An orthosteric/allosteric bivalent peptide agonist comprising covalently linked protease-activated receptor-derived peptides mimics in vitro and in vivo activities of activated protein C.

BACKGROUND: Activated protein C (APC) has anticoagulant and cytoprotective cell-signaling activities, which often require protease-activated receptor (PAR) 1 and PAR3 and PAR cleavages at noncanonical sites (R46-N47 and R41-G42, respectively). Some PAR1-derived (P1) peptides and PAR3-derived (P3) peptides, eg, P1-47-66 and P3-42-65, mimic APC's cell signaling. In anti-inflammatory assays, these 2 peptides at low concentrations synergistically attenuate cellular inflammation. OBJECTIVES: To determine whether a P1 peptide covalently linked to a P3 peptide mimics APC's anti-inflammatory and endothelial barrier stabilization activities. METHODS: Anti-inflammatory assays employed stimulated THP-1 cells and caspase-1 measurements. Cultured human EA.hy926 or murine aortic endothelial cells (ECs) exposed to thrombin were monitored for transendothelial electrical resistance. Bivalent covalently linked P1:P3 peptides were studied for APC-like activities. RESULTS: In anti-inflammatory assays, P1-47-55 was as active as P1-47-66 and some P3 peptides (eg, P3-44-54 and P3-51-65) were as active as P3-42-65. The bivalent P1:P3 peptide comprising P1-47-55-(Gly[10 residues])-P3-51-65 (designated "G10 peptide") was more potently anti-inflammatory than the P1 or P3 peptide alone. In transendothelial electrical resistance studies of thrombin-challenged ECs, P1-47-55 and the G10 peptide mimicked APC's protective actions. In dose-response studies, the G10 peptide was more potent than the P1-47-55 peptide. In murine EC studies, the murine PAR-sequence-derived G10 peptide mimicked murine APC's activity. Anti-PAR1 and anti-PAR3 antibodies, but not anti-endothelial protein C receptor antibodies, abated G10's cytoprotection, showing that G10's actions involve PAR1:PAR3. G10 significantly increased survival in murine endotoxemia. CONCLUSION: The PAR-sequence-derived G10 peptide is a bivalent agonist that mimics APC's cytoprotective, anti-inflammatory, and endothelial barrier-stabilizing actions and APC's protection against endotoxemic mortality.

C1-inhibitor influence on platelet activation by thrombin receptors agonists.

INTRODUCTION: Protease activated receptors 1 (PAR1) and 4 (PAR4) agonists are used to study platelet activation. Data on platelet activation are extrapolated across experimental settings. C1-inhibitor (C1INH) is a protease inhibitor present in plasma but not in isolated platelet suspensions. Here we show that C1INH affects platelet activation through PAR1 and PAR4 agonists. METHODS: Platelets were isolated from healthy donor whole blood and then labeled with anti-CD62P and PAC1 antibodies. The platelet suspensions were exposed to PAR1 agonists SFLLRN, TFLLR and TFLLRN; PAR4 agonists AYPGKF and GYPGQV; ADP and thrombin. Flow-cytometric measurements were performed in 5, 10 and 15 min after activation. RESULTS: 0.25 mg/ml C1INH addition made platelets to faster expose CD62P and glycoprotein IIb/IIIa complex after activation with PAR1 agonists. Conversely, C1INH addition led to inhibition of platelet activation with PAR4 agonists and thrombin. Activation with ADP was not affected by C1INH. CONCLUSIONS: Our results suggest that C1INH can modify platelet activation in the presence of synthetic PAR agonists used in platelet research. These observations may be relevant to the development of new methods to assess platelet function.

PAR-4/Ca2+-calpain pathway activation stimulates platelet-derived microparticles in hyperglycemic type 2 diabetes.

BACKGROUND: Patients with type 2 diabetes (T2DM) have a prothrombotic state that needs to be fully clarified; microparticles (MPs) have emerged as mediators and markers of this condition. Thus, we investigate, in vivo, in T2DM either with good (HbA1c ≤ 7.0%; GGC) or poor (HbA1c > 7.0%; PGC) glycemic control, the circulating levels of MPs, and in vitro, the molecular pathways involved in the release of MPs from platelets (PMP) and tested their pro-inflammatory effects on THP-1 transformed macrophages. METHODS: In 59 T2DM, and 23 control subjects with normal glucose tolerance (NGT), circulating levels of CD62E+, CD62P+, CD142+, CD45+ MPs were determined by flow cytometry, while plasma levels of ICAM-1, VCAM-1, IL-6 by ELISA. In vitro, PMP release and activation of isolated platelets from GGC and PGC were investigated, along with their effect on IL-6 secretion in THP-1 transformed macrophages. RESULTS: We found that MPs CD62P+ (PMP) and CD142+ (tissue factor-bearing MP) were significantly higher in PGC T2DM than GGC T2DM and NGT. Among MPs, PMP were also correlated with HbA1c and IL-6. In vitro, we showed that acute thrombin exposure stimulated a significantly higher PMP release in PGC T2DM than GGC T2DM through a more robust activation of PAR-4 receptor than PAR-1 receptor. Treatment with PAR-4 agonist induced an increased release of PMP in PGC with a Ca2+-calpain dependent mechanism since this effect was blunted by calpain inhibitor. Finally, the uptake of PMP derived from PAR-4 treated PGC platelets into THP-1 transformed macrophages promoted a marked increase of IL-6 release compared to PMP derived from GGC through the activation of the NF-kB pathway. CONCLUSIONS: These results identify PAR-4 as a mediator of platelet activation, microparticle release, and inflammation, in poorly controlled T2DM.

Enhanced endothelial barrier function by monoclonal antibody activation of vascular endothelial cadherin.

Excessive vascular permeability occurs in inflammatory disease processes. Vascular endothelial cadherin (VE-cadherin) is an adhesion protein that controls vascular permeability. We identified monoclonal antibodies (mAbs) to human VE-cadherin that activate cell adhesion and inhibit the increased permeability of endothelial cell monolayers induced by thrombin receptor activator peptide-6 (TRAP-6). Two mAbs, 8A12c and 3A5a, reduce permeability, whereas an inhibitory mAb, 2E11d, enhances permeability. Activating mAbs also reduce permeability induced by tumor necrosis factor-α (TNF-α) and vascular endothelial cell growth factor (VEGF). The activating mAbs also stabilize the organization of the adherens junctions that are disrupted by TRAP-6, VEGF, or TNF-α. The activating mAbs act directly on the adhesive function of VE-cadherin because they did not block the accumulation of actin filaments stimulated by TRAP-6 and enhance physical cell-cell adhesion of VE-cadherin-expressing tissue culture cells. Therefore, VE-cadherin function can be regulated at the cell surface to control endothelial permeability.NEW & NOTEWORTHY Excessive vascular permeability is a serious complication of many inflammatory disease conditions. We have developed monoclonal antibodies that inhibit increases in endothelial monolayer permeability induced by several signaling factors by activating VE-cadherin mediated adhesion and stabilizing cell junctions. These antibodies and/or the mechanisms they reveal may lead to important therapeutics to treat vascular leakiness and inflammation.

Proteinase-Activated Receptor 4 Activation Triggers Cell Membrane Blebbing through RhoA and ß-Arrestin.

Proteinase-activated receptors (PARs) are a four-member family of G-protein-coupled receptors that are activated via proteolysis. PAR4 is a member of this family that is cleaved and activated by serine proteinases such as thrombin, trypsin, and cathepsin-G. PAR4 is expressed in a variety of tissues and cell types, including platelets, vascular smooth muscle cells, and neuronal cells. In studying PAR4 signaling and trafficking, we observed dynamic changes in the cell membrane, with spherical membrane protrusions that resemble plasma membrane blebbing. Since nonapoptotic membrane blebbing is now recognized as an important regulator of cell migration, cancer cell invasion, and vesicular content release, we sought to elucidate the signaling pathway downstream of PAR4 activation that leads to such events. Using a combination of pharmacological inhibition and CRISPR/CRISPR-associated protein 9 (Cas9)-mediated gene editing approaches, we establish that PAR4-dependent membrane blebbing occurs independently of the Gα q/11- and Gα i-signaling pathways and is dependent on signaling via the ß-arrestin-1/2 and Ras homolog family member A (RhoA) signaling pathways. Together these studies provide further mechanistic insight into PAR4 regulation of cellular function. SIGNIFICANCE STATEMENT: We find that the thrombin receptor PAR4 triggers cell membrane blebbing in a RhoA-and ß-arrestin-dependent manner. In addition to identifying novel cellular responses mediated by PAR4, these data provide further evidence for biased signaling in PAR4 since membrane blebbing was dependent on some, but not all, signaling pathways activated by PAR4.

Molecular basis for activation and biased signaling at the thrombin-activated GPCR proteinase activated receptor-4 (PAR4).

Proteinase-activated receptor (PAR)-4 is a member of the proteolytically-activated PAR family of G-protein-coupled receptors (GPCR) that represents an important target in the development of anti-platelet therapeutics. PARs are activated by proteolytic cleavage of their receptor N terminus by enzymes such as thrombin, trypsin, and cathepsin-G. This reveals the receptor-activating motif, termed the tethered ligand that binds intramolecularly to the receptor and triggers signaling. However, PARs are also activated by exogenous application of synthetic peptides derived from the tethered-ligand sequence. To better understand the molecular basis for PAR4-dependent signaling, we examined PAR4-signaling responses to a peptide library derived from the canonical PAR4-agonist peptide, AYPGKF-NH2, and we monitored activation of the Gαq/11-coupled calcium-signaling pathway, ß-arrestin recruitment, and mitogen-activated protein kinase (MAPK) pathway activation. We identified peptides that are poor activators of PAR4-dependent calcium signaling but were fully competent in recruiting ß-arrestin-1 and -2. Peptides that were unable to stimulate PAR4-dependent calcium signaling could not trigger MAPK activation. Using in silico docking and site-directed mutagenesis, we identified Asp230 in the extracellular loop-2 as being critical for PAR4 activation by both agonist peptide and the tethered ligand. Probing the consequence of biased signaling on platelet activation, we found that a peptide that cannot activate calcium signaling fails to cause platelet aggregation, whereas a peptide that is able to stimulate calcium signaling and is more potent for ß-arrestin recruitment triggered greater levels of platelet aggregation compared with the canonical PAR4 agonist peptide. These findings uncover molecular determinants critical for agonist binding and biased signaling through PAR4.

Spinal macrophage migration inhibitory factor and high mobility group box 1 mediate persistent bladder pain.

Repeated intravesical PAR4 (protease activated receptor 4) activation elicits persistent bladder pain lasting 5 days after the last treatment. Persistent bladder pain was fully reversed by a systemic HMGB1 (high mobility group box 1) inhibitor while a MIF (macrophage migration inhibitory factor) antagonist partly reversed it. Since there is growing evidence that spinal MIF and HMGB1 mediate inflammatory and neuropathic pain we examined whether there were spinal changes occurring during persistent bladder pain that may be responsible for maintaining bladder pain. In addition, we tested whether we could modulate persistent bladder pain with spinal MIF or HMGB1 antagonists. Persistent bladder pain was elicited in female C57 mice by repeated (3x) intravesical instillation of PAR4-activating peptide while control animals received scramble peptide treatment. On day 4, spinal cord (L6-S1) changes in c-fos (non-specific marker of spinal activation) was assessed with immunofluorescence while MIF and HMGB1 were assessed with immunofluorescence, western blotting and real-time PCR. On day 7, mice received an intrathecal injection of a neutralizing MIF monoclonal antibody (15 µg in 5 µl PBS) or a HMGB1 inhibitor glycyrrhizin (25 µg in 5 µl of 5% alcohol in PBS) and abdominal mechanical threshold was tested. On day 9, mice were treated with vehicle or control and abdominal mechanical threshold was tested. Immunofluorescence showed that c-fos and MIF in the dorsal horn, dorsal grey commissure and intermediolateral areas significantly increased in PAR4-treated mice while HMGB1 was decreased. In addition, intrathecal treatment with MIF neutralizing mAb or glycyrrhizin significantly alleviated abdominal mechanical hypersensitivity at 1 and 2 h and the analgesic effect diminished at 6 h. Vehicle or control treatment had no effect. Persistent bladder pain is associated with spinal changes in MIF and HMGB1 levels. Furthermore, spinal treatment with MIF monoclonal antibody and HMGB1 inhibitor temporarily reversed bladder pain. Our findings suggest that spinal MIF and HMGB1 participate in persistent bladder pain induced by repeated intravesical PAR4 and may be potential therapeutic targets in chronic bladder pain conditions.

Thrombin and the Protease-Activated Receptor-1 in Organophosphate-Induced Status Epilepticus.

Organophosphates (OP) are a major threat to the health of soldiers and civilians due to their use as chemical weapons in war and in terror attacks. Among the acute manifestations of OP poisoning, status epilepticus (SE) is bearing the highest potential for long-term damages. Current therapies do not prevent brain damage and seizure-related brain injuries in OP-exposed humans. Thrombin is a serine protease known to have a fundamental function in the clotting cascade. It is highly expressed in the brain where we have previously found that it regulates synaptic transmission and plasticity. In addition, we have found that an excess of thrombin in the brain leads to hyperexcitability and therefore seizures through a glutamate-dependent mechanism. In the current study, we carried out in vitro, ex vivo, and in vivo experiments in order to determine the role of thrombin and its receptor PAR-1 in paraoxon-induced SE. Elevated thrombin activity was found in the brain slices from mice that were treated (in vitro and in vivo) with paraoxon. Increased levels of PAR-1 and pERK proteins and decreased prothrombin mRNA were found in the brains of paraoxon-treated mice. Furthermore, ex vivo and in vivo electrophysiological experiments showed that exposure to paraoxon causes elevated electrical activity in CA1 and CA3 regions of the hippocampus. Moreover, a specific PAR-1 antagonist (SCH79797) reduced this activity. Altogether, these results reveal the importance of thrombin and PAR-1 in paraoxon poisoning. In addition, the results indicate that thrombin and PAR-1 may be a possible target for the treatment of paraoxon-induced status epilepticus.

The protease-activated receptor 4 Ala120Thr variant alters platelet responsiveness to low-dose thrombin and protease-activated receptor 4 desensitization, and is blocked by non-competitive P2Y12 inhibition.

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.

RGS10 shapes the hemostatic response to injury through its differential effects on intracellular signaling by platelet agonists.

Platelets express ≥2 members of the regulators of G protein signaling (RGS) family. Here, we have focused on the most abundant, RGS10, examining its impact on the hemostatic response in vivo and the mechanisms involved. We have previously shown that the hemostatic thrombi formed in response to penetrating injuries consist of a core of fully activated densely packed platelets overlaid by a shell of less-activated platelets responding to adenosine 5'-diphosphate (ADP) and thromboxane A2 (TxA2). Hemostatic thrombi formed in RGS10-/- mice were larger than in controls, with the increase due to expansion of the shell but not the core. Clot retraction was slower, and average packing density was reduced. Deleting RGS10 had agonist-specific effects on signaling. There was a leftward shift in the dose/response curve for the thrombin receptor (PAR4) agonist peptide AYPGKF but no increase in the maximum response. This contrasted with ADP and TxA2, both of which evoked considerably greater maximum responses in RGS10-/- platelets with enhanced Gq- and Gi-mediated signaling. Shape change, which is G13-mediated, was unaffected. Finally, we found that free RGS10 levels in platelets are actively regulated. In resting platelets, RGS10 was bound to 2 scaffold proteins: spinophilin and 14-3-3γ. Platelet activation caused an increase in free RGS10, as did the endothelium-derived platelet antagonist prostacyclin. Collectively, these observations show that RGS10 serves as an actively regulated node on the platelet signaling network, helping to produce smaller and more densely packed hemostatic thrombi with a greater proportion of fully activated platelets.

Safety and efficacy of thrombopoeitin mimetics for refractory immune thrombocytopenia purpura in patients with systemic lupus erythematosus or antiphospholipid syndrome: a case series.

Background While thrombopoeitin (TPO) agonists that act to simulate platelet production have been approved for use in steroid-refractory chronic immune thrombocytopenia purpura (ITP), there are few data on the safety and efficacy of these medications in patients with concurrent systemic lupus erythematosus (SLE) or antiphospholipid syndrome (APS). Given that these agents can increase all hematopoietic cell lineages, it is unclear if there is an increased risk for exacerbation of the underlying lymphocyte-driven autoimmune disease in this population. Case summaries This case series includes four patients with SLE, one with concurrent APS, who were treated for steroid-refractory ITP with TPO mimetics at the University of Virginia between 2005 and 2015. In three of the four cases the medication was successful in improving platelet counts and preventing bleeding events. In addition, none of the patients experienced thrombosis or worsening of their underlying autoimmune disease. Conclusions This case series suggests that TPO mimetics are safe and moderately effective in patients with ITP in the setting of SLE or APS and do not contribute to increased disease activity.

Protease-activated receptor-4 (PAR4) variant influences on platelet reactivity induced by PAR4-activating peptide through altered Ca2+ mobilization and ERK phosphorylation in healthy Japanese subjects.

BACKGROUND: Thrombin belongs to the most potent platelet agonists and activates human platelets through GPIbα and two protease activated receptors (PARs), PAR1 and PAR4. However, the details of thrombin receptor system, especially the role of PAR4 on human platelet activation is still not clear. OBJECTIVES: We found a significant difference in PAR4-activating peptide (PAR4-AP)-induced, but not PAR1-AP, platelet aggregation between healthy Japanese subjects. Sequencing analysis revealed a single nucleotide change in PAR4 gene F2RL3 (SNP rs773902) leading to Ala120Thr variant. To elucidate the role of PAR4 in human platelet activation, we examined if platelet activation induced by PAR4-AP may be associated with PAR4 genotype. METHODS: Platelets from 202 healthy Japanese volunteers were genetically analyzed and determined the genotype frequency of rs773902. Agonist induced platelet aggregation, integrin αIIbß3 activation, granule release, Ca2+ mobilization, and activation of ERK and MLC were evaluated. The specificity of effects observed in platelets was confirmed in 293T cells transfected PAR4-Thr120 or Ala120. RESULTS: The frequencies of PAR4 variant Thr/Thr120, Ala/Thr120, and Ala/Ala 120 were 5.9, 37.1, and 57.0%, respectively. Platelets with Thr/Thr120 showed significantly higher reactivity in PAR4-AP-induced platelet aggregation, αIIbß3 activation and granule release compared to platelets with Ala/Ala120. PAR4-AP induced higher Ca2+ mobilization and ERK activation in platelets with Thr/Thr120 than Ala/Ala120. Ca2+ mobilization and ERK activation were also increased in 293T cells transfected with PAR4-Thr120 compared to Ala120. CONCLUSION: Our data suggested that PAR4-AP-induced platelet reactivity between PAR4 rs773902 was associated with altered intensity of Ca2+ mobilization and ERK activation.

Complement-activation fragment C4a mediates effector functions by binding as untethered agonist to protease-activated receptors 1 and 4.

C4a is a small protein released from complement component C4 upon activation of the complement system's classical and lectin pathways, which are important constituents of innate immune surveillance. Despite the structural similarity between C4a and well-described anaphylatoxins C3a and C5a, the binding partner and biological function of C4a have remained elusive. Using a cell-based reporter assay, we screened C4a against a panel of both known and orphan G protein-coupled receptors and now provide evidence that C4a is a ligand for protease-activated receptor (PAR)1 and PAR4. Whereas C4a showed no activity toward known anaphylatoxin receptors, it acted as an agonist for both PAR1 and PAR4 with nanomolar activity. In human endothelial cells, ERK activation by C4a was mediated through both PAR1 and PAR4 in a Gαi-independent signaling pathway. Like other PAR1 activators, C4a induced calcium mobilization through the PAR1/Gαq/PLCß signaling axis. Moreover, C4a increased stress fiber formation and enhanced endothelial permeability, both of which were reduced by PAR1 antagonists. In sum, our study identifies C4a as an untethered agonist for PAR1 and PAR4 with effects on cellular activation and endothelial permeability, thereby revealing another instance of cross-talk between the complement system and other host defense pathways.

Protease-activated receptor-4 and purinergic receptor P2Y12 dimerize, co-internalize, and activate Akt signaling via endosomal recruitment of ß-arrestin.

Vascular inflammation and thrombosis require the concerted actions of several different agonists, many of which act on G protein-coupled receptors (GPCRs). GPCR dimerization is a well-established phenomenon that can alter protomer function. In platelets and other cell types, protease-activated receptor-4 (PAR4) has been shown to dimerize with the purinergic receptor P2Y12 to coordinate ß-arrestin-mediated Akt signaling, an important mediator of integrin activation. However, the mechanism by which the PAR4-P2Y12 dimer controls ß-arrestin-dependent Akt signaling is not known. We now report that PAR4 and P2Y12 heterodimer internalization is required for ß-arrestin recruitment to endosomes and Akt signaling. Using bioluminescence resonance energy transfer, immunofluorescence microscopy, and co-immunoprecipitation in cells expressing receptors exogenously and endogenously, we demonstrate that PAR4 and P2Y12 specifically interact and form dimers expressed at the cell surface. We also found that activation of PAR4 but not of P2Y12 drives internalization of the PAR4-P2Y12 heterodimer. Remarkably, activated PAR4 internalization was required for recruitment of ß-arrestin to endocytic vesicles, which was dependent on co-expression of P2Y12. Interestingly, stimulation of the PAR4-P2Y12 heterodimer promotes ß-arrestin and Akt co-localization to intracellular vesicles. Moreover, activated PAR4-P2Y12 internalization is required for sustained Akt activation. Thus, internalization of the PAR4-P2Y12 heterodimer is necessary for ß-arrestin recruitment to endosomes and Akt signaling and lays the foundation for examining whether blockade of PAR4 internalization reduces integrin and platelet activation.

Trade-off of myocardial infarction vs. bleeding types on mortality after acute coronary syndrome: lessons from the Thrombin Receptor Antagonist for Clinical Event Reduction in Acute Coronary Syndrome (TRACER) randomized trial.

AIMS: Dual antiplatelet therapy reduces non-fatal ischaemic events after acute coronary syndrome (ACS) but increases bleeding to a similar extent. We sought to determine the prognostic impact of myocardial infarction (MI) vs. bleeding during an extended follow-up period to gain insight into the trade-off between efficacy and safety among patients after ACS. METHODS AND RESULTS: In 12 944 patients with non-ST-segment elevation ACS from the Thrombin Receptor Antagonist for Clinical Event Reduction in Acute Coronary Syndrome (TRACER) trial, we investigated the relative impact of MI and bleeding occurring >30 days post-ACS and subsequent all-cause mortality. Bleeding was graded according to Bleeding Academic Research Consortium (BARC) criteria. MI was associated with a five-fold increase in mortality. BARC type 2 and 3, but not type 1, bleeding had a significant impact on mortality. MI was associated with a greater risk of mortality compared with BARC 2 [relative risk (RR) 3.5; 95% confidence interval (CI) 2.08-4.77; P < 0.001] and BARC 3a bleeding (RR 2.23; 95% CI 1.36-3.64; P = 0.001), and a risk similar to BARC 3b bleeding (RR 1.37; 95% CI 0.81-2.30; P = 0.242). Risk of death after MI was significantly lower than after BARC 3c bleeding (RR 0.22; 95% CI 0.13-0.36; P < 0.001). MI and bleeding had similar time-associations with mortality, which remained significant for several months, still being higher early after the event. CONCLUSION: In patients treated with antiplatelet therapy after ACS, both MI and bleeding significantly impacted mortality with similar time-dependency. Although BARC 2 and 3a bleeding were less prognostic for death than MI, the risk of mortality was equivalent between BARC 3b bleeding and MI, and was higher following BARC 3c bleeding.

Effect of Vorapaxar Alone and in Combination with Aspirin on Bleeding Time and Platelet Aggregation in Healthy Adult Subjects.

The effect of the protease-activated receptor-1 (PAR-1) antagonist vorapaxar on human bleeding time is not known. This was a randomized, two-period, open-label trial in healthy men (n = 31) and women (n = 5). In period 1, subjects received 81 mg aspirin q.d. or a vorapaxar regimen achieving steady-state plasma concentrations equivalent to chronic 2.5 mg q.d. doses, for 7 days. In period 2, each group added 7 days of the therapy alternate to that of period 1 without washout. Bleeding time and platelet aggregation using arachidonic acid, ADP, and TRAP agonists were assessed. Bleeding time geometric mean ratio (90% CI) for vorapaxar/baseline was 1.01 (0.88-1.15), aspirin/baseline was 1.32 (1.15-1.51), vorapaxar + aspirin/vorapaxar was 1.47 (1.26-1.70), and vorapaxar + aspirin/aspirin was 1.12 (0.96-1.30). Unlike aspirin, vorapaxar did not prolong bleeding time compared with baseline. Bleeding time following administration of vorapaxar with aspirin was similar to that following aspirin alone.

Protease-activated receptor 4 deficiency offers cardioprotection after acute ischemia reperfusion injury.

Protease-activated receptor (PAR)4 is a low affinity thrombin receptor with less understood function relative to PAR1. PAR4 is involved in platelet activation and hemostasis, but its specific actions on myocyte growth and cardiac function remain unknown. This study examined the role of PAR4 deficiency on cardioprotection after myocardial ischemia-reperfusion (IR) injury in mice. When challenged by in vivo or ex vivo IR, PAR4 knockout (KO) mice exhibited increased tolerance to injury, which was manifest as reduced infarct size and a more robust functional recovery compared to wild-type mice. PAR4 KO mice also showed reduced cardiomyocyte apoptosis and putative signaling shifts in survival pathways in response to IR. Inhibition of PAR4 expression in isolated cardiomyocytes by shRNA offered protection against thrombin and PAR4-agonist peptide-induced apoptosis, while overexpression of wild-type PAR4 significantly enhanced the susceptibility of cardiomyocytes to apoptosis, even under low thrombin concentrations. Further studies implicate Src- and epidermal growth factor receptor-dependent activation of JNK on the proapoptotic effect of PAR4 in cardiomyocytes. These findings reveal a pivotal role for PAR4 as a regulator of cardiomyocyte survival and point to PAR4 inhibition as a therapeutic target offering cardioprotection after acute IR injury.

A Double-Chambered Protein Nanocage Loaded with Thrombin Receptor Agonist Peptide (TRAP) and γ-Carboxyglutamic Acid of Protein C (PC-Gla) for Sepsis Treatment.

New protein nanocages are designed bearing two functional proteins, γ-carboxyglutamic acid of protein C (PC-Gla) and thrombin receptor agonist peptide (TRAP), and have an anti-septic response. These nanoparticles reduce sepsis-induced organ injury and septic mortality in vivo. Noting that there are currently no medications for severe sepsis, these results show that novel nanoparticles can be used to treat sepsis.

Modulating platelet reactivity through control of RGS18 availability.

Most platelet agonists activate platelets by binding to G-protein-coupled receptors. We have shown previously that a critical node in the G-protein signaling network in platelets is formed by a scaffold protein, spinophilin (SPL), the tyrosine phosphatase, Src homology region 2 domain-containing phosphatase-1 (SHP-1), and the regulator of G-protein signaling family member, RGS18. Here, we asked whether SPL and other RGS18 binding proteins such as 14-3-3γ regulate platelet reactivity by sequestering RGS18 and, if so, how this is accomplished. The results show that, in resting platelets, free RGS18 levels are relatively low, increasing when platelets are activated by thrombin. Free RGS18 levels also rise when platelets are rendered resistant to activation by exposure to prostaglandin I2 (PGI2) or forskolin, both of which increase platelet cyclic adenosine monophosphate (cAMP) levels. However, the mechanism for raising free RGS18 is different in these 2 settings. Whereas thrombin activates SHP-1 and causes dephosphorylation of SPL tyrosine residues, PGI2 and forskolin cause phosphorylation of SPL Ser94 without reducing tyrosine phosphorylation. Substituting alanine for Ser94 blocks cAMP-induced dissociation of the SPL/RGS/SHP-1 complex. Replacing Ser94 with aspartate prevents formation of the complex and produces a loss-of-function phenotype when expressed in mouse platelets. Together with the defect in platelet function we previously observed in SPL(-/-) mice, these data show that (1) regulated sequestration and release of RGS18 by intracellular binding proteins provides a mechanism for coordinating activating and inhibitory signaling networks in platelets, and (2) differential phosphorylation of SPL tyrosine and serine residues provides a key to understanding both.

Specific activation, signalling and secretion profiles of human platelets following PAR-1 and PAR-4 stimulation.

Blood platelets play a central haemostatic function; however, they also play a role in inflammation and are capable of secreting various cytokines, chemokines and related products. The purpose of this study was to identify subtle variations in platelet physiology using proteomics. We compared the levels of membrane proteins (n = 3), α and δ granule proteins (n = 18), and signalling proteins (n = 30) from unstimulated platelets with those of protease-activated receptor (PAR)-1- and PAR-4-stimulated platelets (n = 10). The vast majority of these proteins responded similarly to PAR-1 or PAR-4 engagement. However, differences were observed within membrane CD40L expressed, and α granule GRO-α and MDC secreted proteins.