Platelet matrix metalloprotease-1 mediates thrombogenesis by activating PAR1 at a cryptic ligand site.

Matrix metalloproteases (MMPs) play important roles in normal and pathological remodeling processes including atherothrombotic disease, inflammation, angiogenesis, and cancer. MMPs have been viewed as matrix-degrading enzymes, but recent studies have shown that they possess direct signaling capabilities. Platelets harbor several MMPs that modulate hemostatic function and platelet survival; however their mode of action remains unknown. We show that platelet MMP-1 activates protease-activated receptor-1 (PAR1) on the surface of platelets. Exposure of platelets to fibrillar collagen converts the surface-bound proMMP-1 zymogen to active MMP-1, which promotes aggregation through PAR1. Unexpectedly, MMP-1 cleaves PAR1 at a distinct site that strongly activates Rho-GTP pathways, cell shape change and motility, and MAPK signaling. Blockade of MMP1-PAR1 curtails thrombogenesis under arterial flow conditions and inhibits thrombosis in animals. These studies provide a link between matrix-dependent activation of metalloproteases and platelet-G protein signaling and identify MMP1-PAR1 as a potential target for the prevention of arterial thrombosis.

PAR2 promotes impaired glucose uptake and insulin resistance in NAFLD through GLUT2 and Akt interference.

BACKGROUND AND AIMS: Insulin resistance and poor glycemic control are key drivers of the development of NAFLD and have recently been shown to be associated with fibrosis progression in NASH. However, the underlying mechanisms involving dysfunctional glucose metabolism and relationship with NAFLD/NASH progression remain poorly understood. We set out to determine whether protease-activated receptor 2 (PAR2), a sensor of extracellular inflammatory and coagulation proteases, links NAFLD and NASH with liver glucose metabolism. APPROACH AND RESULTS: Here, we demonstrate that hepatic expression of PAR2 increases in patients and mice with diabetes and NAFLD/NASH. Mechanistic studies using whole-body and liver-specific PAR2-knockout mice reveal that hepatic PAR2 plays an unexpected role in suppressing glucose internalization, glycogen storage, and insulin signaling through a bifurcating Gq -dependent mechanism. PAR2 activation downregulates the major glucose transporter of liver, GLUT2, through Gq -MAPK-FoxA3 and inhibits insulin-Akt signaling through Gq -calcium-CaMKK2 pathways. Therapeutic dosing with a liver-homing pepducin, PZ-235, blocked PAR2-Gq signaling and afforded significant improvements in glycemic indices and HbA1c levels in severely diabetic mice. CONCLUSIONS: This work provides evidence that PAR2 is a major regulator of liver glucose homeostasis and a potential target for the treatment of diabetes and NASH.

Lipid Receptor GPR31 (G-Protein-Coupled Receptor 31) Regulates Platelet Reactivity and Thrombosis Without Affecting Hemostasis.

OBJECTIVE: 12-LOX (12-lipoxygenase) produces a number of bioactive lipids including 12(S)-HETE that are involved in inflammation and platelet reactivity. The GPR31 (G-protein-coupled receptor 31) is the proposed receptor of 12(S)-HETE; however, it is not known whether the 12(S)-HETE-GPR31 signaling axis serves to enhance or inhibit platelet activity. Approach and Results: Using pepducin technology and biochemical approaches, we provide evidence that 12(S)-HETE-GPR31 signals through Gi to enhance PAR (protease-activated receptor)-4-mediated platelet activation and arterial thrombosis using both human platelets and mouse carotid artery injury models. 12(S)-HETE suppressed AC (adenylyl cyclase) activity through GPR31 and resulted in Rap1 (Ras-related protein 1) and p38 activation and low but detectable calcium flux but did not induce platelet aggregation. A GPR31 third intracellular (i3) loop-derived pepducin, GPR310 (G-protein-coupled receptor 310), significantly inhibited platelet aggregation in response to thrombin, collagen, and PAR4 agonist, AYPGKF, in human and mouse platelets but relative sparing of PAR1 agonist SFLLRN in human platelets. GPR310 treatment gave a highly significant 80% protection (P=0.0018) against ferric chloride-induced carotid artery injury in mice by extending occlusion time, without any effect on tail bleeding. PAR4-mediated dense granule secretion and calcium flux were both attenuated by GPR310. Consistent with these results, GPR310 inhibited 12(S)-HETE-mediated and PAR4-mediated Rap1-GTP and RASA3 translocation to the plasma membrane and attenuated PAR4-Akt and ERK activation. GPR310 caused a right shift in thrombin-mediated human platelet aggregation, comparable to the effects of inhibition of the Gi-coupled P2Y12 receptor. Co-immunoprecipitation studies revealed that GPR31 and PAR4 form a heterodimeric complex in recombinant systems. CONCLUSIONS: The 12-LOX product 12(S)-HETE stimulates GPR31-Gi-signaling pathways, which enhance thrombin-PAR4 platelet activation and arterial thrombosis in human platelets and mouse models. Suppression of this bioactive lipid pathway, as exemplified by a GPR31 pepducin antagonist, may provide beneficial protective effects against platelet aggregation and arterial thrombosis with minimal effect on hemostasis.

Deficiency of MMP1a (Matrix Metalloprotease 1a) Collagenase Suppresses Development of Atherosclerosis in Mice: Translational Implications for Human Coronary Artery Disease.

[Figure: see text].

PAR1 (Protease-Activated Receptor 1) Pepducin Therapy Targeting Myocardial Necrosis in Coronary Artery Disease and Acute Coronary Syndrome Patients Undergoing Cardiac Catheterization: A Randomized, Placebo-Controlled, Phase 2 Study.

OBJECTIVE: Arterial thrombosis leading to ischemic injury worsens the prognosis of many patients with cardiovascular disease. PZ-128 is a first-in-class pepducin that reversibly inhibits PAR1 (protease-activated receptor 1) on platelets and other vascular cells by targeting the intracellular surface of the receptor. The TRIP-PCI (Thrombin Receptor Inhibitory Pepducin in Percutaneous Coronary Intervention) trial was conducted to assess the safety and efficacy of PZ-128 in patients undergoing cardiac catheterization with intent to perform percutaneous coronary intervention. Approach and Results: In this randomized, double-blind, placebo-controlled, phase 2 trial, 100 patients were randomly assigned (2:1) to receive PZ-128 (0.3 or 0.5 mg/kg), or placebo in a 2-hour infusion initiated just before the start of cardiac catheterization, on top of standard oral antiplatelet therapy. Rates of the primary end point of bleeding were not different between the combined PZ-128 doses (1.6%, 1/62) and placebo group (0%, 0/35). The secondary end points of major adverse coronary events at 30 and 90 days did not significantly differ but were numerically lower in the PZ-128 groups (0% and 2% in the PZ-128 groups, 6% and 6% with placebo, p=0.13, p=0.29, respectively). In the subgroup of patients with elevated baseline cardiac troponin I, the exploratory end point of 30-day major adverse coronary events + myocardial injury showed 83% events in the placebo group versus 31% events in the combined PZ-128 drug groups, an adjusted relative risk of 0.14 (95% CI, 0.02-0.75); P=0.02. CONCLUSIONS: In this first-in-patient experience, PZ-128 added to standard antiplatelet therapy appeared to be safe, well tolerated, and potentially reduced periprocedural myonecrosis, thus providing the basis for further clinical trials. Registration: URL: https://www.clinicaltrials.gov. Unique identifier: NCT02561000.

Trace amine-associated receptor 1 (TAAR1) promotes anti-diabetic signaling in insulin-secreting cells.

Pancreatic ß-cell failure in type 2 diabetes mellitus is a serious challenge that results in an inability of the pancreas to produce sufficient insulin to properly regulate blood glucose levels. Trace amine-associated receptor 1 (TAAR1) is a G protein-coupled receptor expressed by ß-cells that has recently been proposed as a potential target for improving glycemic control and suppressing binge eating behaviors. We discovered that TAAR1 is coupled to Gαs-signaling pathways in insulin-secreting ß-cells to cause protein kinase A (PKA)/exchange protein activated by cAMP (Epac)-dependent release of insulin, activation of RAF proto-oncogene, Ser/Thr kinase (Raf)-mitogen-activated protein kinase (MAPK) signaling, induction of cAMP response element-binding protein (CREB)-insulin receptor substrate 2 (Irs-2), and increased ß-cell proliferation. Interestingly, TAAR1 triggered cAMP-mediated calcium influx and release from internal stores, both of which were required for activation of a MAPK cascade utilizing calmodulin-dependent protein kinase II (CaMKII), Raf, and MAPK/ERK kinase 1/2 (MEK1/2). Together, these data identify TAAR1/Gαs-mediated signaling pathways that promote insulin secretion, improved ß-cell function and proliferation, and highlight TAAR1 as a promising new target for improving ß-cell health in type 2 diabetes mellitus.

'Role reversal' for the receptor PAR1 in sepsis-induced vascular damage.

Sepsis is a deadly disease characterized by considerable derangement of the proinflammatory, anti-inflammatory and coagulation responses. Protease-activated receptor 1 (PAR1), an important regulator of endothelial barrier function and blood coagulation, has been proposed to be involved in the lethal sequelae of sepsis, but it is unknown whether activation of PAR1 is beneficial or harmful. Using a cell-penetrating peptide (pepducin) approach, we provide evidence that PAR1 switched from being a vascular-disruptive receptor to a vascular-protective receptor during the progression of sepsis in mice. Unexpectedly, we found that the protective effects of PAR1 required transactivation of PAR2 signaling pathways. Our results suggest therapeutics that selectively activate PAR1-PAR2 complexes may be beneficial in the treatment of sepsis.

Atorvastatin Decreases Renal Menaquinone-4 Formation in C57BL/6 Male Mice.

BACKGROUND: Menaquinone-4 (MK4), a vitamin K metabolite, is converted from phylloquinone through a process that requires intermediates of endogenous cholesterol production. Recent evidence suggests that MK4 is involved in kidney function. OBJECTIVE: The purpose of this study was to determine the effect of atorvastatin treatment on MK4 formation in young and old male mice. METHODS: C57BL/6 male mice (4-mo-old and 20-mo-old) were randomly assigned to either a diet containing 300 mg atorvastatin/kg with 3 mg phylloquinone/kg or a control diet containing 3 mg phylloquinone/kg for 8 wk. During week 8, all mice received deuterium-labeled phylloquinone in the diet. Labeled and unlabeled phylloquinone and MK4 in liver, kidney, brain, and intestine were measured by atmospheric pressure chemical ionization LC/MS. 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase gene expression was quantified by reverse transcriptase-PCR. Tissue MK4 and phylloquinone concentrations were compared between atorvastatin treatment groups with use of general linear models. RESULTS: There was no age-treatment interaction on MK4 tissue concentrations. In atorvastatin-treated mice, total MK4 and percentage of deuterium-labeled MK4 in kidney were both approximately 45% lower compared to values in mice not given atorvastatin (all P < 0.05). MK4 concentrations did not differ between groups in any other tissue measured. CONCLUSION: In male mice, atorvastatin reduced endogenous MK4 formation in the kidney, but not other organs. These observations are consistent with our hypothesis that cholesterol metabolism is involved in the generation of MK4. Further research is needed to understand potential regulatory mechanisms and the unique functions of MK4 in the kidney.

Noncanonical Matrix Metalloprotease 1-Protease-Activated Receptor 1 Signaling Drives Progression of Atherosclerosis.

OBJECTIVE: Protease-activated receptor-1 (PAR1) is classically activated by thrombin and is critical in controlling the balance of hemostasis and thrombosis. More recently, it has been shown that noncanonical activation of PAR1 by matrix metalloprotease-1 (MMP1) contributes to arterial thrombosis. However, the role of PAR1 in long-term development of atherosclerosis is unknown, regardless of the protease agonist. APPROACH AND RESULTS: We found that plasma MMP1 was significantly correlated (R=0.33; P=0.0015) with coronary atherosclerotic burden as determined by angiography in 91 patients with coronary artery disease and acute coronary syndrome undergoing cardiac catheterization or percutaneous coronary intervention. A cell-penetrating PAR1 pepducin, PZ-128, currently being tested as an antithrombotic agent in the acute setting in the TRIP-PCI study (Thrombin Receptor Inhibitory Pepducin-Percutaneous Coronary Intervention), caused a significant decrease in total atherosclerotic burden by 58% to 70% (P<0.05) and reduced plaque macrophage content by 54% (P<0.05) in apolipoprotein E-deficient mice. An MMP1 inhibitor gave similar beneficial effects, in contrast to the thrombin inhibitor bivalirudin that gave no improvement on atherosclerosis end points. Mechanistic studies revealed that inflammatory signaling mediated by MMP1-PAR1 plays a critical role in amplifying tumor necrosis factor α signaling in endothelial cells. CONCLUSIONS: These data suggest that targeting the MMP1-PAR1 system may be effective in tamping down chronic inflammatory signaling in plaques and halting the progression of atherosclerosis.

Protease-Activated Receptor 1 as Therapeutic Target in Breast, Lung, and Ovarian Cancer: Pepducin Approach.

The G-protein coupled receptors (GPCRs) belong to a large family of diverse receptors that are well recognized as pharmacological targets. However, very few of these receptors have been pursued as oncology drug targets. The Protease-activated receptor 1 (PAR1), which is a G-protein coupled receptor, has been shown to act as an oncogene and is an emerging anti-cancer drug target. In this paper, we provide an overview of PAR1's biased signaling role in metastatic cancers of the breast, lungs, and ovaries and describe the development of PAR1 inhibitors that are currently in clinical use to treat acute coronary syndromes. PAR1 inhibitor PZ-128 is in a Phase II clinical trial and is being developed to prevent ischemic and thrombotic complication of patients undergoing cardiac catheterization. PZ-128 belongs to a new class of cell-penetrating, membrane-tethered peptides named pepducins that are based on the intracellular loops of receptors targeting the receptor G-protein interface. Application of PZ-128 as an anti-metastatic and anti-angiogenic therapeutic agent in breast, lung, and ovarian cancer is being reviewed.

Reversal of murine alcoholic steatohepatitis by pepducin-based functional blockade of interleukin-8 receptors.

OBJECTIVE: Alcoholic steatohepatitis is a life-threatening condition with short-term mortality up to 40%. It features hepatic neutrophil infiltration and blood neutrophilia, and may evolve from ethanol-induced breakdown of the enteric barrier and consequent bacteraemia. Signalling through CXCR1/2 G-protein-coupled-receptors (GPCRs), the interleukin (IL)-8 receptors, is critical for the recruitment and activation of neutrophils. We have developed short lipopeptides (pepducins), which inhibit post-ligand GPCR activation precisely targeting individual GPCRs. DESIGN: Experimental alcoholic liver disease was induced by administering alcohol and a Lieber-DeCarli high-fat diet. CXCR1/2 GPCRs were blocked via pepducins either from onset of the experiment or after disease was fully established. Hepatic inflammatory infiltration, hepatocyte lipid accumulation and overall survival were assessed as primary outcome parameters. Neutrophil activation was assessed by myeloperoxidase activity and liver cell damage by aspartate aminotransferase and alanine aminotransferase plasma levels. Chemotaxis assays were performed to identify chemoattractant signals derived from alcohol-exposed hepatocytes. RESULTS: Here, we show that experimental alcoholic liver disease is driven by CXCR1/2-dependent activation of neutrophils. CXCR1/2-specific pepducins not only protected mice from liver inflammation, weight loss and mortality associated with experimental alcoholic liver disease, but therapeutic administration cured disease and prevented further mortality in fully established disease. Hepatic neutrophil infiltration and triglyceride accumulation was abrogated by CXCR1/2 blockade. Moreover, CXCL-1 plasma levels were decreased with the pepducin therapy as was the transcription of hepatic IL-1ß mRNA. CONCLUSIONS: We propose that high circulating IL-8 in human alcoholic hepatitis may cause pathogenic overzealous neutrophil activation, and therapeutic blockade via pepducins merits clinical study.

Targeting Liver Fibrosis with a Cell-penetrating Protease-activated Receptor-2 (PAR2) Pepducin.

Chronic liver inflammation and fibrosis in nonalcoholic steatohepatitis can lead to cirrhosis and liver failure for which there are currently no approved treatments. Protease-activated receptor-2 (PAR2) is an emerging new target expressed on liver stellate cells and hepatocytes that regulates the response to liver injury and inflammation. Here, we identified a pepducin to block the deleterious actions of PAR2 in promoting liver fibrosis. Non-alcoholic fatty liver disease and early fibrosis were induced by the methionine-choline-deficient diet in mice. Fibrotic liver disease was induced by administering carbon tetrachloride for 8 weeks. Mice were treated with the pepducin PZ-235 either from onset of the experiment or after fibrosis was established. Hepatic fibrosis, collagen content, inflammatory cytokines, steatosis, triglycerides, and NAFLD activity score were assessed as primary outcome parameters depending on the model. The activity of the PAR2 pepducin on cultured stellate cell activation and hepatocyte reactive oxygen species production was evaluated. PZ-235 significantly suppressed liver fibrosis, collagen deposition, inflammatory cytokines, NAFLD activity score, steatosis, triglycerides, aspartate transaminase, alanine transaminase, and stellate cell proliferation by up to 50-100%. The PAR2 inhibitor afforded significant protective effects against hepatocellular necrosis and attenuated PAR2-mediated reactive oxygen species production in hepatocytes. PZ-235 was distributed to liver and other mouse tissues and was found to form a well structured α-helix that closely resembles the juxtamembrane helical region of the analogous TM6 and third intracellular region of the intact receptor that is critical for coupling to internal G proteins. The ability of PZ-235 to effectively suppress fibrosis, hepatocellular necrosis, reactive oxygen species production, steatosis, and inflammation indicates the potential for PAR2 pepducin inhibitors to be broadly efficacious in the treatment of liver fibrosis.

Cell-Penetrating Pepducin Therapy Targeting PAR1 in Subjects With Coronary Artery Disease.

OBJECTIVE: Pepducins are membrane-tethered, cell-penetrating lipopeptides that target the cytoplasmic surface of their cognate receptor. Here, we report the first human use of a protease-activated receptor-1-based pepducin, which is intended as an antiplatelet agent to prevent ischemic complications of percutaneous coronary interventions. APPROACH AND RESULTS: PZ-128 was administered by 1 to 2 hours continuous intravenous infusion (0.01-2 mg/kg) to 31 subjects with coronary artery disease or multiple coronary artery disease risk factors. Safety, antiplatelet efficacy, and pharmacokinetics were assessed at baseline and 0.5, 1, 2, 6, 24 hours, and 7 to 10 days postdosing. The inhibitory effects of PZ-128 on platelet aggregation stimulated by the protease-activated receptor-1 agonist SFLLRN (8 µmol/L) at 30 minutes to 6 hours were dose dependent with 20% to 40% inhibition at 0.3 mg/kg, 40% to 60% at 0.5 mg/kg, and ≥ 80% to 100% at 1 to 2 mg/kg. The subgroup receiving aspirin in the 0.5 and 1-mg/kg dose cohorts had 65% to 100% inhibition of final aggregation to SFLLRN at 30 minutes to 2 hours and 95% to 100% inhibition by 6 hours. The inhibitory effects of 0.5 mg/kg PZ-128 were reversible with 50% recovery of aggregation to SFLLRN by 24 hours. There were no significant effects of PZ-128 on aggregation induced by AYPGKF, ADP, or collagen, indicating that the observed effects were specific to protease-activated receptor-1. The plasma half-life was 1.3 to 1.8 hours, and PZ-128 was nondetectable in urine. There were no effects on bleeding, coagulation, clinical chemistry, or ECG parameters. CONCLUSIONS: PZ-128 is a promising antiplatelet agent that provides rapid, specific, dose dependent, and reversible inhibition of platelet protease-activated receptor-1 through a novel intracellular mechanism. CLINICAL TRIAL REGISTRATION: URL: http://www.clinicaltrials.gov. Unique identifier: NCT01806077.

Allosteric Activation of a G Protein-coupled Receptor with Cell-penetrating Receptor Mimetics.

G protein-coupled receptors (GPCRs) are remarkably versatile signaling systems that are activated by a large number of different agonists on the outside of the cell. However, the inside surface of the receptors that couple to G proteins has not yet been effectively modulated for activity or treatment of diseases. Pepducins are cell-penetrating lipopeptides that have enabled chemical and physical access to the intracellular face of GPCRs. The structure of a third intracellular (i3) loop agonist, pepducin, based on protease-activated receptor-1 (PAR1) was solved by NMR and found to closely resemble the i3 loop structure predicted for the intact receptor in the on-state. Mechanistic studies revealed that the pepducin directly interacts with the intracellular H8 helix region of PAR1 and allosterically activates the receptor through the adjacent (D/N)PXXYYY motif through a dimer-like mechanism. The i3 pepducin enhances PAR1/Gα subunit interactions and induces a conformational change in fluorescently labeled PAR1 in a very similar manner to that induced by thrombin. As pepducins can potentially be made to target any GPCR, these data provide insight into the identification of allosteric modulators to this major drug target class.

Tissue Concentrations of Vitamin K and Expression of Key Enzymes of Vitamin K Metabolism Are Influenced by Sex and Diet but Not Housing in C57Bl6 Mice.

BACKGROUND: There has been limited characterization of biological variables that impact vitamin K metabolism. This gap in knowledge can limit the translation of data obtained from preclinical animal studies to future human studies. OBJECTIVE: The purpose of this study was to determine the effects of diet, sex, and housing on serum, tissue, and fecal vitamin K concentrations and gene expression in C57BL6 mice during dietary vitamin K manipulation. METHODS: C57BL6 4-mo-old male and female mice were randomly assigned to conventional or suspended-wire cages and fed control [1400 ± 80 µg phylloquinone (PK)/kg] or deficient (31 ± 0.45 µg PK/kg) diets for 28 d in a factorial design. PK and menaquinone (MK) 4 plasma and tissue concentrations were measured by HPLC. Long-chain MKs were measured in all matrices by LC-atmospheric pressure chemical ionization-mass spectrometry. Gene expression was quantified by reverse transcriptase-polymerase chain reaction in the liver, brain, kidney, pancreas, and adipose tissue. RESULTS: Male and female mice responded differently to dietary manipulation in a tissue-dependent manner. In mice fed the control diet, females had ∼3-fold more MK4 in the brain and mesenteric adipose tissue than did males and 100% greater PK concentrations in the liver, kidney, and mesenteric adipose tissue than did males. In mice fed the deficient diet, kidney MK4 concentrations were ∼4-fold greater in females than in males, and there were no differences in other tissues. Males and females differed in the expression of vitamin K expoxide reductase complex 1 (Vkorc1) in mesenteric adipose tissue and the pancreas and ubiA domain-containing protein 1 (Ubiad1) in the kidney and brain. There was no effect of housing on serum, tissue, or fecal concentrations of any vitamin K form. CONCLUSIONS: Vitamin K concentrations and expression of key metabolic enzymes differ between male and female mice and in response to the dietary PK concentration. Identifying factors that may impact study design and outcomes of interest is critical to optimize study parameters examining vitamin K metabolism in animal models.

G-protein-coupled receptors signaling pathways in new antiplatelet drug development.

Platelet G-protein-coupled receptors influence platelet function by mediating the response to various agonists, including ADP, thromboxane A2, and thrombin. Blockade of the ADP receptor, P2Y12, in combination with cyclooxygenase-1 inhibition by aspirin has been among the most widely used pharmacological strategies to reduce cardiovascular event occurrence in high-risk patients. The latter dual pathway blockade strategy is one of the greatest advances in the field of cardiovascular medicine. In addition to P2Y12, the platelet thrombin receptor, protease activated receptor-1, has also been recently targeted for inhibition. Blockade of protease activated receptor-1 has been associated with reduced thrombotic event occurrence when added to a strategy using P2Y12 and cyclooxygenase-1 inhibition. At this time, the relative contributions of these G-protein-coupled receptor signaling pathways to in vivo thrombosis remain incompletely defined. The observation of treatment failure in ≈10% of high-risk patients treated with aspirin and potent P2Y12 inhibitors provides the rationale for targeting novel pathways mediating platelet function. Targeting intracellular signaling downstream from G-protein-coupled receptor receptors with phosphotidylionisitol 3-kinase and Gq inhibitors are among the novel strategies under investigation to prevent arterial ischemic event occurrence. Greater understanding of the mechanisms of G-protein-coupled receptor-mediated signaling may allow the tailoring of antiplatelet therapy.

Matrix metalloproteases and PAR1 activation.

Cardiovascular diseases, including atherothrombosis, are the leading cause of morbidity and mortality in the United States, Europe, and the developed world. Matrix metalloproteases (MMPs) have recently emerged as important mediators of platelet and endothelial function, and atherothrombotic disease. Protease-activated receptor-1 (PAR1) is a G protein-coupled receptor that is classically activated through cleavage of the N-terminal exodomain by the serine protease thrombin. Most recently, 2 MMPs have been discovered to have agonist activity for PAR1. Unexpectedly, MMP-1 and MMP-13 cleave the N-terminal exodomain of PAR1 at noncanonical sites, which result in distinct tethered ligands that activate G-protein signaling pathways. PAR1 exhibits metalloprotease-specific signaling patterns, known as biased agonism, that produce distinct functional outputs by the cell. Here we contrast the mechanisms of canonical (thrombin) and noncanonical (MMP) PAR1 activation, the contribution of MMP-PAR1 signaling to diseases of the vasculature, and the therapeutic potential of inhibiting MMP-PAR1 signaling with MMP inhibitors, including atherothrombotic disease, in-stent restenosis, heart failure, and sepsis.

Noncanonical matrix metalloprotease-1-protease-activated receptor-1 signaling triggers vascular smooth muscle cell dedifferentiation and arterial stenosis.

Vascular injury that results in proliferation and dedifferentiation of vascular smooth muscle cells (SMCs) is an important contributor to restenosis following percutaneous coronary interventions or plaque rupture. Protease-activated receptor-1 (PAR1) has been shown to play a role in vascular repair processes; however, little is known regarding its function or the relative roles of the upstream proteases thrombin and matrix metalloprotease-1 (MMP-1) in triggering PAR1-mediated arterial restenosis. The goal of this study was to determine whether noncanonical MMP-1 signaling through PAR1 would contribute to aberrant vascular repair processes in models of arterial injury. A mouse carotid arterial wire injury model was used for studies of neointima hyperplasia and arterial stenosis. The mice were treated post-injury for 21 days with a small molecule inhibitor of MMP-1 or a direct thrombin inhibitor and compared with vehicle control. Intimal and medial hyperplasia was significantly inhibited by 2.8-fold after daily treatment with the small molecule MMP-1 inhibitor, an effect that was lost in PAR1-deficient mice. Conversely, chronic inhibition of thrombin showed no benefit in suppressing the development of arterial stenosis. Thrombin-PAR1 signaling resulted in a supercontractile, differentiated phenotype in SMCs. Noncanonical MMP-1-PAR1 signaling resulted in the opposite effect and led to a dedifferentiated phenotype via a different G protein pathway. MMP-1-PAR1 significantly stimulated hyperplasia and migration of SMCs, and resulted in down-regulation of SMC contractile genes. These studies provide a new mechanism for the development of vascular intimal hyperplasia and suggest a novel therapeutic strategy to suppress restenosis by targeting noncanonical MMP-1-PAR1 signaling in vascular SMCs.

Mouse matrix metalloprotease-1a (Mmp1a) gives new insight into MMP function.

Matrix metalloprotease-1 (MMP1) has been implicated in many human disease processes, however the lack of a well characterized murine homologue has significantly limited the study of MMP1 and the development of MMP-targeted therapeutics. The discovery of murine Mmp1a in 2001, the functional mouse homologue of MMP1, offers a valuable tool for modeling MMP1-mediated processes in mice. Variation in physiologic expression levels of Mmp1a in mice as compared to MMP1 in humans highlights the importance of understanding the similarities and differences between the homologues. Recent studies have demonstrated tumor growth-, invasion-, and angiogenesis-promoting functions of Mmp1a in lung cancer models, consistent with the analogous functions observed for human MMP1. Biochemical investigations have shown that point mutations in the pro-domain of mouse Mmp1a weaken docking between the pro- and catalytic domains, generating an unstable zymogen primed for activation. The difficulty to effectively maintain Mmp1a in the zymogen form may account for the tight control of Mmp1a expression and reduced expression in normal tissue as compared to inflammatory states or cancer. This discovery raises important questions about the activation mechanisms and regulation of the MMP family in general.