THROMBIN RECEPTOR-G PROTEIN SIGNALING MECHANISMS
Athan Kuliopulos, Associate Professor
Tufts Medical Center, 800 Washington St, Boston, Ma 02111-1526
Grant 5R01HL064701-09 from National Heart, Lung, And Blood Institute
Abstract: Proteolytic cleavage of the G protein-coupled protease-activated receptors activates an extraordinarily diverse array of physiologic responses. These include platelet activation and aggregation, cellular proliferation/apoptosis, cell migration and protease-homing, angiogenesis, and the hemostatic and inflammatory responses to vascular injury. Four protease-activated receptors have been identified PAR1, PAR2, PARS, and PAR4. Matrix metalloproteases have also emerged as important proteases in vascular biology. Initially described as extracellular matrix remodeling enzymes involved in tissue repair and cancer invasion, a renewed focus has centered on MMPs and the related metalloprotease disintegrins because of their prominence in atherothrombotic disease and platelet hemostasis. Quite recently, we found that a matrix metalloprotease, MMP-1, can also function as a protease agonist of PAR1 cleaving the receptor to generate PAR1-dependent Ca2+ signals, migration, platelet activation and cell shape changes. MMP-1 is expressed in high levels in platelets and endothelium. Circulating levels of MMP-1 have been shown to be significantly elevated in plasma from patients following acute myocardial infarction pointing to activated platelets and endothelium as potential sources for MMP-1. However, it is not clear what role(s) MMP1-PAR1 signaling plays in hemostasis, thrombosis and the control of vascular integrity or whether MMP1 activates different G protein pathways as compared to thrombin. PAR1, has been shown to couple to Gq, Gi, Gi2/i3, and (3y under a variety of in vitro conditions. The specifics of how PAR1 interacts with the G proteins and the relative importance and temporal ordering of differential G protein activation under in vivo conditions is still unknown. Far less is known about the identity of non-G protein effectors that might also interact with the intracellular loops of the PARs. The major goals of these studies are 1) to investigate the molecular basis of G protein activation by PAR1 under in vivo conditions, 2) to understand the mechanism and physiologic relevance of MMP1-PAR1 signaling in platelets during arterial thrombus formation 3) to investigate the mechanism of endothelial MMP1 activation of PAR1 and the functional consequences of MMP1-PAR1 signaling on endothelial function during sepsis, and 4) to determine how the newly identified PAR1 effector, BicD1, modulates PAR1 signaling, signal termination, and cell motility. Platelet activation has been shown to be heightened in the setting of angioplasty and stenting which may cause clinical complications including acute heart attacks and death. The PAR1 receptor has long been recognized as an obvious candidate for therapeutic intervention of patients with heart conditions, the work proposed here may lead to the development of novel treatments for patients with arterial blood clots and those suffering from life- threatening sepsis
Keywords: ADP-Ribosyltransferase (Polymerizing); Acute; Acute myocardial infarct; Acute myocardial infarction; Agonist; Angioplasty; Apoptosis; Apoptosis Pathway; Biology; Bizzozero`s corpuscle/cell; Blood Clot; Blood Clotting; Blood Plasma; Blood Platelet Disorders; Blood Platelets; Blood Vessels; Blood coagulation; CF2R; CLG; Cancers; Cardiac infarction; Cell Communication and Signaling; Cell Death, Programmed; Cell Growth in Number; Cell Locomotion; Cell Migration; Cell Movement; Cell Multiplication; Cell Proliferation; Cell Shape; Cell Signaling; Cell-Extracellular Matrix; Cellular Migration; Cellular Proliferation; Cessation of life; Cleaved cell; Clinical; Coagulation Factor II Receptor; Coupling; Death; Deetjeen`s body; Development; Disintegrins; ECM; Endopeptidase-Activated Receptors; Endothelium; Enzymes; Esteroproteases; Extracellular Matrix; F2R; F2R gene; Fibroblast Collagenase; G-Proteins; GTP-Binding Proteins; GTP-Regulatory Proteins; Goals; Guanine Nucleotide Coupling Protein; Guanine Nucleotide Regulatory Proteins; Hayem`s elementary corpuscle; Heart; Hemostasis; Hemostatic Agents; Hemostatic function; Hemostatics; In Vitro; Inflammatory Response; Injury; Interstitial Collagenase; Intracellular Communication and Signaling; Lead; Life; MMP-1; MMP-1Fibroblast Collagenase; MMP1; MMPs; Malignant Neoplasms; Malignant Tumor; Marrow platelet; Matrix Metalloproteinase-1; Matrix Metalloproteinases; Metallopeptidases; Metalloproteases; Metalloproteinases; Molecular; Motility; Motility, Cellular; Myocardial Infarct; Myocardial Infarction; NAD+[{..}]poly(adenosine diphosphate D-ribose)-acceptor ADP-D-ribosyltransferase; PAR-1 Receptor; PAR1; PAR1 Receptor; PAR4; PARP Polymerase; PARS; PAWR; PAWR gene; Pathway interactions; Patients; Pb element; Peptidases; Peptide Hydrolases; Physiologic; Physiological; Plasma; Platelet Activation; Platelets; Play; Poly(ADP-Ribose) Synthase; Poly(ADP-Ribose) Transferase; Poly(ADP-ribose) Polymerases; Poly(ADPR) Polymerase; Poly(ADPribose) Polymerase; Protease-Activated Receptor 1; Protease-Activated Receptors; Proteases; Proteinase-Activated Receptor 1; Proteinase-Activated Receptors; Proteinases; Proteolytic Enzymes; Receptor Protein; Receptor, PAR-1; Receptors, Proteinase-Activated; Relative; Relative (related person); Reticuloendothelial System, Platelets; Reticuloendothelial System, Serum, Plasma; Role; Sepsis; Serine Endopeptidases; Serine Protease; Serine Protein Hydrolases; Serine Proteinases; Serum, Plasma; Signal Transduction; Signal Transduction Systems; Signaling; Source; Therapeutic Intervention; Thrombase; Thrombin; Thrombin Receptor; Thrombocytes; Thrombocytopathy; Thrombosis; Thrombus; Work; Wound Healing; Wound Repair; angiogenesis; base; biological signal transduction; bloodstream infection; cardiac infarct; cell motility; cleaved; coronary attack; coronary infarct; coronary infarction; fibrinogenase; heart attack; heart infarct; heart infarction; heavy metal Pb; heavy metal lead; in vivo; intervention therapy; intraluminal angioplasty; malignancy; metalloproteinase (general); migration; neoplasm/cancer; novel; par-4; pathway; platelet disease; platelet disorder; poly ADP polymerase; poly ADP ribose synthetase; protein activation; receptor; response; social role; thrombocyte/platelet; tissue repair; trafficking; vascular
Project start date: 2000-04-01
Project end date: 2012-03-31
Budget start date: 1-APR-2010
Budget end date: 31-MAR-2011
5R01HL064701-09 (2010): $402500
Sponsored Links Excellgen http://Excellgen.com
THROMBIN RECEPTOR-G PROTEIN SIGNALING MECHANISMS
Athan Kuliopulos, Associate Professor
Tufts Medical Center, 800 Washington St, Boston, Ma 02111-1526
Grant 5R01HL064701-08 from National Heart, Lung, And Blood Institute
Abstract: Proteolytic cleavage of the G protein-coupled protease-activated receptors activates an extraordinarily diverse array of physiologic responses. These include platelet activation and aggregation, cellular proliferation/apoptosis, cell migration and protease-homing, angiogenesis, and the hemostatic and inflammatory responses to vascular injury. Four protease-activated receptors have been identified PAR1, PAR2, PARS, and PAR4. Matrix metalloproteases have also emerged as important proteases in vascular biology. Initially described as extracellular matrix remodeling enzymes involved in tissue repair and cancer invasion, a renewed focus has centered on MMPs and the related metalloprotease disintegrins because of their prominence in atherothrombotic disease and platelet hemostasis. Quite recently, we found that a matrix metalloprotease, MMP-1, can also function as a protease agonist of PAR1 cleaving the receptor to generate PAR1-dependent Ca2+ signals, migration, platelet activation and cell shape changes. MMP-1 is expressed in high levels in platelets and endothelium. Circulating levels of MMP-1 have been shown to be significantly elevated in plasma from patients following acute myocardial infarction pointing to activated platelets and endothelium as potential sources for MMP-1. However, it is not clear what role(s) MMP1-PAR1 signaling plays in hemostasis, thrombosis and the control of vascular integrity or whether MMP1 activates different G protein pathways as compared to thrombin. PAR1, has been shown to couple to Gq, Gi, Gi2/i3, and (3y under a variety of in vitro conditions. The specifics of how PAR1 interacts with the G proteins and the relative importance and temporal ordering of differential G protein activation under in vivo conditions is still unknown. Far less is known about the identity of non-G protein effectors that might also interact with the intracellular loops of the PARs. The major goals of these studies are 1) to investigate the molecular basis of G protein activation by PAR1 under in vivo conditions, 2) to understand the mechanism and physiologic relevance of MMP1-PAR1 signaling in platelets during arterial thrombus formation 3) to investigate the mechanism of endothelial MMP1 activation of PAR1 and the functional consequences of MMP1-PAR1 signaling on endothelial function during sepsis, and 4) to determine how the newly identified PAR1 effector, BicD1, modulates PAR1 signaling, signal termination, and cell motility. Platelet activation has been shown to be heightened in the setting of angioplasty and stenting which may cause clinical complications including acute heart attacks and death. The PAR1 receptor has long been recognized as an obvious candidate for therapeutic intervention of patients with heart conditions, the work proposed here may lead to the development of novel treatments for patients with arterial blood clots and those suffering from life- threatening sepsis
Keywords: ADP-Ribosyltransferase (Polymerizing); Acute; Acute myocardial infarct; Acute myocardial infarction; Agonist; Angioplasty; Apoptosis; Apoptosis Pathway; Biology; Bizzozero`s corpuscle/cell; Blood Clot; Blood Clotting; Blood Plasma; Blood Platelet Disorders; Blood Platelets; Blood Vessels; Blood coagulation; CF2R; CLG; Cancers; Cardiac infarction; Cell Communication and Signaling; Cell Death, Programmed; Cell Growth in Number; Cell Locomotion; Cell Migration; Cell Movement; Cell Multiplication; Cell Proliferation; Cell Shape; Cell Signaling; Cell-Extracellular Matrix; Cellular Migration; Cellular Proliferation; Cessation of life; Cleaved cell; Clinical; Coagulation Factor II Receptor; Coupling; Death; Deetjeen`s body; Development; Disintegrins; ECM; Endopeptidase-Activated Receptors; Endothelium; Enzymes; Esteroproteases; Extracellular Matrix; F2R; F2R gene; Fibroblast Collagenase; G-Proteins; GTP-Binding Proteins; GTP-Regulatory Proteins; Goals; Guanine Nucleotide Coupling Protein; Guanine Nucleotide Regulatory Proteins; Hayem`s elementary corpuscle; Heart; Hemostasis; Hemostatic Agents; Hemostatic function; Hemostatics; In Vitro; Inflammatory Response; Injury; Interstitial Collagenase; Intracellular Communication and Signaling; Lead; Life; MMP-1; MMP-1Fibroblast Collagenase; MMP1; MMPs; Malignant Neoplasms; Malignant Tumor; Marrow platelet; Matrix Metalloproteinase-1; Matrix Metalloproteinases; Metallopeptidases; Metalloproteases; Metalloproteinases; Molecular; Motility; Motility, Cellular; Myocardial Infarct; Myocardial Infarction; NAD+[{..}]poly(adenosine diphosphate D-ribose)-acceptor ADP-D-ribosyltransferase; PAR-1 Receptor; PAR1; PAR1 Receptor; PAR4; PARP Polymerase; PARS; PAWR; PAWR gene; Pathway interactions; Patients; Pb element; Peptidases; Peptide Hydrolases; Physiologic; Physiological; Plasma; Platelet Activation; Platelets; Play; Poly(ADP-Ribose) Synthase; Poly(ADP-Ribose) Transferase; Poly(ADP-ribose) Polymerases; Poly(ADPR) Polymerase; Poly(ADPribose) Polymerase; Protease-Activated Receptor 1; Protease-Activated Receptors; Proteases; Proteinase-Activated Receptor 1; Proteinase-Activated Receptors; Proteinases; Proteolytic Enzymes; Receptor Protein; Receptor, PAR-1; Receptors, Proteinase-Activated; Relative; Relative (related person); Reticuloendothelial System, Platelets; Reticuloendothelial System, Serum, Plasma; Role; Sepsis; Serine Endopeptidases; Serine Protease; Serine Protein Hydrolases; Serine Proteinases; Serum, Plasma; Signal Transduction; Signal Transduction Systems; Signaling; Source; Therapeutic Intervention; Thrombase; Thrombin; Thrombin Receptor; Thrombocytes; Thrombocytopathy; Thrombosis; Thrombus; Work; Wound Healing; Wound Repair; angiogenesis; base; biological signal transduction; bloodstream infection; cardiac infarct; cell motility; cleaved; coronary attack; coronary infarct; coronary infarction; fibrinogenase; heart attack; heart infarct; heart infarction; heavy metal Pb; heavy metal lead; in vivo; intervention therapy; intraluminal angioplasty; malignancy; metalloproteinase (general); migration; neoplasm/cancer; novel; par-4; pathway; platelet disease; platelet disorder; poly ADP polymerase; poly ADP ribose synthetase; protein activation; receptor; response; social role; thrombocyte/platelet; tissue repair; trafficking; vascular
Project start date: 2000-04-01
Project end date: 2012-03-31
Budget start date: 1-APR-2009
Budget end date: 31-MAR-2010
5R01HL064701-08 (2009): $402500
5R01HL064701-05 (2005): $355500
5R01HL064701-04 (2004): $355500
5R01HL064701-03 (2003): $355500
5R01HL064701-02 (2002): $355500
Grants awarded to Athan Kuliopulos
Mechanism Of Thrombin Receptor Activation/Deactivation
Athan Kuliopulos, Associate Professor
New England Medical Center Hospitals 750 Washington St Boston, Ma 021111533
Grant 5R01HL057905-08 from National Heart, Lung, And Blood Institute IRG: HEM
Abstract: When a blood vessel is cut, the coagulation protease, thrombin, quickly activates platelets and fibrinogen to form a hemostatic plug. Thrombin activation of human platelets is mediated by dual protease-activated receptors--a high affinity PAR1 thrombin receptor and a low affinity PAR4 thrombin receptor. PAR1 and PAR4 are specifically tuned to be cleaved at very different rates which gives rise to their respective roles in the initiation and propagation phases of platelet aggregation. Chronic stimulation of PAR1 in the pro-thrombotic milieu of an atherosclerotic plaque has also been implicated in smooth muscle cell proliferation and in restenosis following acute coronary interventions in humans and animal model systems. More recently, it has been shown that PAR1 expression is upregulated in malignant cells and controls the invasion of epithelial-derived breast cancer cells and the metastasis of melanoma cells. Thrombin exerts these diverse cellular effects by cleaving the PAR exodomains to create a new N-terminus that binds to the extracellular loops of the receptor in an unusual intramolecular liganding mode. The first goal of these studies is to determine the underlying functional and structural basis of the differences in intramoleular liganding of PAR1 in extracellular loops e2-e4 and PAR4 in the N-terminal exodomain using NMR and biochemical approaches. We will also determine the structure of a non-cleavable PAR1 exodomain substrate in complex with thrombin. Our recent kinetic and NMR structural studies demonstrated that the cleaved PAR1 exodomain product retains high-affinity binding to exosite I of thrombin via its hirudin-like sequence but leaves the active site and exosite II (heparin-binding site) of thrombin freely accessible to other large macromolecules such as PAR4 and GPIb. The second aim focuses on the thrombin binding and cleavage reactions occurring with intact PAR1 and PAR4 on the surface of platelets and mammalian cells. We will determine whether platelet PAR1 and the GPIb-IX-V von Willebrand factor receptor serve as thrombin-binding cofactors for PAR4. The formation of hetero- and homo-dimers of PAR1 and PAR4 will be detected by fluorescence resonance energy transfer (FRET) of PAR-YFP and PAR-CFP pairs, and by biochemical analysis of epitope-tagged receptors. In the third aim, we will determine what role tissue factor plays in the in situ generation of thrombin and cleavage of PARs on the surface of cancer cells. We anticipate that these studies will lead to the identification of novel intra- and inter-molecular interactions between the various thrombin receptors and accessory proteins that may be critical for the regulation of thrombin signaling during hemostasis, thrombosis, and metastasis.
Keywords: protein structure function, receptor binding, receptor expression, thrombin, thrombin receptor, binding site, blood coagulation, chemical cleavage, conformation, enzyme activity, intermolecular interaction, neoplastic cell, platelet activation, platelet aggregation, thromboplastin, vascular endothelium, von Willebrand factor, X ray crystallography, clinical research, fluorescence resonance energy transfer, human subject, nuclear magnetic resonance spectroscopy, tissue /cell culture
Project start date: 1999-01-01
Project end date: 2007-08-20
5R01HL057905-08 (2006): $355935
5R01HL057905-07 (2005): $364500
5R01HL057905-06 (2004): $364500
2R01HL057905-05 (2003): $364500
CTRIP: MMP1-PAR1-BASED INTERVENTIONS IN ARTERIAL THROMBOSIS
Athan Kuliopulos
Tufts Medical Center, 800 Washington St, Boston, Ma 02111-1526
Grant 5RC2HL101783-02 from Office Of The Director, National Institutes Of Health
Abstract: The proposed studies of this GO grant application are designed to translate our recent discovery of a new therapeutic target, MMP1-PAR1 on platelets. Using various animal models and blood samples from humans, we identified a blood clotting mechanism that is driven by matrix metalloprotease-1 (MMP-1) on the platelet surface. We found that MMP-1 activates protease-activated receptor-1 (PAR1) in an autocrine manner after platelets are exposed to collagen from the blood vessel wall. Treatments that block the MMP1-PAR1 pathway prevented blood clots from forming in the presence of collagen, suggesting that drugs targeting this metalloprotease-receptor system could offer a new way to treat patients with atherothrombotic disease and acute coronary syndromes. In this application we propose to use our novel Pepducin technology as a new treatment to prevent collagen-MMP1-PAR1 arterial thrombosis in the acute setting. Pepducins are lipidated peptides which target the cytoplasmic surface of their cognate receptor and interrupt signaling to internally-located G proteins. One of these PAR1-based pepducins, PZ-128 (P1pal-7), has been extensively tested in animals and proven to be highly effective in inhibiting PAR1-dependent platelet activation, collagen-driven arterial thrombosis, and atherosclerosis. PZ-128 has been shown to be safe and well tolerated in rodents when administered daily at high doses for 40-70 days. In the first stage of this CTRIP program, IND-enabling studies will be conducted to assess efficacy of PZ-128 in guinea pigs and non-human primates, and safety and toxicology in two other species with GMP material under GLP conditions. Clinical trials will be designed to evaluate the safety and efficacy of PZ-128 in normal volunteers and in patients with coronary artery disease. These studies will be conducted in collaboration with multiple academic, clinical, and CRO research laboratories across the United States. The major milestone at the end of the 24 month grant period will be an investigator-initiated IND submission to the FDA. If successful, we will then conduct phase I and II clinical studies as a five-year Stage 2 CTRIP award in normal volunteers and patients with acute coronary syndromes. In the most recent data supplied by the American Heart Association, cardiovascular disease remained the major underlying cause of death in the United States with the majority of these deaths being due to coronary heart disease and stroke. Given the high prevalence of atherothrombotic disease and high MI and death rates, and incidence of adverse effects (bleeding and other safety issues), there remains a high unmet need for new therapeutics as exemplified by PZ-128, that can target both collagen and thrombin-dependent activation of platelets without unduly affecting hemostasis
Keywords: Acute; Adverse effects; Affect; American; Animal Model; Animal Models and Related Studies; Animals; Apoplexy; Applications Grants; Arterial Fatty Streak; Atheroma; Atheromatous; Atheromatous degeneration; Atheromatous plaque; Atheroscleroses; Atherosclerosis; Atherosclerotic Cardiovascular Disease; Autocrine Systems; Award; Baboons; Bizzozero`s corpuscle/cell; Bleeding; Blood Clot; Blood Clotting; Blood Platelets; Blood Sample; Blood Vessels; Blood coagulation; Blood specimen; CLG; CSBP1; CSBP2; CSPB1; Canine Species; Canis familiaris; Cardiac infarction; Cardiovascular; Cardiovascular Body System; Cardiovascular Diseases; Cardiovascular system; Cardiovascular system (all sites); Cause of Death; Cavia; Cell Communication and Signaling; Cell Signaling; Cerebral Stroke; Cerebrovascular Apoplexy; Cerebrovascular Stroke; Cerebrovascular accident; Cessation of life; Clinical; Clinical Protocols; Clinical Research; Clinical Study; Clinical Trials; Clinical Trials, Phase II; Clinical Trials, Unspecified; Coagulation Factor II Receptor; Collaborations; Collagen; Common Rat Strains; Conduct Clinical Trials; Coronary Arteriosclerosis; Coronary Artery Disease; Coronary Artery Disorder; Coronary Atherosclerosis; Coronary Disease; Coronary heart disease; Data; Death; Death Rate; Deetjeen`s body; Disease; Disorder; Dogs; Dose; Drug Delivery; Drug Delivery Systems; Drug Formulations; Drug Kinetics; Drug Targeting; Drug Targetings; EXIP; Effects, Longterm; Event; F2R; Fibroblast Collagenase; Formulation; Formulations, Drug; Funding; G-Proteins; GTP-Binding Proteins; GTP-Regulatory Proteins; Grant; Grant Proposals; Grants, Applications; Guanine Nucleotide Coupling Protein; Guanine Nucleotide Regulatory Proteins; Guinea Pigs; Hayem`s elementary corpuscle; Health Benefit; Heart; Hemorrhage; Hemostasis; Hemostatic function; High Prevalence; Human; Human, General; In Vitro; Incidence; Individual; Infusion; Infusion procedures; Interstitial Collagenase; Intervention; Intervention Strategies; Intracellular Communication and Signaling; Investigators; L-Leucine, D-phenylalanyl-L-prolyl-L-arginyl-L-prolylglycylglycylglycylglycyl-L-asparaginylglycyl-L-alpha-aspartyl-L-phenylalanyl-L-alpha-glutamyl-L-alpha-glutamyl-L-isoleucyl-L-prolyl-L-alpha-glutamyl-L-alpha-glutamyl-L-tyrosyl-; Laboratories; Laboratory Research; Lead; Life; Long-Term Effects; MAPK Inhibitors; MAPK14; MAPK14 gene; MMP-1; MMP-1Fibroblast Collagenase; MMP1; Mammals, Dogs; Mammals, Guinea Pigs; Mammals, Rats; Mammals, Rodents; Man (Taxonomy); Man, Modern; Marrow platelet; Maryland; Massachusetts; Matrix Metalloproteinase-1; Metallopeptidases; Metalloproteases; Metalloproteinases; Michigan; Mitogen-Activated Protein Kinase Inhibitor; Modeling; Mxi2; Myocardial Infarct; Myocardial Infarction; N-(butylsulfonyl)-O-(4-(4-piperidyl)butyl)-L-tyrosine; NIH; National Institutes of Health; National Institutes of Health (U.S.); Oregon; Organ System, Cardiovascular; PAR-1 Receptor; PAR1 Receptor; PRKM14; PRKM15; Papio; Papios; Pathway interactions; Patients; Pb element; Peptides; Pharmaceutical Agent; Pharmaceuticals; Pharmacodynamics; Pharmacokinetics; Pharmacologic Substance; Pharmacological Substance; Pharmacology; Phase; Phase 2 Clinical Trials; Phase I/II Trial; Phase II Clinical Trials; Phe-Pro-Arg-Pro-(Gly)4 desulfato-Tyr63`-hirugen; Phe-Pro-Arg-Pro-(Gly)4-Asn-Gly-Asp-Phe-Glu-Glu-Ile-Pro-Glu-Glu-Tyr-Leu; Phe-Pro-Arg-Pro-(Gly)4-desulfohirudin-(53-64); Platelet Activation; Platelets; Plavix; Production; Programs (PT); Programs [Publication Type]; Protease-Activated Receptor 1; Proteinase-Activated Receptor 1; Public Health; Rat; Rattus; Receptor Protein; Receptor, PAR-1; Research Contracts; Research Design; Research Personnel; Researchers; Reticuloendothelial System, Platelets; Rodent; Rodentia; Rodentias; Rupture; SAPK2A; Safety; Savanna Baboons; Signal Transduction; Signal Transduction Systems; Signaling; South Carolina; Staging; Streaks, Arterial Fatty; Stroke; Study Type; Surface; System; System, LOINC Axis 4; Technology; Testing; Therapeutic; Thrombase; Thrombin; Thrombocytes; Thrombosis; Thrombus; Toxic effect; Toxicities; Toxicology; Translating; Translatings; Treatment Side Effects; United States; United States National Institutes of Health; Vascular Accident, Brain; Vascular, Heart; Wisconsin; ing; acute coronary syndrome; atheromatosis; atherosclerosis plaque; atherosclerotic lesions; atherosclerotic plaque; atherosclerotic vascular disease; autocrine; base; biological signal transduction; bivalirudin; blood loss; brain attack; canine; cardiac infarct; cardiovascular disorder; cerebral vascular accident; circulatory system; clinical investigation; coronary attack; coronary disorder; coronary infarct; coronary infarction; design; designing; disease/disorder; domestic dog; fibrinogenase; heart attack; heart infarct; heart infarction; heavy metal Pb; heavy metal lead; in vivo; inhibitor; inhibitor/antagonist; interventional strategy; language translation; metalloproteinase (general); model organism; neurobehavioral; new therapeutic target; new therapeutics; next generation therapeutics; non-human primate; nonhuman primate; novel; novel therapeutics; p38; p38 MAPK Gene; p38Alpha; pathway; percutaneous coronary intervention; phase 2 study; phase 2 trial; phase II trial; prevent; preventing; programs; protocol, phase II; public health medicine (field); receptor; respiratory; safety study; side effect; small molecule; stroke; study design; study, phase II; therapy adverse effect; thrombocyte/platelet; tirofiban; treatment adverse effect; vascular; volunteer; vulnerable plaque
Relevance: Relevance In the most recent data supplied by the American Heart Assocation, cardiovascular disease remained the major underlying cause of death in the United States with the majority of these deaths being due to coronary heart disease and stroke. Given the high prevalence of atherothrombotic disease and high MI and death rates, and incidence of adverse effects (bleeding and other safety issues), there remains a high unmet need for new therapeutics as exemplified by PZ-128, that can target both collagen and thrombin-dependent activation of platelets without unduly affecting hemostasis
Project start date: 2009-09-30
Project end date: 2011-08-31
Budget start date: 1-SEP-2010
Budget end date: 31-AUG-2011
PFA/PA: RFA-OD-09-004
5RC2HL101783-02 (2010): $601043
1RC2HL101783-01 (2009): $1098405
SIGNAL TRANSDUCTION OF A G PROTEIN COUPLED RECEPTOR
Athan Kuliopulos, Associate Professor
New England Medical Center Hospitals 750 Washington St Boston, Ma 021111533
Grant 5R29GM052926-05 from National Institute Of General Medical Sciences IRG: CBY
Abstract: The alphaFactor receptor is the first component of a signal transduction pathway that leads to cellular differentiation and mitotic growth arrest in Saccharomyces cerevisiae. This receptor is a member of a super-family of 7-transmembrane receptors involved in such diverse physiological processes as endocrine signaling, neurotransmission, vision, and blood- clotting. The function of these receptors is to sense low abundance signals impinging from outside the cell and to relay that signal to a tightly associated G-protein. Disruption of these G-protein coupled receptor complexes by mutation is known to cause a number of human diseases such as neonatal severe hyperparathyroidism, precocious puberty, retinitis pigmentosa, and various neoplasms. The yeast pheromone-response pathway is activated when a secreted 13-amino acid residue peptide (alphaFactor) binds its cognate receptor. The mechanism by which the alphaFactor receptor recognizes this peptide ligand and activates the internally-located G-alpha subunit is not known. The purpose of this investigation is to define the peptide binding site on the receptor and to study the physical properties of the receptor-ligand complex in both activated and unactivated states. The alphaFactor receptor will be overexpressed in yeast with an epitope-His6 tag appended to the C- terminus. Mapping of the peptide binding site will be done with a combination of N-bromoacetyl- and benzoylphenylalanine-alphaFactor peptide affinity labels. N-terminally truncated alphaFactor peptide affinity labels are antagonists and will serve as probes of the unactivated peptide-receptor complex. The labeled-receptor-His6 protein will be retrieved from crude membrane preparations by Ni-chelation chromatography and preparative polyacrylamide gel electrophoresis using techniques established by our group. Localization of the site of labeling will be done by epitope mapping, electrospray mass spectrometry, and sequencing of proteolytic fragments. Site-specific mutagenesis of the receptor will confirm these mapping studies. The receptor will be purified from yeast membranes by peptide affinity columns and by affinity tags incorporated into the receptor itself. Biophysical analysis of receptor-ligand macromolecular complexes will define the conformational change that occurs upon ligand activation. These studies include determination of the orientation and solvent accessibility of bound peptide-fluorophores, and detection of the receptor conformational change in real time using fluorescent donor-acceptor tags on both peptide and the third intracellular loop of the receptor. Capillary electrophoresis will serve as a highly sensitive means to quantitate peptide-receptor binding in solution and to detect gross conformational changes upon ligand activation. These studies should provide the basis for understanding how this receptor activates the downstream signaling pathway and will serve as a general model for other G protein/receptor mutant-related human diseases.
Keywords: G protein, biological signal transduction, hormone receptor, receptor binding, receptor coupling, chemical binding, conformation, pheromone, protein structure /function, receptor expression, Saccharomyces cerevisiae, affinity labeling, capillary electrophoresis, electrospray ionization mass spectrometry, epitope mapping, gel electrophoresis, high performance liquid chromatography, nuclear magnetic resonance spectroscopy, nucleic acid sequence, polymerase chain reaction, site directed mutagenesis
Project start date: 1995-08-01
Project end date: 2001-01-31
5R29GM052926-05 (1999): $85452
5R29GM052926-04 (1998): $127562
5R29GM052926-03 (1997): $122653
Thrombin Receptor-G Protein Signaling Mechanisms
Athan Kuliopulos, Associate Professor
New England Medical Center Hospitals 750 Washington St Boston, Ma 021111533
Grant 2R01HL064701-06A2 from National Heart, Lung, And Blood Institute IRG: HT
Abstract: Proteolytic cleavage of the G protein-coupled protease-activated receptors activates an extraordinarily diverse array of physiologic responses. These include platelet activation and aggregation, cellular proliferation/apoptosis, cell migration and protease-homing, angiogenesis, and the hemostatic and inflammatory responses to vascular injury. Four protease-activated receptors have been identified PAR1, PAR2, PARS, and PAR4. Matrix metalloproteases have also emerged as important proteases in vascular biology. Initially described as extracellular matrix remodeling enzymes involved in tissue repair and cancer invasion, a renewed focus has centered on MMPs and the related metalloprotease disintegrins because of their prominence in atherothrombotic disease and platelet hemostasis. Quite recently, we found that a matrix metalloprotease, MMP-1, can also function as a protease agonist of PAR1 cleaving the receptor to generate PAR1-dependent Ca2+ signals, migration, platelet activation and cell shape changes. MMP-1 is expressed in high levels in platelets and endothelium. Circulating levels of MMP-1 have been shown to be significantly elevated in plasma from patients following acute myocardial infarction pointing to activated platelets and endothelium as potential sources for MMP-1. However, it is not clear what role(s) MMP1-PAR1 signaling plays in hemostasis, thrombosis and the control of vascular integrity or whether MMP1 activates different G protein pathways as compared to thrombin. PAR1, has been shown to couple to Gq, Gi, Gi2/i3, and (3y under a variety of in vitro conditions. The specifics of how PAR1 interacts with the G proteins and the relative importance and temporal ordering of differential G protein activation under in vivo conditions is still unknown. Far less is known about the identity of non-G protein effectors that might also interact with the intracellular loops of the PARs. The major goals of these studies are 1) to investigate the molecular basis of G protein activation by PAR1 under in vivo conditions, 2) to understand the mechanism and physiologic relevance of MMP1-PAR1 signaling in platelets during arterial thrombus formation 3) to investigate the mechanism of endothelial MMP1 activation of PAR1 and the functional consequences of MMP1-PAR1 signaling on endothelial function during sepsis, and 4) to determine how the newly identified PAR1 effector, BicD1, modulates PAR1 signaling, signal termination, and cell motility. Platelet activation has been shown to be heightened in the setting of angioplasty and stenting which may cause clinical complications including acute heart attacks and death. The PAR1 receptor has long been recognized as an obvious candidate for therapeutic intervention of patients with heart conditions, the work proposed here may lead to the development of novel treatments for patients with arterial blood clots and those suffering from life- threatening sepsis.
Keywords: G protein, biological signal transduction, collagenase, protein structure function, thrombin receptor, cell motility, chemical cleavage, enzyme activity, platelet aggregation, protein protein interaction, protein transport, receptor coupling, septicemia, thrombosis, clinical research, genetically modified animal, human subject, immunoprecipitation, laboratory mouse
Project start date: 2000-04-01
Project end date: 2012-03-31
2R01HL064701-06A2 (2007): $402500
Sponsored Links Excellgen http://Excellgen.com
MECHANISM OF THROMBIN RECEPTOR ACTIVATION/DEACTIVATION
Athan Kuliopulos, Associate Professor
New England Medical Center Hospitals
750 Washington St
boston, Ma 021111533
Grant 5R01HL057905-03 from National Heart, Lung, And Blood Institute IRG: HEM
Abstract: One of the greatest challenges facing hematologic researchers today is understanding how normal physiologic processes such as blood clotting and wound repair become dysfunctional and contribute to coronary artery disease and atherosclerosis. New therapeutic agents are urgently needed that can specifically target the abnormal accumulation of smooth muscle cells, inflammatory cells, and fibrin deposits that comprise these lesions without disrupting normal blood clotting. Currently used thrombolytic therapies can have effects at non-thrombotic loci which lead to hemorrhagic complications. One potential target is the thrombin receptor. The thrombin receptor links the coagulation events occurring within the lumen of the blood vessel with the cellular signaling pathways that mediate platelet aggregation, cellular proliferation, and wound healing. Thrombin activates the receptor by a unique proteolytic cleavage of the extracellular domain. In an incompletely understood sequence of events, the new N-terminus serves as an intramolecular ligand that binds the body of the receptor. This intramolecular complex then activates an internally located G-protein. The cell must then switch off the irreversibly-cleaved thrombin receptor to prevent overstimulation of downstream signaling pathways. The first goal of these studies is to solve the 3-dimensional structure of the extracellular domain in both resting and activated states by NMR. Another NMR project will study the exodomain in complex with receptor extracellular loops and with thrombin. Capillary electrophoresis and inhibition of small chromogenic substrate hydrolysis will quantitate binding of thrombin to non-cleavable exodomains. A second goal is to create a genetic system to study thrombin receptor activation and G- protein coupling in isolation from all other mammalian proteins. We will capitalize on our recent expression and purification of affinity-tagged thrombin receptor in yeast to produce sufficient quantities of receptor for mapping the ligand binding surface. Functional coupling of the receptor to the yeast G-protein signaling pathway will allow testing of the mechanism of intramolecular ligand-receptor activation and the specificity of G-protein interactions. This genetic system which responds to soluble ligands will be a useful tool in the screening and development of novel anti-thrombin receptor agents. A third goal is to explore the role of anticoagulant serum proteases in truncation of the receptor exodomain. Previous experiments that determined cleavage sites will be correlated with loss of platelet function and cleavage of receptors on platelets and endothelium in a thombolytic animal model
Keywords: G protein, biological signal transduction, protein structure /function, receptor, thrombin anticoagulant, binding site, blood coagulation, conformation, crosslink, endopeptidase, enzyme activity, extracellular matrix protein, intermolecular interaction, membrane structure, plasmin, platelet aggregation, proteolysis, receptor coupling, receptor expression, receptor sensitivity, vascular endothelium nuclear magnetic resonance spectroscopy, tissue /cell culture, yeast
Project start date: 1999-01-01
Project end date: 2002-12-31
5R01HL057905-03 (2001): $297792
5R01HL057905-02 (2000): $289118
1R01HL057905-01A2 (1999): $282030
MECHANISM OF THROMBIN RECEPTOR ACTIVATION DEACTIVATION
Athan Kuliopulos
Boston University Medical Campus 715 Albany St, 560 Boston, Ma 021182394
Grant 5P41RR010888-050038 from National Center For Research Resources
Abstract: My lab is interested in the structure and function of f-actin binding proteins. We arecurrently determining the structure of the "headpiece" domain of the f-actin bundfing protein villin by NMR and X-ray crystallography. We have expressed a series of mutants of headpiece in E. coli and require mass spectral analysis to insure the product that we purify is the correct sequence and has not been modified during purification. Recently, we have developed conditions where we can reproducibly get crystals of headpiece that diffiract well in the X-ray beam (to beyond 1.8 A resolution). In order to the obtain phase information necessary to solve the high resolution X-ray crystal structure of villin headpiece, we turned to the multiple anomolous dispersion (MAD) method. MAD phasing requires data to be collected at a synchrotron source with headpiece derivatives labeled with selenomethionine. Data collection time at the Brookhaven Synchrotron Source is limited and must be applied for. Therefore, it was essential to have our selenium-labeled crystal prepared and characterized in advance. Is was important for us to insure that our labeling was effective and, in addition, that the selenium was not oxidized or lost during thepurification, before our visit to the synchrotron. The mass spectral analysis performed by the center was critical in insuring our crystal contained the expected selenium and was therefore suitable for synchrotron data collection and MAD phasing. No other method is as effective as mass spectral analysis in providing proof of the presence and percent occupancy of the selenium in our protein crystals.
Keywords: biological product, biomedical resource, biotechnology, connective tissue, immunology, inflammation, protein, spectrometry, structural biology, technology /technique, virus
INTER-CELLULAR SIGNALING OF INVASTION RECEPTORS IN THE TUMOR MICROENVIRONMENT
Athan Kuliopulos, Associate Professor
Tufts Medical Center, 800 Washington St, Boston, Ma 02111-1526
Grant 5R01CA122992-04 from National Cancer Institute
Abstract: G protein-coupled receptors (GPCRs) are the largest family of cell surface receptors with more than 1000 members yet only a few GPCRs have been found to be oncogenes. Among these, the protease-activated receptor 1 (PAR1) has been identified as a potent oncogene and confers invasive behavior to pre-cancerous breast cells. In this grant we test the hypothesis that PAR1 is critical for cancer-host communication by stimulating production of paracrine and angiogenesis factors in the tumor environment. We recently identified host fibroblast-derived matrix metalloprotease-1 (MMP-1) as a novel agonist that can cleave and activate PAR1 in breast and ovarian tumors. MMP-1 is highly expressed in stromal cells and is a predictive marker for poor prognosis in breast, colorectal and other tumors. Targeting PAR1 with the novel cell-penetrating pepducins described here blocks the pathway downstream of MMP-1 and receptor inhibiting cancer growth and invasion. PAR1 pepducins also resulted in pronounced reduction of stromal infiltration and angiogenesis of breast and ovarian cancers. We will test the hypothesis that stromal-derived PAR1 may have a distinct role from cancer cell-derived PAR1 in tumor biology and stimulation of paracrine factors that promote invasion and angiogenesis. We will utilize two cancer-stromal co-culturing model systems and in vivo cancer- stromal coimplantation xenografts. Activation of PAR1 (and PAR2) have been shown to produce IL-8, Gro-?, VEGF, IL-6 and GM-CSF in a variety of cell types including prostate cancer, but the role of PAR1 in paracrine communication in cancer has not been directly addressed. We hypothesize that activation of PAR1 on cancer cells leads to production of chemokines which stimulate endothelial cells resulting in increased angiogenesis and tumor growth. Pepducin technology will be used to define the role of endothelial chemokine CXCR1 and CXCR2 receptors in blood vessel formation and validate our preliminary data that PAR1 and potentially CXCR1/2 pepducins can block angiogenesis and extend survival in ovarian and breast xenograft animal models. Lastly, we will follow-up our recent discovery of a novel PAR1-effector, BicD1, which acts as a suppressor of PAR1-dependent migration and invasion of breast cancer cells. Knock-down of BicD1 expression greatly prolongs mitogen-activated kinase signaling suggesting that BicD1 may regulate MAPK phosphatase (MKP) activity in breast cancer cells. We will test the hypothesis that upregulation of PAR1 expression or stimulation of PAR1 by MMP-1 or thrombin regulates BicD1 and MKP expression and that these downstream effectors in turn control MAPK-dependent invasion and proliferation of breast cancer. The pepducin approach has the prospect to significantly change our understanding of the role of cross-talk between receptors such PAR1 and CXCR1/2 in cancer growth and blood vessel formation. As envisioned, these studies will develop the first inhibitors of these invasion and chemokine receptors for the potential treatment of advanced breast and ovarian cancers
Keywords: 3-10C; AMCF-I; Active Follow-up; Address; Agonist; Angiogenic Factor; Animal Model; Animal Models and Related Studies; B cell differentiation factor; B cell stimulating factor 2; B-Cell Differentiation Factor-2; B-Cell Stimulatory Factor-2; BCDF; BSF-2; BSF2; BSF2 (B cell stimulating factor 2); Behavior; Biological Models; Blood Vessels; Breast; Breast Cancer Cell; Breast Cancer Model; Breast Carcinoma; Breast Neoplasms; Breast Tumors; C-C-CKR-1; CDw128a; CDw128b Antigens; CLG; CMKAR1; CSBP1; CSBP2; CSPB1; CXC Chemokine Receptor 2; CXCL8; CXCR1; CXCR2 Protein; Cancer Cell Growth; Cancer Genes; Cancer of Breast; Cancer of Prostate; Cancer of the Ovary; Cancer-Promoting Gene; Cancerous; Cancers; Carcinoma Cell; Cell Communication; Cell Communication and Signaling; Cell Interaction; Cell Locomotion; Cell Migration; Cell Movement; Cell Signaling; Cell Surface Receptors; Cell-to-Cell Interaction; Cells; Cellular Migration; Chemokine (C-X-C) Receptor 2; Cleaved cell; Co-culture; Coagulation Factor II Receptor; Cocultivation; Coculture; Coculture Techniques; Colon or Rectum; Colorectal; Communication; Cytokines, Chemotactic; Data; Differentiation Factor, B-Cell; EC 2.7; ERK 2; EXIP; Employee Strikes; Endothelial Cells; Environment; Esteroproteases; Exhibits; Extracellular Signal-Regulated Kinase 2; F2R; Factor, Angiogenesis; Family; Fibroblast Collagenase; Fibroblasts; Forecast of outcome; G Protein-Complex Receptor; G-Protein-Coupled Receptors; G-Proteins; GCP-1; GCP1; GM-CSF; GMCSF; GRO/MGSA Receptor; GTP-Binding Proteins; GTP-Regulatory Proteins; Generalized Growth; Grant; Granulocyte-Macrophage Colony-Stimulating Factor; Growth; Guanine Nucleotide Coupling Protein; Guanine Nucleotide Regulatory Proteins; HPGF; Hepatocyte-Stimulating Factor; Heterograft; High Affinity Interleukin 8 Receptor Type B; Histamine-Producing Cell-Stimulating Factor; Homologous Chemotactic Cytokines; Human Breast Cancer Cell; Hybridoma Growth Factor; IFN-beta 2; IFNB2; IL-6; IL-8; IL-8RB; IL-8Rbeta; IL6 Protein; IL8; IL8 Receptor Type 2; IL8 gene; IL8R1; IL8RA; IL8RA gene; IL8RB; IL8RBA; Infiltration; Inflammatory; Intercrines; Interleukin 6 (Interferon, Beta 2); Interleukin 8 Receptor Beta; Interleukin 8 Receptor Type 2; Interleukin-6; Interleukin-8 Receptor Type B; Interleukin-8 Receptors B; Interleukin-8B Receptor; Interstitial Collagenase; Intracellular Communication and Signaling; K60; Kinases; LECT; LUCT; LYNAP; Ligands; MAP Kinase 1; MAP Kinase 2; MAPK phosphatase; MAPK1; MAPK1 Mitogen-Activated Protein Kinase; MAPK14; MAPK14 gene; MAPK2 Mitogen-Activated Protein Kinase; MCF-7; MCF-7 Cell; MCF7; MCF7 cell; MDNCF; MGI-2; MMP-1; MMP-1Fibroblast Collagenase; MMP1; MONAP; MTGN; Malignant Cell; Malignant Epithelial Cell; Malignant Neoplasms; Malignant Ovarian Neoplasm; Malignant Ovarian Tumor; Malignant Tumor; Malignant Tumor of the Breast; Malignant Tumor of the Ovary; Malignant Tumor of the Prostate; Malignant neoplasm of breast; Malignant neoplasm of ovary; Malignant neoplasm of prostate; Malignant prostatic tumor; Mammary Cancer; Mammary Carcinoma; Mammary Neoplasms; Matrix Metalloproteinase-1; Metallopeptidases; Metalloproteases; Metalloproteinases; Metastasis; Metastasize; Metastatic Neoplasm; Metastatic Tumor; Mitogen Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 2; Mitogens; Model System; Models, Biologic; Molgramostin; Motility; Motility, Cellular; Mxi2; Myeloid Differentiation-Inducing Protein; NAF; Neoplasm Metastasis; Oncogenes; Oncogenesis; Oncogenic; Ovarian; Ovarian Tumor; Ovary Neoplasms; P42MAPK; PAR-1 Receptor; PAR1 Receptor; PRKM1; PRKM14; PRKM15; Paracrine Communication; Paracrine Signaling; Pathway interactions; Peptidases; Peptide Hydrolases; Phosphatases; Phosphohydrolases; Phosphomonoesterases; Phosphoric Monoester Hydrolases; Phosphotransferases; Plasmacytoma Growth Factor; Production; Prognosis; Prostate CA; Prostate Cancer; Prostatic Cancer; Protease-Activated Receptor 1; Proteases; Proteinase-Activated Receptor 1; Proteinases; Proteolytic Enzymes; Receptor Protein; Receptor, PAR-1; Receptors, CXCR2; Regulation; Role; SAPK2A; SCYB8; SIS cytokines; Secondary Neoplasm; Secondary Tumor; Signal Transduction; Signal Transduction Systems; Signaling; Strikes; Strikes, Employee; Stromal Cells; Stromal Neoplasm; Stromal Tumor; System; System, LOINC Axis 4; TC-GM-CSF; TSG-1; Technology; Testing; Thrombase; Thrombin; Tissue Growth; Transforming Genes; Transphosphorylases; Transplantation, Heterologous; Tube; Tumor Angiogenesis; Tumor Biology; Tumor Cell Invasion; Tumor Cell Migration; Tumor Invasion; Tumor of the Ovary; Tumor-Cell Human GM Colony-Stimulating Factor; Up-Regulation; Up-Regulation (Physiology); Upregulation; VEGFs; Vascular Endothelial Growth Factors; Vegf; Xenograft; Xenograft Model; Xenograft procedure; Xenotransplantation; angiogenesis; b-ENAP; biological signal transduction; cancer cell; cancer metastasis; cancer progression; cell motility; cell type; chemoattractant cytokine; chemokine; chemokine receptor; cleaved; fibrinogenase; follow-up; granulocyte macrophage colony stimulating factor; in vivo; inhibitor; inhibitor/antagonist; interferon beta 2; knock-down; malignancy; malignant breast neoplasm; mammary cancer model; mammary tumor; mammary tumor model; member; metalloproteinase (general); migration; model organism; mouse model; neoplasm progression; neoplasm/cancer; neoplastic progression; new therapeutic target; novel; ontogeny; outcome forecast; ovarian cancer; ovarian neoplasm; p38; p38 MAPK Gene; p38Alpha; p42 MAP Kinase; p42 MAPK; p42(Mapk) Kinase; p42(Mitogen-Activated Protein Kinase); paracrine; pathway; prevent; preventing; receptor; response; rho; scaffold; scaffolding; social role; tumor; tumor growth; tumor progression; tumorigenesis; vascular
Project start date: 2007-09-19
Project end date: 2011-07-31
Budget start date: 1-AUG-2010
Budget end date: 31-JUL-2011
5R01CA122992-04 (2010): $305900
5R01CA122992-03 (2009): $305900
1R01CA122992-01A1 (2007): $305900
PROTEASE-ACTIVATED RECEPTORS IN VASCULAR INTEGRITY AND DYSFUNCTION
Athan Kuliopulos, Associate Professor
Tufts Medical Center, 800 Washington St, Boston, Ma 02111-1526
Grant 5R01HL057905-12 from National Heart, Lung, And Blood Institute
Abstract: Emerging evidence suggests that matrix metalloproteases (MMPs) play a pivotal role in vascular integrity and wound healing, however, inappropriate MMP activity may underlie the pathogenesis of several diseases including restenosis, atherosclerosis, and myocardial dysfunction. Selectively controlling defined aspects of MMP activity in restenosis and end-stage coronary artery disease may be a valid target for therapy. We recently made the unanticipated discovery that the matrix metalloprotease MMP-1, cleaves and activates protease-activated receptor 1 (PAR1) in a variety of cells including endothelium, vascular SMCs, platelets and carcinoma cells. Both MMP-1 and PAR1 are highly upregulated by several cell types in the blood vessel wall in the context of intimal hyperplasia and inflammatory states. However, it is completely unknown what role(s), MMP1-PAR1 signaling plays in either normal blood vessel biology or in vascular disease. Moreover, activation of PAR1 can lead to decreased barrier function, dysregulated vasomotor tone and control of SMC functions through inter-cellular communication pathways. In aim 1, we will investigate the molecular mechanism and (patho)physiologic relevance of MMP1-PAR1 signaling in SMC proliferation, migration and invasion, survival and de-differentiation in the arterial wall. Modulation of PAR1 signaling with cell- penetrating pepducins will be used to evaluate the function(s) of MMP1-PAR1 signaling in in vivo arterial injury and restenosis models using wild-type mice and strains deficient in PAR1, PAR2 and PAR4. Furthermore, prior studies suggested that PAR1 promotes the conversion of the endothelium to a pro- thrombotic and pro-inflammatory state. We made the recent observation that PAR1 can also confer protective effects in endothelial barrier function and survival in animal models of systemic inflammation by transactivation of the structurally-related PAR2. Our preliminary data suggest that the signaling outputs of PAR1 and PAR2 are entwined and need to be examined as a potential functional unit. Aim 2 will use a combination of biochemical, pharmacological and RNA interference techniques to determine how PAR1 and PAR2 regulate Rac versus Rho signaling. We will delineate the components and kinetics of downstream pro- inflammatory versus anti-inflammatory signaling pathways emanating from PAR1 and PAR2 in the context of barrier disruption and leukocyte transmigration. Critical downstream chemokine pathways known to mediate both autocrine and paracrine signaling during these processes will be interrupted with our newly validated chemokine receptor pepducins. Lastly, we will investigate the molecular basis of PAR1-PAR2 interactions and liganding using biochemical and genetic approaches. Relevance to public health The studies outlined here may lead to the eventual discovery of novel therapeutics that may prevent heart attacks and stroke in patients with heart disease, and could be used in other conditions such as life-threatening sepsis
Keywords: Animal Model; Animal Models and Related Studies; Anti-Inflammatories; Anti-Inflammatory Agents; Anti-inflammatory; Antiinflammatories; Antiinflammatory Agents; Apoplexy; Arterial Injury; Atheroscleroses; Atherosclerosis; Atherosclerotic Cardiovascular Disease; Autocrine Systems; Biochemical; Biochemical Genetics; Biology; Bizzozero`s corpuscle/cell; Blood Platelets; Blood Vessels; Blood leukocyte; Carcinoma Cell; Cardiac Diseases; Cardiac Disorders; Cardiac infarction; Cell Communication and Signaling; Cell Function; Cell Growth in Number; Cell Multiplication; Cell Process; Cell Proliferation; Cell Signaling; Cell physiology; Cells; Cellular Function; Cellular Physiology; Cellular Process; Cellular Proliferation; Cerebral Stroke; Cerebrovascular Apoplexy; Cerebrovascular Stroke; Cerebrovascular accident; Cleaved cell; Coagulation Factor II Receptor; Communication; Coronary Arteriosclerosis; Coronary Artery Disease; Coronary Artery Disorder; Coronary Atherosclerosis; Cytokines, Chemotactic; Data; Deetjeen`s body; Disease; Disorder; Dysfunction; Endopeptidase-Activated Receptors; Endothelial Cells; Endothelium; F2R; Functional disorder; Genetic, Biochemical; Hayem`s elementary corpuscle; Heart Diseases; Homologous Chemotactic Cytokines; Hyperplasia; Hyperplastic; INFLM; Inflammation; Inflammatory; Intercrines; Intracellular Communication and Signaling; Kinetic; Kinetics; Lead; Leiomyocyte; Leukocytes; Life; Ligands; Malignant Epithelial Cell; Marrow leukocyte; Marrow platelet; Mediating; Metallopeptidases; Metalloproteases; Metalloproteinases; Methods and Techniques; Methods, Other; Modeling; Molecular; Mouse Strains; Myocardial Infarct; Myocardial Infarction; Myocardial depression; Myocardial dysfunction; Myocytes, Smooth Muscle; Output; PAR-1 Receptor; PAR1 Receptor; PAR4; PAWR; PAWR gene; Paracrine Communication; Paracrine Signaling; Pathogenesis; Pathway interactions; Patients; Pb element; Physiologic; Physiological; Physiopathology; Platelets; Play; Post-Transcriptional Gene Silencing; Post-Transcriptional Gene Silencings; Posttranscriptional Gene Silencing; Posttranscriptional Gene Silencings; Process; Protease-Activated Receptor 1; Protease-Activated Receptors; Proteinase-Activated Receptor 1; Proteinase-Activated Receptors; Public Health; Quelling; RNA Interference; RNA Silencing; RNA Silencings; RNAi; Receptor, PAR-1; Receptors, Proteinase-Activated; Reticuloendothelial System, Leukocytes; Reticuloendothelial System, Platelets; Role; SIS cytokines; Sepsis; Sequence-Specific Posttranscriptional Gene Silencing; Signal Pathway; Signal Transduction; Signal Transduction Systems; Signaling; Smooth Muscle Cells; Smooth Muscle Myocytes; Smooth Muscle Tissue Cell; Staging; Stroke; Subcellular Process; Techniques; Thrombocytes; Trans-Activation (Genetics); Transactivation; Vascular Accident, Brain; Vascular Diseases; Vascular Disorder; Vascular Endothelium; Vasomotor; White Blood Cells; White Cell; Wild Type Mouse; Wound Healing; Wound Repair; atheromatosis; atherosclerotic vascular disease; autocrine; base; biological signal transduction; blood vessel disorder; bloodstream infection; brain attack; cardiac infarct; cell type; cerebral vascular accident; chemoattractant cytokine; chemokine; chemokine receptor; cleaved; coronary attack; coronary infarct; coronary infarction; disease/disorder; heart attack; heart disorder; heart infarct; heart infarction; heavy metal Pb; heavy metal lead; in vivo; injury and repair; metalloproteinase (general); migration; model organism; new therapeutics; next generation therapeutics; novel; novel therapeutics; par-4; pathophysiology; pathway; prevent; preventing; protective effect; public health medicine (field); restenosis; rho; social role; stroke; thrombocyte/platelet; tissue repair; vascular; white blood cell; white blood corpuscle
Project start date: 1999-01-01
Project end date: 2011-06-30
Budget start date: 1-JUL-2010
Budget end date: 30-JUN-2011
5R01HL057905-12 (2010): $402500
Sponsored Links Excellgen http://Excellgen.com
5R01HL057905-11 (2009): $402500
2R01HL057905-09 (2007): $402500
THROMBIN RECEPTOR-G PROTEIN SIGNALING MECHANISMS
Athan Kuliopulos, Associate Professor
New England Medical Center Hospitals
750 Washington St
boston, Ma 021111533
Grant 1R01HL064701-01A1 from National Heart, Lung, And Blood Institute IRG: HEM
Abstract: Applicant´s Verbatim) Thrombin cleavage of the thrombin receptors activates an extraordinarily diverse array of physiologic responses. These include platelet aggregation, cellular proliferation/apoptosis, cell-cell adhesion and inflammation, and potentially the invasive and tissue-reorganizing processes involved in cancer. Activation of platelet thrombin receptors is likely to play a major role in the initiation and maintenance of pathological arterial and venous thromboses and the development of atherosclerotic lesions. Four G protein-coupled, protease-activated receptors have been identified PAR1, PAR2, PAR3, and PAR4. The prototypical protease-activated receptor, PAR1, has been shown to couple to Gq., Gi(betagamma), and G12/13 under a variety of in vitro conditions. The specifics of how PAR1 interacts with the G proteins and the relative importance and temporal ordering of differential G protein activation is still unknown. Far less is known about the identity of the G proteins that couple to the newly discovered PAR3 and PAR4 thrombin receptors. The goals of these studies are 1) to investigate the mechanistic basis of differential G protein activation by PAR1 under in vivo conditions, 2) to understand the underlying differences between PAR4 and PAR1 G-protein dependent signaling and signal termination, and 3) to expand the repertoire of intracellular proteins that might interact with the class of protease-activated receptors. In the first specific aim we will exploit our newly discovered class of cell-penetrating pepducins. These reagents rapidly penetrate the plasma membrane of intact cells such as platelets and fibroblasts and cause specific activation and/or inhibition of receptor-dependent intracellular signal transduction under in vivo conditions. We have demonstrated that these pepducins exhibit properties that are normally attributable to activated PAR1. These include full platelet aggregation and shape change, stimulation of regulated Ca++ fluxes, activation of phospholipase C, and desensitization of thrombin receptor responses. We anticipate that the use of the pepducins to study intracellular signal transduction will open new avenues of experimental research in cells previously not amenable to molecular techniques. Ultimately, these may prove to be the first therapeutically useful agents that are targeted at receptor-G protein interfaces. The second aim will explore the mechanistic basis of PAR4 versus PARI-activation of intracellular signaling pathways using a variety of genetic and biochemical techniques. We will expand on recent work done in our lab that demonstrated that PAR1 and PAR4 have distinct kinetics of Ca++ signaling in platelets. In the third aim, we will study coexpressed PAR1 and mammalian G proteins in yeast and will identify novel proteins which specifically interact with PAR1 and PAR4 intracellular domains
Keywords: G protein, biological signal transduction, thrombin receptor G protein coupled receptor kinase, calcium flux, mutant, phospholipase C, platelet aggregation, protein protein interaction, protein structure /function, receptor coupling, receptor expression human tissue, molecular cloning, yeast, yeast two hybrid system
Project start date: 2000-12-20
Project end date: 2005-11-30
1R01HL064701-01A1 (2001): $355500
STRUCTURE & ACTIVITY OF HUMAN THROMBIN RECEPTOR
Athan Kuliopulos
Institution:
Grant 1P41RR010888-01A10032 from National Center For Research Resources
Abstract: A project on the activation of the 7-transmembrane/G-protein coupled human thrombin receptor and its role in platelet aggregation is being done by Dr. Athan Kulopulost. Dr. Kullopulos is studying how platelet aggregation is stimulated by alpha-thrombin which exerts its action by recognizing and cleaving the N-terminal extracellular domain of the receptor located on the platelet membrane. The autolysis products of the extracellular domain have been identified using Edman sequencing and MALDI-TOF-MS. Further work concerns the identification of reaction products generated by thrombin cleavage of a recombinant extracellular domain of thrombin for which MALDI- TOF-MS or ESI-MS will be required.
SIGNAL TRANSDUCTION OF A G PROTEIN COUPLED RECEPTOR
Athan Kuliopulos, Associate Professor
New England Medical Center Hospitals 750 Washington St Boston, Ma 021111533
Grant 5R29GM052926-02 from National Institute Of General Medical Sciences IRG: CBY
Project start date: 1995-08-01
Project end date: 2000-07-31
5R29GM052926-02 (1996): $117936
VITAMIN K DEPENDENT CARBOXLYASE--MOLECULAR STUDIES
Athan Kuliopulos, Associate Professor
Biological Chemistryharvard University (medical School)
medical School Campus
boston, Ma 02115
Grant 1F32HL008882-01 from National Heart, Lung, And Blood Institute IRG: CLIN
1F32HL008882-01 (1993): $31200