals via protease‐activated receptor‐1 (PAR1). APC enhances endothelial cytoprotection through cleavage of PAR1 at an N‐terminal arginine (R)‐46 site, that is distinct from the canonical thrombin cleavage site at R‐41. This results in the generation of a distinct tethered ligand that promotes PAR1 bias signaling. We discovered that APC/PAR1 signals preferentially via b‐arrestin2 (b‐arr2), rather than heterotrimeric G proteins to promote endothelial barrier protection. The objective of this study is to develop a mechanistic understanding of how APC/PAR1 generates b‐arr2 cytoprotective signaling. G protein‐coupled receptor kinases (GRKs) phosphorylate thrombin‐activated PAR1 and facilitate β‐arrestin‐mediated desensitization. However, contribution of GRKs to β‐arrestin driven APC/PAR1‐induced cytoprotection is unclear. We hypothesize that compartmentalization of APC/PAR1 together with GRK5 is essential to initiate b‐arr2 recruitment and biased signaling. Here we will present studies examining the function and mechanism by which GRKs regulate endothelial cytoprotective responses using GRK‐specific siRNA to assess function as well as confocal immunofluorescence imaging and biochemical sucrose fraction to examine GRK localization and compartmentalization in caveolae. The work is expected to provide important new insight into the role of GRKs in regulating APC/PAR1 cytoprotection in human cultured endothelial cells.

Influence of PAR-1 in patients with non-valvular atrial fibrillation: The antiplatelet effect of dabigatran

IntroductionDabigatran, a direct thrombin inhibitor, has been widely used in patients with non-valvular atrial fibrillation (NVAF) and is considered to have an antiplatelet effect. However, the mechanisms remain unclear. We evaluated protease-activated receptor-1 (PAR-1) expression and activation by thrombin on platelets from NVAF patients, before and after dabigatran treatment, in addition to the expression of platelet activation marker CD62P. Materials and methodsThe study included 18 NVAF patients. We used flow cytometry to measure the binding of PAR-1 monoclonal antibodies (SPAN12 and WEDE15) and the expression of CD62P with and without thrombin stimulation, before, 14 days after, and 28 days after treatment with dabigatran. Coagulation fibrinolysis markers were also measured. ResultsPAR-1 expression was significantly lower in NVAF patients than in healthy controls (HC); it was further reduced by thrombin stimulation. CD62P expression was almost absent on the platelets in NVAF patients, but was significantly increased by thrombin stimulation. PAR-1 expression was not significantly different before and after treatment; CD62P expression was inhibited by dabigatran. The levels of coagulation markers were significantly higher in NVAF patients than in HC, and decreased after treatment. ConclusionsLower expression of PAR-1 in NVAF patients resulted from the cleavage of PAR-1 on some platelets, by exposure to small amounts of thrombin in vivo. The therapeutic effect of dabigatran in NVAF patients was demonstrated by inhibition of CD62P expression on the platelet upon thrombin stimulation in vitro. Our results indicate that dabigatran may reveal antithrombotic activity with antiplatelet and anticoagulant effects.

Platelet-derived calpain cleaves the endothelial protease-activated receptor 1 to induce vascular inflammation in diabetes

Diabetes mellitus is a major risk factor for cardiovascular disease. Platelets from diabetic patients are hyperreactive and release microparticles that carry activated cysteine proteases or calpains. Whether platelet-derived calpains contribute to the development of vascular complications in diabetes is unknown. Here we report that platelet-derived calpain1 (CAPN1) cleaves the protease-activated receptor 1 (PAR-1) on the surface of endothelial cells, which then initiates a signaling cascade that includes the activation of the tumor necrosis factor (TNF)-α converting enzyme (TACE). The latter elicits the shedding of the endothelial protein C receptor and the generation of TNF-α, which in turn, induces intracellular adhesion molecule (ICAM)-1 expression to promote monocyte adhesion. All of the effects of CAPN1 were mimicked by platelet-derived microparticles from diabetic patients or from wild-type mice but not from CAPN1−/− mice, and were not observed in PAR-1-deficient endothelial cells. Importantly, aortae from diabetic mice expressed less PAR-1 but more ICAM-1 than non-diabetic mice, effects that were prevented by treating diabetic mice with a calpain inhibitor as well as by the platelet specific deletion of CAPN1. Thus, platelet-derived CAPN1 contributes to the initiation of the sterile vascular inflammation associated with diabetes via the cleavage of PAR-1 and the release of TNF-α from the endothelial cell surface.

Biased PAR-1 Signaling Regulates Thrombo-Inflammation in a Mouse Model of Sickle Cell Disease

Thrombin and activated protein C (APC) are serine proteases involved in coagulation and inflammatory responses. Protease activated receptor-1 (PAR-1) is a G-protein coupled receptor (GPCR) activated by proteolytic cleavage of the extracellular N-terminus. PAR-1 cleavage at R41 by thrombin or at R46 by APC creates unique tethered ligands that activate different signaling pathways. On endothelial cells, thrombin employs PAR-1 to promote endothelial barrier disruption, vascular leakage, and inflammation. In contrast, APC employs PAR-1 to stabilize endothelial barrier and provides anti-inflammatory and anti-apoptotic activities. These opposing effects of PAR-1 activation are a classic example of "GPCR biased signaling". We have previously demonstrated that PAR-1 deficiency in all hematopoietic cells reduces heme-induced vascular stasis in sickle mice. In contrast it does not affect the elevated plasma levels of IL-6 or thrombin-antithrombin (TAT) complexes observed in sickle cell disease (SCD) mice compared to non-SCD controls during steady state. In this study, we investigated the effects of thrombin- and APC-induced PAR-1 activation on thromboinflammation in the well-characterized Townes mouse model of SCD. Procoagulant and proinflammatory responses were analyzed in control (AA) and sickle (SS) mice at steady state (saline injection) and 5 hours after TNFα (2 µg/kg, i.p.) to mimic the pro-inflammatory milieu that occurs during painful crisis in SCD patients. As previously described, SS mice had elevated plasma levels of TAT compared to AA mice (8.6± 2.4 vs 3.5±0.7ng/mL; p<0.05), that were further enhanced in SS mice by TNFα injection (21.5 ± 4.2 ng/mL, p<0.001). A similar pattern was observed for plasma levels of IL-6 (AA: 2.4±2 vs SS: 18±7.8 ng/mL, p<0.05; SS+TNFα: 231 ± 32 ng/mL, p<0.0001 vs SS). Using these experimental conditions, we first investigated the role of endogenous APC in SCD. We used a rat anti-mouse protein C mAb (SPC-54), which inhibits the active site of APC, thereby blocking all its enzymatic activities, including PAR-1-dependent signaling and anticoagulant actions. SS mice were injected with anti-APC SPC54 (10 mg/kg) or control IgG antibodies. Eighteen hours later both groups of mice were injected with either saline or TNFα and plasma samples were collected 5 hours later. We TNFαSSdata suggest that endogenous APC has anticoagulant and anti-inflammatory roles for both the steady state and after TNFα challenge. To address the individual roles of thrombin and APC signaling on vascular inflammation in SCD, we used mice with point mutations in PAR-1 at R41 or R46. Mutation of R41 to Glu (R41Q) renders PAR-1 insensitive to thrombin while preserving APC-mediated cleavage at R46. Mutation of R46 to Glu (R46Q) ablates APC's cytoprotective effects. Bone marrow (BM) from SS mice was transplanted into PAR-1WT, PAR-1R41Q, or PAR-1R46Q mice to generate SSBM mice with either normal PAR-1 (SSBM/PAR-1WT) or the mutated form of this receptor on all non-hematopoietic cells (SSBM/PAR-1R41Q and SSBM/PAR-1R46Q). Four months after BM transplantation, plasma levels of TAT, IL-6, and HMGB1 were analyzed in all 3 groups at steady state or 5 hours after TNFα challenge. Neither the PAR-1R41Q nor PAR-1R46Q mutation on non-hematopoietic cells affected plasma levels of TAT, IL-6, or HMGB1 in SS mice at steady state. Interestingly, plasma levels of IL-6 were significantly elevated in SSBM/PAR-1R46Q mice and reduced in SSBM/PAR-1R41Q mice compared to SSBM/PAR-1WT mice after TNFα challenge, with a statistically significant difference between the two PAR-1 mutant groups. Furthermore plasma levels of TAT and HMGB1 were significantly reduced in SSBM/PAR-1R41Q but not changed in SSBM/PAR-1R46Q mice after TNFα challenge (Figure 2A-C). These data suggest that PAR-1 activation at R41 regulates TAT, IL-6, and HMGB1, whereas activation of R46 negatively regulates IL-6 in sickle mice during TNFα challenge. Our data imply that pharmacological approaches aiming to eliminate thrombin-dependent, while preserving APC-dependent PAR-1 activation, may be effective in attenuating thrombo-inflamatory complications associated with SCD. Studies evaluating the effects of R41Q and R46Q mutations on microvascular stasis in SS mice are currently ongoing. Disclosures No relevant conflicts of interest to declare.

KLK6 expression in skin induces PAR1-mediated psoriasiform dermatitis and inflammatory joint disease

Kallikrein-related peptidase 6 (KLK6) is a secreted serine protease hypothesized to promote inflammation via cleavage of protease-activated receptor 1 (PAR1) and PAR2. KLK6 levels are elevated in multiple inflammatory and autoimmune conditions, but no definitive role in pathogenesis has been established. Here, we show that skin-targeted overexpression of KLK6 causes generalized, severe psoriasiform dermatitis with spontaneous development of debilitating psoriatic arthritis-like joint disease. The psoriatic skin and joint phenotypes are reversed by normalization of skin KLK6 levels and attenuated following genetic elimination of PAR1 but not PAR2. Conservation of this regulatory pathway was confirmed in human psoriasis using vorapaxar, an FDA-approved PAR1 antagonist, on explanted lesional skin from patients with psoriasis. Beyond defining a critical role for KLK6/PAR1 signaling in promoting psoriasis, our results demonstrate that KLK6/PAR1-mediated inflammation in the skin alone is sufficient to drive inflammatory joint disease. Further, we identify PAR1 as a promising cytokine-independent target in therapy of psoriasis and psoriatic arthritis.

C‐terminal residues of activated protein C light chain contribute to its anticoagulant and cytoprotective activities

BackgroundActivated protein C (APC) is an important homeostatic blood coagulation protease that conveys anticoagulant and cytoprotective activities. Proteolytic inactivation of factors Va and VIIIa facilitated by cofactor protein S is responsible for APC's anticoagulant effects, whereas cytoprotective effects of APC involve primarily the endothelial protein C receptor (EPCR), protease activated receptor (PAR)1 and PAR3. ObjectiveTo date, several binding exosites in the protease domain of APC have been identified that contribute to APC's interaction with its substrates but potential contributions of the C‐terminus of the light chain have not been studied in detail. MethodsSite‐directed Ala‐scanning mutagenesis of six positively charged residues within G142‐L155 was used to characterize their contributions to APC's anticoagulant and cytoprotective activities. Results and ConclusionsK151 was involved in protein S dependent‐anticoagulant activity of APC with some contribution of K150. 3D structural analysis supported that these two residues were exposed in an extended protein S binding site on one face of APC. Both K150 and K151 were important for PAR1 and PAR3 cleavage by APC, suggesting that this region may also mediate interactions with PARs. Accordingly, APC's cytoprotective activity as determined by endothelial barrier protection was impaired by Ala substitutions of these residues. Thus, both K150 and K151 are involved in APC's anticoagulant and cytoprotective activities. The differential contribution of K150 relative to K151 for protein S‐dependent anticoagulant activity and PAR cleavage highlights that binding exosites for protein S binding and for PAR cleavage in the C‐terminal region of APC's light chain overlap.

Phospho‐proteomic Analysis of Protease‐activated Receptor‐1 Biased Signaling Reveals Novel Modulators of Endothelial Barrier Function

Dysregulation of the vascular endothelium is a hallmark of inflammation and promotes barrier disruption, vascular leakage and tissue edema. Vascular endothelial dysfunction is mediated by vasoactive agents and inflammatory mediators, many of which signal through G protein‐coupled receptors (GPCRs). A key GPCR inflammatory mediator is thrombin, a procoagulant protease generated in response to vascular injury and inflammation. Thrombin activates protease‐activated receptor‐1 (PAR1) in endothelial cells to promote inflammatory responses and endothelial dysfunction. In contrast, activated protein C (APC) is an anticoagulant protease that activates PAR1 through a distinct mechanism and promotes endothelial barrier stabilization. The ability of distinct tethered ligands formed through cleavage of PAR1 by different proteases results in stabilization of unique receptor active conformational states that promote opposite signaling effects is termed biased signaling. Despite progress in understanding PAR1 bias signaling in endothelial cells, the proteins and pathways by which thrombin and APC regulate the endothelial barrier integrity are largely unknown. Here, we report the global phosphoproteome of thrombin and APC signaling using tandem‐mass‐tag (TMT) mass spectrometry and computational analyses. Our results reveal unique temporal dynamics profiles of thrombin and APC signaling and the identification of multiple enriched phosphoproteome associated biological functions including microtubules, adherens junctions, actin and signaling, all key modulators of endothelial barrier permeability. We selected candidates that showed statistically significant changes in phosphorylation following thrombin or APC stimulation, and validated the function of four proteins including: afadin (AFDN), CDC42 binding protein kinase alpha (BPA), adducin 1 (ADD1), and GPCR interacting protein 1 (GIT1) using an siRNA‐targeted approach. Using target‐specific siRNAs to deplete candidate proteins in endothelial cells, we found that depletion of afadin and adducin had statistically significant effects on endothelial barrier permeability. Afadin depletion led to an increase in thrombin‐induced permeability, while adducin depletion virtually ablated thrombin‐stimulated permeability. Immunofluorescence microscopy imaging of morphological changes in F‐actin stress fiber formation supported these results and revealed corresponding changes in thrombin‐induced gap formation consistent with the observed changes in permeability. In addition, depletion of candidate proteins also modulated thrombin‐stimulated p38 pro‐inflammatory signaling and APC‐induced AKT cytoprotective signaling. These studies define distinct thrombin and APC dynamic signaling profiles and a rich array of proteins and biological pathways that engender PAR1 bias signaling in endothelial cells.Support or Funding InformationThis work was supported by NIH/NIGMS R01 GM116597 and R35 GM127121 (J.T.), UC Tobacco‐related Disease Research Program Predoctoral Fellowship (C.C.R), and NIH/NIGMS K12 GM068524 (O.M.I., J.D.L.)

FVIIa (Factor VIIa) Induces Biased Cytoprotective Signaling in Mice Through the Cleavage of PAR (Protease-Activated Receptor)-1 at Canonical Arg41 (Arginine41) Site.

Recent studies showed that FVIIa (factor VIIa), upon binding to EPCR (endothelial cell protein C receptor), elicits endothelial barrier stabilization and anti-inflammatory effects via activation of PAR (protease-activated receptor)-1-mediated signaling. It is unknown whether FVIIa induces PAR1-dependent cytoprotective signaling through cleavage of PAR1 at the canonical site or a noncanonical site, similar to that of APC (activated protein C). Approach and Results: Mouse strains carrying homozygous R41Q (canonical site) or R46Q (noncanonical site) point mutations in PAR1 (QQ41-PAR1 and QQ46-PAR1 mice) were used to investigate in vivo mechanism of PAR1-dependent pharmacological beneficial effects of FVIIa. Administration of FVIIa reduced lipopolysaccharide-induced inflammation, barrier permeability, and VEGF (vascular endothelial cell growth factor)-induced barrier disruption in wild-type (WT) and QQ46-PAR1 mice but not in QQ41-PAR1 mice. In vitro signaling studies performed with brain endothelial cells isolated from WT, QQ41-PAR1, and QQ46-PAR1 mice showed that FVIIa activation of Akt (protein kinase B) in endothelial cells required R41 cleavage site in PAR1. Our studies showed that FVIIa cleaved endogenous PAR1 in endothelial cells, and FVIIa-cleaved PAR1 was readily internalized, unlike APC-cleaved PAR1 that remained on the cell surface. Additional studies showed that pretreatment of endothelial cells with FVIIa reduced subsequent thrombin-induced signaling. This process was dependent on β-arrestin1. Our results indicate that in vivo pharmacological benefits of FVIIa in mice arise from PAR1-dependent biased signaling following the cleavage of PAR1 at the canonical R41 site. The mechanism of FVIIa-induced cytoprotective signaling is distinctly different from that of APC. Our data provide another layer of complexity of biased agonism of PAR1 and signaling diversity.

Phosphoproteomic analysis of protease-activated receptor-1 biased signaling reveals unique modulators of endothelial barrier function

Thrombin, a procoagulant protease, cleaves and activates protease-activated receptor-1 (PAR1) to promote inflammatory responses and endothelial dysfunction. In contrast, activated protein C (APC), an anticoagulant protease, activates PAR1 through a distinct cleavage site and promotes anti-inflammatory responses, prosurvival, and endothelial barrier stabilization. The distinct tethered ligands formed through cleavage of PAR1 by thrombin versus APC result in unique active receptor conformations that bias PAR1 signaling. Despite progress in understanding PAR1 biased signaling, the proteins and pathways utilized by thrombin versus APC signaling to induce opposing cellular functions are largely unknown. Here, we report the global phosphoproteome induced by thrombin and APC signaling in endothelial cells with the quantification of 11,266 unique phosphopeptides using multiplexed quantitative mass spectrometry. Our results reveal unique dynamic phosphoproteome profiles of thrombin and APC signaling, an enrichment of associated biological functions, including key modulators of endothelial barrier function, regulators of gene transcription, and specific kinases predicted to mediate PAR1 biased signaling. Using small interfering RNA to deplete a subset of phosphorylated proteins not previously linked to thrombin or APC signaling, a function for afadin and adducin-1 actin binding proteins in thrombin-induced endothelial barrier disruption is unveiled. Afadin depletion resulted in enhanced thrombin-promoted barrier permeability, whereas adducin-1 depletion completely ablated thrombin-induced barrier disruption without compromising p38 signaling. However, loss of adducin-1 blocked APC-induced Akt signaling. These studies define distinct thrombin and APC dynamic signaling profiles and a rich array of proteins and biological pathways that engender PAR1 biased signaling in endothelial cells.

Factor VIIa Induces Biased Cytoprotective Signaling through the Cleavage of Protease Activated Receptor 1 at Canonical Arg41 Site

Endothelial cell protein C receptor (EPCR) interacts with diverse ligands, in addition to its known ligands protein C and activated protein C (APC). We reported earlier that procoagulant clotting factor VIIa (FVIIa) binds EPCR with the same affinity as APC. FVIIa binding to EPCR leads to the down regulation of the EPCR-mediated anticoagulation pathway. Our recent studies showed that FVIIa, like APC, induces EPCR-dependent cytoprotective signaling through activation of protease activated receptor 1 (PAR1). Recent studies of Griffin, Mosnier and their colleagues revealed that APC noncanonical cleavage of PAR1 at Arg46 site that generates a novel tethered ligand is responsible for APC-induced β-arrestin2-dependent PAR1 biased signaling. It is unknown at present whether FVIIa follows a similar mechanism as APC in inducing PAR1 biased signaling. PAR1 reporter constructs - wild-type and cleavage site-specific mutants - were routinely used to investigate PAR1 cleavage by thrombin, APC, or other proteases, and to determine protease-specific cleavage sites in PAR1. Unfortunately, this approach was not useful in determining any FVIIa cleavage site in PAR1. In contrast to thrombin or APC, FVIIa treatment failed to show a detectable cleavage (over the background) of transfected wild-type PAR1 reporter constructs expressed in cultured endothelial cells. However, in other studies, FVIIa was shown to cleave endogenous PAR1 in endothelial cells as assessed by the loss of cleavage-specific PAR1 mAb binding. The recent generation of transgenic mice strains carrying R41Q or R46Q homozygous point mutations in PAR1 has allowed us in the present study to investigate in vivo mechanisms for PAR1-dependent cytoprotective signaling of FVIIa. We employed two murine injury models, LPS-induced inflammation and VEGF-induced barrier disruption. Murine brain endothelial cells isolated from the PAR1 mutated strains and primary human endothelial cells were used to validate in vivo findings and extend the mechanistic studies. Our studies show that administration of rFVIIa (250 µg/kg body weight) reduced LPS-induced cytokine elaboration and neutrophil infiltration in the lung tissues of wild-type (WT) PAR1 and QQ46-PAR1 mice but not in QQ41-PAR1 mice. Similarly, FVIIa suppression of the VEGF-induced barrier disruption was abolished in the QQ41-PAR1 mice but not in WT and the QQ46-PAR1 mice. Parallel experiments conducted with APC showed, as expected, that it protected WT and QQ41-PAR1 mice but not QQ46-PAR1 mice against LPS-induced inflammation and VEGF-mediated barrier destabilization. In vitro signaling studies performed with brain endothelial cells isolated from WT, QQ41-PAR1 and QQ46-PAR1 mice showed that FVIIa activation of Akt in endothelial cells required Arg41 in PAR1. Additional studies showed that FVIIa-cleaved endogenous PAR1 was readily internalized, whereas APC-cleaved PAR1 remained on the cell surface. Very low concentrations of thrombin (< 1 nM) mimicked FVIIa in inducing PAR1-dependent cytoprotective signaling. However, very low concentration thrombin-induced cytoprotective signaling differed from EPCR-FVIIa-induced cytoprotective signaling in the isoform of ß-arrestin required for the protective effect. EPCR-FVIIa-induced PAR1-mediated cytoprotective signaling was soley mediated via the β-arrestin1-dependent pathway whereas very low dose thrombin-induced cytoprotective effects appear to be mediated by either β-arrestin1 or β-arrestin2. Silencing of ß-arrestin1 or ß-arrestin2 alone did not affect the protective effects of very low doses of thrombin, but the silencing both ß-arrestin1 and ß-arrestin2 together completely prevented a low dose of thrombin-induced protective effect. In summary, our data strongly support the hypothesis that the in vivo mechanism of action for FVIIa's pharmacologic benefits in curbing inflammation and endothelial barrier disruption involves biased signaling of PAR1 due to cleavage at Arg41. Moreover, our studies demonstrate that very low concentrations of thrombin are also capable of inducing PAR1 biased beneficial -cytoprotective signaling by coupling to either ß-arrestin1 or 2. These results emphasize the striking diversity of PAR1's conformational states and interactomes that initiate canonical and biased signaling. Disclosures No relevant conflicts of interest to declare.

Recent Advances in the Understanding of Thrombosis.

Recent Advances in the Understanding of Thrombosis.

Activated protein C inhibits lipopolysaccharide‐mediated acetylation and secretion of high‐mobility group box 1 in endothelial cells

Essentials APC elicits cytoprotective responses in endothelial cells via EPCR-dependent cleavage of PAR1. APC inhibits LPS-mediated translocation and extracellular secretion of HMGB1 in endothelial cells. Signaling activity of APC inhibits LPS-mediated acetylation of HMGB1 by epigenetic mechanisms. APC inhibits LPS-mediated HMGB1 expression in CD31-positive endothelial cells in cremaster muscle. SUMMARY: Background Activated proteinC (APC) inhibits high-mobility group box1 (HMGB1) signaling and its lipopolysaccharide (LPS)-mediated release by endothelial proteinC receptor (EPCR)-dependent activation of protease-activated receptor1 (PAR1) in endothelial cells. Post-translational acetylation is known to modulate the subcellular localization of HMGB1, and its hyperacetylated form is translocated to the cytoplasm of innate immune cells before being secreted into the extracellular space. Objective To determine whether APC inhibits LPS-mediated HMGB1 secretion from endothelial cells by modulating its acetylation status. Methods The subcellular localization of HMGB1 in LPS-treated endothelial cells was monitored in the absence and presence of APC by western blot analysis of fractionated cell lysates and confocal immunofluorescence microscopy. Results Both western blot and immunofluorescence data indicated that APC effectively inhibits LPS-mediated translocation of HMGB1 from the nucleus to the cytoplasm by EPCR-dependent and PAR1-dependent mechanisms. When EPCR was ligated by the Gla-domain of proteinC/APC, thrombin also inhibited LPS-mediated HMGB1 translocation. Further studies revealed that APC inhibits the translocation of HMGB1 from the nucleus to the cytoplasm by inhibiting LPS-mediated hyperacetylation of HMGB1 by (de)acetylating enzymes. Furthermore, the translocated HMGB1 was found to be associated with lysosome-associated membrane protein1 in LPS-treated endothelial cells. The invivo relevance of these findings was investigated in the mouse cremaster muscle, and this demonstrated that both wild-type APC and a signaling-selective mutant of APC inhibit LPS-mediated HMGB1 expression and translocation in CD31-positive endothelial cells. Conclusion These results suggest that APC inhibits LPS-mediated cytoplasmic translocation and secretion of HMGB1 in endothelial cells by epigenetic mechanisms.

Thrombin and the Coag-Inflammatory Nexus in Neurotrauma, ALS, and Other Neurodegenerative Disorders.

This review details our current understanding of thrombin signaling in neurodegeneration, with a focus on amyotrophic lateral sclerosis (ALS, Lou Gehrig's disease) as well as future directions to be pursued. The key factors are multifunctional and involved in regulatory pathways, namely innate immune and the coagulation cascade activation, that are essential for normal nervous system function and health. These two major host defense systems have a long history in evolution and include elements and regulators of the coagulation pathway that have significant impacts on both the peripheral and central nervous system in health and disease. The clotting cascade responds to a variety of insults to the CNS including injury and infection. The blood brain barrier is affected by these responses and its compromise also contributes to these detrimental effects. Important molecules in signaling that contribute to or protect against neurodegeneration include thrombin, thrombomodulin (TM), protease activated receptor 1 (PAR1), damage associated molecular patterns (DAMPs), such as high mobility group box protein 1 (HMGB1) and those released from mitochondria (mtDAMPs). Each of these molecules are entangled in choices dependent upon specific signaling pathways in play. For example, the particular cleavage of PAR1 by thrombin vs. activated protein C (APC) will have downstream effects through coupled factors to result in toxicity or neuroprotection. Furthermore, numerous interactions influence these choices such as the interplay between HMGB1, thrombin, and TM. Our hope is that improved understanding of the ways that components of the coagulation cascade affect innate immune inflammatory responses and influence the course of neurodegeneration, especially after injury, will lead to effective therapeutic approaches for ALS, traumatic brain injury, and other neurodegenerative disorders.