Phosphorylation of protein kinase Cδ Tyr311 positively regulates thromboxane generation in platelets.

Platelets are key mediators of physiological hemostasis and pathological thrombosis, whose function must be carefully balanced by signaling downstream of receptors such as protease-activated receptor (PAR)4. Protein kinase C (PKC) is known to regulate various aspects of platelet function. For instance, PKCδ is known to regulate dense granule secretion, which is important for platelet activation. However, the mechanism by which PKCδ regulates this process as well as other facets of platelet activity is unknown. We speculated that the way PKCδ regulates platelet function may be because of the phosphorylation of tyrosine residues on PKCδ. We investigated phosphorylation of PKCδ following glycoprotein VI-mediated and PAR4-mediated platelet activation and found that Y311 is selectively phosphorylated when PAR4 is activated in human platelets. Therefore, we generated PKCδ Y311F knock-in mice, which are viable and have no gross abnormalities. However, PKCδY311F mice have significantly enhanced tail-bleeding times compared with WT littermate controls, which means hemostasis is interrupted. Furthermore, PKCδY311F mice exhibit longer time to carotid artery occlusion compared with WT control using a ferric chloride in vivo thrombosis model, indicating that the phosphorylation of PKCδ Y311 is prothrombotic. Washed platelets from PKCδY311F mice have reduced reactivity after stimulation with a PAR-4 agonist indicating its importance in platelet signaling. The phenotype observed in Y311F mouse platelets is because of reduced thromboxane generation, as an inhibitor of thromboxane generation equalizes the PKCδY311F platelet response to that of WT. Therefore, phosphorylation of PKCδ on Y311 is important for regulation of platelet function and specifically thromboxane generation, which reinforces platelet activation.

The enzymology of the human prostanoid pathway.

Prostanoids are short-lived autocrine and paracrine signaling molecules involved in a wide range of biological functions. They have been shown to be intimately involved in many different disease states when their regulation becomes dysfunctional. In order to fully understand the progression of any disease state or the biological functions of the well state, a complete evaluation of the genomics, proteomics, and metabolomics of the system is necessary. This review is focused on the enzymology for the enzymes involved in the synthesis of the prostanoids (prostaglandins, prostacyclins and thromboxanes). In particular, the isolation and purification of the enzymes, their enzymatic parameters and catalytic mechanisms are presented.

Thromboxane Governs the Differentiation of Adipose-Derived Stromal Cells Toward Endothelial Cells In Vitro and In Vivo.

RATIONALE: Autologous adipose-derived stromal cells (ASCs) offer great promise as angiogenic cell therapy for ischemic diseases. Because of their limited self-renewal capacity and pluripotentiality, the therapeutic efficacy of ASCs is still relatively low. Thromboxane has been shown to play an important role in the maintenance of vascular homeostasis. However, little is known about the effects of thromboxane on ASC-mediated angiogenesis. OBJECTIVE: To explore the role of the thromboxane-prostanoid receptor (TP) in mediating the angiogenic capacity of ASCs in vivo. METHODS AND RESULTS: ASCs were prepared from mouse epididymal fat pads and induced to differentiate into endothelial cells (ECs) by vascular endothelial growth factor. Cyclooxygenase-2 expression, thromboxane production, and TP expression were upregulated in ASCs on vascular endothelial growth factor treatment. Genetic deletion or pharmacological inhibition of TP in mouse or human ASCs accelerated EC differentiation and increased tube formation in vitro, enhanced angiogenesis in in vivo Matrigel plugs and ischemic mouse hindlimbs. TP deficiency resulted in a significant cellular accumulation of ß-catenin by suppression of calpain-mediated degradation in ASCs. Knockdown of ß-catenin completely abrogated the enhanced EC differentiation of TP-deficient ASCs, whereas inhibition of calpain reversed the suppressed angiogenic capacity of TP re-expressed ASCs. Moreover, TP was coupled with Gαq to induce calpain-mediated suppression of ß-catenin signaling through calcium influx in ASCs. CONCLUSION: Thromboxane restrained EC differentiation of ASCs through TP-mediated repression of the calpain-dependent ß-catenin signaling pathway. These results indicate that TP inhibition could be a promising strategy for therapy utilizing ASCs in the treatment of ischemic diseases.

Defective acid hydrolase secretion in RUNX1 haplodeficiency: Evidence for a global platelet secretory defect.

BACKGROUND: RUNX1 haplodeficiency is associated with thrombocytopenia, platelet dysfunction and a predisposition to acute leukaemia. Platelets possess three distinct types of granules and secretory processes involving dense granules (DG), α-granules and vesicles or lysosomes containing acid hydrolases (AH). Dense granules and granule deficiencies have been reported in patients with RUNX1 mutations. Little is known regarding the secretion from AH-containing vesicles. METHODS AND RESULTS: We studied two related patients with a RUNX1 mutation, easy bruising, and mild thrombocytopenia. Platelet aggregation and 14 C serotonin in platelet-rich plasma (PRP) were impaired in response to ADP, epinephrine, collagen and arachidonic acid. Contents of DG (ATP, ADP), α-granules (ß-thromboglobulin) and AH-containing vesicles (ß-glucuronidase, ß-hexosaminidase, α-mannosidase) were normal or minimally decreased. Dense granules secretion on stimulation of gel-filtered platelets with thrombin and divalent ionophore A23187 (4-12 µmol L-1 ) were diminished. ß-thromboglobulin and AH secretion was impaired in response to thrombin or A23187. We studied thromboxane-related pathways. The incorporation of 14 C -arachidonic acid into phospholipids and subsequent arachidonic acid release on thrombin activation was normal. Platelet thromboxane A2 production in whole blood serum and on thrombin stimulation of PRP was normal, suggesting that the defective secretion was not due to impaired thromboxane production. CONCLUSIONS: These studies provide the first evidence in patients with a RUNX1 mutation for a defect in AH (lysosomal) secretion, and for a global defect in secretion involving all three types of platelet granules that is unrelated to a granule content deficiency. They highlight the pleiotropic effects and multiple platelet defects associated with RUNX1 mutations.

No effects of atorvastatin (10 mg/d or 80 mg/d) on nitric oxide, prostacyclin, thromboxane and oxidative stress in type 2 diabetes mellitus patients of the DALI study.

The present study describes the effects of atorvastatin on whole body synthesis of nitric oxide (NO), prostacyclin (PGI2), and thromboxane A2 (TxA2), on oxidative stress and nitrite/nitrate-related renal carbonic anhydrase (CA) activity in patients with type 2 diabetes mellitus (T2DM). A double-blind, randomized, placebo-controlled parallel-group trial (the DALI study group) on 217 patients with T2DM and dyslipidemia was performed. Urinary samples were collected before and after administration of a standard dose (10 mg/d, n=73), a maximal dose atorvastatin (80 mg/d, n=72) or placebo (n=72) for 30 weeks. Urinary nitrite and nitrate were measured to assess whole body NO synthesis. The urinary molar ratio of nitrate to nitrite (UNOxR) served as a measure of renal CA activity. Free radical- and cyclooxygenase (COX)-catalyzed lipid peroxidation was estimated by measuring urinary 8-iso-prostaglandin F2α (8-iso-PGF2α). In subgroups, systemic PGI2 and TxA2 synthesis was assessed by measuring their major urinary metabolites 2,3-dinor-6-keto-prostaglandin F1α and 2,3-dinor-thromboxane B2, respectively. All biochemical parameters were measured by GC-MS and GC-MS/MS methods. T2DM patients had elevated levels of nitrate, nitrite, UNOxR, and 8-iso-PGF2α compared to healthy non-diabetic and normolipidemic subjects. Thirty-week treatment with atorvastatin (10 or 80 mg/d) did not significantly alter NO, PGI2, TxA2 and 8-iso-PGF2α synthesis and did not improve the renal reabsorption of nitrite which is considered an important reservoir of NO. Our study suggests that atorvastatin (10 or 80 mg/d) does not provide cardiovascular benefit beyond its cholesterol lowering effect in patients with T2DM.

The effect of kisspeptin on the regulation of vascular tone.

Kisspeptin has been implicated in cardiovascular control. Eicosanoids play a crucial role in the activation of platelets and the regulation of vascular tone. In the present study, we investigated the effect of kisspeptins on eicosanoid synthesis in platelets and aorta in vitro. Platelets and aorta were isolated from Wistar-Kyoto rats. After preincubation with different doses of kisspeptin, samples were incubated with [1-(14)C]arachidonic acid (0.172 pmol/mL) in tissue culture Medium 199. The amount of labeled eicosanoids was measured with liquid scintillation, after separation with overpressure thin-layer chromatography. Kisspeptin-13 stimulated the thromboxane synthesis. The dose-response curve was bell-shaped and the most effective concentration was 2.5 × 10(-8) mol/L, inducing a 27% increase. Lipoxygenase products of platelets displayed a dose-dependent elevation up to the dose of 5 × 10(-8) mol/L. In the aorta, kisspeptin-13 induced a marked elevation in the production of 6-keto-prostaglandin F1α, the stable metabolite of prostacyclin, and lipoxygenase products. Different effects of kisspeptin on cyclooxygenase and lipoxygenase products indicate that beyond intracellular Ca(2+) mobilization, other signaling pathways might also contribute to its actions. Our data suggest that kisspeptin, through the alteration of eicosanoid synthesis in platelets and aorta, may play a physiologic and (or) pathologic role in the regulation of vascular tone.

The effects of an inhibitor of diglyceride lipase on collagen-induced platelet activation.

Human platelet activation by collagen occurs in a dose-dependent manner. High concentrations of collagen bind to a pair of receptors, the α2ß1 integrin and glycoprotein (GP)VI/Fc-receptor γ-chain (FcRγ), which stimulate a cascade of events including Syk, LAT, Btk, Gads, and phospholipase Cγ2, leading to calcium release and protein kinase C (PKC) activation. Calcium and PKC are responsible for a range of platelet responses including exocytosis and aggregation, as well as the cytosolic phospholipase A2 (cPLA2)-mediated release of arachidonic acid, which is converted to thromboxane (Tx)A2. In contrast, low concentrations of collagen are acutely aspirin-sensitive, and calcium release and aggregation are TxA2-dependent. Under these conditions, cPLA2 is not involved and it has been suggested that phospholipase C generates 1,2-diacylglycerol (DG) from which arachidonic acid is liberated by diglyceride lipase (DGL). Here a novel DGL blocker (OMDM-188) inhibited collagen-, but not arachidonic acid-induced aggregation and TxA2 synthesis. Furthermore, OMDM-188 inhibited collagen-induced arachidonic acid release. Finally OMDM-188 inhibited collagen-induced p38(MAPK) phosphorylation, but not extracellular signal-regulated kinase (ERK) phosphorylation, with no effect on the phosphorylation of either enzyme in response to arachidonic acid. Taken together, these data suggest a role for a pathway involving phospholipase C liberating DG from membrane phospholipids in response to minimally activating concentrations of collagen. The DG serves as a substrate for DGL, potentially under the regulations of p38(MAPK), to release arachidonic acid, which is subsequently converted to TxA2, which mediates the final platelet response.

Aspirin and clopidogrel resistance: possible mechanisms and clinical relevance. Part II: Potential causes and laboratory tests.

Recent meta-analyses have indicated that patients with vascular disease demonstrated by laboratory tests to be aspirin or clopidogrel-resistant are at an increased risk of major vascular events. The suggested mechanisms of aspirin resistance include genetic polymorphism, alternative pathways of platelet activation, aspirin-insensitive thromboxane biosynthesis, drug interactions, or a low aspirin dose. Clopidogrel resistance is likely to develop as a result of a decreased bioavailability of the active metabolite, due to genetic variation or concomitant drug treatment. Additional work is required to improve and validate laboratory tests of platelet function, so that they may become useful tools for selection of the most appropriate antiplatelet therapy for an individual patient. Improvements in antiplatelet treatment strategies in the future should lead to a reduction in premature vascular events.

The contribution of cyclooxygenase-1 and -2 to persistent thromboxane biosynthesis in aspirin-treated essential thrombocythemia: implications for antiplatelet therapy.

We tested whether cyclooxygenase 2 (COX-2) expression and unacetylated COX-1 in newly formed platelets might contribute to persistent thromboxane (TX) biosynthesis in aspirin-treated essential thrombocythemia (ET). Forty-one patients on chronic aspirin (100 mg/day) and 24 healthy subjects were studied. Platelet COX-2 expression was significantly increased in patients and correlated with thiazole orange-positive platelets (r = 0.71, P < .001). The rate of TXA(2) biosynthesis in vivo, as reflected by urinary 11-dehydro-TXB(2) (TXM) excretion, and the maximal biosynthetic capacity of platelets, as reflected by serum TXB(2), were higher in patients compared with aspirin-treated healthy volunteers. Serum TXB(2) was significantly reduced by the selective COX-2 inhibitor NS-398 added in vitro. Patients were randomized to adding the selective COX-2 inhibitor, etoricoxib, or continuing aspirin for 7 days. Etoricoxib significantly reduced by approximately 25% TXM excretion and serum TXB(2). Fourteen of the 41 patients were studied again 21 (+/- 7) months after the first visit. Serum TXB(2) was consistently reduced by approximately 30% by adding NS398 in vitro, while it was completely suppressed with 50 microM aspirin. Accelerated platelet regeneration in most aspirin-treated ET patients may explain aspirin-persistent TXA(2) biosynthesis through enhanced COX-2 activity and faster renewal of unacetylated COX-1. These findings may help in reassessing the optimal antiplatelet strategy in ET.

Laboratory effect on platelet activity within 24 h of the first 300-mg oral dose of aspirin given in hospital during the acute phase of ischemic cerebral events.

BACKGROUND AND PURPOSE: Aspirin is the most commonly used antiplatelet treatment during the acute phase of cerebral ischemic events. It inhibits the production of thromboxane (TX) A(2), a powerful platelet activator. Despite this protection, early ischemic recurrences are frequent and considered clinical failures of this therapy. Only a few trials have focused on the use of antiplatelet therapy during this phase, and none has described the laboratory effect of the first dose of aspirin given after an ischemic cerebral event. However, this study may help clinicians to understand the mechanisms of early recurrences, and to design new therapeutic strategies, in particular for patients already treated with a daily dose of aspirin. METHOD: We studied laboratory parameters of the first 300-mg oral dose of aspirin given within 48 h after an ischemic cerebral event. Two blood samples were taken from all of the patients: the first during the third hour following aspirin intake (T1) and the second during the twenty-fourth hour (T2). For patients already treated with a daily dose of aspirin, a supplementary sample was taken before aspirin intake (T0). Platelet reactivity was studied on the basis of serum TXB(2) levels, a metabolite of TXA(2), and light transmission aggregometry after stimulation of platelet-rich plasma by arachidonic acid and by two concentrations of collagen, i.e. 2 µg/ml (Col2), dependent on the TXA(2) pathway, and 20 µg/ml (Col20), independent of the TXA(2) pathway. Results with Col2 were related to results with Col20 (Col2/20 ratio) to limit the impact of variations induced by the effects of preanalytical conditions. RESULTS: Fifty patients were included. TXB(2) values (p < 0.001) and relative values of the Col2/20 ratio (p = 0.037) were significantly higher at T2 compared to T1. For patients already treated with aspirin, TXB(2) levels (p < 0.001) were significantly lower at T1 compared to T0, and the Col2/20 ratio tended to decrease (p = 0.096). CONCLUSION: Platelet reactivity recovers within 24 h following the first 300-mg oral dose of aspirin during the acute phase of a cerebral ischemic event as demonstrated by an increase in TXB(2) levels, and the Col2/20 ratio, at T2 compared to T1. This would favor early ischemic recurrences. However, for patients already treated with aspirin, this dose is able to decrease TXB(2) levels and to complete the inhibition of the TXA(2) pathway, which shows the utility of this prescription in this case.

Platelets contribute to allograft rejection through glutamate receptor signaling.

Platelets recruit leukocytes and mediate interactions between leukocytes and endothelial cells. Platelets have been long described as markers of transplant rejection, but the contribution of platelets to transplant rejection has not been critically examined. We demonstrate in this study that following T cell initiation of allograft rejection, platelets contribute to T cell recruitment and increased plasma inflammatory mediators and accelerate T cell-meditated skin graft rejection. Prior work from our laboratory has shown that platelets secrete glutamate when activated, which then induces platelet thromboxane production by signaling through platelet-expressed ionotropic glutamate receptors. Glutamate receptor antagonists therefore represent, to our knowledge, novel inhibitors of platelet-accelerated inflammation. We have found that plasma glutamate is increased in mice that receive skin grafts and that mice treated with glutamate receptor antagonists have improved graft survival and decreased plasma thromboxane, platelet factor 4 (CXCL4), and IFN-γ. Taken together, our work now demonstrates that subsequent to T cell initiation of skin graft rejection, platelets contribute to further T cell recruitment and that by blunting glutamate-mediated platelet activation, graft survival is improved.

Aspirin resistance and platelet turnover: a 25-year old issue.

The evidence of an incomplete inhibition of platelet function by aspirin, despite therapeutic doses of the drug proved to be clinically effective are employed, was first reported in the '80s, in the frame of studies devoted to platelet turnover. Because inhibition of platelet aggregation by aspirin is irreversible, the return after an interval of time of the ability to form thromboxane by platelets in circulating blood should reflect the entry into the circulation of platelets whose cyclooxygenase activity has not been affected by aspirin. Based on this concept, the possibility of monitoring the entry of newly formed platelets into the circulation after aspirin ingestion was documented by measuring the return of thromboxane biosynthesis by platelets challenged in vitro by pairs of aggregating agents. The data obtained showed that platelets with intact cyclooxygenase activity could be detected into the circulation of control individuals as early as 4-6 h after aspirin ingestion, and at shorter time intervals in diabetic angiopathy. In the latter setting,the data allowed to conclude that "schedules of aspirin which may suffice in normals are not effective in patients with diabetic angiopathy, presumably because these patients have a high rate of entry of new platelets into the circulation".

Thromboxane generation by human peripheral blood polymorphonuclear leukocytes.

Human peripheral blood polymorphonuclear leukocytes were stimulated to generate thromboxane B2 in a time- and concentration-dependent fashion upon exposure to serum-treated zymosan particles. Conversion by stimulated PMN of [14C] arachidonic acid to [14C]thromboxane B2 was confirmed by thin-layer radiochromatography, radio-gas chromatography, and mass spectrometry. Generation of thromboxane B2 was independent of platelet contamination and could be inhibited by the cyclooxygenase inhibitor, indomethacin. Cells rendered incapable of ingesting particles by treatment with cytochalasin B generated comparable amounts of thromboxane B2. These results suggest that human peripheral blood polymorphonuclear leukocytes synthesize thromboxanes in response to surface stimulation independently of phagocytosis.

Thromboxane generation by human monocytes enhances platelet function.

Human monocytes potentiate the ADP-stimulated aggregation of autologous platelets through a fourfold increased binding of 125I-fibrinogen to the platelet surface. The enhancement of platelet function is rapid, relatively transient and is due to thromboxane (Tx) synthesized by monocytes under these conditions. Tx generation by monocytes is triggered by the interaction between fibrinogen and the specific monocyte membrane receptor. These data suggest that the monocyte enhancement of platelet function combined with the clot-promoting activity of these cells might unbalance normal hemostasis.

Circulating endothelial progenitor cells and residual in vivo thromboxane biosynthesis in low-dose aspirin-treated polycythemia vera patients.

Polycythemia vera (PV) is associated with high morbidity and mortality for thrombosis. We hypothesized that in PV altered sensitivity to aspirin might be related to dysfunction of the endothelial repair and/or of the nitric oxide (NO) system. Urinary thromboxane (TX) A(2) metabolite (TXM), endothelial colony-forming cells (ECFCs), plasma asymmetric dimethylarginine (ADMA) and von Willebrand factor (VWF) were measured in 37 PV patients on low-dose aspirin and 12 healthy controls. Patients showed an approximately 2-fold increase in median TXM and plasma ADMA levels (P < .001), while ECFC numbers were reduced by approximately 7-fold (P < .001) as compared with non-aspirinated control. These differences were more pronounced in patients with previous thrombosis. An 8-week course of aspirin did not affect ECFCs in 6 controls. VWF and TXM correlated directly with ADMA, and inversely with ECFCs. By multiple regression analysis, lower ECFC quartiles (beta = -0.39; SE = 0.17; P = .028) and higher VWF levels (beta = 0.338, SE = 0.002, P = .034) were independent predictors of higher TXM quartiles (R(2) = 0.39). Serum TXB(2), measured in 22 patients, was approximately 10-fold higher than aspirin-treated controls. PV patients appear to have an unbalanced ECFC/NO axis, and an apparent altered sensitivity of platelet TXA(2) production, all potentially contributing to aspirin-insensitive TXM formation. Thus, additional antithrombotic strategies may be beneficial in PV.

Activity of platelet hemostasis in children with spinal deformities.

An increase of adhesion and aggregation functions of platelets in vivo and in vitro was detected in 5-6-year-old children with scoliosis. These disorders were caused by hyperproduction of von Willebrand's factor in the vascular wall and intensification of thromboxane production in blood platelets. Activation of thromboxane formation is the main cause of platelet hyperactivity in children with scoliosis. Correction of platelet hemostasis may include pathogenetically substantiated complex of therapeutic exercises, swimming, and massage.

Incomplete inhibition of thromboxane biosynthesis by acetylsalicylic acid: determinants and effect on cardiovascular risk.

BACKGROUND: Incomplete inhibition of platelet thromboxane generation, as measured by elevated urinary 11-dehydro thromboxane B(2) concentrations, has been associated with an increased risk of cardiovascular events. We aimed to determine the external validity of this association in aspirin-treated patients enrolled in the Clopidogrel for High Atherothrombotic Risk and Ischemic Stabilization, Management and Avoidance (CHARISMA) trial and to determine whether there are any modifiable factors or interventions that lower urinary 11-dehydro thromboxane B(2) concentrations that could thereby reduce cardiovascular risk. METHODS AND RESULTS: Urinary 11-dehydro thromboxane B(2) concentrations were measured in 3261 aspirin-treated patients at least 1 month after they had been randomly assigned to placebo or clopidogrel. Baseline urinary 11-dehydro thromboxane B(2) concentrations in the highest quartile were associated with an increased risk of stroke, myocardial infarction, or cardiovascular death compared with the lowest quartile (adjusted hazard ratio 1.66, 95% CI 1.06 to 2.61, P=0.03). Increasing age, female sex, history of peripheral artery disease, current smoking, and oral hypoglycemic or angiotensin-converting enzyme inhibitor therapy were independently associated with higher urinary concentrations of 11-dehydro thromboxane B(2), whereas aspirin dose > or =150 mg/d, history of treatment with nonsteroidal antiinflammatory drugs, history of hypercholesterolemia, and statin treatment were associated with lower concentrations. Randomization to clopidogrel (versus placebo) did not reduce the hazard of cardiovascular events in patients in the highest quartile of urinary 11-dehydro thromboxane B(2) levels. CONCLUSIONS: In aspirin-treated patients, urinary concentrations of 11-dehydro thromboxane B(2) are an externally valid and potentially modifiable determinant of stroke, myocardial infarction, or cardiovascular death in patients at risk for atherothrombotic events.

Estrogen receptor-beta mediates cyclooxygenase-2 expression and vascular prostanoid levels in human placental villous endothelial cells.

OBJECTIVE: Regulation of fetoplacental blood flow is likely mediated by factors such as prostanoids. Estrogen and its receptors affect prostanoid biosynthesis. Previously, we demonstrated that villous endothelial cells express estrogen receptor-beta (ESR2), and we sought to determine its role in the mediation of fetoplacental vascular function. STUDY DESIGN: Villous endothelial cells from uncomplicated pregnancies were isolated, cultured, and treated with estrogen. RNA interference, real-time polymerase chain reaction, Western blotting, and enzyme immunoassays were performed. RESULTS: Cyclooxygenase-2 (COX-2) expression levels were not altered consistently by estrogen. RNA interference of ESR2 led to a concomitant decrease in COX-2 messenger RNA (P < .0001) and protein (P < .05) in the presence and absence of estradiol. ESR2 knock-down also led to diminished prostacyclin and thromboxane concentrations in the absence of estradiol (P < .005). CONCLUSION: ESR2 mediates COX-2 expression levels and both prostacyclin and thromboxane concentrations in the basal state, which suggests the possibility of ligand-independent regulation of COX-2 activity and prostaglandin H2 substrate availability. Further investigation regarding ESR2 regulation of prostanoid biosynthesis and its effects on the fetoplacental vasculature is warranted.

Parecoxib does not suppress thromboxane synthesis in newborn piglets with group B streptococcal sepsis.

Group B streptococci (GBS) cause fatal sepsis in newborns. Strong activation of thromboxane synthesis is assumed to correlate with severe pulmonary hypertension. In this study we compared the impact of indomethacin versus parecoxib on hemodynamics and outcome and investigated the pharmacological effects on thromboxane synthesis and EP-3 receptor gene expression. Whereas both parecoxib and indometacin reduced expression of thromboxane synthase and EP-3 receptor in infected lung tissue, parecoxib did not suppress urine levels of thromboxane like indometacin. We presume that COX-2 inhibition in GBS sepsis is associated with enhanced thrombogenicity.

Structure, stability, and antiplatelet activity of O-acyl derivatives of salicylic acid and lipophilic esters of acetylsalicylate.

The anti-thrombotic activity of acetylsalicylic acid (ASA) has been shown to be due to specific irreversible acetylation of blood platelet cyclooxygenase. The aim of our study was to investigate the associations between the antiplatelet activities of derivatives of both ASA and salicylic acid (SA), as well as the structure, stability, and molecular properties of these compounds. Homologous series of O-acyl derivatives of salicylic acid (propionyl-, butyrylsalicylic acids, PSA, BSA) and lipophilic dodecyl (C12)-, hexadecyl (C16)-, and cholesteryl acetylsalicylates were synthesized and tested for structure-activity relationships. The molecular properties (heat of formation, molecular surface area, dipole moment) of ASA and SA derivatives obtained by theoretical calculations changed with the increasing length of the acyl or alkyl residue. The inhibition of whole blood platelet aggregation and the reduction in thromboxane (TX) generation by O-acyl derivatives were concentration-dependent and decreased along with increasing the length of acyl hain. These effects correlated with the extent of platelet reactivity and P-selectin expression inhibition in collagen-activated platelets. In contrast to ASA and O-acyl derivatives of SA, none of the lipophilic ASA derivatives had a significant inhibitory effect on platelet aggregation. In conclusion, all SA and ASA derivatives studied under in vitro conditions showed much lower antiplatelet activities than ASA itself, despite their higher affinity to plasma proteins or membrane components and their equivalent ability to acetylate protein free amino groups.We suggest the significance of the carboxylic group, dipole moment, geometry, and size of these pharmaceuticals in their ability to bind to the active site of cyclooxygenase and their antiplatelet efficacy.