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Targeted drug delivery to disease-associated activated neutrophils can provide novel therapeutic opportunities while avoiding systemic effects on immune functions. We created a nanomedicine platform that uniquely utilizes an α(1)-antitrypsin-derived peptide to confer binding specificity to neutrophil elastase on activated neutrophils. Surface decoration with this peptide enabled specific anchorage of nanoparticles to activated neutrophils and platelet–neutrophil aggregates, in vitro and in vivo. Nanoparticle delivery of a model drug, hydroxychloroquine, demonstrated significant reduction of neutrophil activities in vitro and a therapeutic effect on murine venous thrombosis in vivo. This innovative approach of cell-specific and activation-state-specific targeting can be applied to several neutrophil-driven pathologies.

Platelet-neutrophil interactions regulate ischemic vascular injury. Platelets are activated by serine proteases that cleave protease-activated receptor (PAR) amino termini, resulting in an activating tethered ligand. Neutrophils release cathepsin G (CatG) at sites of injury and inflammation, which activates PAR4 but not PAR1, although the molecular mechanism of CatG-induced PAR4 activation is unknown. We show that blockade of the canonical PAR4 thrombin cleavage site did not alter CatG-induced platelet aggregation, suggesting CatG cleaves a different site than thrombin. Mass spectrometry analysis using PAR4 N-terminus peptides revealed CatG cleavage at Ser67-Arg68. A synthetic peptide, RALLLGWVPTR, representing the tethered ligand resulting from CatG proteolyzed PAR4, induced PAR4-dependent calcium flux and greater platelet aggregation than the thrombin-generated GYPGQV peptide. Mutating PAR4 Ser67or Arg68 reduced CatG-induced calcium flux without affecting thrombin-induced calcium flux. Dog platelets, which contain a conserved CatG PAR4 Ser-Arg cleavage site, aggregated in response to human CatG and RALLLGWVPTR, while mouse (Ser-Gln) and rat (Ser-Glu) platelets were unresponsive. Thus, CatG amputates the PAR4 thrombin cleavage site by cleavage at Ser67-Arg68 and activates PAR4 by generating a new functional tethered ligand. These findings support PAR4 as an important CatG signaling receptor and suggest a novel therapeutic approach for blocking platelet-neutrophil-mediated pathophysiologies.

Objective Plasminogen/plasmin is a serine protease system primarily responsible for degrading fibrin within blood clots. Plasminogen mediates its functions by interacting with plasminogen receptors on the cell surface. H2B, one such plasminogen receptor, is found on the surface of several cell types including macrophages. Both basic and clinical studies support the role of plasminogen in the process of foam cell formation (FCF), a hallmark of atherosclerosis. Growing evidence also implicates serine protease-activated receptors (PARs) in atherosclerosis. These receptors are also found on macrophages, and plasmin is capable of activating PAR1 and PAR4. The goal of this study was to determine the extent of H2B's contribution to plasminogen-mediated FCF by macrophages and if PARs are involved in this process. Approach and results Treating macrophages with plasminogen increases their oxidized low-density lipoprotein uptake and plasminogen-mediated foam cell formation (Plg-FCF) significantly. The magnitude of Plg-FCF correlates with cell-surface expression of the H2B level. H2B blockade or downregulation reduces Plg-FCF, whereas its overexpression or high endogenous levels increases Plg-FCF. Modulating PAR1 level in mouse macrophages affects Plg-FCF. Activation/overexpression of PAR1 increases and its blockade/knockdown reduces this response. Confocal imaging indicates that both H2B and PAR1 colocalize with clathrin coated pits on the surface of macrophages, and reducing expression of clathrin or interfering with the clathrin-coated pits integrity reduces Plg-FCF. Conclusion Our data indicate that the magnitude of Plg-FCF by macrophages is proportional to the H2B levels and demonstrate for the first time that PAR1 is involved in this process and that the integrity of clathrin-coated pits is required for the full effect of Plg-induced FCF.

Background Protease‐activated receptor (PAR) 1 and PAR4 are key thrombin signal mediators for human platelet activation and aggregation in response to vascular injury. They are primarily activated by thrombin cleavage of the N‐terminus to expose a tethered ligand. In addition to the canonical activation by thrombin, a growing panel of proteases can also elicit PAR1‐ or PAR4‐mediated signal transduction. Recently, complement factor C4a was reported as the first endogenous agonist for both PAR1 and PAR4. Further, it is the first endogenous nontethered ligand that activates PAR1 and PAR4. These studies were conducted with human microvascular cells; the impact of C4a on platelet PARs is unknown. Objectives The goal of this study was to interrogate PAR1 and PAR4 activation by C4a on human platelets. Methods Platelet‐rich plasma (PRP) was isolated from healthy donors. PRP was stimulated with C4a, and the platelet aggregation was measured. Human embryonic kidney (HEK) 293 Flp‐In T‐rex cells were used to further test if C4a stimulation can initiate PAR1‐ or PAR4‐mediated Gαq signaling, which was measured by intracellular calcium mobilization. Results C4a failed to elicit platelet aggregation via PAR1‐ or PAR4‐mediated manner. In addition, no PAR1‐ or PAR4‐mediated calcium mobilization was observed upon C4a stimulation on HEK293 cells. Conclusions Complement factor C4a does not activate PAR1 or PAR4 on human platelets. These data show that PAR1 and PAR4 activation by C4a on microvascular cells likely requires a cofactor, which reinforces the concept of cell type–specific regulation of protease signaling.

Protease activated receptors (PARs) are G-protein coupled receptors (GPCRs) that have a unique activation mechanism. Unlike other GPCRs that can be activated by free ligands, under physiological conditions, PARs are activated by the tethered ligand, which is a part of their N-terminus that is unmasked by proteolysis. It has been 30 years since the first member of the family, PAR1, was identified. In this review, we will discuss this unique tethered ligand mediate receptor activation of PARs in detail: how they interact with the proteases, the complex structural rearrangement of the receptors upon activation, and the termination of the signaling. We also summarize the structural studies of the PARs and how single nucleotide polymorphisms impact the receptor reactivity. Finally, we review the current strategies for inhibiting PAR function with therapeutic targets for anti-thrombosis. The focus of this review is PAR1 and PAR4 as they are the thrombin signal mediators on human platelets and therapeutics targets. We also include the structural studies of PAR2 as it informs the mechanism of action for PARs in general.

Proteases are important regulators of cell behavior, survival, and apoptosis. They communicate to cells directly through a special class of G‐protein–coupled receptors known as protease‐activated receptors (PARs). N‐terminal PAR proteolysis unmasks a neo‐N‐terminus, which serves as a tethered ligand to activate PARs. Using this unique irreversible activation mechanism, PARs relay information across cell membranes. The year 2020 is the 30th year since discovery of the first member of this family, PAR1. In this illustrated review, we highlight achievements in the PAR field over the past 3 decades. Additionally, the known expression profiles of PARs in human tissues and across species are portrayed. We also illustrate the tethered ligand activation mechanism, which is unique to PARs, and PAR regulatory mechanisms. PAR1 was originally named “thrombin receptor” because thrombin was the first protease identified to activate PAR1. However, over the past 30 years, a growing number of proteases have been found to cleave PARs and trigger differential downstream signaling depending on cleavage site, cell type, and species. We exemplify the diversity of PAR1‐mediated signaling outcomes in platelets and endothelial cells as pertinent examples to the hemostasis, thrombosis, and vascular biology fields. Further, the termination and regulation of PAR signaling via endocytosis and currently available pharmacologic approaches are depicted. We conclude with portrayal of clinically translational aspects of PAR biology including pharmacologic manipulation and single‐nucleotide polymorphisms.

Background Platelet adhesion is the critical process mediating stable thrombus formation. Previous reports of cadherin‐6 on human platelets have demonstrated its role in platelet aggregation and thrombus formation. Objectives We aimed to further characterize the importance of cadherin‐6 in thrombosis in vivo. Methods Cadherin‐6 platelet expression was evaluated by western blotting, flow cytometry, and immunoprecipitation. Thrombosis was evaluated using the FeCl3 and Rose Bengal carotid artery models in C57Bl6 mice treated with anti–cadherin‐6 or IgG and wild‐type or Cdh6−/− mice. Platelet function was compared in wild‐type and Cdh6−/− mice using tail‐clip assays, aggregometry, and flow cytometry. Results Human platelet expression of cadherin‐6 was confirmed at ~3000 copies per platelet. Cdh6−/− mice or those treated with anti–cadherin‐6 antibody showed an increased time to occlusion in both thrombosis models. Cadherin‐6 was not expressed on mouse platelets, and there were no differences in tail bleeding times, platelet aggregation, or platelet activation in wild‐type versus Cdh6−/− mice. Conclusions Cadherin‐6 plays an essential role in thrombosis in vivo. However, cadherin‐6 is not expressed on murine platelets. These data are in contrast to human platelets, which express a functional cadherin‐6/catenin complex. The essential, platelet‐independent role for cadherin‐6 in hemostasis may allow it to be an effective and safe therapeutic target.

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.

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.

Targeted drug delivery to disease-associated activated neutrophils can provide novel therapeutic opportunities while avoiding systemic effects on immune functions. We created a nanomedicine platform that uniquely utilizes an α

The protease activated receptor (PAR) family is a group of G-protein coupled receptors (GPCRs) activated by proteolytic cleavage of the extracellular domain. PARs are expressed in a variety of cell types with crucial roles in homeostasis, immune responses, inflammation, and pain. PAR3 is the least researched of the four PARs, with little known about its expression and function. We sought to better understand its potential function in the peripheral sensory nervous system. Mouse single-cell RNA sequencing data demonstrates that PAR3 is widely expressed in dorsal root ganglion (DRG) neurons. Co-expression of PAR3 mRNA with other PARs was identified in various DRG neuron subpopulations, consistent with its proposed role as a coreceptor of other PARs. We developed a lipid tethered PAR3 agonist, C660, that selectively activates PAR3 by eliciting a Ca2+ response in DRG and trigeminal neurons. In vivo, C660 induces mechanical hypersensitivity and facial grimacing in WT but not PAR3-/- mice. We characterized other nociceptive phenotypes in PAR3-/- mice and found a loss of hyperalgesic priming in response to IL-6, carrageenan, and a PAR2 agonist, suggesting that PAR3 contributes to long-lasting nociceptor plasticity in some contexts. To examine the potential role of PAR3 in regulating the activity of other PARs in sensory neurons, we administered PAR1, PAR2, and PAR4 agonists and assessed mechanical and affective pain behaviors in WT and PAR3-/- mice. We observed that the nociceptive effects of PAR1 agonists were potentiated in the absence of PAR3. Our findings suggest a complex role of PAR3 in the physiology and plasticity of nociceptors. PERSPECTIVE: We evaluated the role of PAR3, a G-protein coupled receptor, in nociception by developing a selective peptide agonist. Our findings suggest that PAR3 contributes to nociception in various contexts and plays a role in modulating the activity of other PARs.

Introduction Nephrotic syndrome (NS) is associated with an acquired hypercoagulopathy that drives its strong predilection for life-threatening thrombosis. We previously demonstrated that hypercoagulopathy is proportional to NS disease severity in animal models. Therefore, hypercoagulopathy and disease severity may inform thrombosis risk and better guide therapeutic decision making. The objective of this study was thus to establish the relationship between disease severity and hypercoagulopathy in human NS. Materials and methods Thrombin generation assays (TGA) were performed on biorepository plasma samples from a prospective longitudinal NS cohort study. TGA was also determined on a separate cohort of incident NS patients. Multivariable regression was used to build NS-hypercoagulopathy relationship models. Results Endogenous thrombin potential (ETP) was the TGA parameter most strongly correlated with NS severity and was proportional to conventional measures of NS disease activity including proteinuria, hypercholesterolemia, and hypoalbuminemia. The overall disease activity model was well correlated with ETP (R2 = 0.38). The relationship with disease activity was confirmed in the second cohort. These models further revealed that ETP is related to disease activity in a manner dependent on remission status. Conclusion Consistent with our previously reported animal model observations, we found that the combination of proteinuria, hypercholesterolemia, and hypoalbuminemia correlated with ETP-defined hypercoagulopathy. Hypercoagulopathy improved significantly with partial or complete NS remission. These data are expected to inform studies designed to stratify thrombotic risk for patients with NS.

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.

BackgroundProtease activated receptor 1 (PAR1) and PAR4 are key thrombin signal mediators for human platelet activation and aggregation in response to vascular injury. They are primarily activated by thrombin cleavage of the N-terminus to expose a tethered ligand. In addition to the canonical activation by thrombin, a growing panel of proteases can also elicit PAR1- or PAR4-mediate signal transduction. Recently, complement factor C4a was reported as the first endogenous agonist for both PAR1 and PAR4. Further, it is the first endogenous non-tethered ligand that activates PAR1 and PAR4. These studies were conducted with human microvascular cells; the impact of C4a on platelet PARs is unknown.ObjectivesThe goal of this study was to interrogate PAR1 and PAR4 activation by C4a on human platelets.MethodsPlatelet rich plasma (PRP) were isolated from healthy donors. PRP was stimulated with C4a and the platelet aggregation was measured. HEK293 Flp-In T-rex cells were used to further test if C4a stimulation can initiate PAR1- or PAR4-mediated Gαqsignaling, which was measured by intracellular calcium mobilization.ResultsC4a failed to elicit platelet aggregation via PAR1- or PAR4-mediated manner. In addition, no PAR1- or PAR4-mediated calcium mobilization was observed upon C4a stimulation on HEK293 cells.ConclusionsComplement factor C4a does not activate PAR1 or PAR4 on human platelets. These data show that PAR1 and PAR4 activation by C4a on microvascular cells likely requires a cofactor, which re-enforces the concept of cell-type specific regulation of protease signaling.EssentialsC4a is an agonist for both PAR1 and PAR4 on human microvascular cells.We sought to determine if C4a activates human platelets through PAR1 or PAR4.C4a does not activate PAR1 and PAR4 on human platelets.This re-enforces the concept of cell-type specific regulation of PAR signaling.

BackgroundPlatelet adhesion is the critical process mediating stable thrombus formation. Previous reports of cadherin-6 on human platelets have demonstrated its role in platelet aggregation and thrombus formation.ObjectivesWe aimed to further characterize the importance of cadherin-6 in thrombosis in vivo.MethodsCadherin-6 platelet expression was evaluated by western blotting, flow cytometry and immunoprecipitation. Thrombosis was evaluated using the FeCl3 and Rose Bengal carotid artery models in C57Bl6 mice treated with anti-cadherin-6 or IgG and wild-type or Cdh6-/- mice. Platelet function was compared in wild type and Cdh6-/- mice using tail-clip assays and aggregometry.ResultsHuman platelet expression of cadherin-6 was confirmed at ~3,000 copies per platelet. Cdh6-/- mice or those treated with anti-Cadherin-6 antibody showed an increased time to occlusion in both thrombosis models. Cadherin-6 was not expressed on mouse platelets, and there were no differences in tail bleeding times or platelet aggregation in wild-type versus Cdh6-/- mice.ConclusionsCadherin-6 plays an essential role in thrombosis in vivo. However, cadherin-6 is not expressed on murine platelets. These data are in contrast to human platelets, which express a functional cadherin-6/catenin complex. The essential, platelet-independent role for cadherin-6 in hemostasis may allow it to be an effective and safe therapeutic target.EssentialsCadherin-6 function in thrombus formation was investigated in vivo using two murine models of thrombosis.Blocking or deleting cadherin-6 significantly delayed time to occlusionHuman platelets express cadherin-6, but murine platelets do not.

Nephrotic syndrome (NS) is associated with an acquired hypercoagulopathy and strong predilection for life-threatening thrombotic complications. Current anticoagulant prophylaxis guidelines are based upon controversial hypoalbuminemia thresholds. Anticoagulant prophylaxis is thus inconsistently implemented due to a lack of high-grade safety and efficacy data. Development of evidence-based clinical parameters that define thrombosis risk may thus refine safe and effective anticoagulant use. Endogenous thrombin potential (ETP) is a recognized measure of hypercoagulopathy and established predictor of both incident and recurrent thrombosis. This study utilized biorepository samples from a prospective longitudinal cohort study to demonstrate that ETP is proportional to NS disease activity, resulting in multivariable models that are significantly correlated with ETP. The relationship with disease activity was confirmed in a separate cohort. These models revealed that ETP is related to disease activity in a manner dependent on remission status and that proteinuria and hypercholesterolemia exert the strongest influence on ETP. In contrast to prior epidemiology studies, which did not include hypercoagulopathy measures, we found that hypoalbuminemia was less predictive of ETP. These findings are consistent with our previously reported animal model observations and are expected to inform the design of clinical trials that will generate high-grade evidence to guide more effective and safer anticoagulant use and thus reduce life-threatening thrombotic events in patients with NS.

Thrombosis and inflammation have been commonly identified as linked processes. While neutrophils are one of the first responders to areas of inflammation, they are also the first cells to arrive and collaborate with platelets in thrombosis. Recently, the crosstalk between platelets and neutrophils has been recognized in a growing number of pathophysiological processes known as thrombo-inflammation. Neutrophil and platelet functions are tightly regulated by G-protein coupled receptors (GPCRs), because excessive signaling can cause tissue injury and pathological thrombi, respectively. In this chapter, we describe the GPCRs expressed on neutrophils and platelets and their response to thrombo-inflammatory stimuli, as well as the regulators of this coordinated response. We also highlight pharmacological agents and structural studies that may inform future efforts to target these GPCRs therapeutically.

Essentials The rs773902 SNP results in differences in platelet protease-activated receptor (PAR4) function. The functional consequences of rs773902 were analyzed in human platelets and stroke patients. rs773902 affects thrombin-induced platelet function, PAR4 desensitization, stroke association. Enhanced PAR4 Thr120 effects on platelet function are blocked by ticagrelor. SUMMARY: Background F2RL3 encodes protease-activated receptor (PAR) 4 and harbors an A/G single-nucleotide polymorphism (SNP) (rs773902) with racially dimorphic allelic frequencies. This SNP mediates an alanine to threonine substitution at residue 120 that alters platelet PAR4 activation by the artificial PAR4-activation peptide (PAR4-AP) AYPGKF. Objectives To determine the functional effects of rs773902 on stimulation by a physiological agonist, thrombin, and on antiplatelet antagonist activity. Methods Healthy human donors were screened and genotyped for rs773902. Platelet function in response to thrombin was assessed without and with antiplatelet antagonists. The association of rs773902 alleles with stroke was assessed in the Stroke Genetics Network study. Results As compared with rs773902 GG donors, platelets from rs773902 AA donors had increased aggregation in response to subnanomolar concentrations of thrombin, increased granule secretion, and decreased sensitivity to PAR4 desensitization. In the presence of PAR1 blockade, this genotype effect was abolished by higher concentrations of or longer exposure to thrombin. We were unable to detect a genotype effect on thrombin-induced PAR4 cleavage, dimerization, and lipid raft localization; however, rs773902 AA platelets required a three-fold higher level of PAR4-AP for receptor desensitization. Ticagrelor, but not vorapaxar, abolished the PAR4 variant effect on thrombin-induced platelet aggregation. A significant association of modest effect was detected between the rs773902 A allele and stroke. Conclusion The F2RL3 rs773902 SNP alters platelet reactivity to thrombin; the allelic effect requires P2Y12 , and is not affected by gender. Ticagrelor blocks the enhanced reactivity of rs773902 A platelets. PAR4 encoded by the rs773902 A allele is relatively resistant to desensitization and may contribute to stroke risk.

Serotonin receptors (5-HT3AR) directly regulate gut movement, and drugs that inhibit 5-HT3AR function are used to control emetic reflexes associated with gastrointestinal pathologies and cancer therapies. The 5-HT3AR function involves a finely tuned orchestration of three domain movements that include the ligand-binding domain, the pore domain, and the intracellular domain. Here, we present the structure from the full-length 5-HT3AR channel in the apo-state determined by single-particle cryo-electron microscopy at a nominal resolution of 4.3 Å. In this conformation, the ligand-binding domain adopts a conformation reminiscent of the unliganded state with the pore domain captured in a closed conformation. In comparison to the 5-HT3AR crystal structure, the full-length channel in the apo-conformation adopts a more expanded conformation of all the three domains with a characteristic twist that is implicated in gating.

Non-compressible uncontrolled hemorrhage remains a major cause of mortality from traumatic injuries. Additionally, patients with congenital, disease-associated or drug-induced hemostatic dysfunctions, may often be at risk of excessive bleeding. Therefore, treatments that render rapid hemostasis are clinically significant in potentially saving lives. The clinical gold standard for this is the transfusion of whole blood (WB) or blood components (e.g. controlled ratios of platelets, RBCs, and plasma), as evidenced by several clinical studies (e.g. PROPPR, PROMMTT and PAMPer). However, the availability of such blood products is donor-dependent, their shelf-life is limited due to contamination risks, and, their portability and storage is often challenging. While extensive research efforts are currently being focused on addressing these challenges, e.g. using low titer Group O whole blood, cold-storage and freeze-drying of platelets and plasma, in vitro generation of platelets from iPSCs etc., a parallel research focus has emerged in designing biomaterials-based I.V.-administrable technologies (nanoparticles, polymers etc.) that can provide specific functional attributes of hemostasis while allowing donor-independent manufacturing, scale-up, and on-demand availability. Prominent examples of these are 'synthetic platelet' (SynthoPlate) nanoparticles that recapitulate platelet's binding interactions with von Willebrand Factor (vWF), collagen and active platelet integrin GPIIb-IIIa, flexible platelet-like particles (PLP) that bind fibrin to recapitulate platelet's biomechanical properties, fibrinogen function-mimicking nanoparticles that amplify the aggregation of active platelets, peptide-modified synthetic polymers (e.g. PolySTAT, HAPPI etc.) that render clot stabilization etc. In this framework, we present the design and evaluation of I.V.-administrable unique platelet-inspired nanoparticles that render injury site-targeted, enzyme-responsive direct delivery of thrombin, to site-specifically augment fibrin generation for hemostasis. Our design is inspired by platelets' crucial hemostatic mechanisms of : (i) rapidly accumulating at the injury site to form a plug and (ii) serving as a coagulation amplifier via presenting anionic phospholipids on the activated platelet surface to render tenase and prothrombinase factor assemblies leading to thrombin (FIIa) burst, which can then site-specifically convert fibrinogen to fibrin. Thrombin delivery to augment hemostasis is clinically well-accepted, as exemplified by products like Tisseel where thrombin and fibrinogen are co-delivered by syringe directly at wound site. Researchers have also studied thrombin-loaded topical dressings and topical administration of thrombin-loaded particles on wounds to mitigate bleeding, but these cannot be used intravenously. A recent interesting study has explored encapsulation of thrombin-loaded nanoparticles inside actual platelets with the idea of the particles being released (analogous to granule secretion) upon platelet activation, but this was only demonstrated in vitro because optimizing this complex strategy for consistent in vivo function may be challenging. Our approach circumvents these challenges by: (i) loading consistent amount of thrombin in I.V.-administrable lipid nanoparticles (LNPs), (ii) directly targeting the thrombin-loaded LNPs (TLNPs) to the injury site via specific binding to vWF and collagen, and (iii) releasing the loaded thrombin via particle destabilization by the action of injury site-specific enzyme phospholipase A2 for in situ fibrin production. We evaluated the TLNPs in vitro in human blood and plasma where hemostatic defects were created by platelet depletion and anticoagulant treatment. Spectrophotometric studies of fibrin generation, rotational thromboelastometry (ROTEM) based studies of clot characteristics and BioFlux microfluidics based real-time imaging of fibrin generation under simulated vascular flow conditions, confirmed the ability of TLNPs to restore fibrin generation in hemostatic dysfunction settings. Subsequently, the in vivo feasibility of these TLNPs was tested in a mouse tail-clip bleeding model where a combination of platelet depletion plus anticoagulant treatment was used to render significant hemostatic defect. TLNPs were able to effectively reduce tail-bleeding in mice. Figure 1 Figure 1. Disclosures Sen Gupta: Haima Therapeutics: Other: Co-founder, Patents & Royalties: US 9107845, US 9107963.

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.

Thrombin, the most potent platelet agonist in vivo, activates human platelets through PAR1 (protease-activated receptor 1) and PAR4 (protease-activated receptor 4) by proteolysis of the N terminus, which exposes the tethered ligands.1 Although these receptors are activated by the same mechanism, the overall sequence identity is only ≈33%, and each has a distinct mode of interaction with thrombin, which impacts the subsequent rates of proteolysis.2–4 PAR1 is an efficient substrate and responds to subnanomolar thrombin. In contrast, PAR4 requires ≈10-fold more thrombin for proteolysis. This difference led to the hypothesis that PAR4 was a redundant backup thrombin receptor, which limited the interest in developing PAR4 antagonists. See accompanying article on page 448 On further examination, the dual receptor system offers the intriguing possibility of pharmacologically fine-tuning thrombin signaling in platelets by taking advantage of the individual contributions of PAR1 and PAR4.5 Both receptors initiate signaling through Gq and G12/13 pathways but with distinct kinetics. PAR1 activation results in a rapid transient signal. In contrast, PAR4 mediates prolonged signaling that is required for stable thrombus formation (Figure [A]). Blocking the sustained signaling from PAR4 may limit thrombosis, while leaving the transient PAR1 signaling mechanism available to initiate hemostasis and limit bleeding (Figure [B]). With this goal in mind, PAR4 has been targeted with inhibitory antibodies, intracellular peptides, and small molecules.6–8 Several compounds that achieve PAR4 antagonism, such as YD-3 and ML354, are currently used as tools to study PAR4 …

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.

Thrombin, a serine protease, is a potent platelet agonist that signals primarily through the protease-activated receptors (PARs). The PAR family contains four G-protein coupled receptors (GPCRs), named PARs1–4, that are activated by enzymatic cleavage of the N-terminus to expose a tethered ligand that intramolecularly activates the receptor. The expression profile of PARs on platelets is species-specific; PAR1 and PAR4 are expressed on human platelets and are the focus of this chapter. PAR1 and PAR4 interact with thrombin through distinct mechanisms. Once activated, PARs mediate signaling through overlapping pathways with distinct kinetics. PAR1 signaling is rapid and short-lived, while PAR4 initiates slower but sustained signaling. PARs have become important targets for antiplatelet therapies and the development of PAR antagonists has facilitated a greater understanding of their molecular structure and physiological roles in vivo.

F2RL3 encodes protease-activated receptor 4 (PAR4) and harbors a A/G SNP (rs773902) with racially dimorphic allelic frequencies. This SNP mediates an alanine to threonine substitution at residue 120 that alters platelet PAR4 activation by the artificial PAR4-activation peptide (PAR4-AP), AYPGKF, which does not occur in vivo. Platelet function studies from healthy human donors demonstrated that compared to rs773902 GG donors, platelets from rs773902 AA donors had increased aggregation to low concentrations (2+ signaling, increased granule secretion and decreased PAR4 desensitization. The increased thrombin sensitivity of rs773902 AA platelets required both PAR4 and PAR1. Whereas receptor cleavage, dimerization, or lipid raft localization were not different between platelets from the two F2RL3 genotypes, rs773902 AA platelets required more than 3-fold amounts of PAR4-AP for receptor desensitization. PAR4-AP stimulation of PAR4-transfected COS-7 cells increased PAR4 levels on the cell surface. Thrombin-stimulated platelets from rs773902 AA donors displayed reduced inhibition by P2Y12 and PAR1 blockers, but not aspirin. Finally, the association of rs773902 alleles with stroke was assessed using the Stroke Genetics Network (SiGN) study. Patients carrying at least one copy of the rs773902 A allele had an increased risk of both large vessel stroke and diabetes in the SiGN dataset. In summary, both PAR4 and PAR1 are required for platelet activation to sub-nanomolar thrombin concentrations. Compared to rs773902 GG platelets, rs773902 AA platelets have a lower EC50 for thrombin and are relatively resistant to receptor desensitization, perhaps due to anterograde trafficking of internal PAR4 to the plasma membrane post-stimulation. The association of the rs773902 A variant with large vessel stroke, diabetes and partial resistance to PAR1 and P2Y12 inhibition must be considered for development of personalized anti-platelet agents.

Platelets are the primary component of pathological arterial thrombi. As such, antiplatelet therapy is a key strategy for treatment of acute coronary syndrome (ACS). The recent publication by Wong and colleagues earlier this year reported a compound, BMS-986120, that targets protease activated receptor 4 (PAR4) in a reversible manner (1). BMS-986120 effectively inhibits thrombus formation with minimal bleeding complications in their animal model (1). Their paper demonstrates the potential of targeting PAR4 as a promising antiplatelet therapeutic approach (1).

Coagulation factor XII (FXII) deficiency is associated with decreased neutrophil migration, but the mechanisms remain uncharacterized. Here, we examine how FXII contributes to the inflammatory response. In 2 models of sterile inflammation, FXII-deficient mice (F12-/-) had fewer neutrophils recruited than WT mice. We discovered that neutrophils produced a pool of FXII that is functionally distinct from hepatic-derived FXII and contributes to neutrophil trafficking at sites of inflammation. FXII signals in neutrophils through urokinase plasminogen activator receptor-mediated (uPAR-mediated) Akt2 phosphorylation at S474 (pAktS474). Downstream of pAkt2S474, FXII stimulation of neutrophils upregulated surface expression of αMβ2 integrin, increased intracellular calcium, and promoted extracellular DNA release. The sum of these activities contributed to neutrophil cell adhesion, migration, and release of neutrophil extracellular traps in a process called NETosis. Decreased neutrophil signaling in F12-/- mice resulted in less inflammation and faster wound healing. Targeting hepatic F12 with siRNA did not affect neutrophil migration, whereas WT BM transplanted into F12-/- hosts was sufficient to correct the neutrophil migration defect in F12-/- mice and restore wound inflammation. Importantly, these activities were a zymogen FXII function and independent of FXIIa and contact activation, highlighting that FXII has a sophisticated role in vivo that has not been previously appreciated.

The Case Western Reserve University School of Medicine medical education curriculum includes a series of supplemental online pharmacology modules (“PharmWeb”) that run concurrently with the standard curriculum to introduce first-year students to fundamental principles of pharmacology. Although this voluntary online pharmacology curriculum correlated with higher test scores during the formal, required portion of pharmacology learning in the curriculum, the utilization among medical students of the PharmWeb resource was low. We hypothesized that improving the design and accessibility of the online curriculum using commercial design principles could promote adoption of this voluntary curriculum. The modules were redesigned with a cleaner interface, and convenience of access to each module was enhanced by adding links to relevant modules within required, formative weekly pharmacology multiple choice questions. We report here an increased use of this optional online introduction to pharmacological principles and increased satisfaction in learning pharmacology that correlates with the design intervention.

Protease activated receptor 4 (PAR4) is a G protein coupled receptor (GPCR) which is activated by proteolytic cleavage of its N-terminal exodomain. This generates a tethered ligand that activates the receptor and triggers downstream signaling events. With the current focus in the development of anti-platelet therapies shifted towards PARs, new reagents are needed for expanding the field's knowledge on PAR4. Currently, there are no PAR4 reagents which are able to detect activation of the receptor.Monoclonal PAR4 antibodies were purified from hybridomas producing antibody that were generated by fusing splenocytes with NS-1 cells. Immunoblotting, immunofluorescence, and flow cytometry were utilized to detect the epitope for each antibody and to evaluate the interaction of the antibodies with cells.Here, we report the successful generation of three monoclonal antibodies to the N-terminal extracellular domain of PAR4: 14H6, 5F10, and 2D6. We mapped the epitope on PAR4 of 14H6, 5F10, and 2D6 antibodies to residues (48-53), (41-47), and (73-78), respectively. Two of the antibodies (14H6 and 5F10) interacted close to the thrombin cleavage and were sensitive to α-thrombin cleavage of PAR4. In addition, 5F10 was able to partially inhibit the cleavage of PAR4 expressed in HEK293 cells by α-thrombin.These new antibodies provide a means to monitor endogenous PAR4 expression and activation by proteases on cells.

Serotonin receptors (5-HT3AR) directly regulate gut movement, and drugs that inhibit 5-HT3AR function are used to control emetic reflexes associated with gastrointestinal pathologies and cancer therapies. The 5-HT3AR function involves a finely tuned orchestration of three domain movements that include the ligand-binding domain, the pore domain, and the intracellular domain. Here, we present the structure from the full-length 5-HT3AR channel in the apo-state determined by single-particle cryo-electron microscopy at a nominal resolution of 4.3 Å. In this conformation, the ligand-binding domain adopts a conformation reminiscent of the unliganded state with the pore domain captured in a closed conformation. In comparison to the 5-HT3AR crystal structure, the full-length channel in the apo-conformation adopts a more expanded conformation of all the three domains with a characteristic twist that is implicated in gating., Serotonin receptor (5-HT3AR), a pentameric ligand-gated ion channel, regulates numerous gastrointestinal functions. Here the authors provide a cryo-electron microscopic structure from the full-length 5-HT3AR in the apo-state which corresponds to a resting conformation of the channel.

Essentials ClotChip is a novel microsensor for comprehensive assessment of ex vivo hemostasis. Clinical samples show high sensitivity to detecting the entire hemostatic process. ClotChip readout exhibits distinct information on coagulation factor and platelet abnormalities. ClotChip has potential as a point-of-care platform for comprehensive hemostatic analysis. SUMMARY Background Rapid point-of-care (POC) assessment of hemostasis is clinically important in patients with a variety of coagulation factor and platelet defects who have bleeding disorders. Objective To evaluate a novel dielectric microsensor, termed ClotChip, which is based on the electrical technique of dielectric spectroscopy for rapid, comprehensive assessment of whole blood coagulation. Methods The ClotChip is a three-dimensional, parallel-plate, capacitive sensor integrated into a single-use microfluidic channel with miniscule sample volume (

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.

The serine protease thrombin is a potent activator of platelets. It binds to two classes of receptors, the GPIb-IX complex and protease-activated receptors (PARs). PARs constitute a family of four G protein-coupled receptors named PARs 1–4 that mediate protease signaling in a wide variety of cells. In this chapter we describe the genomic organization and expression of PARs in platelets from humans and other species. Thrombin is the primary activator of PARs in platelets. We focus on the factors that determine the specificity and rate of cleavage PARs by thrombin, which are the initiating events of thrombin-induced platelet activation. Human platelets express PAR1 and PAR4, which have both overlapping and distinct signaling pathways. These differences have become increasingly important as therapeutics targeting PAR1 and PAR4 are developed. In addition to thrombin-PAR interactions, the activation and downstream signaling of PAR subtypes is influenced by dimerization with one another and other platelet GPCRs. We also discuss the recent identification of genetic variations that impact PAR4 signaling in humans. Finally, we highlight the differences in PAR expression on platelets across species that impact how animal models can be used as preclinical tools.

Background Human platelet activation and aggregation is a complex process. To date, many therapies have been developed targeting proteins that mediate this process to prevent unwanted activation. However, the current standard of care for acute coronary syndromes still has limitations, including bleeding risk. Objective To evaluate the protease-activated receptor 4 (PAR4) anionic cluster as a viable antiplatelet target by using a polyclonal antibody (CAN12). Methods We used western blotting, aggregation and secretion ex vivo to evaluate the ability of CAN12 to interact with PAR4 and inhibit platelet activation. The effects of CAN12 in vivo were evaluated with the Rose Bengal arterial thrombosis model and two models of hemostasis. Results CAN12 was able to interact with human PAR4 and delay PAR4 cleavage. In addition, CAN12 inhibited thrombin-induced human platelet aggregation and secretion in a dose-dependent manner. The specificity of CAN12 was agonist-dependent. In vivo, we determined that CAN12 was able to inhibit arterial thrombosis, and, using two independent methods, we found that CAN12 did not influence hemostasis. Conclusion Targeting the extracellular anionic cluster on PAR4 is a viable novel strategy as an antiplatelet therapy.

RAP GTPases, important regulators of cellular adhesion, are abundant signaling molecules in the platelet/megakaryocytic lineage. However, mice lacking the predominant isoform, RAP1B, display a partial platelet integrin activation defect and have a normal platelet count, suggesting the existence of a RAP1-independent pathway to integrin activation in platelets and a negligible role for RAP GTPases in megakaryocyte biology. To determine the importance of individual RAP isoforms on platelet production and on platelet activation at sites of mechanical injury or vascular leakage, we generated mice with megakaryocyte-specific deletion (mKO) of Rap1a and/or Rap1b. Interestingly, Rap1a/b-mKO mice displayed a marked macrothrombocytopenia due to impaired proplatelet formation by megakaryocytes. In platelets, RAP isoforms had redundant and isoform-specific functions. Deletion of RAP1B, but not RAP1A, significantly reduced α-granule secretion and activation of the cytoskeleton regulator RAC1. Both isoforms significantly contributed to thromboxane A(2) generation and the inside-out activation of platelet integrins. Combined deficiency of RAP1A and RAP1B markedly impaired platelet aggregation, spreading, and clot retraction. Consistently, thrombus formation in physiological flow conditions was abolished in Rap1a/b-mKO, but not Rap1a-mKO or Rap1b-mKO, platelets. Rap1a/b-mKO mice were strongly protected from experimental thrombosis and exhibited a severe defect in hemostasis after mechanical injury. Surprisingly, Rap1a/b-mKO platelets were indistinguishable from controls in their ability to prevent blood–lymphatic mixing during development and hemorrhage at sites of inflammation. In summary, our studies demonstrate an essential role for RAP1 signaling in platelet integrin activation and a critical role in platelet production. Although important for hemostatic/thrombotic plug formation, platelet RAP1 signaling is dispensable for vascular integrity during development and inflammation.