ABSTRACT
Platelets are best known for their vasoprotective responses to injury and inflammation. Here, we have asked whether they also support vascular integrity when neither injury nor inflammation is present. Changes in vascular barrier function in dermal and meningeal vessels were measured in real time in mouse models using the differential extravasation of fluorescent tracers as a biomarker. Severe thrombocytopenia produced by two distinct methods caused increased extravasation of 40-kDa dextran from capillaries and postcapillary venules but had no effect on extravasation of 70-kDa dextran or albumin. This reduction in barrier function required more than 4 h to emerge after thrombocytopenia was established, reverting to normal as the platelet count recovered. Barrier dysfunction was also observed in mice that lacked platelet-dense granules, dense granule secretion machinery, glycoprotein (GP) VI, or the GPVI signaling effector phospholipase C (PLC) γ2. It did not occur in mice lacking α-granules, C type lectin receptor-2 (CLEC-2), or protease activated receptor 4 (PAR4). Notably, although both meningeal and dermal vessels were affected, intracerebral vessels, which are known for their tighter junctions between endothelial cells, were not. Collectively, these observations 1) highlight a role for platelets in maintaining vascular homeostasis in the absence of injury or inflammation, 2) provide a sensitive biomarker for detecting changes in platelet-dependent barrier function, 3) identify which platelet processes are required, and 4) suggest that the absence of competent platelets causes changes in the vessel wall itself, accounting for the time required for dysfunction to emerge.
Subject(s)
Blood Platelets/immunology , Blood Vessels/immunology , Hemostasis , Homeostasis , Animals , Blood Vessels/injuries , Blood Vessels/physiopathology , Female , Lectins, C-Type/genetics , Lectins, C-Type/immunology , Male , Meninges/blood supply , Meninges/immunology , Mice , Phospholipase C gamma/genetics , Phospholipase C gamma/immunology , Skin/blood supply , Skin/immunologyABSTRACT
Protease-activated receptor 4 (PAR4) mediates sustained thrombin signaling in platelets and is required for a stable thrombus. PAR4 is activated by proteolysis of the N terminus to expose a tethered ligand. The structural basis for PAR4 activation and the location of its ligand binding site (LBS) are unknown. Using hydrogen/deuterium exchange (H/D exchange), computational modeling, and signaling studies, we determined the molecular mechanism for tethered ligand-mediated PAR4 activation. H/D exchange identified that the LBS is composed of transmembrane 3 (TM3) domain and TM7. Unbiased computational modeling further predicted an interaction between Gly48 from the tethered ligand and Thr153 from the LBS. Mutating Thr153 significantly decreased PAR4 signaling. H/D exchange and modeling also showed that extracellular loop 3 (ECL3) serves as a gatekeeper for the interaction between the tethered ligand and LBS. A naturally occurring sequence variant (P310L, rs2227376) and 2 experimental mutations (S311A and P312L) determined that the rigidity conferred by prolines in ECL3 are essential for PAR4 activation. Finally, we examined the role of the polymorphism at position 310 in venous thromboembolism (VTE) using the International Network Against Venous Thrombosis (INVENT) consortium multi-ancestry genome-wide association study (GWAS) meta-analysis. Individuals with the PAR4 Leu310 allele had a 15% reduction in relative risk for VTE (odds ratio, 0.85; 95% confidence interval, 0.77-0.94) compared with the Pro310 allele. These data are consistent with our H/D exchange, molecular modeling, and signaling studies. In conclusion, we have uncovered the structural basis for PAR4 activation and identified a previously unrecognized role for PAR4 in VTE.
Subject(s)
Cell Membrane/chemistry , Proline/metabolism , Receptors, Thrombin/metabolism , Threonine/metabolism , Venous Thrombosis/pathology , Cell Membrane/metabolism , Genome-Wide Association Study , Humans , Proline/chemistry , Proline/genetics , Protein Conformation , Protein Domains , Receptors, Thrombin/chemistry , Receptors, Thrombin/genetics , Threonine/chemistry , Threonine/genetics , Venous Thrombosis/genetics , Venous Thrombosis/metabolismABSTRACT
Venous thromboembolism is a major cause of morbidity and mortality. The impact of the US Surgeon General's The Surgeon General's Call to Action to Prevent Deep Vein Thrombosis and Pulmonary Embolism in 2008 has been lower than expected given the public health impact of this disease. This scientific statement highlights future research priorities in venous thromboembolism, developed by experts and a crowdsourcing survey across 16 scientific organizations. At the fundamental research level (T0), researchers need to identify pathobiological causative mechanisms for the 50% of patients with unprovoked venous thromboembolism and to better understand mechanisms that differentiate hemostasis from thrombosis. At the human level (T1), new methods for diagnosing, treating, and preventing venous thromboembolism will allow tailoring of diagnostic and therapeutic approaches to individuals. At the patient level (T2), research efforts are required to understand how foundational evidence impacts care of patients (eg, biomarkers). New treatments, such as catheter-based therapies, require further testing to identify which patients are most likely to experience benefit. At the practice level (T3), translating evidence into practice remains challenging. Areas of overuse and underuse will require evidence-based tools to improve care delivery. At the community and population level (T4), public awareness campaigns need thorough impact assessment. Large population-based cohort studies can elucidate the biological and environmental underpinnings of venous thromboembolism and its complications. To achieve these goals, funding agencies and training programs must support a new generation of scientists and clinicians who work in multidisciplinary teams to solve the pressing public health problem of venous thromboembolism.
Subject(s)
Hemostasis/physiology , Thrombosis/diagnosis , Venous Thromboembolism/diagnosis , American Heart Association , Biomarkers , Evidence-Based Medicine , Expert Testimony , Humans , Practice Guidelines as Topic , Research , Translational Research, Biomedical , United States , Venous Thromboembolism/therapyABSTRACT
Protease-activated receptors (PARs) are G-protein-coupled receptors that are activated by proteolysis of the N-terminus, which exposes a tethered ligand that interacts with the receptor. Numerous studies have focused on the signaling pathways mediated by PARs. However, the structural basis for initiation of these pathways is unknown. Here, we describe a strategy for the expression and purification of PAR4. This is the first PAR family member to be isolated without stabilizing modifications for biophysical studies. We monitored PAR4 activation with histidine hydrogen-deuterium exchange. PAR4 has nine histidines that are spaced throughout the protein, allowing a global view of solvent accessible and nonaccessible regions. Peptides containing each of the nine His residues were used to determine the t1/2 for each His residue in apo or thrombin-activated PAR4. The thrombin-cleaved PAR4 exhibited a 2-fold increase (p > 0.01) in t1/2 values observed for four histidine residues (His180, His229, His240, and His380), demonstrating that these regions have decreased solvent accessibility upon thrombin treatment. In agreement, thrombin-cleaved PAR4 also was resistant to thermolysin digestion. In contrast, the rate of thermolysin proteolysis following stimulation with the PAR4 activation peptide was the same as that of unstimulated PAR4. Further analysis showed the C-terminus is protected in thrombin-activated PAR4 compared to uncleaved or agonist peptide-treated PAR4. The studies described here are the first to examine the tethered ligand activation mechanism for a PAR family member biophysically and shed light on the overall conformational changes that follow activation of PARs by a protease.
Subject(s)
Deuterium Exchange Measurement , Histidine/analysis , Receptors, Thrombin/analysis , Humans , Mass Spectrometry , Receptors, Thrombin/genetics , Receptors, Thrombin/isolation & purificationABSTRACT
BACKGROUND: The tumor suppressor actions of hexamethylene bis-acetamide (HMBA)-inducible protein 1 (HEXIM1) in the breast, prostate, melanomas, and AML have been reported by our group and others. Increased HEXIM1 expression caused differentiation and inhibited proliferation and metastasis of cancer cells. Historically, HEXIM1 has been experimentally induced with the hybrid polar compound HMBA, but HMBA is a poor clinical candidate due to lack of a known target, poor pharmacological properties, and unfavorable ADMETox characteristics. Thus, HEXIM1 induction is an intriguing therapeutic approach to cancer treatment, but requires better chemical tools than HMBA. METHODS: We identified and verified KDM5B as a target of HEXIM1 inducers using a chemical proteomics approach, biotin-NeutrAvidin pull-down assays, surface plasmon resonance, and molecular docking. The regulation of HEXIM1 by KDM5B and KDM5B inhibitors was assessed using chromatin immunoprecipitation assays, RT-PCR, western blotting, and depletion of KDM5B with shRNAs. The regulation of breast cancer cell phenotype by KDM5B inhibitors was assessed using western blots, differentiation assays, proliferation assays, and a mouse model of breast cancer metastasis. The relative role of HEXIM1 in the action of KDM5B inhibitors was determined by depleting HEXIM1 using shRNAs followed by western blots, differentiation assays, and proliferation assays. RESULTS: We have identified a highly druggable target, KDM5B, which is inhibited by small molecule inducers of HEXIM1. RNAi knockdown of KDM5B induced HEXIM1 expression, thus validating the specific negative regulation of tumor suppressor HEXIM1 by the H3K4me3/2 demethylase KDM5B. Known inhibitors of KDM5B were also able to induce HEXIM1 expression, inhibit cell proliferation, induce differentiation, potentiate sensitivity to cancer chemotherapy, and inhibit breast tumor metastasis. CONCLUSION: HMBA and 4a1 induce HEXIM1 expression by inhibiting KDM5B. Upregulation of HEXIM1 expression levels plays a critical role in the inhibition of proliferation of breast cancer cells using KDM5B inhibitors. Based on the novel molecular scaffolds that we identified which more potently induced HEXIM1 expression and data in support that KDM5B is a target of these compounds, we have opened up new lead discovery and optimization directions.
Subject(s)
Gene Expression Regulation, Neoplastic , Jumonji Domain-Containing Histone Demethylases/antagonists & inhibitors , Jumonji Domain-Containing Histone Demethylases/metabolism , Nuclear Proteins/antagonists & inhibitors , Nuclear Proteins/metabolism , RNA-Binding Proteins/genetics , Repressor Proteins/antagonists & inhibitors , Repressor Proteins/metabolism , Transcription Factors/genetics , Biomarkers, Tumor , Breast Neoplasms/etiology , Breast Neoplasms/metabolism , Breast Neoplasms/mortality , Breast Neoplasms/pathology , Cell Differentiation , Cell Line, Tumor , Cell Proliferation , Female , Gene Expression Regulation, Neoplastic/drug effects , Histones/metabolism , Humans , Jumonji Domain-Containing Histone Demethylases/chemistry , Kaplan-Meier Estimate , Models, Molecular , Neoplasm Staging , Nuclear Proteins/chemistry , Promoter Regions, Genetic , Protein Binding , RNA-Binding Proteins/chemistry , Recurrence , Repressor Proteins/chemistry , Structure-Activity Relationship , Transcription Factors/chemistryABSTRACT
Platelet-derived microparticles (PMPs) are associated with enhancement of metastasis and poor cancer outcomes. Circulating PMPs transfer platelet microRNAs (miRNAs) to vascular cells. Solid tumor vasculature is highly permeable, allowing the possibility of PMP-tumor cell interaction. Here, we show that PMPs infiltrate solid tumors in humans and mice and transfer platelet-derived RNA, including miRNAs, to tumor cells in vivo and in vitro, resulting in tumor cell apoptosis. MiR-24 was a major species in this transfer. PMP transfusion inhibited growth of both lung and colon carcinoma ectopic tumors, whereas blockade of miR-24 in tumor cells accelerated tumor growth in vivo, and prevented tumor growth inhibition by PMPs. Conversely, Par4-deleted mice, which had reduced circulating microparticles (MPs), supported accelerated tumor growth which was halted by PMP transfusion. PMP targeting was associated with tumor cell apoptosis in vivo. We identified direct RNA targets of platelet-derived miR-24 in tumor cells, which included mitochondrial mt-Nd2, and Snora75, a noncoding small nucleolar RNA. These RNAs were suppressed in PMP-treated tumor cells, resulting in mitochondrial dysfunction and growth inhibition, in an miR-24-dependent manner. Thus, platelet-derived miRNAs transfer in vivo to tumor cells in solid tumors via infiltrating MPs, regulate tumor cell gene expression, and modulate tumor progression. These findings provide novel insight into mechanisms of horizontal RNA transfer and add multiple layers to the regulatory roles of miRNAs and PMPs in tumor progression. Plasma MP-mediated transfer of regulatory RNAs and modulation of gene expression may be a common feature with important outcomes in contexts of enhanced vascular permeability.
Subject(s)
Blood Platelets/metabolism , Cell-Derived Microparticles/metabolism , Colonic Neoplasms/metabolism , Lung Neoplasms/metabolism , MicroRNAs/metabolism , Animals , Cell-Derived Microparticles/transplantation , Colonic Neoplasms/genetics , Colonic Neoplasms/pathology , Colonic Neoplasms/therapy , Humans , Lung Neoplasms/genetics , Lung Neoplasms/pathology , Lung Neoplasms/therapy , Mice , NADH Dehydrogenase/genetics , NADH Dehydrogenase/metabolism , Neoplasm Metastasis , Neoplasm Proteins/genetics , Neoplasm Proteins/metabolism , Receptors, Proteinase-ActivatedABSTRACT
Protease signaling in cells elicits multiple physiologically important responses via protease-activated receptors (PARs). There are 4 members of this family of G-protein-coupled receptors (PAR1-4). PARs are activated by proteolysis of the N terminus to reveal a tethered ligand. The rate-limiting step of PAR signaling is determined by the efficiency of proteolysis of the N terminus, which is regulated by allosteric binding sites, cofactors, membrane localization, and receptor dimerization. This ultimately controls the initiation of PAR signaling. In addition, these factors also control the cellular response by directing signaling toward G-protein or ß-arrestin pathways. PAR1 signaling on endothelial cells is controlled by the activating protease and heterodimerization with PAR2 or PAR3. As a consequence, the genetic and epigenetic control of PARs and their cofactors in physiologic and pathophysiologic conditions have the potential to influence cellular behavior. Recent studies have uncovered polymorphisms that result in PAR4 sequence variants with altered reactivity that interact to influence platelet response. This further demonstrates how interactions within the plasma membrane can control the physiological output. Understanding the structural rearrangement following PAR activation and how PARs are allosterically controlled within the plasma membrane will determine how best to target this family of receptors therapeutically. The purpose of this article is to review how signaling from PARs is influenced by alternative cleavage sites and the physical interactions within the membrane. Going forward, it will be important to relate the altered signaling to the molecular arrangement of PARs in the cell membrane and to determine how these may be influenced genetically.
Subject(s)
Cell Membrane/metabolism , Endothelial Cells/metabolism , Hemostasis/physiology , Protein Multimerization/physiology , Receptors, Proteinase-Activated/metabolism , Signal Transduction/physiologyABSTRACT
Human platelets contain microRNAs (miRNAs) and miRNA processing machinery, but their contribution to platelet function remains incompletely understood. Here, we show that murine megakaryocyte (MK)-specific knockdown of Dicer1, the ribonuclease that cleaves miRNA precursors into mature miRNAs, reduces the level of the majority of miRNAs in platelets. This leads to altered platelet messenger RNA (mRNA) expression profiles and mild thrombocytopenia. Fibrinogen receptor subunits Itga2b (αIIb) and Itgb3 (ß3) mRNAs were among the differentially expressed transcripts that are increased in platelets lacking Dicer1. Argonaute 2 (Ago2), a member of the miRNA silencing complex, co-immunoprecipitated with αIIband ß3mRNAs in wild-type platelets. Furthermore, co-immunoprecipitation experiments suggested reduced αIIb/ß3/Ago2 complexes in miRNA-deficient platelets. These results suggested that miRNAs regulate both integrin subunits. Subsequent 3' untranslated region luciferase reporter assays confirmed that the translation of both αIIband ß3mRNAs can be regulated by miRNAs miR-326, miR-128, miR-331, and miR-500. Consistent with these molecular changes, the deletion ofDicer1resulted in increased surface expression of integrins αIIband ß3, and enhanced platelet binding to fibrinogen in vivo and in vitro. Heightened platelet reactivity, shortened tail-bleeding time, and reduced survival following collagen/epinephrine-induced pulmonary embolism were also observed in Dicer1-deficient animals. CombinedPf4-cre-mediated deletion of Drosha and Dicer1 did not significantly exacerbate phenotypes observed in single Dicer1 knockout mice. In summary, these findings indicate that Dicer1-dependent generation of mature miRNAs in late-stage MKs and platelets modulates the expression of target mRNAs important for the hemostatic and thrombotic function of platelets.
Subject(s)
Blood Platelets/metabolism , DEAD-box RNA Helicases/metabolism , MicroRNAs/metabolism , RNA Processing, Post-Transcriptional/physiology , RNA, Messenger/metabolism , Ribonuclease III/metabolism , Animals , Argonaute Proteins/genetics , Argonaute Proteins/metabolism , DEAD-box RNA Helicases/genetics , Humans , Integrin alpha2/biosynthesis , Integrin alpha2/genetics , Integrin beta3/biosynthesis , Integrin beta3/genetics , Mice , Mice, Knockout , MicroRNAs/genetics , Pulmonary Embolism/chemically induced , Pulmonary Embolism/genetics , Pulmonary Embolism/metabolism , RNA, Messenger/genetics , Ribonuclease III/geneticsABSTRACT
Nephrotic syndrome is characterized by massive proteinuria and injury of specialized glomerular epithelial cells called podocytes. Studies have shown that, whereas low-concentration thrombin may be cytoprotective, higher thrombin concentrations may contribute to podocyte injury. We and others have demonstrated that ex vivo plasma thrombin generation is enhanced during nephrosis, suggesting that thrombin may contribute to nephrotic progression. Moreover, nonspecific thrombin inhibition has been shown to decrease proteinuria in nephrotic animal models. We thus hypothesized that thrombin contributes to podocyte injury in a protease-activated receptor-specific manner during nephrosis. Here, we show that specific inhibition of thrombin with hirudin reduced proteinuria in two rat nephrosis models, and thrombin colocalized with a podocyte-specific marker in rat glomeruli. Furthermore, flow cytometry immunophenotyping revealed that rat podocytes express the protease-activated receptor family of coagulation receptors in vivo High-concentration thrombin directly injured conditionally immortalized human and rat podocytes. Using receptor-blocking antibodies and activation peptides, we determined that thrombin-mediated injury depended upon interactions between protease-activated receptor 3 and protease-activated receptor 4 in human podocytes, and between protease-activated receptor 1 and protease-activated receptor 4 in rat podocytes. Proximity ligation and coimmunoprecipitation assays confirmed thrombin-dependent interactions between human protease-activated receptor 3 and protease-activated receptor 4, and between rat protease-activated receptor 1 and protease-activated receptor 4 in cultured podocytes. Collectively, these data implicate thrombinuria as a contributor to podocyte injury during nephrosis, and suggest that thrombin and/or podocyte-expressed thrombin receptors may be novel therapeutic targets for nephrotic syndrome.
Subject(s)
Kidney Glomerulus/metabolism , Nephrosis/metabolism , Podocytes/pathology , Receptor, PAR-1/metabolism , Receptors, Thrombin/metabolism , Thrombin/metabolism , Animals , Antithrombins/pharmacology , Cell Survival , Cells, Cultured , Disease Models, Animal , Gene Expression , Hirudins/pharmacology , Humans , Immunophenotyping , Nephrosis/complications , Nephrosis/pathology , Nephrosis/urine , Podocytes/metabolism , Proteinuria/etiology , Rats , Receptor, PAR-1/genetics , Receptors, Thrombin/genetics , Signal Transduction , Thrombin/antagonists & inhibitors , Thrombin/pharmacology , Thrombin/urineABSTRACT
Protease-activated receptor-4 (PAR4) is a G protein-coupled receptor (GPCR) for thrombin and is proteolytically activated, similar to the prototypical PAR1. Due to the irreversible activation of PAR1, receptor trafficking is intimately linked to signal regulation. However, unlike PAR1, the mechanisms that control PAR4 trafficking are not known. Here, we sought to define the mechanisms that control PAR4 trafficking and signaling. In HeLa cells depleted of clathrin by siRNA, activated PAR4 failed to internalize. Consistent with clathrin-mediated endocytosis, expression of a dynamin dominant-negative K44A mutant also blocked activated PAR4 internalization. However, unlike most GPCRs, PAR4 internalization occurred independently of ß-arrestins and the receptor's C-tail domain. Rather, we discovered a highly conserved tyrosine-based motif in the third intracellular loop of PAR4 and found that the clathrin adaptor protein complex-2 (AP-2) is important for internalization. Depletion of AP-2 inhibited PAR4 internalization induced by agonist. In addition, mutation of the critical residues of the tyrosine-based motif disrupted agonist-induced PAR4 internalization. Using Dami megakaryocytic cells, we confirmed that AP-2 is required for agonist-induced internalization of endogenous PAR4. Moreover, inhibition of activated PAR4 internalization enhanced ERK1/2 signaling, whereas Akt signaling was markedly diminished. These findings indicate that activated PAR4 internalization requires AP-2 and a tyrosine-based motif and occurs independent of ß-arrestins, unlike most classical GPCRs. Moreover, these findings are the first to show that internalization of activated PAR4 is linked to proper ERK1/2 and Akt activation.
Subject(s)
Adaptor Protein Complex 2/metabolism , MAP Kinase Signaling System/physiology , Megakaryocytes/metabolism , Receptors, Thrombin/metabolism , beta-Arrestins/metabolism , Adaptor Protein Complex 2/genetics , Amino Acid Motifs , Animals , HeLa Cells , Humans , Megakaryocytes/cytology , Mice , Protein Transport/physiology , Proto-Oncogene Proteins c-akt/genetics , Proto-Oncogene Proteins c-akt/metabolism , Receptors, Thrombin/genetics , beta-Arrestins/geneticsABSTRACT
In this issue of Blood, Aisiku et al describe a novel class of protease-activated receptor-1 (PAR1) inhibitors that block proinflammatory pathways but spare cytoprotective signaling in endothelial cells. These compounds, parmodulins, target the cytoplasmic face of PAR1, where they selectively interfere with Gαq, but not Gα12/13. This strategy of blocking specific pathways provides the ability to modulate the activity of receptors with multiple functions (such as PAR1) and may have therapeutic advantages.
Subject(s)
Endothelium, Vascular/injuries , Lactones/adverse effects , Pyridines/adverse effects , Receptor, PAR-1/antagonists & inhibitors , Thrombosis/drug therapy , Thrombosis/prevention & control , Animals , HumansABSTRACT
Thrombotic disease, a major life-threatening complication of nephrotic syndrome, has been associated with proteinuria and hypoalbuminemia severity. However, it is not fully understood how disease severity correlates with severity of the acquired hypercoagulopathy of nephrotic syndrome. Without this knowledge, the utility of proteinuria and/or hypoalbuminemia as biomarkers of thrombotic risk remains limited. Here, we show that two well established ex vivo hypercoagulopathy assays, thrombin generation and rotational thromboelastometry, are highly correlated with proteinuria and hypoalbuminemia in the puromycin aminonucleoside and adriamycin rat models of nephrotic syndrome. Notably, in the puromycin aminonucleoside model, hyperfibrinogenemia and antithrombin deficiency were also correlated with proteinuria severity, consistent with reports in human nephrotic syndrome. Importantly, although coagulation was not spontaneously activated in vivo with increasing proteinuria, vascular injury induced a more robust thrombotic response in nephrotic animals. In conclusion, hypercoagulopathy is highly correlated with nephrotic disease severity, but overt thrombosis may require an initiating insult, such as vascular injury. Our results suggest that proteinuria and/or hypoalbuminemia could be developed as clinically meaningful surrogate biomarkers of hypercoagulopathy to identify patients with nephrotic syndrome at highest risk for thrombotic disease and potentially target them for anticoagulant pharmacoprophylaxis.
Subject(s)
Blood Coagulation Disorders/blood , Blood Coagulation Disorders/urine , Hypoalbuminemia/blood , Nephrotic Syndrome/blood , Proteinuria/blood , Thrombosis/etiology , Animals , Antithrombins/blood , Biomarkers/blood , Biomarkers/urine , Blood Coagulation/physiology , Blood Coagulation Disorders/etiology , Disease Models, Animal , Doxorubicin , Elasticity , Fibrinogen/metabolism , Hemostasis , Male , Nephrotic Syndrome/chemically induced , Nephrotic Syndrome/complications , Puromycin Aminonucleoside , Rats , Rats, Wistar , Serum Albumin/metabolism , Severity of Illness Index , Thrombin/biosynthesisSubject(s)
Blood Platelets , Signal Transduction , Animals , Mice , Protein Isoforms , ThrombopoiesisABSTRACT
Bradykinin B2 receptor-deleted mice (Bdkrb2(-/-)) have delayed carotid artery thrombosis times and prolonged tail bleeding time resulting from elevated angiotensin II (AngII) and angiotensin receptor 2 (AT2R) producing increased plasma nitric oxide (NO) and prostacyclin. Bdkrb2(-/-) also have elevated plasma angiotensin-(1-7) and messenger RNA and protein for its receptor Mas. Blockade of Mas with its antagonist A-779 in Bdkrb2(-/-) shortens thrombosis times (58 ± 4 minutes to 38 ± 4 minutes) and bleeding times (170 ± 13 seconds to 88 ± 8 seconds) and lowers plasma nitrate (22 ± 4 µM to 15 ± 5 µM), and 6-keto-PGF1α (259 ± 103 pg/mL to 132 ± 58 pg/mL). Bdkrb2(-/-) platelets express increased NO, guanosine 3',5'-cyclic monophosphate, and cyclic adenosine monophosphate with reduced spreading on collagen, collagen peptide GFOGER, or fibrinogen. In vivo A-779 or combined L-NAME and nimesulide treatment corrects it. Bdkrb2(-/-) platelets have reduced collagen-related peptide-induced integrin α2bß3 activation and P-selectin expression that are partially corrected by in vivo A-779, nimesulide, or L-NAME. Bone marrow transplantations show that the platelet phenotype and thrombosis time depends on the host rather than donor bone marrow progenitors. Transplantation of wild-type bone marrow into Bdkrb2(-/-) hosts produces platelets with a spreading defect and delayed thrombosis times. In Bdkrb2(-/-), combined AT2R and Mas overexpression produce elevated plasma prostacyclin and NO leading to acquired platelet function defects and thrombosis delay.
Subject(s)
Angiotensin I/blood , Blood Platelets/metabolism , Epoprostenol/blood , Nitric Oxide/blood , Peptide Fragments/blood , Platelet Membrane Glycoproteins/metabolism , Proto-Oncogene Proteins/blood , Receptors, G-Protein-Coupled/blood , Angiotensin II/analogs & derivatives , Angiotensin II/pharmacology , Animals , Bleeding Time , Blood Platelets/drug effects , Bone Marrow Transplantation , Cyclic AMP/blood , Cyclic GMP/blood , Immunoblotting , Mice , Mice, 129 Strain , Mice, Knockout , NG-Nitroarginine Methyl Ester/pharmacology , Peptide Fragments/pharmacology , Platelet Aggregation Inhibitors/pharmacology , Proto-Oncogene Mas , Proto-Oncogene Proteins/genetics , Receptor, Angiotensin, Type 2/blood , Receptor, Bradykinin B2/deficiency , Receptor, Bradykinin B2/genetics , Receptors, G-Protein-Coupled/genetics , Reverse Transcriptase Polymerase Chain Reaction , Sulfonamides/pharmacology , Thrombosis/blood , Time FactorsABSTRACT
Thrombin is a potent platelet agonist that activates platelets and other cells of the cardiovascular system by cleaving its G-protein-coupled receptors, protease-activated receptor 1 (PAR1), PAR4, or both. We now show that cleaving PAR1 and PAR4 with α-thrombin induces heterodimer formation. PAR1-PAR4 heterodimers were not detected when unstimulated; however, when the cells were stimulated with 10 nm α-thrombin, we were able to detect a strong interaction between PAR1 and PAR4 by bioluminescence resonance energy transfer. In contrast, activating the receptors without cleavage using PAR1 and PAR4 agonist peptides (TFLLRN and AYPGKF, respectively) did not enhance heterodimer formation. Preventing PAR1 or PAR4 cleavage with point mutations or hirugen also prevented the induction of heterodimers. To further characterize the PAR1-PAR4 interactions, we mapped the heterodimer interface by introducing point mutations in transmembrane helix 4 of PAR1 or PAR4 that prevented heterodimer formation. Finally, we show that mutations in PAR1 or PAR4 at the heterodimer interface prevented PAR1-assisted cleavage of PAR4. These data demonstrate that PAR1 and PAR4 require allosteric changes induced via receptor cleavage by α-thrombin to mediate heterodimer formation, and we have determined the PAR1-PAR4 heterodimer interface. Our findings show that PAR1 and PAR4 have dynamic interactions on the cell surface that should be taken into account when developing and characterizing PAR antagonists.
Subject(s)
Protein Multimerization/physiology , Proteolysis , Receptor, PAR-1/metabolism , Receptors, Thrombin/metabolism , Thrombin/metabolism , Allosteric Regulation/drug effects , Animals , COS Cells , Chlorocebus aethiops , HEK293 Cells , HeLa Cells , Hirudins/pharmacology , Humans , Peptide Fragments/pharmacology , Point Mutation , Protein Multimerization/drug effects , Protein Structure, Quaternary , Protein Structure, Secondary , Receptor, PAR-1/genetics , Receptors, Thrombin/genetics , Thrombin/geneticsABSTRACT
BACKGROUND: Protease-activated receptor 4 (PAR4) mediates thrombin signaling on platelets and other cells. Our recent structural studies demonstrated that a single nucleotide polymorphism in extracellular loop 3 and PAR4-P310L (rs2227376) leads to a hyporeactive receptor. OBJECTIVES: The goal of this study was to determine how the hyporeactive PAR4 variant in extracellular loop 3 impacts platelet function in vivo using a novel knock-in mouse model (PAR4-322L). METHODS: A point mutation was introduced into the PAR4 gene F2rl3 via CRISPR/Cas9 to create PAR4-P322L, the mouse homolog to human PAR4-P310L. Platelet response to PAR4 activation peptide (AYPGKF), thrombin, ADP, and convulxin was monitored by αIIbß3 integrin activation and P-selectin translocation using flow cytometry or platelet aggregation. In vivo responses were determined by the tail bleeding assay and the ferric chloride-induced carotid artery injury model. RESULTS: PAR4-P/L and PAR4-L/L platelets had a reduced response to AYPGKF and thrombin measured by P-selectin translocation or αIIbß3 activation. The response to ADP and convulxin was unchanged among genotypes. In addition, both PAR4-P/L and PAR4-L/L platelets showed a reduced response to thrombin in aggregation studies. There was an increase in the tail bleeding time for PAR4-L/L mice. The PAR4-P/L and PAR4-L/L mice both showed an extended time to arterial thrombosis. CONCLUSION: PAR4-322L significantly reduced platelet responsiveness to AYPGKF and thrombin, which is in agreement with our previous structural and cell signaling studies. In addition, PAR4-322L had prolonged arterial thrombosis time. Our mouse model provides a foundation to further evaluate the role of PAR4 in other pathophysiological contexts.
Subject(s)
Blood Platelets , Mice, Inbred C57BL , Platelet Aggregation , Platelet Glycoprotein GPIIb-IIIa Complex , Receptors, Thrombin , Thrombin , Animals , Blood Platelets/metabolism , Receptors, Thrombin/genetics , Receptors, Thrombin/metabolism , Thrombin/metabolism , Platelet Glycoprotein GPIIb-IIIa Complex/metabolism , Platelet Glycoprotein GPIIb-IIIa Complex/genetics , Disease Models, Animal , Crotalid Venoms/pharmacology , Crotalid Venoms/toxicity , Adenosine Diphosphate/metabolism , Adenosine Diphosphate/pharmacology , P-Selectin/metabolism , P-Selectin/genetics , Point Mutation , Gene Knock-In Techniques , Signal Transduction , Thrombosis/genetics , Thrombosis/blood , Male , Chlorides , Mice , Platelet Activation , CRISPR-Cas Systems , Humans , Phenotype , Ferric Compounds , Oligopeptides , Lectins, C-Type , Receptors, Proteinase-ActivatedABSTRACT
Chronic kidney disease (CKD) is a leading cause of death, and its progression is driven by glomerular podocyte injury and loss, manifesting as proteinuria. Proteinuria includes urinary loss of coagulation zymogens, cofactors, and inhibitors. Importantly, both CKD and proteinuria significantly increase the risk of thromboembolic disease. Prior studies demonstrated that anticoagulants reduced proteinuria in rats and that thrombin injured cultured podocytes. Herein we aimed to directly determine the influence of circulating prothrombin on glomerular pathobiology. We hypothesized that (pro)thrombin drives podocytopathy, podocytopenia, and proteinuria. Glomerular proteinuria was induced with puromycin aminonucleoside (PAN) in Wistar rats. Circulating prothrombin was either knocked down using a rat-specific antisense oligonucleotide or elevated by serial intravenous infusions of prothrombin protein, which are previously established methods to model hypo- (LoPT) and hyper-prothrombinemia (HiPT), respectively. After 10 days (peak proteinuria in this model) plasma prothrombin levels were determined, kidneys were examined for (pro)thrombin co-localization to podocytes, histology, and electron microscopy. Podocytopathy and podocytopenia were determined and proteinuria, and plasma albumin were measured. LoPT significantly reduced prothrombin colocalization to podocytes, podocytopathy, and proteinuria with improved plasma albumin. In contrast, HiPT significantly increased podocytopathy and proteinuria. Podocytopenia was significantly reduced in LoPT vs. HiPT rats. In summary, prothrombin knockdown ameliorated PAN-induced glomerular disease whereas hyper-prothrombinemia exacerbated disease. Thus, (pro)thrombin antagonism may be a viable strategy to simultaneously provide thromboprophylaxis and prevent podocytopathy-mediated CKD progression.
ABSTRACT
Thrombin activates platelets by binding and cleaving protease-activated receptors 1 and 4 (PAR1 and PAR4). Because of the importance of PAR4 activation on platelets in humans and mice and emerging roles for PAR4 in other tissues, experiments were done to characterize the interaction between PAR4 homodimers. Bimolecular fluorescence complementation and bioluminescence resonance energy transfer (BRET) were used to examine the PAR4 homodimer interface. In bimolecular fluorescence complementation experiments, PAR4 formed homodimers that were disrupted by unlabeled PAR4 in a concentration-dependent manner, but not by rhodopsin. In BRET experiments, the PAR4 homodimers showed a specific interaction as indicated by a hyperbolic BRET signal in response to increasing PAR4-GFP expression. PAR4 did not interact with rhodopsin in BRET assays. The threshold maximum BRET signal was disrupted in a concentration-dependent manner by unlabeled PAR4. In contrast, rhodopsin was unable to disrupt the BRET signal, indicating that the disruption of the PAR4 homodimer is not due to nonspecific interactions. A panel of rho-PAR4 chimeras and PAR4 point mutants has mapped the dimer interface to hydrophobic residues in transmembrane helix 4. Finally, mutations that disrupted dimer formation had reduced calcium mobilization in response to the PAR4 agonist peptide. These results link the loss of dimer formation to a loss of PAR4 signaling.