Thromboxane A2 Synthesis Research Articles

Ozagrel for Patients With Noncardioembolic Ischemic Stroke: A Propensity Score-Matched Analysis

Ozagrel sodium (ozagrel), a thromboxane A2 synthesis inhibitor, is used for ischemic stroke patients in several countries, despite a lack of strict evidence of its benefits. We investigated whether ozagrel was beneficial for patients with atherothrombotic stroke or lacunar infarction. This was a retrospective observational study using the Diagnosis Procedure Combination database in Japan. We identified patients with atherothrombotic stroke or lacunar infarction who were admitted to 781 hospitals from July 1, 2010 to March 31, 2012. Propensity score-matched analyses were performed separately for patients with atherothrombotic stroke and those with lacunar infarction, which balanced differences in baseline characteristics between patients who received ozagrel (ozagrel group) and those who did not (control group) in each stroke subtype. The modified Rankin Scale scores at discharge and occurrence of hemorrhagic complications after admission were compared between the ozagrel and control groups. After the propensity score matching, 2726 pairs of patients with atherothrombotic stroke and 1612 pairs of patients with lacunar infarction were analyzed. Ordinal logistic regression analyses showed that ozagrel use was not significantly associated with modified Rankin Scale score at discharge in patients with atherothrombotic stroke (odds ratio: .99; 95% confidence interval: .88-1.11) or in those with lacunar infarction (odds ratio: 1.00; 95% confidence interval: .87-1.16). The occurrence of hemorrhagic complications did not differ significantly between the ozagrel and control groups. The present study suggested that ozagrel was safe to use but did not improve functional outcomes in patients with atherothrombotic or lacunar infarction.

The effectiveness of antioxidant therapy in aspirin resistance, diabetes population for prevention of thrombosis.

Thrombosis as the main complication of coronary heart disease (CHD) represents the primary cause of morbidity and mortality in patients with diabetes mellitus (DM). In the course of diabetes mellitus some coagulation abnormalities occur, that may result in a thrombogenic propensity. Aspirin (ASA) as a platelet-inhibiting agent through inactivation of Cyclooxygenase-1 (COX-1) is mostly used for the prevention and treatment of atherothrombotic disorders. ASA inhibits the COX-1 enzyme and therefore blocks platelet thromboxane A2 (TXA2) synthesis. However, some of the serious vascular events in high-risk vascular patients are attributable to a failure of ASA to suppress platelet aggregation. The consumption of antioxidant or antioxidant rich foods such as vitamin C, E, and polyphenols might impart anti-thrombotic and cardiovascular protective effects via their inhibition of platelet hyper-activation or aggregation similar to the action of aspirin. This review will discuss the risk of thrombosis in diabetes, what aspirin resistance means, and the effectiveness of antioxidant therapy in the prevention and possible treatment of atherothrombotic disorders.

초법에 따른 당귀의 항산화 및 항혈전 효과

Objectives： Arachidonic acid is control the thromboxane A2 (TXA2) and prostacycline (PGI2) synthesis, TXA2 increase lead to thrombus produced by induces platelet aggregation and vasoconstriction. Angelicae gigantis radix (RAR) is mainly used blood deficiency and stagnation. In previous studies, RAR has been reported that a vasodilating and blood clotting delay effects. In this study, investigate that anti-oxidant and anti-thrombotic effects of RAR by heat-process.Methods： The heated angelicae gigantis radix sample were made by 140, 180, and 220 ℃ and 4, 6, 9 and 12 min using water or 30% ethanol. The anti-oxidant effects were measured by total polyphenol, total flavonoid, DPPH and ABTS radical scavening activation. Anti-thrombotic effect conducted in samples that are determined to be effective through the anti-oxidant experiment such as angelicae gigantis radix roasted 180℃, and 220℃ and angelicae gigantis radix roasted with 30% ethanol 180℃, and 220℃.Results： Anti-oxidant parameters were efficacious in high temperature roasted AR. Also AR and EAR increased a inhibitory activity of FXa compared with RAR. The blood coagulation time of administration groups were significantly increased compare with control group. The TXB2 was significantly decreased in AR and EAR.Conclusions : We confirmed that whether AR and EAR administration has anti-oxidant and anti-thrombotic effect or not. As the results, AR and EAR were improved anti-oxidant effects and blood biochemistry compare with control group. This study provides scientific evidence that AR and EAR are have an anti-oxidant effect and anti-thrombotic effect, it expected that there is no difference between the two.

Involvement of Nitric Oxide on Calcium Mobilization and Arachidonic Acid Pathway Activation during Platelet Aggregation with different aggregating agonists

Platelet aggregation by different aggregating agonists is essential in the normal blood coagulation process, the excess of which caused acute coronary syndrome (ACS). In all cases, the activation of arachidonic acid by cycloxygenase was needed for the synthesis of thromboxane A2 (TXA2) but the mechanism of arachidonic acid release in platelets remains obscure. Studies were conducted to determine the role of nitric oxide (NO), if any, on the release of arachidonic acid in platelets. The cytosolic Ca2+ was visualized and quantitated by fluorescent spectroscopy by using QUIN-2. NO was measured by methemoglobin method. Arachidonic acid was determined by HPLC. TXA2 was measured as ThromboxaneB2 (TXB2) by ELISA. Treatment of platelets in platelet-rich plasma (PRP) with different aggregating agents resulted in the inhibition of nitric oxide synthase (NOS) which inhibited the production of NO synthesis and increased TXA2 synthesis. Furthermore, the treatment of washed PRP with different platelet aggregating agents resulted in the increase of [Ca2+] in nM ranges. In contrast, the pre-treatment of washed PRP with aspirin increased platelet NO level and inhibited the Ca2+ mobilization and TXA2 synthesis. These results indicated that the aggregation of platelets by different aggregating agonists was caused by the cytosolic Ca2+ mobilization due to the inhibition of NOS.

Oxidative Stress-Dependent Coronary Endothelial Dysfunction in Obese Mice.

Obesity is involved in several cardiovascular diseases including coronary artery disease and endothelial dysfunction. Endothelial Endothelium vasodilator and vasoconstrictor agonists play a key role in regulation of vascular tone. In this study, we evaluated coronary vascular response in an 8 weeks diet-induced obese C57BL/6 mice model. Coronary perfusion pressure in response to acetylcholine in isolated hearts from obese mice showed increased vasoconstriction and reduced vasodilation responses compared with control mice. Vascular nitric oxide assessed in situ with DAF-2 DA showed diminished levels in coronary arteries from obese mice in both basal and acetylcholine-stimulated conditions. Also, released prostacyclin was decreased in heart perfusates from obese mice, along with plasma tetrahydrobiopterin level and endothelium nitric oxide synthase dimer/monomer ratio. Obesity increased thromboxane A2 synthesis and oxidative stress evaluated by superoxide and peroxynitrite levels, compared with control mice. Obese mice treated with apocynin, a NADPH oxidase inhibitor, reversed all parameters to normal levels. These results suggest that after 8 weeks on a high-fat diet, the increase in oxidative stress lead to imbalance in vasoactive substances and consequently to endothelial dysfunction in coronary arteries.

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 (10mg/d, n=73), a maximal dose atorvastatin (80mg/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 80mg/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 80mg/d) does not provide cardiovascular benefit beyond its cholesterol lowering effect in patients with T2DM.

Assessment of Platelet Function in Acute Ischemic Stroke Patients Previously Treated with Aspirin

Platelet inhibition measured by platelet function tests could be critical to understand the reasons for early recurrence and to guide therapeutic recommendations. We assess the platelet function during the acute phase of ischemic stroke in patients pretreated with aspirin who continue their treatment with aspirin only, are started on clopidogrel only, or add clopidogrel to aspirin. Sixty-four patients were taking aspirin before the stroke. Depending on the administered antiplatelet, 3 groups were defined: ASA: patients who continued on aspirin orally or intravenous acetylsalicylate of lysine, n = 30; CLO: patients who discontinued aspirin and were started on clopidogrel, n = 16; and ASA + CLO: patients who were prescribed both aspirin and clopidogrel, n = 10. Collagen-induced thromboxane A2 (TXA2) synthesis, ADP (adenosine diphosphate)-induced aggregation, and occlusion time (PF-100) were measured. CLO group only had a marked elevation of TXA2 (17.44 ± 15.62 ng/mL, P = .000) and a shortening of the platelet function analyzer (PFA)-100 closure time (157.13 ± 88 seconds, P = .047) compared with the other 2 groups (ASA: TXA2, .62 ± 1.59 ng/mL; ASA + CLO: TXA2 1.79 ± 4.59 ng/mL). They achieved a small (13%) but significant reduction of ADP-induced aggregation (87.00 ± 23.06 mm, P = .008) compared with the ASA group (102.82 ± 22.38 seconds). Stopping aspirin intake within the first 72 hours of the acute stroke drastically increases TXA2 synthesis. During the same time window, the freshly prescribed clopidogrel manages to reduce the ADP-induced aggregation only slightly (13%). This study offers analytic proof that the common practice of replacing aspirin with clopidogrel does not leave stroke patients fully protected during the first days after an ischemic stroke. Possible solutions could be to preserve aspirin during a few days or to use loading doses of clopidogrel at hospital admission.

The role of inhibition of nitric oxide synthesis in the aggregation of platelets due to the stimulated production of thromboxane A2.

The aggregation of platelets by ADP is reported to be mediated through prostaglandin synthesis. In contrast, nitric oxide is known to inhibit platelet aggregation through the synthesis of cyclic AMP and cyclic GMP. Studies were conducted to determine the role of ADP, if any, on the synthesis of nitric oxide in platelets. Both normal male and female volunteers between the ages of 30 and 45 years participated in the study. Thromboxane A2 (TXA2) was measured as thromboxane B2 by ELISA. Nitric oxide was measured by methhaemoglobin method. It was found that the treatment of platelet-rich plasma (PRP) with different concentrations of ADP (0-8.0 μmol/l) resulted in increased platelet aggregation, and at 8.0 μmol/l ADP, the basal nitric oxide level was found to be maximally decreased from 0.3 ± 0.10 nmol/10 platelets to 0 nmol/10 platelets in PRP (P < 0.0001; n = 10). Line-weaver-Burk plot of nitric oxide synthase (NOS) activity in the presence of 2.0 μmol/l ADP reduced the Vmax from 6.662 to 2.22 nmol nitric oxide/h per mg protein compared with control. Inhibition of nitric oxide synthesis by N-methyl-L-arginine acetate ester (L-NAME), an inhibitor of NOS, was found to aggregate platelets due to the reduction of platelet nitric oxide level (Pearson's coefficient of correlation, r = -0.986, P < 0.001, n = 10). The treatment of PRP to L-NAME was found to increase TXA2 synthesis to 1.679 ± 0.05 from 0 pmol/10 platelets. These results suggested that inhibition of NOS in platelets resulted in platelet aggregation through TXA2 synthesis in PRP through a novel pathway.

C0426: Partial Inhibition of Platelet Thromboxane A2 Synthesis at the Early Onset of Acute Myocardial Infarction is Associated with Cardiac Death at One Year Follow-Up

C0426: Partial Inhibition of Platelet Thromboxane A2 Synthesis at the Early Onset of Acute Myocardial Infarction is Associated with Cardiac Death at One Year Follow-Up

Platelets, Cyclooxygenases, and Colon Cancer

A growing body of evidence supports the central role of platelets in early events of tumorigenesis and metastasis. Activated platelets, in response to tissue damage, induce a proinflammatory program involving the aberrant expression of cyclooxygenase (COX)-2, which leads to increased tissue concentrations of the proinflammatory and protumorigenic prostaglandin E2. The central role of platelet activation in cancer development is sustained by the analysis of clinical studies with aspirin showing an anti-cancer efficacy by the drug, even at the low doses used for the prevention of atherothrombosis. Low-dose aspirin acts as an antiplatelet agent by causing an irreversible inactivation of platelet COX-1 activity and the synthesis of thromboxane A2. Further experimental and clinical studies are ongoing to confirm the central role of platelets in the development of inflammation and cancer. The corroboration of this hypothesis will open new opportunities for the prevention and treatment of cancer. In addition to the possible use of traditional antithrombotic agents, the recent identification of novel molecular determinants involved in the cross-talk between platelets and other cellular player of tumorigenesis and metastasis has led to the suggestion of novel therapeutic strategies in oncology.

Centrally administered CDP-choline induced cardiovascular responses are mediated by activation of the central phospholipase-prostaglandin signaling cascade

The present study was designed to determine the involvement of central prostaglandin synthesis on the pressor and bradycardic effect of cytidine 5′-diphosphocholine (CDP-choline). Intracerebroventricular (i.c.v.) administration of CDP-choline was made and blood pressure and heart rate were recorded in male Sprague Dawley rats throughout this study. Microdialysis and immunohistochemical studies were performed to measure extracellular total prostaglandin concentration and to show cyclooxygenase-1 and -2 (COX-1 and -2) immunoreactivities, respectively, in the posterior hypothalamic area. Moreover, rats were pretreated (i.c.v) with mepacrine [a phospholipase A2 (PLA2) inhibitor], ibuprofen [a nonselective COX inhibitor], neomycine [a phospholipase C (PLC) inhibitor] or furegrelate [a thromboxane A2 (TXA2) synthesis inhibitor] 5min prior to the injection of CDP-choline to determine the effects of these inhibitors on cardiovascular responses to CDP-choline. Control rats were pretreated (i.c.v) with saline.CDP-choline caused a dose- and time-dependent increase in blood pressure and decrease in heart rate. Immunohistochemical studies showed that CDP-choline increased COX-1 and -2 immunoreactivities in the posterior hypothalamic area. CDP-choline also elevated hypothalamic extracellular total prostaglandin concentration by 62%, as shown in microdialysis studies. Mepacrine or ibuprofen pretreatments almost completely blocked the pressor and bradycardic responses to CDP-choline while neomycine or furegrelate partially attenuated the drug-induced cardiovascular effects.The results suggest that CDP-choline may stimulate prostaglandin synthesis through the activation of PLA2, cyclooxygenases (COX-1 and -2) and prostaglandins and at least TXA2, may mediate the drug׳s cardiovascular effects.

The effect of centrally injected CDP-choline on respiratory system; involvement of phospholipase to thromboxane signaling pathway

CDP-choline is an endogenous metabolite in phosphatidylcholine biosynthesis. Exogenous administration of CDP-choline has been shown to affect brain metabolism and to exhibit cardiovascular, neuroendocrine neuroprotective actions. On the other hand, little is known regarding its respiratory actions and/or central mechanism of its respiratory effect. Therefore the current study was designed to investigate the possible effects of centrally injected CDP-choline on respiratory system and the mediation of the central cholinergic receptors and phospholipase to thromboxane signaling pathway on CDP-choline-induced respiratory effects in anaesthetized rats.Intracerebroventricularly (i.c.v.) administration of CDP-choline induced dose- and time-dependent increased respiratory rates, tidal volume and minute ventilation of male anaesthetized Spraque Dawley rats. İ.c.v. pretreatment with atropine failed to alter the hyperventilation responses to CDP-choline whereas mecamylamine, cholinergic nicotinic receptor antagonist, mepacrine, phospholipase A2 inhibitor, and neomycin phospholipase C inhibitor, blocked completely the hyperventilation induced by CDP-choline. In addition, central pretreatment with furegrelate, thromboxane A2 synthesis inhibitor, also partially blocked CDP-choline-evoked hyperventilation effects.These data show that centrally administered CDP-choline induces hyperventilation which is mediated by activation of central nicotinic receptors and phospholipase to thromboxane signaling pathway.

Suboptimal Inhibition of Platelet Cyclooxygenase 1 by Aspirin in Systemic Lupus Erythematosus: Association With Metabolic Syndrome

Low-dose aspirin prevents platelet aggregation by suppressing thromboxane A2 (TXA2 ) synthesis. However, in some individuals TXA2 suppression by aspirin is impaired, indicating suboptimal inhibition of platelet cyclooxygenase 1 (COX-1) by aspirin. Because patients with systemic lupus erythematosus (SLE) have increased risk of thrombotic events, many receive aspirin; however, the efficacy of aspirin in SLE has not been determined. We examined the hypothesis that aspirin response is impaired in SLE. We assessed the effect of aspirin by measuring concentrations of the stable metabolite of TXA2 , serum thromboxane B2 (sTXB2 ), before and after treatment with daily aspirin (81 mg) for 7 days in 34 patients with SLE and 36 control subjects. The inability to suppress sTXB2 synthesis to <10 ng/ml represents suboptimal inhibition of platelet COX-1 by aspirin. Aspirin almost completely suppressed sTXB2 in control subjects to median 1.5 ng/ml (interquartile range [IQR] 0.8-2.7) but had less effect in patients with SLE (median 3.1 ng/ml [IQR 2.2-5.3]) (P = 0.002). A suboptimal effect of aspirin was present in 15% (5 of 34) of the patients with SLE but not in control subjects (0 of 36) (P = 0.023). Incomplete responders were more likely to have metabolic syndrome (P = 0.048), obesity (P = 0.048), and higher concentrations of C-reactive protein (CRP) (P = 0.018). The pharmacologic effect of aspirin is suboptimal in 15% of patients with SLE but in none of the control subjects, and the suboptimal response was associated with metabolic syndrome, obesity, and higher CRP concentrations.

Mean platelet volume: interactions with platelet aggregation activity and glycoprotein IIb-IIIa and Ib expression levels

Increased mean platelet volume (MPV) is an independent risk factor of thrombotic events in patients with cardiovascular diseases. Interactions of MPV with platelet aggregation activity and contents of glycoprotein (GP) IIb-IIIa (alphaIIb/beta3 integrin, fibrinogen receptor) and GP Ib (von Willebrand factor receptor) were investigated in this study. Investigation was performed in a group of healthy volunteers (n = 38) and in a group of patients with acute coronary syndrome (ACS). In patients blood was collected at days 1, 3-5 and 8-12 after ACS development. As an antiaggregant therapy all patients received acetylsalicylic acid (ASA, inhibitor of thromboxane A2 synthesis) and most of them--clopidogrel (ADP receptor antagonist) with the exception of part of the patients (n = 44) at day 1 who had not taken clopidogrel before first blood collection. In volunteers platelet aggregation was stimulated by 1.25, 2.5, 5 and 20 M ADP, and in patients--by 5 and 20 M ADP. GP IIb-IIIa and GP Ib content on platelet surface was measured using 125I-labelled monoclonal antibodies. GP IIb-IIIa and GP Ib genetic polymorphisms were determined in ACS patients. In healthy donors significant correlations between MPV and aggregation levels were revealed at 1.25 and 2.5 M ADP (coefficients of correlation (r)--0.396 and 0.373, p < 0.05) and at 5 and 20 those interactions did not reach significant level (r--0.279 and 0.205, p > 0.05). Correlations between MPV and aggregation levels were observed at day 1 of ACS in a subgroup of patients who received ASA but had not started clopidogrel treatment (r--0.526, p < 0.01 and 0.368, p < 0.05 for 5 and 20 M ADP respectively). Interactions between these parameters were not registered upon combined treatment with ASA and clopidogrel. Strong direct correlations between MPV and GP IIb-IIIa and GP Ib contents were detected in healthy donors and ACS patients (at all time points) -r from 0.439 to 0.647 (p < or = 0.001 for all correlations). Genetic polymorphisms of GP IIb-IIIa (GP IIIa Leu33Pro) and GP Ib ((-5)T/C (Kozak) and Thr145Met) identified in ACS patients did not affect expression levels of corresponding glycoproteins. The data obtained indicated that increased MPV values correlate with increased platelet aggregation activity and enhanced GP IIb-IIIa and GP Ib expression.

Platelet Function and Hyperglycemia in Acute Coronary Syndrome

The literature provides strong evidence showing that platelet function is altered in patients with diabetes mellitus (DM) and that the platelet hyperactivation associated with hyperglycemia is produced by a number of thromboxane A2-independent pathways, such as those responsible for the release of adenosine diphosphatase, thrombin, epinephrine, and von Willebrand factor, among others. Being that they are independent of thromboxane A2, none of these platelet activation pathways are affected by the potential beneficial effect of the classical antiplatelet therapy with acetylsalicylic acid (ASA) or more modern compounds like clopidogrel. For this reason, the search for novel therapeutic approaches designed to control platelet reactivity, especially in diabetic individuals and not only in atherosclerosis not associated with DM, has been an object of interest in recent years, particularly if we take into consideration that diabetics have a certain resistance to the antiplatelet effect of ASA. Diabetic thrombocytopathy is due to the activation of multiple pathways that play a role in platelet function, the most relevant of which are the modification of platelet membrane components, the alteration of calcium and magnesium homeostasis (increasing and decreasing mobilization, respectively), increases in arachidonic acid metabolism and in thromboxane A2 synthesis, a decrease in prostacyclin and nitric oxide synthesis, and increased expression of certain adhesion molecules such as glycoprotein IIb/IIIa (GPIIb/IIIa) and P-selectin. The platelets of patients with DM, both type 1 and type 2, exhibit increased reactivity in early stages of the disease, and these phenomena are relevant because they contribute to the excessive rates of cardiovascular morbidity and mortality associated with this disease. Thus, the knowledge of these plateletrelated phenomena from the pathophysiological point of view, and of their eventual normalization, has been of great interest; in fact, the clinical practice guidelines on DM have incorporated, to a greater or lesser extent, recommendations concerning antiplatelet therapy. The platelets of diabetic patients express greater amounts of adhesion molecules such as activated GPIIb/IIIa, lysosomal GP53, thrombospondin, and P-selectin. In addition to supporting greater platelet aggregability, this may play a role in

Thromboxane A2 receptor α promotes tumor growth through an autoregulatory feedback pathway

Tobacco smoking can cause a number of cancers. The role of thromboxane synthase (TxAS) in smoking-related cancers is largely unknown. In this study, 37 pairs of tumor and non-tumor lung tissues of non-small-cell lung cancer, 5 lung cancer cell lines, and a mouse tumor model were used to study TxAS and its related molecules. A mouse model of smoking carcinogen 4-methylnitrosamino-1-3-pyridyl-1-butanone (NNK)-induced lung tumor showed an increase in TxAS. Thromboxane A2 receptor (TP) was aberrant in lung cancer tissues of smokers. TxAS and TP were increased in lung tissues of NNK-treated mice. The in vitro studies showed that TPα rather than TPβ promoted tumor growth, and NNK increased TPα. NNK-induced TxAS, which depended on activation of cyclooxygenase-2 (COX-2), ERK and NF-κB, could be inhibited by miR-34b/c. TPα played a positive role in NNK-induced COX-2/ERK/NF-κB activation, leading to the upregulation of TxAS expression and thromboxane A2 (TxA2) synthesis. The newly synthesized TxA2 could further activate TPα, forming an autoregulatory feedback loop for TPα activation. Collectively, NNK promotes lung tumor growth via inducing TxAS and TPα, which constitutes an auto-positive feedback loop to exaggerate the growth. This study suggests that TPα and TxAS are the ideal targets against smoking-related lung cancer.

TXA2 synthesis and COX1-independent platelet reactivity in aspirin-treated patients soon after acute cerebral stroke or transient ischaemic attack

The pharmacological target of aspirin is the inhibition of cyclooxygenase-1 (COX1) and thromboxane-A2 (TX) synthesis. Very few data are available on TX assessment in patients with stroke. We studied platelet TX synthesis, COX1-independent platelet reactivity, the influence of platelet-erythrocyte interactions and the potential association between platelet responses and the severity of stroke, evaluated with a clinical score (NIHSS). We examined 157 aspirin-treated patients with acute stroke or TIA, 128 aspirin-free and 15 aspirin-treated healthy subjects (HS). Collagen-induced TX, platelet recruitment in whole blood and platelets ± erythrocytes (haematocrit 40%) were assessed in patients on daily-aspirin within three days from onset. Arachidonic-acid-, ADP-, thrombin-receptor activating peptide TRAP-, and collagen-induced aggregation were also evaluated. Partial TX inhibition (<95% inhibition vs aspirin-free controls) was observed in 13% of patients. This was associated with marked increases in COX1-dependent responses (arachidonic-acid- and collagen-induced aggregation and platelet recruitment; P<0.0001) but not with differences in ADP- or TRAP-induced aggregation. Partial TX inhibition was independently associated with severe stroke (NIHSS ≥ 12) at both admission (P<0.05) and discharge (P<0.05). Among patients with fully blocked TX, those with elevated COX1-independent platelet reactivity (mean+2SD of aspirin-treated HS) were most likely to suffer severe stroke (P<0.05). Platelet-erythrocyte interactions enhanced platelet reactivity in these patients by COX1-dependent and -independent mechanisms (P<0.0001). TX inhibition by aspirin varied across patients. Partial TX inhibition and COX1-independent platelet hyperfunction were associated with more-severe stroke.

LIM kinase-1 selectively promotes glycoprotein Ib-IX–mediated TXA2 synthesis, platelet activation, and thrombosis

AbstractCurrent antithrombotic drugs have an adverse effect on bleeding, highlighting the need for new molecular targets for developing antithrombotic drugs that minimally affect hemostasis. Here we show that LIMK1−/− mice have defective arterial thrombosis in vivo but do not differ from wild-type mice with respect to bleeding time. LIMK1−/− mice show a selective defect in platelet activation induced through the von Willebrand Factor (VWF) receptor, the glycoprotein Ib-IX-V complex (GPIb-IX), but not by GPIb-IX–independent platelet agonists. In fact, LIMK1−/− platelets show an enhanced reaction to certain GPIb-IX–independent agonists. The defect of LIMK1−/− platelets in GPIb-IX–mediated platelet activation is attributed to a selective inhibition in VWF/GPIb-IX–induced phosphorylation of cytosolic phospholipase A2 (cPLA2) and consequent thromboxane A2 (TXA2) production. Supplementing a TXA2 analog, U46619, corrected the defect of LIMK1−/− platelets in VWF-induced stable platelet adhesion. Although LIMK1−/− platelets also showed reduced actin polymerization after GPIb-IX–mediated platelet aggregation, actin polymerization inhibitors did not reduce TXA2 generation, but rather accelerated platelet aggregation, suggesting that the role of LIMK1 in GPIb-mediated platelet activation is independent of actin polymerization. Thus, LIMK1 plays a novel role in selectively mediating GPIb-IX–dependent TXA2 synthesis and thrombosis and represents a potential target for developing antithrombotic drugs with minimal bleeding side effect.

Association of plasma concentrations of salicylic acid and high on ASA platelet reactivity in type 2 diabetes patients

The objective of this study was to investigate the association between plasma concentrations of salicylic acid (SA) and other minor acetylsalicylic acid (ASA) metabolites and high on ASA platelet reactivity assessed with different methods in type 2 diabetic patients (T2DM). Study cohort consisted of 293 T2DM patients on chronic ASA therapy. Platelet function inhibition was analyzed using measurements of serum thromboxane B2 (S-TxB2), VerifyNow Aspirin and Platelet Function Analyzer (PFA)-100 assays. The concentration of ASA metabolites in plasma was measured with a high-performance liquid chromatography (HPLC). In logistic regression analysis both ASA dose/kg of body weight and plasma SA concentration were found to be predictive of S-TxB2 concentrations above 0.72 ng/mL cut-off point (OR 16.9, 95% CI 2.29-125.8, p = 0.006 and OR 5.34, 95% CI 2.67-10.68, p < 0.001, respectively). When using the VerifyNow Aspirin Assay, the concentrations of SA were significantly lower (p = 0.007) in the group with high on ASA platelet reactivity when compared with the group with normal on ASA platelet reactivity. In logistic regression analysis plasma SA concentration was found to be predictive of VerifyNow Aspirin Reaction Units (ARU) ≥ 550 (OR 3.86, 95% CI 1.86-8.00, p < 0.001). Our study suggests that disturbances of pharmacokinetic mechanisms might contribute to lower plasma SA levels, and subsequently incomplete inhibition of thromboxane A2 synthesis as measured with S-TxB2 concentrations and increased platelet reactivity measured with VerifyNow in T2DM patients.

Reduction of platelet cytosolic phospholipase A2 activity by atorvastatin and simvastatin: Biochemical regulatory mechanisms

Statins have demonstrated effects beyond reducing cholesterol level that may contribute to their clinical benefit, including effects on platelet biochemistry and function. ObjectivesTo explore and compare the antiplatelet effect of two lipophilic statins (atorvastatin and simvastatin) and one hydrophilic statin (pravastatin) concerning: a) collagen-induced platelet aggregation and thromboxane A2 (TXA2) synthesis; b) the additive effect of statins on TXA2 synthesis in platelets treated with a submaximally effective concentration of aspirin and c) the biochemical mechanisms involved. Methods and ResultsWashed human platelets were incubated with statins (1–20μM), and stimulated with collagen (1μg/ml) or arachidonic acid (AA) (200μM) and TXB2 was quantified by ELISA. Incubation with simvastatin or atorvastatin reduced (36.2% and 31.0%, respectively) collagen-induced TXB2 synthesis (p<0.05) and platelet aggregation (p<0.001), whereas pravastatin had no effects. Simultaneous incubation with a submaximally effective concentration of aspirin (1μM) and atorvastatin or simvastatin significantly increased the inhibition of TXB2 synthesis by aspirin by 4.4- and 4.1-fold, respectively. Statins did not affect AA-induced TXB2 synthesis, excluding an effect on COX-1/TXA2 synthase activities. Atorvastatin and simvastatin concentration-dependently inhibited the collagen-induced increase in cytosolic calcium and the kinetics of cPLA2 phosphorylation. Lipophilic statins reduced phosphorylation of both ERK1/2 and p38 MAPK, which regulate cPLA2 phosphorylation and calcium movement. ConclusionWe report for the first time a direct downregulation by atorvastatin and simvastatin of platelet cPLA2 activity through effects on calcium and MAPK, which reduce collagen-induced TXA2 synthesis. These mechanisms might contribute to their beneficial effects, even in aspirin-treated patients.