Eugenol Suppresses Platelet Activation and Mitigates Pulmonary Thromboembolism in Humans and Murine Models.

Platelets assume a pivotal role in the pathogenesis of cardiovascular diseases (CVDs), emphasizing their significance in disease progression. Consequently, addressing CVDs necessitates a targeted approach focused on mitigating platelet activation. Eugenol, predominantly derived from clove oil, is recognized for its antibacterial, anticancer, and anti-inflammatory properties, rendering it a valuable medicinal agent. This investigation delves into the intricate mechanisms through which eugenol influences human platelets. At a low concentration of 2 µM, eugenol demonstrates inhibition of collagen and arachidonic acid (AA)-induced platelet aggregation. Notably, thrombin and U46619 remain unaffected by eugenol. Its modulatory effects extend to ATP release, P-selectin expression, and intracellular calcium levels ([Ca2+]i). Eugenol significantly inhibits various signaling cascades, including phospholipase Cγ2 (PLCγ2)/protein kinase C (PKC), phosphoinositide 3-kinase/Akt/glycogen synthase kinase-3ß, mitogen-activated protein kinases, and cytosolic phospholipase A2 (cPLA2)/thromboxane A2 (TxA2) formation induced by collagen. Eugenol selectively inhibited cPLA2/TxA2 phosphorylation induced by AA, not affecting p38 MAPK. In ADP-treated mice, eugenol reduced occluded lung vessels by platelet thrombi without extending bleeding time. In conclusion, eugenol exerts a potent inhibitory effect on platelet activation, achieved through the inhibition of the PLCγ2-PKC and cPLA2-TxA2 cascade, consequently suppressing platelet aggregation. These findings underscore the potential therapeutic applications of eugenol in CVDs.

Increased Coronary Contraction to Thromboxane A2 in Cardiac Surgery Patients With Poorly Controlled Hypertension.

INTRODUCTION: Cardioplegia and cardiopulmonary bypass (CP/CPB) alters coronary arteriolar response to thromboxane A2 (TXA2) in patients undergoing cardiac surgery. Comorbidities, including hypertension (HTN), can further alter coronary vasomotor tone. This study investigates the effects of HTN on coronary arteriolar response to TXA2 pre and post-CP/CPB and cardiac surgery. MATERIALS AND METHODS: Coronary arterioles pre and post-CP/CPB were dissected from atrial tissue samples in patients with no HTN (NH, n = 9), well-controlled HTN (WC, n = 12), or uncontrolled HTN (UC, n = 12). In-vitro coronary microvascular reactivity was examined in the presence of TXA2 analog U46619 (10-9-10-4M). Protein expression of TXA2 receptor in the harvested right atrial tissue samples were measured by immunoblotting. RESULTS: TXA2 analog U46619 induced dose-dependent contractile responses of coronary arterioles in all groups. Pre-CPB contractile responses to U46619 were significantly increased in microvessels in the UC group compared to the NH group (P < 0.05). The pre-CP/CPB contractile responses of coronary arterioles were significantly diminished post-CP/CPB among the three groups (P < 0.05), but there remained an increased contractile response in the microvessels of the UC group compared to the WC and NH groups (P < 0.05). There were no significant differences in U46619-induced vasomotor tone between patients in the NH and WC groups (P > 0.05). There were no differences in expression of TXA2R among groups. CONCLUSIONS: Poorly controlled HTN is associated with increased contractile response of coronary arterioles to TXA2. This alteration may contribute to worsened recovery of coronary microvascular function in patients with poorly controlled HTN after CP/CPB and cardiac surgery.

Reprogramming Megakaryocytes for Controlled Release of Platelet-like Particles Carrying a Single-Chain Thromboxane A2 Receptor-G-Protein Complex with Therapeutic Potential.

In this study, we reported that novel single-chain fusion proteins linking thromboxane A2 (TXA2) receptor (TP) to a selected G-protein α-subunit q (SC-TP-Gαq) or to α-subunit s (SC-TP-Gαs) could be stably expressed in megakaryocytes (MKs). We tested the MK-released platelet-linked particles (PLPs) to be used as a vehicle to deliver the overexpressed SC-TP-Gαq or the SC-TP-Gαs to regulate human platelet function. To understand how the single-chain TP-Gα fusion proteins could regulate opposite platelet activities by an identical ligand TXA2, we tested their dual functions-binding to ligands and directly linking to different signaling pathways within a single polypeptide chain-using a 3D structural model. The immature MKs were cultured and transfected with cDNAs constructed from structural models of the individual SC-TP-Gαq and SC-TP-Gαs, respectively. After transient expression was identified, the immature MKs stably expressing SC-TP-Gαq or SC-TP-Gαs (stable cell lines) were selected. The stable cell lines were induced into mature MKs which released PLPs. Western blot analysis confirmed that the released PLPs were carrying the recombinant SC-TP-Gαq or SC-TP-Gαs. Flow cytometry analysis showed that the PLPs carrying SC-TP-Gαq were able to perform the activity by promoting platelet aggregation. In contrast, PLPs carrying SC-TP-Gαs reversed Gq to Gs signaling to inhibit platelet aggregation. This is the first time demonstrating that SC-TP-Gαq and SC-TP-Gαs were successfully overexpressed in MK cells and released as PLPs with proper folding and programmed biological activities. This bio-engineering led to the formation of two sets of biologically active PLP forms mediating calcium and cAMP signaling, respectively. As a result, these PLPs are able to bind to identical endogenous TXA2 with opposite activities, inhibiting and promoting platelet aggregation as reprogrammed for therapeutic process. Results also demonstrated that the nucleus-free PLPs could be used to deliver recombinant membrane-bound GPCRs to regulate cellular activity in general.

Preparing to strike: Acute events in signaling by the serpentine receptor for thromboxane A2.

Over the last two decades, awareness of the (patho)physiological roles of thromboxane A2 signaling has been greatly extended. From humble beginnings as a short-lived stimulus that activates platelets and causes vasoconstriction to a dichotomous receptor system involving multiple endogenous ligands capable of modifying tissue homeostasis and disease generation in almost every tissue of the body. Thromboxane A2 receptor (TP) signal transduction is associated with the pathogenesis of cancer, atherosclerosis, heart disease, asthma, and host response to parasitic infection amongst others. The two receptors mediating these cellular responses (TPα and TPß) are derived from a single gene (TBXA2R) through alternative splicing. Recently, knowledge about the mechanism(s) of signal propagation by the two receptors has undergone a revolution in understanding. Not only have the structural relationships associated with G-protein coupling been established but the modulation of that signaling by post-translational modification to the receptor has come sharply into focus. Moreover, the signaling of the receptor unrelated to G-protein coupling has become a burgeoning field of endeavor with over 70 interacting proteins currently identified. These data are reshaping the concept of TP signaling from a mere guanine nucleotide exchange factors for Gα activation to a nexus for the convergence of diverse and poorly characterized signaling pathways. This review summarizes the advances in understanding in TP signaling, and the potential for new growth in a field that after almost 50 years is finally coming of age.

Reduction in SOCE and Associated Aggregation in Platelets from Mice with Platelet-Specific Deletion of Orai1.

Calcium signalling in platelets through store operated Ca2+ entry (SOCE) or receptor-operated Ca2+ entry (ROCE) mechanisms is crucial for platelet activation and function. Orai1 proteins have been implicated in platelet's SOCE. In this study we evaluated the contribution of Orai1 proteins to these processes using washed platelets from adult mice from both genders with platelet-specific deletion of the Orai1 gene (Orai1flox/flox; Pf4-Cre termed as Orai1Plt-KO) since mice with ubiquitous Orai1 deficiency show early lethality. Platelet aggregation as well as Ca2+ entry and release were measured in vitro following stimulation with collagen, collagen related peptide (CRP), thromboxane A2 analogue U46619, thrombin, ADP and the sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) inhibitor thapsigargin, respectively. SOCE and aggregation induced by Thapsigargin up to a concentration of 0.3 µM was abrogated in Orai1-deficient platelets. Receptor-operated Ca2+-entry and/or platelet aggregation induced by CRP, U46619 or thrombin were partially affected by Orai1 deletion depending on the gender. In contrast, ADP-, collagen- and CRP-induced aggregation was comparable in Orai1Plt-KO platelets and control cells over the entire concentration range. Our results reinforce the indispensability of Orai1 proteins for SOCE in murine platelets, contribute to understand its role in agonist-dependent signalling and emphasize the importance to analyse platelets from both genders.

Platelet thromboxane inhibition by low-dose aspirin in polycythemia vera: Ex vivo and in vivo measurements and in silico simulation.

Low-dose aspirin is currently recommended for patients with polycythemia vera (PV), a myeloproliferative neoplasm with increased risk of arterial and venous thromboses. Based on aspirin pharmacodynamics in essential thrombocythemia, a twice-daily regimen is recommended for patients with PV deemed at particularly high thrombotic risk. We investigated the effects of low-dose aspirin on platelet cyclooxygenase activity and in vivo platelet activation in 49 patients with PV, as assessed by serum thromboxane (TX) B2 and urinary TXA2 /TXB2 metabolite (TXM) measurements, respectively. A previously described pharmacokinetic-pharmacodynamic in silico model was used to simulate the degree of platelet TXA2 inhibition by once-daily (q.d.) and twice-daily (b.i.d.) aspirin, and to predict the effect of missing an aspirin dose during q.d. and b.i.d. regimens. Serum TXB2 averaged 8.2 (1.6-54.7) ng/ml and significantly correlated with the platelet count (γ = 0.39) and urinary TXM (γ = 0.52) in multivariable analysis. One-third of aspirin-treated patients with PV displayed less-than-maximal platelet TXB2 inhibition, and were characterized by significantly higher platelet counts and platelet-count corrected serum TXB2 than those with adequate inhibition. Eight patients with PV were sampled again after 12 ± 4 months, and had reproducible serum TXB2 and urinary TXM values. The in silico model predicted complete inhibition of platelet-derived TXB2 by b.i.d. aspirin, a prediction verified in a patient with PV with the highest TXB2 value while on aspirin q.d. and treated short-term with a b.i.d. regimen. In conclusion, one in three patients with PV on low-dose aspirin display less-than-maximal inhibition of platelet TXA2 production. Serum TXB2 measurement can be a valuable option to guide precision dosing of antiplatelet therapy in patients with PV.

The Functional Interaction of EGFR with AT1R or TP in Primary Vascular Smooth Muscle Cells Triggers a Synergistic Regulation of Gene Expression.

In vivo, cells are simultaneously exposed to multiple stimuli whose effects are difficult to distinguish. Therefore, they are often investigated in experimental cell culture conditions where stimuli are applied separately. However, it cannot be presumed that their individual effects simply add up. As a proof-of-principle to address the relevance of transcriptional signaling synergy, we investigated the interplay of the Epidermal Growth Factor Receptor (EGFR) with the Angiotensin-II (AT1R) or the Thromboxane-A2 (TP) receptors in murine primary aortic vascular smooth muscle cells. Transcriptome analysis revealed that EGFR-AT1R or EGFR-TP simultaneous activations led to different patterns of regulated genes compared to individual receptor activations (qualitative synergy). Combined EGFR-TP activation also caused a variation of amplitude regulation for a defined set of genes (quantitative synergy), including vascular injury-relevant ones (Klf15 and Spp1). Moreover, Gene Ontology enrichment suggested that EGFR and TP-induced gene expression changes altered processes critical for vascular integrity, such as cell cycle and senescence. These bioinformatics predictions regarding the functional relevance of signaling synergy were experimentally confirmed. Therefore, by showing that the activation of more than one receptor can trigger a synergistic regulation of gene expression, our results epitomize the necessity to perform comprehensive network investigations, as the study of individual receptors may not be sufficient to understand their physiological or pathological impact.

Antiplatelet Agents Affecting GPCR Signaling Implicated in Tumor Metastasis.

Metastasis requires that cancer cells survive in the circulation, colonize distant organs, and grow. Despite platelets being central contributors to hemostasis, leukocyte trafficking during inflammation, and vessel stability maintenance, there is significant evidence to support their essential role in supporting metastasis through different mechanisms. In addition to their direct interaction with cancer cells, thus forming heteroaggregates such as leukocytes, platelets release molecules that are necessary to promote a disseminating phenotype in cancer cells via the induction of an epithelial-mesenchymal-like transition. Therefore, agents that affect platelet activation can potentially restrain these prometastatic mechanisms. Although the primary adhesion of platelets to cancer cells is mainly independent of G protein-mediated signaling, soluble mediators released from platelets, such as ADP, thromboxane (TX) A2, and prostaglandin (PG) E2, act through G protein-coupled receptors (GPCRs) to cause the activation of more additional platelets and drive metastatic signaling pathways in cancer cells. In this review, we examine the contribution of the GPCRs of platelets and cancer cells in the development of cancer metastasis. Finally, the possible use of agents affecting GPCR signaling pathways as antimetastatic agents is discussed.

Anti-Thrombotic Effects of Artesunate through Regulation of cAMP and PI3K/MAPK Pathway on Human Platelets.

Normal activation of platelets and their aggregation are crucial for proper hemostasis. It appears that excessive or abnormal aggregation of platelets may bring about cardiovascular diseases such as stroke, atherosclerosis, and thrombosis. For this reason, finding a substance that can regulate platelet aggregation or suppress aggregation will aid in the prevention and treatment of cardiovascular diseases. Artesunate is a compound extracted from the plant roots of Artemisia or Scopolia, and its effects have shown to be promising in areas of anticancer and Alzheimer's disease. However, the role and mechanisms by which artesunate affects the aggregation of platelets and the formation of a thrombus are currently not understood. This study examines the ways artesunate affects the aggregation of platelets and the formation of a thrombus on platelets induced by U46619. As a result, cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) production were increased significantly by artesunate relative to the doses, as well as phosphorylated vasodilator-stimulated phosphoprotein (VASP) and inositol 1,4,5-trisphosphate receptor (IP3R), substrates to cAMP-dependent kinase and cGMP-dependent kinase, in a significant manner. The Ca2+, normally mobilized from the dense tubular system, was inhibited due to IP3R phosphorylation from artesunate, and phosphorylated VASP aided in inhibiting platelet activity via αIIb/ß3 platelet membrane inactivation and inhibiting fibrinogen binding. In addition, MAPK and PI3K/Akt phosphorylation was inhibited via artesunate in a significant manner, causing the production of TXA2 and intracellular granular secretion (serotonin and ATP release) to be reduced. Therefore, we suggest that artesunate has value as a substance that inhibits platelet aggregation and thrombus formation through an antiplatelet mechanism.

Tetrahydrocurcumin Downregulates MAPKs/cPLA2 Signaling and Attenuates Platelet Thromboxane A2 Generation, Granule Secretion, and Thrombus Growth.

Platelet granule secretion plays a key role in atherothrombosis. Curcumin, a natural polyphenol compound derived from turmeric, exerts multiple biological activities. The current study sought to investigate the efficacy of tetrahydrocurcumin (THC, the major active metabolite of curcumin) on platelet granule secretion in vitro and thrombus formation in vivo. We found that THC significantly attenuated agonist-induced granule secretion in human gel-filtered platelets in vitro, including CD62P and CD63 expression and platelet factor 4, CCL5, and adenosine triphosphate release. These inhibitory effects of THC were partially mediated by the attenuation of cytosolic phospholipase A2 (cPLA2) phosphorylation, leading to a decrease in thromboxane A2 (TxA2) generation. Moreover, the MAPK (Erk1/2, JNK1/2, and p38 MAPK) signaling pathways were downregulated by THC treatment, resulting in reduced cPLA2 activation, TxA2 generation, and granule secretion. Additionally, THC and curcumin attenuated murine thrombus growth in a FeCl3-induced mesenteric arteriole thrombosis model in C57BL/6J mice without prolonging the tail bleeding time. THC exerted more potent inhibitory effects on thrombosis formation than curcumin. Through blocking cyclooxygenase-1 activity and thus inhibiting platelet TxA2 synthesis and granule secretion with aspirin, we found that THC did not further decrease the inhibitory effects of aspirin on thrombosis formation. Thus, through inhibiting MAPKs/cPLA2 signaling, and attenuating platelet TxA2 generation, granule secretion, and thrombus formation, THC may be a potent cardioprotective agent.

Kupffer cell activation by different microbial lysates: Toll-like receptor-2 plays pivotal role on thromboxane A2 production in mice and humans.

Thromboxane (TX) A2 has been identified as an important intrahepatic vasoconstrictor upon Kupffer cell (KC) activation during infections such as spontaneous bacterial peritonitis (SBP). The study aimed to investigate the role of TLRs in the TXA2 increase in liver nonparenchymal cells and their related mechanisms. Here, we identified TLR-2 as a common pathway for different microbials: microbial lysates including Gram-positive bacteria, Gram-negative bacteria, and fungi all increased TXA2 secretion via activation of TLR-2 in human KCs, accompanied by increased expression and phosphorylation of Myd88-related pathway. Of all TLR agonists, only TLR-1, -2, and -4 agonists increased TXA2 in human KCs. These results were further confirmed by mouse liver nonparenchymal cells. Comparing the effects of TLR-1, -2, and -4 antagonists, only TLR-2 antagonist showed inhibitory effects with all tested microbial lysates. Pretreatment with TLR-2 antagonist in human KCs blocked the secretion of IL-10, CXCL-10, TNF-α, and IL-6 induced by Gram-positive and Gram-negative bacterial stimulation. IL-23 and IL-1ß were only induced by Gram-negative bacteria. Thus, TLR-2 might be a potential marker and an attractive target for future treatment of patients with SBP. In addition, IL-23 and IL-1ß might distinguish early between Gram-positive and Gram-negative SBP.

Mechanism of thromboxane receptor-induced vasoconstriction in human saphenous vein.

Saphenous vein (SV) is one of the most widely used graft material in patients undergoing coronary artery bypass graft surgery (CABG). Thromboxane A2 (TXA2) is implicated in graft failure by inducing vasoconstriction and platelet aggregation. The aim of this study is to investigate the mechanism involved in TXA2-induced vasoconstriction in human SV. The role of different inhibitors and blockers on U46619 (TXA2-mimetic)-induced vasoconstriction is investigated by using an isolated organ bath system. Relaxation responses to several mediators are evaluated in SV pre-contracted with U46619 and compared with those pre-contracted with phenylephrine. Our results demonstrate that U46619-induced contraction is completely blocked by myosin light chain kinase inhibitor ML-9 or TP receptor antagonist BAY u3405. Furthermore, U46619-induced contraction is partially inhibited by phospholipase C inhibitor U73122, protein kinase C inhibitor calphostin C, Rho-kinase inhibitor Y-27632, L-type calcium channel blocker nifedipine, store-operated channel inhibitor SKF96365 or removal of extracellular calcium. Relaxation responses to NO donor (sodium nitroprusside), guanylate cyclase (GC) stimulator (riociguat), phosphodiesterase (PDE) inhibitors (sildenafil, IBMX), adenylate cyclase (AC) activator (forskolin) and acetylcholine (ACh) are markedly reduced when U46619 is used as a pre-contraction agent. Our results demonstrate that influx of extracellular Ca2+ (through L-type calcium channels and store-operated calcium channels) and intracellular Ca2+ release together with Ca2+ sensitization (through Rho-kinase activation) are necessary components for TXA2-induced vasoconstriction in SV. Moreover, more pronounced decrease in vasorelaxation induced by several mediators (SNP, riociguat, sildenafil, IBMX, forskolin, and ACh) in the presence of U46619 when compared with phenylephrine suggests that there is a crosstalk between the TP receptor signaling pathway and PDE, AC, GC enzymes. We believe that the investigation of mechanism of the TXA2-induced vasoconstriction in SV will provide additional information for the prevention of SV graft failure.

Obeticholic acid ameliorates hepatorenal syndrome in ascitic cirrhotic rats by down-regulating the renal 8-iso-PGF2α-activated COX-TXA2 pathway.

BACKGROUNDS/AIMS: The present study explores the potential of chronic treatment with the Foresaid X receptor (FXR) agonist obeticholic acid (OCA), which inhibits oxidative stress-related pathogenesis, in ascitic cirrhotic rats with hepatorenal syndrome (HRS) developed 6 weeks after bile duct ligation (BDL). METHODS: Systemic, splanchnic, and renal hemodynamics and pathogenic cascades were measured in ascitic BDL and sham rats receiving 2-weeks of either vehicle or OCA treatments (sham-OCA and BDL-OCA groups), and NRK-52E cells, rat kidney tubular epithelial cells. RESULTS: Chronic OCA treatment significantly normalized portal hypertension, glomerular filtration rate, urine output, renal blood flow; decreased ascites, renal vascular resistance, serum creatinine, and the release of renal tubular damage markers, including urinary neutrophil gelatinase-associated lipocalin (uNGAL) and kidney injury moleculae-1 (uKim-1) in BDL-OCA rats. In the BDL group, inhibition of the renal oxidative stress (8-iso-PGF2α)-activated cyclooxygenase-thromboxane A2 [COX-TXA2] pathway, apoptosis, and tubular injury accompanied by a decrease in hyper-responsiveness to the vasoconstrictor 8-iso-PGF2α in perfused kidneys. In vitro experiments revealed that 8-iso-PGF2α induced oxidative stress, release of reactive oxygen species, and cell apoptosis, which were reversed by concomitant incubation with the FXR agonist. CONCLUSIONS: Through the inhibition of renal 8-iso-PGF2α production and the down-regulation of the COX-TXA2 pathway, our study suggests that chronic OCA treatment can ameliorate the HRS in ascitic cirrhotic rats. Thus, OCA is an agent with antioxidative stress, antivasoconstrictive, antiapoptotic properties which benefit ascitic, cirrhotic rats with systemic, hepatic, and renal abnormalities.

Role of GRK6 in the Regulation of Platelet Activation through Selective G Protein-Coupled Receptor (GPCR) Desensitization.

Platelet G protein-coupled receptors (GPCRs) regulate platelet function by mediating the response to various agonists, including adenosine diphosphate (ADP), thromboxane A2, and thrombin. Although GPCR kinases (GRKs) are considered to have the crucial roles in most GPCR functions, little is known regarding the regulation of GPCR signaling and mechanisms of GPCR desensitization by GRKs in platelets. In this study, we investigated the functional role of GRK6 and the molecular basis for regulation of specific GPCR desensitization by GRK6 in platelets. We used GRK6 knockout mice to evaluate the functional role of GRK6 in platelet activation. Platelet aggregation, dense- and -granule secretion, and fibrinogen receptor activation induced by 2-MeSADP, U46619, thrombin, and AYPGKF were significantly potentiated in GRK6-/- platelets compared to the wild-type (WT) platelets. However, collagen-related peptide (CRP)-induced platelet aggregation and secretion were not affected in GRK6-/- platelets. Interestingly, platelet aggregation induced by co-stimulation of serotonin and epinephrine which activate Gq-coupled 5HT2A and Gz-coupled 2A adrenergic receptors, respectively, was not affected in GRK6-/- platelets, suggesting that GRK6 was involved in specific GPCR regulation. In addition, platelet aggregation in response to the second challenge of ADP and AYPGKF was restored in GRK6-/- platelets whereas re-stimulation of the agonist failed to induce aggregation in WT platelets, indicating that GRK6 contributed to P2Y1, P2Y12, and PAR4 receptor desensitization. Furthermore, 2-MeSADP-induced Akt phosphorylation and AYPGKF-induced Akt, extracellular signal-related kinase (ERK), and protein kinase Cδ (PKC) phosphorylation were significantly potentiated in GRK6-/- platelets. Finally, GRK6-/- mice exhibited an enhanced and stable thrombus formation after FeCl3 injury to the carotid artery and shorter tail bleeding times, indicating that GRK6-/- mice were more susceptible to thrombosis and hemostasis. We conclude that GRK6 plays an important role in regulating platelet functional responses and thrombus formation through selective GPCR desensitization.

Inhibition of neurogenic and thromboxane A2 -induced human prostate smooth muscle contraction by the integrin α2ß1 inhibitor BTT-3033 and the integrin-linked kinase inhibitor Cpd22.

INTRODUCTION: Prostate smooth muscle contraction is critical for etiology and treatment of lower urinary tract symptoms in benign prostatic hyperplasia (BPH). Integrins connect the cytoskeleton to membranes and cells to extracellular matrix, what is essential for force generation in smooth muscle contraction. Integrins are composed of different subunits and may cooperate with integrin-linked kinase (ILK). Here, we examined effects of inhibitors for different integrin heterodimers and ILK on contraction of human prostate tissues. METHODS: Prostate tissues were obtained from radical prostatectomy. Integrins and ILK were detected by Western blot, real-time polymerase chain reaction (RT-PCR), and double fluorescence staining. Smooth muscle contractions of prostate strips were studied in an organ bath. Contractions were compared after application of solvent (controls), the ILK inhibitor Cpd22 (N-methyl-3-(1-(4-(piperazin-1-yl)phenyl)-5-(4'-(trifluoromethyl)-[1,1'-biphenyl]-4-yl)-1H-pyrazol-3-yl)propanamide), the integrin α2ß1 inhibitor BTT-3033 (1-(4-fluorophenyl)-N-methyl-N-[4[[(phenylamino)carbonyl]amino]phenyl]-1H-pyrazole-4-sulfonamide), or the integrin α4ß1/α9ß1 inhibitor BOP (N-(benzenesulfonyl)- l-prolyl- l-O-(1-pyrrolidinylcarbonyl)tyrosine sodium salt). RESULTS: Western blot analyses of prostate tissues using antibodies raised against integrins α2b, α4, α9, ß1, and ILK revealed bands matching the expected sizes of corresponding antigens. Expression of integrins and ILK was confirmed by RT-PCR. Individual variations of expression levels occurred independently from divergent degree of BPH, reflected by different contents of prostate-specific antigen. Double fluorescence staining of prostate sections using antibodies raised against integrins α2 and ß1, or against ILK resulted in immunoreactivity colocalizing with calponin, suggesting localization in prostate smooth muscle cells. Electric field stimulation (EFS) induced frequency-dependent contractions, which were inhibited by Cpd22 (3 µM) and BTT-3033 (1 µM) (inhibition around 37% by Cpd22 and 46% by BTT-3033 at 32 Hz). The thromboxane A2 analog U46619-induced concentration-dependent contractions, which were inhibited by Cpd22 and BTT-3033 (around 67% by Cpd22 and 39% by BTT-3033 at 30 µM U46619). Endothelin-1 induced concentration-dependent contractions, which were not affected by Cpd22 or BTT-3033. Noradrenaline and the α1 -adrenergic agonists methoxamine and phenylephrine-induced concentration-dependent contractions, which were not or very slightly inhibited by Cpd22 and BTT-3033. BOP did not change EFS- or agonist-induced contraction. CONCLUSIONS: Integrin α2ß1 and ILK inhibitors inhibit neurogenic and thromboxane A2 -induced prostate smooth muscle contraction in human BPH. A role for these targets for prostate smooth muscle contraction may appear possible.

Mouse laser injury models: variations on a theme.

A confluence of technological advances in genetic manipulation and molecular-based fluorescence imaging has led to the widespread adoption of laser injury models to study hemostasis and thrombosis in mice. In all animal models of hemostasis and thrombosis, detailing the nature of experimentally induced vascular injury is paramount in enabling appropriate interpretation of experimental results. A careful appraisal of the literature shows that direct laser-induced injury can result in variable degrees of vascular damage. This review will compare and contrast models of laser injury utilized in the field, with an emphasis on the mechanism and extent of injury, the use of laser injury in different vascular beds and the molecular mechanisms regulating the response to injury. All of these topics will be discussed in the context of how distinct applications of laser injury models may be viewed as representing thrombosis and/or hemostasis.

Aspirin Reduces the Potentiating Effect of CD40L on Platelet Aggregation via Inhibition of Myosin Light Chain.

Background Antiplatelet therapy with aspirin (acetylsalicylic acid [ASA]) is less efficient in some coronary patients, which increases their risk of developing thrombosis. Elevated blood levels of thromboinflammatory mediators, like soluble CD40L (sCD40L), may explain such variabilities. We hypothesized that in the presence of elevated levels of sCD40L, the efficacy of ASA may vary and aimed to determine the effects of ASA on CD40L signaling and aggregation of platelets. Methods and Results The effects of ASA on CD40L-treated human platelets, in response to suboptimal concentrations of collagen or thrombin, were assessed at levels of aggregation, thromboxane A2 secretion, and phosphorylation of p38 mitogen-activated protein kinase, nuclear factor kappa B, transforming growth factor-ß-activated kinase 1, and myosin light chain. sCD40L significantly elevated thromboxane A2 secretion in platelets in response to suboptimal doses of collagen and thrombin, which was reversed by ASA. ASA did not inhibit the phosphorylation of p38 mitogen-activated protein kinase, nuclear factor kappa B, and transforming growth factor-ß-activated kinase 1, with sCD40L stimulation alone or with platelet agonists. sCD40L potentiated platelet aggregation, an effect completely reversed and partially reduced by ASA in response to a suboptimal dose of collagen and thrombin, respectively. The effects of ASA in sCD40L-treated platelets with collagen were related to inhibition of platelet shape change and myosin light chain phosphorylation. Conclusions ASA does not affect platelet sCD40L signaling but prevents its effect on thromboxane A2 secretion and platelet aggregation in response to collagen, via a mechanism implying inhibition of myosin light chain. Targeting the sCD40L axis in platelets may have a therapeutic potential in patients with elevated levels of sCD40L and who are nonresponsive or less responsive to ASA.

COX-2-derived prostaglandins as mediators of the deleterious effects of nicotine in chronic kidney disease.

Tobacco smoking has been identified as a risk factor in the progression of chronic kidney disease (CKD). In previous studies, we showed that nicotine induces cyclooxygenase (COX)-2 expression in vivo and in vitro and that the administration of nicotine in vivo worsens the severity of renal injury in a model of subtotal renal ablation. In the present study, we tested the role of COX-2-derived prostaglandins on the deleterious effects of nicotine in CKD. Sham and 5/6 nephrectomy (5/6Nx) rats received tap water or nicotine (100 µg/mL) in the drinking water for 12 wk. Additional groups also systemically received the COX-2 inhibitor NS-398 (1.5 mg·kg-1·day-1 via osmotic minipump). The administration of nicotine worsened renal injury and proteinuria in 5/6Nx rats and increased proteinuria in sham rats. 5/6Nx rats had increased cortical production of the prostaglandins PGE2, PGI2, PGD2, and PGF2α and of thromboxane A2. In these rats, nicotine reduced the production of all prostaglandins examined except thromboxane A2. Treatment with the COX-2 inhibitor NS-398 resulted in complete inhibition of all prostaglandins studied and ameliorated renal injury and proteinuria in 5/6Nx rats on nicotine but not in 5/6 Nx rats on tap water. Nicotine also reduced the expression of megalin in all groups examined, and this was partially prevented by COX-2 inhibition. In the present study, we showed that in CKD, nicotine worsens renal injury at least in part by producing an imbalance in the production of prostaglandins. This imbalance in the production of prostaglandins likely plays a role in the deleterious effects of smoking on the progression of CKD.

Targeting the COX1/2-Driven thromboxane A2 pathway suppresses Barrett's esophagus and esophageal adenocarcinoma development.

BACKGROUND: Barrett's esophagus (BE), a complication of gastroesophageal reflux disease (GERD), predisposes patients to esophageal adenocarcinoma (EAC). Reliable biomarkers for early detection and discovery of potential drug targets are urgently needed for improved BE and EAC patient outcomes. METHODS: Patient biopsy samples were evaluated for COX1/2, and thromboxane A2 synthase (TBXAS) expression. Circulating prostaglandins biosynthesis was determined using enzyme immunoassay kits. Anchorage-independent cell growth assay, crystal violet staining assay, and xenograft experiments were conducted to assess BE and EAC cell growth. A surgical mouse model of reflux (i.e., esophagoduodenostomy) was established and samples were analyzed using an enzyme immunoassay kit, immunohistochemistry, immunoblotting, or RT-PCR. Esophageal biopsy samples (pre- and post-intervention) were obtained from a randomized clinical trial in which participants were administered esomeprazole (40 mg) twice daily in combination with an acetylsalicylic acid (ASA) placebo or 81 or 325 mg ASA for 28 days. Esophageal biopsy specimens before and after the intervention period were analyzed. FINDINGS: COX2 and TBXAS are highly expressed in BE and EAC patients accompanied by a pronounced elevation of circulating TXA2 levels. ASA suppressed BE and EAC growth by targeting the TXA2 pathway. Additionally, biopsies from 49 patients (with similar baseline characteristics) showed that ASA substantially decreased serum TXA2 levels, resulting in reduced inflammation. INTERPRETATION: This study establishes the importance of the COX1/2-driven TXA2 pathway in BE and EAC pathophysiology and lays the groundwork for introducing a TXA2-targeting strategy for EAC prevention and early detection. FUNDING: Hormel Foundation, Exact Sciences, Pentax Medical, Intromedic and National Cancer.

Morin hydrate inhibits platelet activation and clot retraction by regulating integrin αIIbß3, TXA2, and cAMP levels.

Morin hydrate is an active constituent of Morus alba L, Prunus dulcis, and Cudrania tricuspidata and has been reported to inhibit platelet activation in vivo and in vitro, but no reports have been issued on its regulation of αIIbß3, a platelet-specific integrin and thromboxane A2 (TXA2), positive feedback molecule. In this study, we investigated the anti-platelet activity of morin hydrate in collagen- and thrombin-induced human platelets and attempted to identify the mechanism responsible for integrin αIIbß3 activation and TXA2 generation. Our results demonstrated that morin hydrate (25-100 µM) inhibited collagen- and thrombin-induced platelet aggregation, granule secretion (P-selectin expression, ATP, and serotonin release), calcium mobilization, TXA2 production, integrin αIIbß3 activation, and clot retraction. Additionally, morin hydrate attenuated the phosphorylations of phospholipase Cγ2 (PLCγ2), cytosolic phospholipase A2 (cPLA2), phosphoinositide 3-kinase (PI3K), Akt, c-Jun N-terminal kinase (JNK), and extracellular signal-regulated kinase (ERK), and enhanced the phosphorylations of inositol trisphosphate receptor (IP3 receptor) and cyclic adenosine monophosphate (cAMP) generation. However, it had no effect on the coagulation pathway. Taken together, these observations indicate morin hydrate inhibits platelet-mediated thrombosis by down-regulating TXA2 production and integrin αIIbß3 activation, and by upregulating cAMP generation, and thus, inhibits clot retraction. These results suggest morin hydrate may have therapeutic potential as a treatment for platelet-activation-related diseases.