Background: Heparin-induced thrombocytopenia (HIT) is an adverse drug reaction that is caused by IgG immune complexes that activate platelets and monocytes through Fc Receptor for IgG IIA (FcγRIIA). These immune complexes also fix complement. Stimulated monocytes produce proinflammatory cytokines including inflammasome dependent IL-1β release, which has not been investigated in HIT. Aims: Platelets have been shown to support monocyte inflammasome cytokine release during inflammation, primarily in microbial infection. This study sought to investigate the role of inflammasome cytokine production in HIT pathology. Methods: We investigated the role of the NLRP3 inflammasome following immune complex (IC) challenge in healthy donor whole blood or immortalized monocyte-like cells. The contribution of inflammasome assembly and cytokine release on HIT pathology was assessed utilizing NLRP3 inhibitor MCC950 in the triple allele ‘HIT’ mouse model. Plasma IL-1β cytokine production was measured using ELISA, and complete blood counts were taken to assess thrombocytopenia. Furthermore, the contribution of complement was assessed using IC challenge of isolated primary blood mononuclear cells suspended in plasma or heat-inactivated plasma. Results: Following human whole blood challenged or isolated mononuclear cell/platelet suspensions with two distinct immune complexes (heat aggregated IgG or heparin/PF4 complexes) we observed a significant increase in inflammasome cytokine production at 22 hours. Mononuclear/platelet cell fractions challenged with heparin/PF4 complexes had a significant increase in inflammasome cytokine secretion that was reduced with pretreatment with an FcγRIIA blocking antibody, or removal of complement with heat-inactivated plasma. We observed that administration of heparin and monoclonal αPF4/heparin antibody KKO to HIT mice resulted in thrombocytopenia and significantly increased circulating IL-1β, whereas mice treated with NLRP3 inflammasome inhibitor MCC950 protected mice from a platelet drop and cytokine production in our humanized mouse model of HIT in vivo. Conclusions: These data demonstrate for the first time that HIT IC mediates the production and secretion of inflammasome cytokines, which is influenced by the presence of platelets in a FcγRIIA dependent manner as well as by complement. Furthermore, the NLRP3 inflammasome is shown in HIT model mice to be a critical component of HIT pathophysiology. These results provide a scientific rationale for exploring mechanisms that drive platelet/monocyte/complement communication and targeting the inflammasome as a possible therapeutic strategy in HIT.

Objective Sepsis, which is the body’s response to overwhelming infection, can lead to septic shock, characterized by thrombocytopenia, hypotension, and organ damage. Polo-like kinase 3 (Plk3) is a ubiquitously expressed serine/threonine kinase, but its exact role in immune function is unknown.Approach and Results We used Plk3−/− and WT mice to evaluate the function of Plk3 in several models of severe sepsis. We found that WT mice die within 48 hours of 100% cecal ligation and puncture (CLP), while Plk3−/− mice survive. Survival following cecal slurry (CS) injection mirrored that of CLP as recipient WT mice succumbed, while recipient Plk3−/− mice survived. Analysis of bacterial load 24 hours after CLP revealed that WT blood and peritonea were loaded with bacteria, but bacteria were virtually undetectable in the peritonea or blood of Plk3−/− mice. To determine if bacteria infiltrate the blood of Plk3−/− mice shortly after infection, we measured bacteria 1 and 3 hours after CS injection. We found a time-dependent increase in bacteria in the blood of WT mice that was not observed in Plk3−/− mice. To determine if the lack of bacteria in the blood of Plk3−/− mice is due to enhanced clearance, we injected E. coli IV into WT and Plk3−/− mice. We found 75% mortality for both WT and Plk3−/− mice within 72 hours following IV injection suggesting that survival of Plk3−/− mice following enteric infection is likely due to reduced bacteremia.Conclusion Collectively our data suggest that Plk3 supports the systemic dissemination of bacteria and subsequent sepsis following enteric infection.### Competing Interest StatementThe authors have declared no competing interest.* Plk3 : polo-like kinase 3 WT : wildtype CLP : cecal ligation and puncture CS : cecal slurry PAMP : pathogen associated molecular pattern LPS : lipopolysaccharide PRR : pattern recognition receptor TLR4 : toll-like receptor 4 GFP : green fluorescent protein IL : interleukin CIB1 : calcium- and integrin-binding protein 1 PE : phycoerythrin AST : aspartate aminotransferase ALT : alanine aminotransferase PBS : phosphate buffered saline GPIX : glycoprotein IX WBC : white blood cell LY : lymphocyte NE : neutrophil MO : monocyte Plt : platelet

Background: Platelet protease activated receptor-4 (PAR4) Thr-120 allele is an activating allele associated with increased platelet reactivity and reduced response to aspirin in vivo. We previously identified this genotype to be associated with an increased odds of preterm birth and placental vascular pathology in an unselected pregnant population. Aspirin is recommended in high risk pregnancies to prevent preeclampsia and preterm birth. It is unclear whether this genotype impacts aspirin effectiveness in pregnancy. Objective: We aimed to compare response to aspirin, as measured by Platelet Function Assay (PFA-100) epinephrine closure time, and perinatal outcomes, by PAR-4 genotype in high risk pregnant singletons recommended 81mg aspirin daily Methods: This is a planned secondary analysis of a prospective cohort study of N=130 high-risk singleton gestations recommended aspirin daily. As part of the primary study, participants were enrolled in the first trimester. They had PFA-100 epinephrine closure time assessed at baseline, and again 2-4 weeks after aspirin initiation (follow up-1) and finally at 28-32 weeks gestation (follow up-2). Participants were included if they had PAR-4 genotyping completed, took 81mg aspirin daily with at least 75% adherence by pill counting, and completed requisite follow up. Primary outcome was PFA-100 epinephrine closure time at follow up-1. Secondary outcomes were PFA-100 at follow up-2, incidence of hypertensive disorders of pregnancy and preterm birth, incidence of placental intervillous thrombosis. Exposure was defined as PAR4 Thr-120 homozygous vs not. Comparison was unadjusted with Mann Whitney U or chi-square, and adjusted with multivariable regression. Results: N=122 had genotyping complete, n=24 (19.6%) were homozygous for PAR-4 Thr120. N=106 completed follow up-1 with >75% adherence. Baseline characteristics were similar except participants homozygous for PAR4 Thr-120 had a significantly higher rate of preterm birth in a prior pregnancy (50.0% vs 16.1%, p=0.004). There was no significant difference in median aspirin response at follow-up 1 in those homozygous vs not: (152.0 [80-300] vs 151[91-300] sec, p=0.90) in unadjusted or adjusted analyses. At follow-up 2 there was a non-significant reduced response: 147.3[66-300] vs 167.0 [76-300]sec, median difference -19 (-43 to 6) sec, p=0.13, and was not significant in adjusted analysis. Rates of preeclampsia and preterm birth were similar. There was a higher rate of placental intervillous thrombosis in PAR4-Thr120 homozygous, but not statistically significant (16.7% vs 3.9%, p=0.08). Conclusion: Patients homozygous for PAR-4 Thr120 had higher incidence of prior preterm birth history, a significant risk factor for poor perinatal outcome. PFA-100 epinephrine closure time as measure of response to 81mg aspirin daily was similar across genotypes, although it is possible we were underpowered to detect a difference in the setting of other known factors (obesity, diabetes). Overall perinatal outcomes were similar, although PAR-4 Thr120 homozygous genotype may be associated with higher rate of placental vascular pathology even with daily aspirin use. Further study is needed to evaluate the role of this genotype in aspirin response, dose based response, and placental vascular pathology in high risk pregnancies.

Mechanisms of proteostasis in anucleate circulating platelets are unknown and may regulate platelet function. We investigated the hypothesis that plasma-borne growth factors/hormones (GFHs) maintain constitutive translation in circulating platelets to facilitate reactivity. Bio-orthogonal noncanonical amino acid tagging (BONCAT) coupled with liquid chromatography-tandem mass spectrometry analysis revealed constitutive translation of a broad-spectrum translatome in human platelets dependent upon plasma or GFH exposure, and in murine circulation. Freshly isolated platelets from plasma showed homeostatic activation of translation-initiation signaling pathways: phosphorylation of p38/ERK upstream kinases, essential intermediate MNK1/2, and effectors eIF4E/4E-BP1. Plasma starvation led to loss of pathway phosphorylation, but it was fully restored with 5-minute stimulation by plasma or GFHs. Cycloheximide or puromycin infusion suppressed ex vivo platelet GpIIb/IIIa activation and P-selectin exposure with low thrombin concentrations and low-to-saturating concentrations of adenosine 5'-diphosphate (ADP) or thromboxane analog but not convulxin. ADP-induced thromboxane generation was blunted by translation inhibition, and secondary-wave aggregation was inhibited in a thromboxane-dependent manner. Intravenously administered puromycin reduced injury-induced clot size in cremaster muscle arterioles, and delayed primary hemostasis after tail tip amputation but did not delay neither final hemostasis after subsequent rebleeds, nor final hemostasis after jugular vein puncture. In contrast, these mice were protected from injury-induced arterial thrombosis and thrombin-induced pulmonary thromboembolism (PE), and adoptive transfer of translation-inhibited platelets into untreated mice inhibited arterial thrombosis and PE. Thus, constitutive plasma GFH-driven translation regulates platelet G protein-coupled receptor reactivity to balance hemostasis and thrombotic potential.

Protease-activated receptors (PARs) are a class of integral membrane proteins that are cleaved by a variety of proteases, most notably thrombin, to reveal a tethered ligand and promote activation. PARs are critical mediators of platelet function in hemostasis and thrombosis, and therefore are attractive targets for anti-platelet therapies. Animal models studying platelet PAR physiology have relied heavily on genetically modified mouse strains, which have provided ample insight but have some inherent limitations. The current review aims to summarize the notable PAR expression and functional differences between the mouse and human, in addition to highlighting some recently developed tools to further study human physiology in mouse models.

Thrombosis with thrombocytopenia syndrome (TTS) is a rare but potentially severe adverse event following immunization with adenovirus vector-based COVID-19 vaccines such as Ad26.COV2.S (Janssen) and ChAdOx1 (AstraZeneca). However, no case of TTS has been reported in over 1.5 million individuals who received a second immunization with Ad26.COV2.S in the United States. Here we utilize transcriptomic and proteomic profiling to compare individuals who receive two doses of Ad26.COV2.S with those vaccinated with BNT162b2 or mRNA-1273. Initial Ad26.COV2.S vaccination induces transient activation of platelet and coagulation and innate immune pathways that resolve by day 7; by contrast, patients with TTS show robust upregulation of these pathways on days 15–19 following initial Ad26.COV2.S vaccination. Meanwhile, a second immunization or a reduced initial dose of Ad26.COV2.S induces lower activation of these pathways than does the full initial dose. Our data suggest a role of coagulation and proinflammatory pathways in TTS pathogenesis, which may help optimize vaccination regimens to reduce TTS risk.

BACKGROUND: Heparin-induced thrombocytopenia (HIT) is a major concern for all individuals that undergo cardiac bypass surgeries or require prolonged heparin exposure. HIT is a life- and limb-threatening adverse drug reaction with an immune response following the formation of ultra-large immune complexes that drive platelet activation through the receptor FcγRIIA. Thrombotic events remain high following the standard of care treatment with anticoagulants, while increasing risk of bleeding complications. This study sought to investigate a novel approach to treatment of HIT. Recent reports demonstrate increased procoagulant activity in HIT; however, these reports required analysis ex vivo, and relevance in vivo remains unclear. METHODS: Using human and mouse model systems, we investigated the cooperativity of PARs (protease-activated receptors) and FcγRIIA in HIT. We challenged humanized FcγRIIA transgenic mice with or without endogenous mouse Par4 (denoted as IIA-Par4 +/+ or IIA-Par4 − /− , respectively) with a well-established model IgG immune complex (anti [α]-CD9). Furthermore, we assessed the procoagulant phenotype and efficacy to treat HIT utilizing inhibitor of 12-LOX (12[S]-lipoxygenase), VLX-1005, previously reported to decrease platelet activation downstream of FcγRIIA and PAR4, using the triple allele HIT mouse model. RESULTS: IIA-Par4 +/+ mice given αCD9 were severely thrombocytopenic, with extensive platelet-fibrin deposition in the lung. In contrast, IIA-Par4 −/− mice had negligible thrombocytopenia or pulmonary platelet-fibrin thrombi. We observed that pharmacological inhibition of 12-LOX resulted in a significant reduction in both platelet procoagulant phenotype ex vivo, and thrombocytopenia and thrombosis in our humanized mouse model of HIT in vivo. CONCLUSIONS: These data demonstrate for the first time the need for dual platelet receptor (PAR and FcγRIIA) stimulation for fibrin formation in HIT in vivo. These results extend our understanding of HIT pathophysiology and provide a scientific rationale for targeting the procoagulant phenotype as a possible therapeutic strategy in HIT.

Vaccine-induced immune thrombotic thrombocytopenia (VITT) is a rare adverse effect of coronavirus disease 2019 (COVID-19) adenoviral vector vaccines. Most cases of VITT have been linked to the ChAdOx1 nCoV-19 (Vaxzevria AstraZeneca) vaccine. 1-3 However, similar clinical and laboratory events occur in patients after vaccination with Ad26.COV2.S (Johnson & Johnson/Janssen). 4 VITT is char-acterized by moderate to severe thrombocytopenia with arterial and/or venous thrombosis, often occurring in unusual locations. 1-3 VITT involves immunoglobulin G (IgG) antibodies binding to platelet factor 4 (PF4/CXCL4) causing intense platelet activation. 1,2,5 The binding site of VITT antibodies after AstraZeneca vaccination has been localized within the heparin-binding region on PF4, an epitope restricted to 8 amino acids. 5 However, the binding site of VITT antibodies on PF4 after the Johnson & Johnson/Janssen vaccine is unknown. In this study, we investigated the binding characteristics of anti-PF4 antibodies in 3 patients with VITT after they received the Johnson & Johnson/Janssen vaccine. Platelet activation, binding response, and epitope determination were measured as previously described. 5-13 All 3 patients received the Johnson & Johnson/Janssen vaccine and did not receive heparin therapy before the

BACKGROUND:Human and mouse platelets both express protease-activated receptor (PAR) 4 but sequence alignment reveals differences in several functional domains. These differences may result in functional disparities between the receptors which make it difficult to translate PAR4 studies using mice to human platelet physiology. OBJECTIVES:To generate transgenic mice that express human, but not mouse, PAR4 and directly compare human and mouse PAR4 function in the same platelet environment. METHODS:Transgenic mice were made using a genomic clone of the F2RL3 gene (encoding PAR4) and backcrossed with Par4 KO mice. For certain experiments, mice were bred with GRK6 KO mice. Tail bleeding time and platelet function in response to PAR4-activating peptide were assessed. RESULTS:Human F2RL3 was successfully integrated into the mouse genome, transgenic mice were crossed to the mPar4 KO background (PAR4 tg/KO), and PAR4 was functionally expressed on platelets. Compared to WT, PAR4 tg/KO mice exhibited shortened tail bleeding time and their platelets were more responsive to PAR4-AP as assessed by α-granule release and integrin activation. The opposite was observed with thrombin. Knocking out GRK6 had no effect on human PAR4-expressing platelets, unlike mouse Par4-expressing platelets. PAR4 tg/KO platelets exhibited greater Ca2+ area under the curve and more robust extracellular vesicle release than WT stimulated with PAR4-AP. CONCLUSION:These data suggest that (1) human PAR4- and mouse Par4-mediated signaling are different and (2) the feedback regulation mechanisms of human and mouse PAR4 are different. These functional differences are important to consider when interpreting PAR4 studies done with mice.

Heparin-induced thrombocytopenia (HIT) is a rare, but deadly disease that occurs in a small percentage of patients following administration of heparin. In these patients, heparin interacts with platelet factor 4 (PF4) resulting in an immune response to the heparin/PF4 complex. This immune complex can then bind to the immune receptor on the platelet, FcγRIIa, to induce platelet activation, clot formation, thrombosis, consumption, and bleeding. This potentially deadly disease results in both a thrombotic event as well as bleeding. Currently, there is only one FDA-approved intervention for HIT, the direct thrombin inhibitor argatroban. However, many patients on argatroban remain at risk for thrombosis, bleeding, and in some cases death. Therefore, we sought to develop a new drug to intervene in patients with HIT by targeting the 12-lipoxygenase enzyme in the platelet which regulates FcγRIIa activity in the platelet. Using mouse models of HIT as well as ex vivo administration of the 12-lipoxygenase inhibitor VLX-1005 (previously known as ML355). In mice expressing the human immune receptor on their platelets, we were able to show that administration of VLX-1005 following induction of HIT in these mice resulted in a blunted thrombocytopenia as well as reduced platelet activation and thrombus formation in the blood. We further demonstrated that coagulation was not impacted by VLX-1005 using thromboelestography while argatroban significantly delayed onset of coagulation and clot formation. Furthermore, bleeding time in these mice was not altered with VLX-1005, while mice on argatroban required cauterization of their tails to stop the bleeding. Finally, Human whole blood was shown in whole blood aggregometry as well as high shear arterial flow chamber experiments to be protected from platelet activation and clot formation in the presence of VLX-1005. The studies presented here demonstrate the potential effectiveness of VLX-1005 in intervention of platelet activation, clot formation, and thrombosis in both mouse models and human blood and support VLX-1005 as a new class of drug for the treatment of HIT in patients without the risk of bleeding.

The critical role of G protein-coupled receptor kinase 2 (GRK2) in regulating cardiac function has been well documented for >3 decades. Targeting GRK2 has therefore been extensively studied as a novel approach to treating cardiovascular disease. However, little is known about its role in hemostasis and thrombosis. We provide here the first evidence that GRK2 limits platelet activation and regulates the hemostatic response to injury. Deletion of GRK2 in mouse platelets causes increased platelet accumulation after laser-induced injury in the cremaster muscle arterioles, shortens tail bleeding time, and enhances thrombosis in adenosine 5'-diphosphate (ADP)-induced pulmonary thromboembolism and in FeCl3-induced carotid injury. GRK2-/- platelets have increased integrin activation, P-selectin exposure, and platelet aggregation in response to ADP stimulation. Furthermore, GRK2-/- platelets retain the ability to aggregate in response to ADP restimulation, indicating that GRK2 contributes to ADP receptor desensitization. Underlying these changes in GRK2-/- platelets is an increase in Ca2+ mobilization, RAS-related protein 1 activation, and Akt phosphorylation stimulated by ADP, as well as an attenuated rise of cyclic adenosine monophosphate levels in response to ADP in the presence of prostaglandin I2. P2Y12 antagonist treatment eliminates the phenotypic difference in platelet accumulation between wild-type and GRK2-/- mice at the site of injury. Pharmacologic inhibition of GRK2 activity in human platelets increases platelet activation in response to ADP. Finally, we show that GRK2 binds to endogenous Gβγ subunits during platelet activation. Collectively, these results show that GRK2 regulates ADP signaling via P2Y1 and P2Y12, interacts with Gβγ, and functions as a signaling hub in platelets for modulating the hemostatic response to injury.

The interindividual variation in the functional response of platelets to activation by agonists is heritable. Genome-wide association studies (GWASs) of quantitative measures of platelet function have identified fewer than 20 distinctly associated variants, some with unknown mechanisms. Here, we report GWASs of pathway-specific functional responses to agonism by adenosine 5'-diphosphate, a glycoprotein VI-specific collagen mimetic, and thrombin receptor-agonist peptides, each specific to 1 of the G protein-coupled receptors PAR-1 and PAR-4, in subsets of 1562 individuals. We identified an association (P = 2.75 x 10(-40)) between a common intronic variant, rs10886430, in the G protein-coupled receptor kinase 5 gene (GRK5) and the sensitivity of platelets to activate through PAR-1. The variant resides in a megakaryocyte-specific enhancer that is bound by the transcription factors GATA1 and MEIS1. The minor allele (G) is associated with fewer GRK5 transcripts in platelets and the greater sensitivity of platelets to activate through PAR-1. We show that thrombin-mediated activation of human platelets causes binding of GRK5 to PAR-1 and that deletion of the mouse homolog Grk5 enhances thrombin-induced platelet activation sensitivity and increases platelet accumulation at the site of vascular injury. This corroborates evidence that the human G allele of rs10886430 is associated with a greater risk for cardiovascular disease. In summary, by combining the results of pathway-specific GWASs and expression quantitative trait locus studies in humans with the results from platelet function studies in Grk5(-/-) mice, we obtain evidence that GRK5 regulates the human platelet response to thrombin via the PAR-1 pathway.

Sepsis and autoimmune diseases remain major causes of morbidity and mortality. The last decade has seen a new appreciation of platelets in host defense, in both immunity and thrombosis. Platelets are first responders in the blood to microbes or non‐microbial antigens. The role of platelets in physiologic immunity is counterbalanced by their role in pathology, for example, microvascular thrombosis. Platelets encounter microbes and antigens via both innate and adaptive immune processes; platelets also help to shape the subsequent adaptive response. FcγRIIA is a receptor for immune complexes opsonized by IgG or pentraxins, and expressed in humans by platelets, granulocytes, monocytes and macrophages. With consideration of the roles of IgG and Fc receptors, the host response to microbes and autoantigens can be called adaptive immunothrombosis. Here we review newer developments involving platelet FcγRIIA in humans and humanized mice in immunity and thrombosis, with special attention to heparin‐induced thrombocytopenia, systemic lupus erythematosus, and bacterial sepsis. Human genetic diversity in platelet receptors and the utility of humanized mouse models are highlighted.

For decades oncogenic RAS proteins were considered undruggable due to a lack of accessible binding pockets on the protein surfaces. Seminal early research in RAS biology uncovered the basic paradigm of post-translational isoprenylation of RAS polypeptides, typically with covalent attachment of a farnesyl group, leading to isoprenylmediated RAS anchorage at the plasma membrane and signal initiation at those sites. However, the failure of farnesyltransferase inhibitors to translate to the clinic stymied anti-RAS therapy development. Over the past ten years, a more complete picture has emerged of RAS protein maturation, intracellular trafficking, and location, positioning and retention in subdomains at the plasma membrane, with a corresponding expansion in our understanding of how these properties of RAS contribute to signal outputs. Each of these aspects of RAS regulation presents a potential vulnerability in RAS function that may be exploited for therapeutic targeting, and inhibitors have been identified or developed that interfere with RAS for nearly all of them. This review will summarize current understanding of RAS membrane targeting with a focus on highlighting development and outcomes of inhibitors at each step.

Clinical management of asthma and chronic obstructive pulmonary disease (COPD) has primarily relied on the use of beta 2 adrenergic receptor agonists (bronchodilators) and corticosteroids, and more recently, monoclonal antibody therapies (biologics) targeting specific cytokines and their functions. Although these approaches provide relief from exacerbations, questions remain on their long-term efficacy and safety. Furthermore, current therapeutics do not address progressive airway remodeling (AR), a key pathological feature of severe obstructive lung disease. Strikingly, agonists of the bitter taste receptors (TAS2Rs) deliver robust bronchodilation, curtail allergen-induced inflammatory responses in the airways and regulate airway smooth muscle (ASM) cell proliferation and mitigate features of AR in vitro and in animal models. The scope of this review is to provide a comprehensive and systematic insight into our current understanding of TAS2Rs with an emphasis on the molecular events that ensue TAS2R activation in distinct airway cell types and expand on the pleiotropic effects of TAS2R targeting in mitigating various pathological features of obstructive lung diseases. Finally, we will discuss specific opportunities that could help the development of selective agonists for specific TAS2R subtypes in the treatment of asthma.

Prostaglandin E2 (PGE(2)) is produced in the airway during allergic lung inflammation and both promotes and inhibits features of asthma pathology. These mixed effects relate to 4 E-prostanoid (EP) receptor subtypes (EP1, 2, 3 and 4) expressed at different levels on different resident and infiltrating airway cells. Although studies have asserted both EP2 and EP4 expression in human airway smooth muscle (HASM), a recent study asserted EP4 to be the functionally dominant EP subtype in HASM. Herein, we employ recently-developed subtype-selective ligands to investigate singular or combined EP2 and EP4 receptor activation in regulating HASM signaling and proliferation. The subtype specificity of ONO-AE1-259-01 (EP2 agonist) and ONO-AE1-329 (EP4 agonist) was first demonstrated in human embryonic kidney 293 cells stably expressing different EP receptor subtypes. EP receptor knockdown and subtype-selective antagonists demonstrated EP2 and EP4 receptor responsiveness in HASM cells to the specific ONO compounds, whereas PGE(2) appeared to preferentially signal via the EP4 receptor. Both singular EP2 and EP4 receptor agonists inhibited HASM proliferation, and combined EP2 and EP4 receptor agonism exhibited positive cooperativity in both chronic G(s)-mediated signaling and inhibiting HASM proliferation. These findings suggest both EP2 and EP4 are functionally important in HASM, and their combined targeting optimally inhibits airway smooth muscle proliferation.

Ovarian cancer G protein-coupled receptor 1 (OGR1) is a recently deorphanized G protein-coupled receptor shown to signal in response to low extracellular pH (↓pHo) or certain benzodiazepines. The pleiotropic nature of OGR1 signaling in human airway smooth muscle (HASM) cells suggests that OGR1 is a potential therapeutic target for the management of obstructive lung diseases. However, the basic pharmacological and regulatory features of OGR1 remain poorly understood. We employed model systems of heterologously expressed [human embryonic kidney 293 (HEK293) cells] or endogenous (HASM) OGR1 to assess changes in expression, subcellular localization, and signaling capabilities following acute or chronic treatment with ↓pHo or the benzodiazepines lorazepam and sulazepam. In HEK293 cells expressing OGR1, treatment with ↓pHo and/or lorazepam, but not sulazepam, caused rapid OGR1 internalization. In HASM cells, acute treatment with ↓pHo or benzodiazepines did not alter abundance of OGR1 mRNA; however, significant downregulation was observed following chronic treatment. Acute and chronic pretreatment of HASM cells with sulazepam or lorazepam resulted in receptor desensitization as demonstrated by reduced phosphorylation of vasodilator-stimulated phosphoprotein (VASP) or p42/p44 upon rechallenge. Acid (acute but not chronic) pretreatment of HASM cells induced desensitization of OGR1-mediated VASP (but not p42/p44) phosphorylation. In contrast to a recent study reporting OGR1 upregulation and sensitization in cardiac tissue subject to ischemic/acidic insult, chronic OGR1 activation in multiple model systems did not increase OGR1 expression or signaling capacity. The ability to induce OGR1 internalization and desensitization was activator dependent, reflecting the ability of different activators to induce specific receptor confirmations and engagement of specific heterotrimeric G proteins.

GPCRs have diverse signaling capabilities, based on their ability to assume various conformations. Moreover, it is now appreciated that certain ligands can promote distinct receptor conformations and thereby bias signaling toward a specific pathway to differentially affect cell function. The recently deorphanized G protein-coupled receptor OGR1 [ovarian cancer G protein-coupled receptor 1 (GPR68)] exhibits diverse signaling events when stimulated by reductions in extracellular pH. We recently demonstrated airway smooth muscle cells transduce multiple signaling events, reflecting a diverse capacity to couple to multiple G proteins. Moreover, we recently discovered that the benzodiazepine lorazepam, more commonly recognized as anagonist of the gamma-aminobutyric acid A(GABA(A)) receptor, canfunctionas an allosteric modulator of OGR1 and, similarly, can promote multiple signaling events. In this study, we demonstrated that different benzodiazepines exhibit a range of biases for OGR1, with sulazepam selectively activating the canonical Gs of the G protein signaling pathway, in heterologous expression systems, as well as in several primary cell types. These findings highlight the potential power of biased ligand pharmacology for manipulating receptor signaling qualitatively, to preferentially activate pathways that are therapeutically beneficial.

Together H-, N- and KRAS mutations are major contributors to ~30% of all human cancers. Thus, Ras inhibition remains an important anti-cancer strategy. The molecular mechanisms of isotypic Ras oncogenesis are still not completely understood. Monopharmacological therapeutics have not been successful in the clinic. These disappointing outcomes have led to attempts to target elements downstream of Ras, mainly targeting either the Phosphatidylinositol 3-Kinase (PI3K) or Mitogen-Activated Protein Kinase (MAPK) pathways. While several such approaches are moderately effective, recent efforts have focused on preclinical evaluation of combination therapies to improve efficacies. This review will detail current understanding of the contributions of plasma membrane microdomain targeting of Ras to mitogenic and tumorigenic signaling and tumor progression. Moreover, this review will outline novel approaches to target Ras in cancers, including targeting schemes for new drug development, as well as putative re-purposing of drugs in current use to take advantage of blunting Ras signaling by interfering with Ras plasma membrane microdomain targeting and retention.