Thromboxane A2 Production Research Articles

Anti-platelet effect of ginkgolide a from Ginkgo biloba

Effects of ginkgolide A (GA) from Ginkgo biloba leaves in collagen (10 μg/mL)-stimulated platelet aggregation were investigated. Zymographic analysis confirmed that pro-matrix metalloproteinase-9 (MMP-9) (92 kDa) was activated by GA to form an activated MMP-9 (86-kDa) on gelatinolytic activities. GA concentration-dependently inhibited platelet aggregation, intracellular Ca2+ mobilization, and thromboxane A2 (TXA2) formation by inhibiting the cyclooxygenase-1 (COX-1) activity in collagen-stimulated platelets. In addition, GA increased the formation of cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP), which have an anti-platelet function in both resting and collagen-stimulated platelets. On the other hand, GA did not prolong prothrombin time (PT) and activated partial thromboplastin time (aPTT) associated with the extrinsic and intrinsic coagulation pathways on human plasma, respectively. Therefore, we suggest that the inhibitory effect of GA on platelet aggregation might involve the following pathway. GA may increase the MMP-9 activity and intracellular cAMP and cGMP production, inhibit intracellular Ca2+ mobilization, and decrease TXA2 production by down-regulating the COX-1, thereby leading to inhibition of platelet aggregation without cytotoxicity. These results strongly indicate that GA is a potent inhibitor of collagen-stimulated platelet aggregation. It may play an important role as a negative regulator during platelet activation.

Nonsteroidal Anti-Inflammatory Drugs and the Heart

Aspirin has been on the market for 115 years. Beginning with the marketing of indomethacin for the treatment of rheumatoid arthritis in 1963, at least 20 other nonsteroidal anti-inflammatory drugs (NSAIDs) with aspirin-like actions have been developed over the past 50 years,1 culminating with the introduction of a new class of selective inhibitors of cyclooxygenase (COX)-2, the coxibs, approximately 15 years ago.2 The NSAIDs represent the single most crowded family of drugs sharing the same therapeutic activities and mechanism of action, perhaps reflecting the unmet therapeutic need in the area of pain management and the large interindividual variability in response to these agents. NSAIDs provide symptomatic relief of pain and inflammation associated with a variety of human disorders, including the rheumatic diseases. Their shared therapeutic actions (ie, analgesic, anti-inflammatory, and antipyretic) are usually accompanied by mechanism-based adverse effects that can, at least in part, be attenuated as a function of individual pharmacokinetic or pharmacodynamic properties.1 Nuances in the tolerability of different NSAIDs had been described in the precoxib era on the basis of clinical trials of a few hundred patients treated for up to a few months with soft end points. More recently, however, important differences in safety have been demonstrated in head-to-head randomized comparisons of individual coxibs and 1 or more traditional NSAIDs that involved tens of thousands of patients treated for up to a few years with hard end points. Even more significantly, the interpretation of the effects of NSAIDs has been greatly enhanced by the availability, for the first time, of large, placebo-controlled trials that aimed to assess the potential for chemoprevention of colorectal cancer by rofecoxib or celecoxib. As a result of these developments, the whole field of NSAIDs has been illuminated during the past 15 years. Although epidemiological studies had previously associated …

MC-002 exhibits positive effects against platelets aggregation and endothelial dysfunction through thromboxane A 2 inhibition

Thromboxane A2 (TXA2) induces platelet aggregation and vasoconstriction, and agents that inhibit TXA2 production or interaction with receptors may exert potential application in stroke therapy. To illustrate the platelet aggregation antagonistic and endothelial protective effect of (E) - 3 - (3 - methoxy - 4 - ((3, 5, 6 - trimethylpyrazin - 2 - yl) methoxy) phenyl) sodium acrylate (MC-002) through TXA2 inhibition and underline mechanisms. Platelets aggregation and thoracic aorta ring contraction of rabbits were induced by U46619. Human umbilical vein endothelial cells (HUVECs) were further applied to explore the protective effect of MC-002 on endothelium when exposed to tumor necrosis factor - α (TNF-α). MTT method was used to assess cell damage, and ELISA analysis was exerted to estimate nitrogen monoxide (NO), endothelin-1 (ET-1), thromboxane B2 (TXB2) and 6-keto-prostaglandin F1α (6-keto-PGF1α) releasing. Fluorescence spectrophotometry was conducted to determine intracellular calcium concentration ([Ca(2+)]i), and western blotting method was applied to evaluate the protein expressions of intracellular adhesion molecule-1 (ICAM-1), P-selectin and nuclear factor-kappa B (NF-κB). TXA2 analog U46619 mediated obvious platelet aggregation and vasoconstriction. MC-002 inhibited platelet aggregation through administration in vivo and incubation with platelet in vitro, and relaxed aorta ring in endothelium dependent manner. MC-002 alleviated cell damage, [Ca(2+)]i overload, ET-1 overexcretion and TXB2 activation, but improved NO availability reduction in HUVECs treated with TNF-α. Furthermore, MC-002 downregulated ICAM-1, P-selectin and NF-κB overexpression induced by TNF-α. In conclusion, MC-002 exerted antiplatelet aggregation effect through TXA2 inhibition and relieved inflammatory injury of endothelial cells through NF-κB signal pathway.

Interactions between thromboxane A2, thromboxane/prostaglandin (TP) receptors, and endothelium-derived hyperpolarization

Endothelium-dependent smooth muscle hyperpolarization (EDH) increasingly predominates over endothelium-derived nitric oxide (NO) as a participant in vasodilation as vessel size decreases. Its underlying nature is highly variable between vessel types, species, disease states, and exact experimental conditions, and is variably mediated by one or more transferable endothelium-derived hyperpolarizing factors and/or the electrotonic spread of endothelial hyperpolarization into the media via gap junctions. Although generally regarded (and studied) as a mechanism that is independent of NO and prostanoids, evidence has emerged that the endothelium-derived contracting factor and prostanoid thromboxane A2 can modulate several signalling components central to EDH, and therefore potentially curtail vasodilation through mechanisms that are distinct from those putatively involved in direct smooth muscle contraction. Notably, vascular production of thromboxane A2 is elevated in a number of cardiovascular disease states that promote endothelial dysfunction. This review will therefore discuss the mechanisms through which thromboxane A2 interacts with and modulates EDH, and will also consider the implications of such cross-talk in vasodilator control in health and disease.

Antiplatelet Effects of Caffeic Acid Due to Ca2＋ MobilizationInhibition Via cAMP-Dependent Inositol-1, 4, 5-Trisphosphate Receptor Phosphorylation

In this study, we investigated the effects of caffeic acid (CAFA), a phenolic acid, on Ca(2＋)-antagonistic cyclic nucleotides associated with the phosphorylation of inositol 1,4,5-trisphosphate receptor (IP3R) and vasodilator-stimulated phosphoprotein (VASP) and the thromboxane A2 (TXA2)-associated microsomal cyclooxygenase-1 (COX-1) activity in collagen (10 μg/mL)-stimulated platelet aggregation. Washed platelets (10(8)/mL) obtained from Sprague-Dawley rats (6-7 weeks old, male) were preincubated for 3 minutes at 37℃ in the presence of 2 mM exogenous CaCl2 with or without CAFA or other materials, stimulated with collagen (10 μg/mL) for 5 minutes, then used to determine the levels of intracellular cytosolic Ca(2＋) ([Ca(2＋)]i), TXA2, cyclic adenosine monophosphate (cAMP), cyclic guanosine monophosphate (cGMP), COX-1 activity, VASP and IP3R phosphorylation. CAFA dose-dependently inhibited collagen-induced platelet aggregation and suppressed the production of TXA2, an aggregation-inducing autacoid associated with the strong inhibition of COX-1 in platelet microsomes exhibiting cytochrome C reductase activity. CAFA dose-dependently inhibited collagen-elevated [Ca(2＋)]i mobilization, which was increased by a cAMP-dependent protein kinase (A-kinase) inhibitor, Rp-8-Br-cAMPS, but not a cGMP-dependent protein kinase (G-kinase) inhibitor, Rp-8-Br-cGMPS. In addition, CAFA significantly increased the formation of cAMP and cGMP, intracellular Ca(2＋)-antagonists that function as aggregation-inhibiting molecules. CAFA increased IP3R (320 kDa) phosphorylation, indicating the inhibition of IP3-mediated Ca(2＋) release from internal stores (i.e. the dense tubular system) via the IP3R on collagen-activated platelets. Furthermore, CAFA-induced IP3R phosphorylation was strongly inhibited by an A-kinase inhibitor, Rp-8-Br-cAMPS, but only mildly inhibited by a G-kinase inhibitor, Rp-8-Br-cGMPS. These results suggest that the inhibition of [Ca(2＋)]i mobilization by CAFA is resulted from the cAMP/A-kinase-dependent phosphorylation of IP3R. CAFA elevated the phosphorylation of VASP-Ser(157), an A-kinase substrate, but not the phosphorylation of VASP-Ser(239), a G-kinase substrate. We demonstrate that CAFA increases cAMP and subsequently phosphorylates both IP3R and VASP-Ser(157) through A-kinase activation to inhibit [Ca(2＋)]i mobilization and TXA2 production via the inhibition of the COX-1 activity. These results strongly indicate that CAFA is a potent beneficial compound that elevates the level of cAMP-dependent protein phosphorylation in collagen-platelet interactions, which may result in the prevention of platelet aggregation-mediated thrombotic diseases.

In vivo prostacyclin biosynthesis and effects of different aspirin regimens in patients with essential thrombocythaemia

Essential thrombocythaemia (ET) is characterised by enhanced platelet generation and thrombosis. Once daily (od) aspirin incompletely inhibits platelet thromboxane (TX)A2 production in ET. A twice daily (bid) dosing is necessary to fully inhibit TXA2. Whether this dosing regimen affects in vivo prostacyclin (PGI2) biosynthesis is unknown. PGI2 biosynthesis was characterised in 50 ET patients on enteric-coated (EC) aspirin 100 mg od, by measuring its urinary metabolite, 2,3-dinor-6-keto-PGF1α (PGI-M). Moreover, in a crossover study 22 patients poorly responsive to standard aspirin based on serum TXB2 levels (≥4 ng/ml) were randomised to different seven-day aspirin regimens: EC aspirin 100 mg od, 100 mg bid, 200 mg od, or plain aspirin 100 mg od. PGI-M measured 24 hours after the last aspirin intake (EC, 100 mg od) was similar in patients and healthy subjects both on (n=10) and off (n=30) aspirin. PGI-M was unrelated to in vivo TXA2 biosynthesis, and not affected by EC aspirin 100 mg bid or 200 mg od as compared to EC 100 mg od. PGI2 biosynthesis in aspirin-treated ET patients appears unrelated to TXA2 biosynthesis, and not affected by an improved aspirin regimen, demonstrating its vascular safety for future trials.

The role of Nox1 and Nox2 in GPVI-dependent platelet activation and thrombus formation

BackgroundActivation of the platelet-specific collagen receptor, glycoprotein (GP) VI, induces intracellular reactive oxygen species (ROS) production; however the relevance of ROS to GPVI-mediated platelet responses remains unclear. ObjectiveThe objective of this study was to explore the role of the ROS-producing NADPH oxidase (Nox)1 and 2 complexes in GPVI-dependent platelet activation and collagen-induced thrombus formation. Methods and resultsROS production was measured by quantitating changes in the oxidation-sensitive dye, H2DCF-DA, following platelet activation with the GPVI-specific agonist, collagen related peptide (CRP). Using a pharmacological inhibitor specific for Nox1, 2-acetylphenothiazine (ML171), and Nox2 deficient mice, we show that Nox1 is the key Nox homolog regulating GPVI-dependent ROS production. Nox1, but not Nox2, was essential for CRP-dependent thromboxane (Tx)A2 production, which was mediated in part through p38 MAPK signaling; while neither Nox1 nor Nox2 was significantly involved in regulating CRP-induced platelet aggregation/integrin αIIbβ3 activation, platelet spreading, or dense granule and α-granule release (ATP release and P-selectin surface expression, respectively). Ex-vivo perfusion analysis of mouse whole blood revealed that both Nox1 and Nox2 were involved in collagen-mediated thrombus formation at arterial shear. ConclusionTogether these results demonstrate a novel role for Nox1 in regulating GPVI-induced ROS production, which is essential for optimal p38 activation and subsequent TxA2 production, providing an explanation for reduced thrombus formation following Nox1 inhibition.

Mitigating the Cardiovascular and Renal Effects of NSAIDs: Table 1

Nonsteroidal anti-inflammatory drugs (NSAIDs) are principal pharmacologic agents for symptom relief in patients with arthritis and other inflammatory conditions. Cardiovascular risk is associated with all NSAIDs, excluding aspirin. Selective inhibition of cyclo-oxygenase-2 (COX)-2 could produce a relative reduction in endothelial production of prostacyclin, while leaving the platelet production of thromboxane A2 (TXA2 ) intact. It has been speculated that this imbalance of homeostatic prostanoids might increase the risk for thrombotic events. The goal of this review is to provide physicians guidelines to mitigate cardiovascular and nephrotoxicity of NSAIDs. We conducted a systematic literature review to determine what information is available to guide treatment decisions in this patient population. Selective inhibition of COX-2 could produce a relative reduction in endothelial production of prostacyclin, while leaving the platelet production of TXA2 intact. Increasing degrees of selectivity for COX-2 are associated with augmented cardiovascular risk, whereas increasing degrees of selectivity for cyclo-oxygenase-2 (COX-1) are associated with augmented gastrointestinal risk. Some NSAIDs (such as ibuprofen) can interfere with the cardioprotective effects of aspirin by competitively binding to COX-1 enzyme, resulting in increased TXA2 production Naproxen may differ from other NSAIDs in sustaining functionally important degrees of inhibition of platelet cyclooxygenase-1 activity throughout the dosing interval. It is of paramount importance to consider individual health factors when choosing therapy with NSAIDs.

Thromboxane A2 Signaling Regulates Heterogeneous Platelet Activation Following Laser-Induced Injury In Mouse Cremaster Arterioles

BackgroundA recent study demonstrated that platelet accumulation following vascular injury in vivo is hierarchically organized resulting in a structure comprised of a core of fully-activated platelets that is overlaid with an unstable shell of less activated platelets (Stalker et al, Blood, 2013). This structure results from different elements of the platelet signaling network giving rise to regions that differ in platelet activation state, packing density, and stability. It was thus proposed that regional differences in platelet activation reflect regional differences in the distribution of platelet agonists. This provides new insights into heterogeneous platelet activation during platelet accumulation in vivo. Thromboxane A2 (TxA2), a dominant prostanoid product of cyclooxygenase 1 (COX-1) generated in platelets, plays an important role in the maintenance of vascular hemostasis and is a major therapeutic target of anti-platelet therapy. But its contribution to the regional architecture of a platelet mass is unknown. ApproachTo determine the contribution of TxA2 activity to the hierarchical organization of a thrombus, multicolor intravital microscopy was used to observe platelet accumulation and activation in thromboxane A2 receptor knockout (TP-/-) and low dose aspirin treated WT mice following laser-induced injury in mouse cremaster arterioles. ResultsTP-/- mice showed reduced total platelet (CD41) accumulation following vascular injury, consistent with a previous report (Yu et al, Sci Transl Med, 2012). The peak CD41 area in TP-/- mice was significantly reduced relative to WT mice (p=0.004). Interestingly, the core area of the thrombus in which the platelets are fully activated (P-selectin+), was not significantly different in TP-/- compared to WT during thrombus formation. This suggests that TxA2 signaling via the TP receptor primarily influences platelet recruitment and retention in the outer shell region of a platelet mass, but not full platelet activation in the core region.Aspirin inhibits TxA2 production through acetylation of COX-1, and is widely used as both primary and secondary prevention of cardiovascular diseases. We treated WT mice with aspirin in their drinking water (30 mg/L) for more than 1 week to mimic the effect of low dose aspirin treatment in humans (Yu et al, J Clin Invest, 2005). Similar to our findings in TP-/- mice, we found that aspirin treatment reduced total platelet accumulation following laser-induced injury in vivo (p<0.05). The decrease in peak platelet accumulation caused by aspirin was observed in the shell region at early time points (up to 2 min post-injury). In contrast to our findings in the TP-/- mice, low dose aspirin also resulted in reduced platelet activation and core region formation at later timepoints (p<0.05), suggesting that COX-1 may contribute to full platelet activation independent of TP receptor signaling. ConclusionOur studies show for the first time the role of TxA2 signaling in producing the hierarchical structure of a platelet mass formed in response to vascular injury. Our data indicate that TxA2signaling is critical for recruitment and/or retention of platelets prior to robust platelet activation including alpha granule secretion. These findings further highlight the importance of discrete spatial localization of platelet agonists within an evolving platelet plug in order to achieve the optimal hemostatic response.(This study was supported by National Natural Science Foundation of China 81170132 to Li Zhu) Disclosures:No relevant conflicts of interest to declare.

Investigation of the long-term patency of a transmural heparinized polycaprolactone and poly(D,L-lactic/glycolic acid) scaffold

BackgroundThe transmural biodegradable polycaprolactone/poly(D,L-lactic/glycolic acid) (PCL/PLGA) scaffold is a promising modality for diffuse coronary atherosclerosis cases that are not suitable for bypass grafting. The purpose of this study was to evaluate the long-term performance of the PCL/PLGA scaffold in vivo in the setting of polymer and heparin degradation. Materials and methodsAfter mechanical drilling through the ventricular wall was performed in the whole ventricular wall, two scaffolds were implanted into the ventricular wall. Animals were grouped into the single drilling group (SD group), the blank scaffold group (BS group), and the heparinized scaffold group (HS group) and were allowed to survived for 6 mo. Next, the patency and integrity of the scaffolds were evaluated by echocardiography and 3D-DOCTOR software. Endothelium coverage of the lumen was evaluated by scanning electron microscopy. Neovessels and collagen fiber within the scaffolds were identified by histologic staining. Metabolite production of prostacyclin (PGI2) and thromboxane A2 (TXA2) in the plasma was measured by an enzyme-linked immunosorbent assay. The expression levels of PGI2 synthase and cyclooxygenase 2 (COX-2) involved in PGI2 production and COX-1 involved in TXA2 production were measured by Western blot analysis. ResultsThe heparinized scaffolds were patent for up to 6 mo and the lumen was covered with confluent endothelial cells. Histologic staining revealed collagen fiber remodeling and reconstruction of the neovascular network immediately surrounding the lumen. The expression of PGI2 synthase and COX-2 in the HS group was significantly higher compared with the SD and BS groups (P < 0.01). The expression of COX-1 was similar in the three groups (P > 0.05). Consistent with synthetase expression, a PGI2 metabolite (6-keto-PGF1a) also showed a significant increase in the HS group relative to the SD and BS groups (P = 0.021 and P = 0.015, respectively). Concomitantly, as a PGI2 antagonist, the TXA2 metabolite (TXB2) did not exhibit a significant difference among the three groups (P = 0.17). ConclusionsDespite polymer and heparin degradation, the scaffold could continuously maintain the structural integrity and lumen patency for up to 6 mo by reinforcement of host collagen fiber and the balance of PGI2/TXA2.

Fatty acids and TxA2 generation, in the absence of platelet-COX-1 activity

Background and aimsOmega-3 fatty acids suppress Thromboxane A2 (TxA2) generation via mechanisms independent to that of aspirin therapy. We sought to evaluate whether baseline omega-3 fatty acid levels influence arachidonic acid proven platelet-cyclooxygenase-1 (COX-1) independent TxA2 generation (TxA2 generation despite adequate aspirin use). Methods and resultsSubjects with acute myocardial infarction, stable CVD or at high risk for CVD, on adequate aspirin therapy were included in this study. Adequate aspirin action was defined as complete inhibition of platelet-COX-1 activity as assessed by <10% change in light transmission aggregometry to ≥1 mmol/L arachidonic acid. TxA2 production was measured via liquid chromatography–tandem mass spectrometry for the stable TxA2 metabolite 11-dehydro-thromboxane B2 (UTxB2) in urine. The relationship between baseline fatty acids, demographics and UTxB2 were evaluated. Baseline omega-3 fatty acid levels were not associated with UTxB2 concentration. However, smoking was associated with UTxB2 in this study. ConclusionBaseline omega-3 fatty acid levels do not influence TxA2 generation in patients with or at high risk for CVD receiving adequate aspirin therapy. The association of smoking and TxA2 generation, in the absence of platelet COX-1 activity, among aspirin treated patients warrants further study.

Effect of Diet and Nutrient on Cell Signaling: Is the Tissue the Main Issue, Proposes Dr. Wilson?

Western diet is characterized with energy dense, refined, ready prepared foods with a high glycemic index (e.g. refined starches; bread, biscuits, candies, cornflakes, pizza, potato chips, cola drinks and sugar) and unhealthy lipids (e.g. trans fats, saturated fat , w-6 rich oils) poor in w-3 fatty acids, phytochemicals and fiber that are abundant in the Mediter- ranean type of diet. Western type of diets are known to predispose inflammation and increase in free fatty acids causing endothelial cell and beta cell dysfunction leading to the epidemic of cardiovascular diseases (CVDs). The cell signaling and the changes in cell membrane properties of the tissues induced by dietary lipids may have important consequences on the development of obesity, atherosclerosis and hypertension. The structural properties and function of cell membrane proteins appear to be modified in hypertensive humans and animal models of hypertension and atherosclerosis. Diet in- duced alterations in membrane lipid composition of hypertensive subjects have been associated with alterations in the transmembrane fluxes of Na + and K + , including Na + -Li + countertransport, which is a marker of essential hypertension, and in cell signaling proteins that participate in the control of blood pressure. It has been demonstrated that dietary lipids have an effect on membrane lipid composition and cell signaling proteins. Since changes in the dietary lipid composition yield to variations in the biophysical properties of the plasma membrane, it is likely that cellular functional changes could result from alterations in the structure of the lipid membrane properties under influence of the diet. Thus, the changes in mem- brane properties induced by dietary lipids may have important consequences on blood pressure regulation. The Mediterra- nean diet has been associated with changes in membrane structure and function. Consumption of olive oil-rich diets in- creases the concentration of oleic acid in plasma membrane lipids of different rat and human cells, with beneficial conse- quences on membrane functionality. In contrast, very little is currently known regarding the effects of nuts, another key ingredient of such diets, on membrane lipid composition and structure. CVD, diabetes mellitus and obesity, that are asso- ciated with increased production of thromboxane A2(TXA2), leukotrienes, prostacyclin, interleukins-1 and 6, tumor ne- crosis factor-alpha and C-reactive proteins in the tissues, is the major issue. Increased dietary intake of w-6 fatty acids is known to enhance all these biomarkers as well as atherogenicity of cholesterol in the tissues which have adverse pro- inflammatory effects resulting in CAD. Mediterranean diet rich in fruits, vegetables, nuts, mustard oil, and olive oil char- acterized with low w-6/w-3 ratio in the diet, can modulate inflammation and endothelial dysfunction of the tissues and may be protective against risk of coronary artery disease (CAD) and all-cause mortality. Inflammation appears to be an important unifying hypothesis, because in the absence of inflammation in the tissues, total cholesterol and other lipids may have neutral effects in the arterial tissues and myocardium. Therefore, it seems that endothelial dysfunction and in- flammation in the tissue is the main issue for treatment.

Aspirin augments carotid-cardiac baroreflex sensitivity during muscle mechanoreflex and metaboreflex activation in humans

Muscle mechanoreflex activation decreases the sensitivity of carotid baroreflex (CBR)-heart rate (HR) control during local metabolite accumulation in humans. However, the contribution of thromboxane A2 (TXA2) toward this response is unknown. Therefore, the effect of inhibiting TXA2 production via low-dose aspirin on CBR-HR sensitivity during muscle mechanoreflex and metaboreflex activation in humans was examined. Twelve young subjects performed two trials during two visits, preceded by 7 days' low-dose aspirin (81 mg) or placebo. One trial involved 3-min passive calf stretch (mechanoreflex) during 7.5-min limb circulatory occlusion (CO). In another trial, CO was preceded by 1.5 min of 70% maximal voluntary contraction isometric calf exercise to accumulate metabolites during CO and stretch (mechanoreflex and metaboreflex). HR (ECG) and mean arterial pressure (Finometer) were recorded. CBR function was assessed using rapid neck pressures ranging from +40 to -80 mmHg. Aspirin significantly decreased baseline thromboxane B2 production by 84 ± 4% (P < 0.05) but did not affect 6-keto prostaglandin F1α. Following aspirin, stretch with metabolite accumulation significantly augmented maximal gain (GMAX) and operating point gain (GOP) of CBR-HR (GMAX; -0.71 ± 0.14 vs. -0.37 ± 0.08 and GOP; -0.69 ± 0.13 vs. -0.35 ± 0.12 beats·min(-1)·mmHg(-1) for aspirin and placebo, respectively; P < 0.05). CBR-HR function curves were reset similarly with aspirin and placebo during stretch with metabolite accumulation. In conclusion, these findings suggest that low-dose aspirin augments CBR-HR sensitivity during concurrent muscle mechanoreflex and metaboreflex activation in humans. This increased sensitivity appears linked to reduced TXA2 production, which likely plays a role in metabolite sensitization of muscle mechanoreceptors.

Assays for quality control of platelets for transfusion

Quality control of platelets relies on the determination of the extent by which platelets are still able to react with known agonists, and here knowledge of the biochemistry of platelet activation may guide to decide which tests are useful. In vivo, platelets will adhere to collagen exposed upon vessel wall damage, and especially under high shear conditions through von Willebrand factor (VWF) that forms a bridge between collagen and platelet glycoprotein (GP) Ib. Binding of GPVI to collagen next results in the activation of a tyrosine kinase cascade, finally resulting in phosphorylation and activation of phospholipase C (PLC) γ2, which leads to an increase in cytosolic Ca2+ levels. High Ca2+ levels trigger the production of thromboxane A2 (TXA2) and release of platelet granules containing among others, adenosine diphosphate (ADP). TXA2 and ADP now will activate their receptors on platelets which are, among others, linked to a G‐protein that activates PLCβ2 with more Ca2+ increase. This ultimately provokes activation of the integrin αIIbβ3 (or GPIIbIIIa), which now can bind the symmetrical fibrinogen, thus allowing cross‐linking of platelets or platelet aggregation. From the above it is clear that testing whether platelets are fully reactive ideally should be looking at as many of the above parameters as possible, while at the same time being simple and fast. Systems in which blood is flown over collagen surfaces mimic best the physiological situation; however, these tests are not fit for stored platelet testing as haematocrit is a critical factor in these experiments. Thromboelastography at best provides a test for G‐protein dependent activation (through thrombin) and integrin αIIbβ3 involvement. Platelet agglutination induced by ristocetin (surrogate for the adhesion phase) next to aggregation (involvement of integrin αIIbβ3) induced by collagen (Tyr kinase pathway) and ADP (G‐protein mediated pathway) can give a comprehensive view on the platelet quality. However, flow cytometry is also an excellent technique to detect (i) bound VWF to platelets in the presence of ristocetin, (ii) collagen‐ or ADP‐induced activation of integrin αIIbβ3 (by determining the binding of fibrinogen or of the activation‐dependent PAC‐1 antibody) and (iii) secretion (by detecting surface expression of P‐selectin).

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.

Protection of vascular endothelium by aspirin in a murine model of chronic Chagas’ disease

Chronic Chagas' disease affects 10-30 % of patients infected with Trypanosoma cruzi, and it mainly manifests as cardiomyopathy. Important pathophysiological mechanisms involved in the cardiac lesions include activation of the endothelium and induced microvascular alterations. These processes involve the production of endothelial adhesion molecules and thromboxane A2, which are involved in inflammatory cell recruitment and platelet aggregation, respectively. Cyclooxygenase inhibitors such as aspirin decrease thromboxane production and alter the course of Chagas' disease, both in the acute and chronic phases. We studied the effects of the administration of low and high doses of aspirin during the early phase of T. cruzi infection, following microvascular damage in the context of a chronic murine model of Chagas' disease. The effects of both schedules were assessed at 24 and 90 days postinfection by evaluating parasitemia, mortality, and cardiac histopathological changes as well as the expression of ICAM, VCAM, and E-selectin in cardiac tissue. Thromboxane A2, soluble ICAM, and E-selectin blood levels were also measured. While aspirin did not affect parasitemia or mortality in the infected mice, it decreased both cardiac inflammatory infiltrates and thromboxane levels. Additionally, at 90 days postinfection, aspirin normalized sICAM and sE-selectin levels. Considering the improved endothelial function induced by aspirin, we propose the possibility of including this drug in clinical therapy to treat chronic Chagas' disease.

A critical role of thrombin/PAR-1 in ADP-induced platelet secretion and the second wave of aggregation

The stable or second wave of platelet aggregation often observed in ADP-stimulated platelet-rich plasma (PRP) with an artificially lowered extracellular calcium level has been attributed to enhanced thromboxane A2 (TXA2 ) generation and inhibition of ectonucleotidase activity. However, the role of thrombin in ADP-induced platelet secretion and the second wave of aggregation is unknown. We employed aggregometry, flow cytometry, immunoblotting and ELISA to determine whether and how thrombin participates in ADP-induced platelet secretion and the second wave of aggregation. ADP induces a phosphoinositide 3-kinase (PI3K) pathway-dependent thrombin generation, presumably resulting from the cleavage of αII b β3 -associated prothrombin. Generated thrombin subsequently activates protease-activated receptor-1 (PAR-1) and mediates dense granule secretion and the second wave of platelet aggregation in ADP-stimulated citrated PRP. Thus, ADP-induced dense granule secretion and the second wave of platelet aggregation in PRP were similarly and non-additively blocked by thrombin inhibitor hirudin, PAR-1 antagonist SCH-79797 or PI3K inhibitor wortmannin. Moreover, ADP stimulation caused the dissociation of prothrombin from αII b β3 and an increased plasma thrombin level; both were prevented by wortmannin. Furthermore, the wortmannin-inhibited second wave of platelet aggregation by ADP was restored by a subaggregation concentration of PAR-1 activating peptide SFLLRN. Blocking TXA2 production with indomethacin or restoring extracellular calcium to physiological concentration did not influence this thrombin/PAR-1 dependence. A PI3K-dependent thrombin generation and the resultant PAR-1 activation serve as an indispensable mechanism to relay the platelet activation process induced by ADP.

Combined Antiplatelet Therapy: Still a Sweeping Combination in Cardiology

Platelets play a key role in the pathogenesis of atherothrombosis, involved in both the development and progression of atherosclerotic heart disease, and the attendant acute thrombotic complications. Antiplatelet therapy constitutes a mainstay therapy for patients with acute coronary syndromes and generally high-risk patients with atherothrombosis. Until recently, dual antiplatelet therapy (DAPT) for the treatment and prevention of the complications of atherothrombotic disease was traditionally limited to aspirin plus clopidogrel. However, a most important pertaining issue emerged, that of the occurrence of drug-resistance or tolerance observed in some patients for both these antithrombotic agents, which limited the efficacy and applicability of this combined therapy.The availability of the newer thienopyridine, prasugrel, and the cyclopentyl-triazolopyrimidine, ticagrelor, represents an important addition to the physician's armamentarium. Dual antiplatelet therapy with aspirin and clopidogrel or one of the newer agents interferes with platelet activation in complementary, but separate pathways. Aspirin irreversibly inhibits cyclooxygenase, thus preventing the production of thromboxane A2, which is a prothrombotic and vasoconstrictive substance. Thienopyridines (clopidogrel/prasugrel) irreversibly and ticagrelor reversibly prevent and inhibit platelet activation by blocking one of the three known adenosine 5'-diphosphate (ADP) receptors (the P2Y12 receptor) on the platelet surface, thus interfering with platelet activation, degranulation and aggregation. Each of these antiplatelet agents has a protective effect against adverse vascular events; classical DAPT with aspirin and clopidogrel has an even stronger antiplatelet effect compared with either agent alone, however DAPT combining aspirin with one of the newer more potent agents translates into superior antithrombotic protection in atherothrobotic vascular disease, albeit at an increased, though not inordinately, risk for bleeding complications. A number of randomized clinical trials have demonstrated and confirmed the incremental benefit and efficacy of DAPT with use of either classical or newer agents, above and beyond that of each antiplatelet agent alone. Data have also been obtained from studies where indications for the use of DAPT continue to expand into other patient groups, rendering and maintaining DAPT a sweeping combination in Cardiology. This article is a comprehensive review of all these data and the landmark trials on the two classical and also the newer antiplatelet agents, the issues involved and the current recommendations for their use in patients with atherosclerotic heart disease and other cardiovascular disorders and procedures.

ASP6537, a novel highly selective cyclooxygenase-1 inhibitor, exerts potent antithrombotic effect without “aspirin dilemma”

IntroductionAspirin inhibits both the cyclooxygenase (COX)-1-dependent production of thromboxane A2 (TXA2) in platelets and COX-2-dependent production of anti-aggregatory prostaglandin I2 (PGI2) in vessel walls, resulting in “aspirin dilemma.” Our objective is to investigate whether ASP6537 can overcome aspirin dilemma and exert a potent antithrombotic effect without a concurrent ulcerogenic effect. MethodsWe evaluated the inhibitory effects of ASP6537 on recombinant human COX-1 (rhCOX-1) and rhCOX-2 activities using a COX-1/2 selectivity test. To determine whether ASP6537 induces aspirin dilemma, we examined the effects of ASP6537 on in vitro TXA2 and PGI2 metabolite production from platelets and isolated aorta of guinea pigs, and on plasma concentrations of TXA2 and PGI2 metabolites in aged rats. Finally, we evaluated the antithrombotic effects and ulcerogenic activity of ASP6537 using an electrically induced carotid arterial thrombosis model and a gastric ulcer model in guinea pigs. ResultsThe IC50 ratios of rhCOX-2 to rhCOX-1 for ASP6537 and aspirin were >142,000 and 1.63 fold, respectively. ASP6537 inhibited TXA2 production more selectively than aspirin in in vitro and in vivo TXA2/PGI2 production studies. ASP6537 exerted a significant antithrombotic effect at ≥3mg/kg, while aspirin tended to inhibit thrombosis at 300mg/kg but it was not statistically significant. Further, ASP6537 did not induce ulcer formation at 100mg/kg, whereas aspirin exhibited an ulcerogenic effect at doses of ≥100mg/kg. ConclusionsASP6537 functions as a highly selective COX-1 inhibitor with a superior ability to aspirin for normalizing TXA2/PGI2 balance, and exerts antithrombotic effect without ulcerogenic effect.

Histamine, carbachol, and serotonin induce hyperresponsiveness to ATP in guinea pig tracheas: involvement of COX-2 pathway

Extracellular ATP promotes an indirect contraction of airway smooth muscle via the secondary release of thromboxane A2 (TXA2) from airway epithelium. Our aim was to evaluate if common contractile agonists modify this response to ATP. Tracheas from sensitized guinea pigs were used to evaluate ATP-induced contractions before and after a transient contraction produced by histamine, carbachol, or serotonin. Epithelial mRNA for COX-1 and COX-2 was measured by RT-PCR and their expression assessed by immunohistochemistry. Compared with the initial response, ATP-induced contraction was potentiated by pretreatment with histamine, carbachol, or serotonin. Either suramin (antagonist of P2X and P2Y receptors) plus RB2 (antagonist of P2Y receptors) or indomethacin (inhibitor of COX-1 and COX-2) annulled the ATP-induced contraction, suggesting that it was mediated by P2Y receptor stimulation and TXA2 production. When COX-2 was inhibited by SC-58125 or thromboxane receptors were antagonized by SQ-29548, just the potentiation was abolished, leaving the basal response intact. Airway epithelial cells showed increased COX-2 mRNA after stimulation with histamine or carbachol, but not serotonin, while COX-1 mRNA was unaffected. Immunochemistry corroborated this upregulation of COX-2. In conclusion, we showed for the first time that histamine and carbachol cause hyperresponsiveness to ATP by upregulating COX-2 in airway epithelium, which likely increases TXA2 production. Serotonin-mediated hyperresponsiveness seems to be independent of COX-2 upregulation, but nonetheless is TXA2 dependent. Because acetylcholine, histamine, and serotonin can be present during asthmatic exacerbations, their potential interactions with ATP might be relevant in its pathophysiology.