Structure-Activity Relationship Studies on Highly Functionalized Pyrazole Hydrazones and Amides as Antiproliferative and Antioxidant Agents.

Aminopyrazoles represent interesting structures in medicinal chemistry, and several derivatives showed biological activity in different therapeutic areas. Previously reported 5-aminopyrazolyl acylhydrazones and amides showed relevant antioxidant and anti-inflammatory activities. To further extend the structure-activity relationships in this class of derivatives, a novel series of pyrazolyl acylhydrazones and amides was designed and prepared through a divergent approach. The novel compounds shared the phenylamino pyrazole nucleus that was differently decorated at positions 1, 3, and 4. The antiproliferative, antiaggregating, and antioxidant properties of the obtained derivatives 10-22 were evaluated in in vitro assays. Derivative 11a showed relevant antitumor properties against selected tumor cell lines (namely, HeLa, MCF7, SKOV3, and SKMEL28) with micromolar IC50 values. In the platelet assay, selected pyrazoles showed higher antioxidant and ROS formation inhibition activity than the reference drugs acetylsalicylic acid and N-acetylcysteine. Furthermore, in vitro radical scavenging screening confirmed the good antioxidant properties of acylhydrazone molecules. Overall, the collected data allowed us to extend the structure-activity relationships of the previously reported compounds and confirmed the pharmaceutical attractiveness of this class of aminopyrazole derivatives.

Investigations of Antioxidant and Anti-Cancer Activities of 5-Aminopyrazole Derivatives.

To further extend the structure-activity relationships (SARs) of 5-aminopyrazoles (5APs) and identify novel compounds able to interfere with inflammation, oxidative stress, and tumorigenesis, 5APs 1-4 have been designed and prepared. Some chemical modifications have been inserted on cathecol function or in aminopyrazole central core; in detail: (i) smaller, bigger, and more lipophilic substituents were introduced in meta and para positions of catechol portion (5APs 1); (ii) a methyl group was inserted on C3 of the pyrazole scaffold (5APs 2); (iii) a more flexible alkyl chain was inserted on N1 position (5APs 3); (iv) the acylhydrazonic linker was moved from position 4 to position 3 of the pyrazole scaffold (5APs 4). All new derivatives 1-4 have been tested for radical scavenging (DPPH assay), anti-aggregating/antioxidant (in human platelets) and cell growth inhibitory activity (MTT assay) properties. In addition, in silico pharmacokinetics, drug-likeness properties, and toxicity have been calculated. 5APs 1 emerged to be promising anti-proliferative agents, able to suppress the growth of specific cancer cell lines. Furthermore, derivatives 3 remarkably inhibited ROS production in platelets and 5APs 4 showed interesting in vitro radical scavenging properties. Overall, the collected results further confirm the pharmaceutical potentials of this class of compounds and support future studies for the development of novel anti-proliferative and antioxidant agents.

Oxidative Stress Induced by Cortisol in Human Platelets.

Hypercortisolism is known to affect platelet function. However, few studies have approached the effect of exogenous cortisol on human platelets, and the results obtained are conflicting and unconvincing. In this study, the effect of exogenous cortisol on several parameters indicative of oxidative status in human platelets has been analysed. We have found that cortisol stimulates ROS production, superoxide anion formation, and lipid peroxidation, with these parameters being in strict correlation. In addition, cortisol decreases GSH and membrane SH-group content, evidencing that the hormone potentiates oxidative stress, depleting platelet antioxidant defence. The involvement of src, syk, PI3K, and AKT enzymes in oxidative mechanisms induced by cortisol is shown. The main sources of ROS in cells can include uncontrolled increase of NADPH oxidase activity and uncoupled aerobic respiration during oxidative phosphorylation. Both mechanisms seem to be involved in ROS formation induced by cortisol, as the NADPH oxidase 1 inhibitor 2(trifluoromethyl)phenothiazine, and rotenone and antimycin A, complex I and III inhibitor, respectively, significantly reduce oxidative stress. On the contrary, the NADPH oxidase inhibitor gp91ds-tat, malate and NaCN, complex II and IV inhibitor, respectively, have a minor effect. It is likely that, in human platelets, oxidative stress induced by cortisol can be associated with venous and arterial thrombosis, greatly contributing to cardiovascular diseases.

Platelet Metabolic Flexibility: A Matter of Substrate and Location.

Platelets are cellular elements that are physiologically involved in hemostasis, inflammation, thrombotic events, and various human diseases. There is a link between the activation of platelets and their metabolism. Platelets possess considerable metabolic versatility. Although the role of platelets in hemostasis and inflammation is known, our current understanding of platelet metabolism in terms of substrate preference is limited. Platelet activation triggers an oxidative metabolism increase to sustain energy requirements better than aerobic glycolysis alone. In addition, platelets possess extra-mitochondrial oxidative phosphorylation, which could be one of the sources of chemical energy required for platelet activation. This review aims to provide an overview of flexible platelet metabolism, focusing on the role of metabolic compartmentalization in substrate preference, since the metabolic flexibility of stimulated platelets could depend on subcellular localization and functional timing. Thus, developing a detailed understanding of the link between platelet activation and metabolic changes is crucial for improving human health.

Insights into the Pharmacological Activity of the Imidazo-Pyrazole Scaffold.

In previous studies, we synthesized different imidazo-pyrazoles 1 and 2 with interesting anticancer, anti-angiogenic and anti-inflammatory activities. To further extend the structure-activity relationships of imidazo-pyrazole scaffold and to identify novel antiproliferative/anti-inflammatory agents potentially active with multi-target mechanisms, a library of compounds 3-5 has been designed and synthesized. The chemical modifications characterizing the novel derivatives include: i) decoration of the catechol ring with groups with different electronic, steric and lipophilic properties (compounds 3); ii) insertion of a methyl group on C-6 of imidazo-pyrazole scaffold (compounds 4); iii) shift of the acylhydrazonic substituent from position 7 to 6 of the imidazo-pyrazole substructure (compounds 5). All synthesized compounds were tested against a panel of cancer and normal cell lines. Derivatives 3 a, 3 e, 4 c, 5 g and 5 h showed IC50 values in the low micromolar range against selected tumor cell lines and proved to have antioxidant properties, being able to inhibit ROS production in human platelet. In silico calculation predicted favourable drug-like and pharmacokinetic properties for the most promising compounds. Furthermore, molecular docking and molecular dynamic simulations suggested the ability of most active derivative 3 e to interact with colchicine binding site in the polymeric tubulin α/tubulin ß/stathmin4 complex.

Inside the Mechanism of Action of Three Pyrazole Derivatives in Human Platelets and Endothelial Cells.

In the effort to obtain multitarget compound interfering with inflammation, oxidative stress, and tumorigenesis, we synthesized a small library of pyrazole compounds, selecting 4a, 4f, and 4g as the most noteworthy being IC50 against platelet ROS production induced by thrombin of about 10 µM. The in vitro antioxidant potential of the three molecules was evaluated, and since they show a remarkable antioxidative activity, their effect on several parameter indicative of oxidative status and on the efficiency of the aerobic metabolism was tested. The three molecules strongly inhibit superoxide anion production, lipid peroxidation, NADPH oxidase activity and almost restore the oxidative phosphorylation efficiency in thrombin-stimulated platelet, demonstrating a protective effect against oxidative stress. This effect was confirmed in endothelial cell in which 4a, 4f, and 4g show an interesting inhibition activity on H2O2-stimulated EA.hy926 cells. At last, antiproliferative activity of 4a, 4f, and 4g was submitted to a large screening at the NCI. The molecules show interesting anticancer activity, among them the most remarkable is 4g able to strongly inhibit the proliferation of both solid tumor and leukemia cells lines. In conclusion, all the three newly synthetized pyrazoles show remarkable antioxidant and antiproliferative effect worthy of further study.

Endocannabinoids effect on oxidative status of human platelets.

Reactive oxygen species (ROS) are known to regulate platelet activation. Since endocannabinoids behave as platelet agonists, we investigated the effect of two endocannabinoids, 2-arachidonoylglycerol (2AG) and anandamide (AEA) on the oxidative status of human platelets. We have demonstrated that 2AG and AEA stimulate ROS production, superoxide anion formation and lipid peroxidation. The effect is dose and time dependent and mainly occurs through the involvement of cannabinoid receptor 1 (CB1) since all tested parameters are greatly reduced by SR141716, the CB1 specific inhibitor. The specific inhibitor of cannabinoid receptor 2 (CB2) SR144528 produces a very small inhibition. The involvement of syk/PI3K/AKT/mTor pathway in oxidative stress induced by endocannabinoids is shown. Nicotinamide adenine dinucleotide phosphate oxidase seems to be poorly involved in the endocannabinoids effect. Concerning the aerobic metabolism, it has been demonstrated that endocannabinoids reduce the oxygen consumption and adenosine triphosphate synthesis, both in the presence of pyruvate + malate or succinate. In addition, endocannabinoids inhibit the activity of respiratory complexes II, III and IV and increase the activity of respiratory complex I. The endocannabinoids effect on aerobic metabolism seems to be also a CB1 mediated mechanism. Thus, in human platelets oxidative stress induced by endocannabinoids, mainly generated in the respiratory chain through the activation of complex I and the inhibition of complex II, III and IV, may lead to thrombotic events, contributing to cardiovascular diseases.

Usefulness of the corporate wellness projects in primary prevention at the population level: a study on the prevalence, awareness, and control of hypertension in the Ferrari company.

The aim of our study was to evaluate the prevalence, awareness, and control of hypertension in an apparently healthy company population. We conducted a cross-sectional study on a total sample of 2058 individuals with a mean age of 38 ± 9 years, enrolled for the first time to the Ferrari corporate wellness program "Formula Benessere". Hypertension was defined as systolic blood pressure (SBP) level ≥140 mmHg or diastolic BP (DBP) ≥90 mmHg or use of antihypertensive medication, whereas BP control was defined as BP level <130/80 mmHg. All 2058 participants were divided into three groups based on age: Group 1 aged <40 years (n = 1177, 57%), Group 2 aged 40-50 years (n = 627, 30%), and Group 3 aged >50 years (n = 254, 13%). Four-hundred and one subjects had BP levels ≥130/80 mmHg (19.5%). Two-hundred and sixty-one individuals (12.7%) had high-normal BP values and 140 subjects had rest SBP ≥140 mmHg and/or DBP ≥90 mmHg (6,8%), of which 41 (29.3%) with grade 2 hypertension. In the overall population, 259 individuals (12.5%) were affected by hypertension, the prevalence increasing with age. Only a minority (51%) was aware of being hypertensive and already treated with antihypertensive medications (45.9%). An adequate BP control was achieved in only 57% of subjects who received BP-lowering therapy. Corporate wellness programs may represent an essential tool in identifying apparently healthy subjects with an inadequate control of cardiovascular (CV) risk factors, such as hypertension. These preventive programs in the workplace may help to improve and spread primary CV prevention at the population level.

Relationship Between Cardiorespiratory Fitness, Baseline Blood Pressure and Hypertensive Response to Exercise in the Ferrari Corporate Population.

AIM: To evaluate the incidence and clinical significance of impaired cardiorespiratory fitness (CRF) and the association with baseline blood pressure (BP) levels and hypertensive response to exercise (HRE). METHODS: A cross-sectional study was conducted on a total sample of 2058 individuals with a mean age of 38 ± 9 years, enrolled for the first time at the Ferrari corporate wellness program "Formula Benessere", including a maximal exercise stress testing (EST). BP and heart rate (HR) values were obtained from EST at rest, during exercise and recovery time. CRF was arbitrarily classified according to estimated VO2 max in optimal, normal, mildly and moderately reduced. RESULTS: One-hundred and thirty-nine individuals of 2058 (6.7%) showed a moderate CRF reduction assessed by EST. Subjects with elevated resting and/or exercise BP showed a worse CRF than those with normal BP levels, also after the adjustment for age, sex, body mass index, smoking habits, peak SBP and DBP. Seventy-seven individuals (3.7%) showed an HRE during EST, with normal baseline BP levels. CONCLUSION: About 7% of a corporate population showed a significantly reduced CRF, assessed by EST. Individuals with lower levels of CRF have higher resting and/or peak exercising BP values after adjusting for co-variables. This study expands the role of EST outside of traditional ischemic CVD evaluation, towards the assessment of reduced CRF and HRE in the general population, as a possible not evaluated CV risk factor.

New Series of Pyrazoles and Imidazo-Pyrazoles Targeting Different Cancer and Inflammation Pathways.

(1) Background: different previously synthesized pyrazoles and imidazo-pyrazoles showed interesting anti-angiogenic action, being able to interfere with ERK1/2, AKT and p38MAPK phosphorylation in different manners and with different potency; (2) Methods: here, a new small compound library, endowed with the same differently decorated chemical scaffolds, has been synthetized to obtain new agents able to inhibit different pathways involved in inflammation, cancer and human platelet aggregation. (3) Results: most of the new synthesized derivatives resulted able to block ROS production, platelet aggregation and p38MAPK phosphorylation both in platelets and Human Umbilical Vein Endothelial cells (HUVEC). This paves the way for the development of new agents with anti-angiogenic activity.

Lectin-induced oxidative stress in human platelets.

Previously we have shown that wheat germ agglutinin (WGA) and, with minor potency, Phaseolus vulgaris agglutinin (PHA), but not lens culinarian agglutinin (LCA), induce platelet aggregation, through the PLCÆ´2 activation by the concerted action of src/syk and PI3K/BTK pathways. In this study, we have investigated platelet oxidative stress induced by lectins. Several parameters indicative of oxidative stress, such as reactive oxygen species (ROS), superoxide anion, lipid peroxidation and the efficiency of the aerobic metabolism, have been measured. It was found that ROS, superoxide anion formation and lipid peroxidation are significantly increased upon platelet treatment with WGA and PHA while LCA is ineffective. WGA is always more effective than PHA in all experimental conditions tested. In addition, the involvement of NADPH oxidase 1, syk and PI3K in oxidative stress induced by WGA and PHA has been shown. Concerning the lectins effect on aerobic metabolism, WGA and PHA, but not LCA, act as uncoupling agents, determining an increase of oxygen consumption and a decrease of ATP synthesis, with a consequent decrease of P/O value. These results are confirmed by the impairment of platelets proton gradient formation, evaluated by membrane potential, in platelets treated with WGA and PHA. In conclusion lectins, especially WGA, induce oxidative stress in platelets and decrease energy availability through modifications of membrane structure leading to the inefficiency of the aerobic machinery that steers platelets toward death as suggested by the decreased metabolic activity of platelets and the increased lactic dehydrogenase release.

New Hybrid Pyrazole and Imidazopyrazole Antinflammatory Agents Able to Reduce ROS Production in Different Biological Targets.

Several anti-inflammatory agents based on pyrazole and imidazopyrazole scaffolds and a large library of substituted catechol PDE4D inhibitors were reported by us in the recent past. To obtain new molecules potentially able to act on different targets involved in inflammation onset we designed and synthesized a series of hybrid compounds by linking pyrazole and imidazo-pyrazole scaffolds to differently decorated catechol moieties through an acylhydrazone chain. Some compounds showed antioxidant activity, inhibiting reactive oxygen species (ROS) elevation in neutrophils, and a good inhibition of phosphodiesterases type 4D and, particularly, type 4B, the isoform most involved in inflammation. In addition, most compounds inhibited ROS production also in platelets, confirming their ability to exert an antiinflammatory response by two independent mechanism. Structure-activity relationship (SAR) analyses evidenced that both heterocyclic scaffolds (pyrazole and imidazopyrazole) and the substituted catechol moiety were determinant for the pharmacodynamic properties, even if hybrid molecules bearing to the pyrazole series were more active than the imidazopyrazole ones. In addition, the pivotal role of the catechol substituents has been analyzed. In conclusion the hybridization approach gave a new serie of multitarget antiinflammatory compounds, characterized by a strong antioxidant activity in different biological targets.

Exercise stress test in apparently healthy individuals - where to place the finish line? The Ferrari corporate wellness programme experience.

AIMS: The aim of our study was to assess the clinical significance of the exercise stress testing endpoints, namely 85% of maximal theoretical heart rate (MTHR), metabolic equivalent of task, and rating of perceived exertion (RPE), and their relation to electrocardiographic (ECG) changes in a healthy adult population. METHODS: A cross-sectional study was conducted on 408 males and 52 females (mean age 39.4 ± 8.6 years) who performed the maximal cycle ergometer exercise stress test until volitional exhaustion, reporting the RPE score at 85% of MTHR and at peak exercise. Metabolic equivalents of task were indirectly calculated from the maximum workload and compared with the predicted values. Sitting torso-lead ECG and blood pressure were recorded at rest, during exercise and during recovery. RESULTS: Of 460 participants, 73% exceeded 85% of MTHR. The RPE score represented the overall most significant endpoint of exercise stress testing, with the median value of 17 at peak exercise. ECG events were detected in 23/124 (18.5%) who reached ≤ 85% of MTHR and in 61/336 (18.2%) who achieved >85% of MTHR ( p = 0.92). In the latter group, 54% of ECG changes occurred at < 85% of MTHR and 46% at > 85% of MTHR ( p = 0.51). If the exercise stress testing had been interrupted at ≤ 85% of MTHR, almost half of the ECG events would have remained undetected and 35% of the cardiovascular abnormalities observed at the diagnostic follow-up would have remained undiagnosed. CONCLUSION: Terminating exercise stress testing before volitional exhaustion and an RPE score of 17 limits the test accuracy and reduces the possibility to detect cardiovascular abnormalities in apparently healthy adult populations.

Reshaped as polyester-based nanoparticles, gallic acid inhibits platelet aggregation, reactive oxygen species production and multi-resistant Gram-positive bacteria with an efficiency never obtained.

Natural polyphenols such as Gallic Acid (GA) form an important class of bioactive chemical entities that, having innumerable biological properties, could represent a safer alternative to common drugs against several disorders, including platelet aggregation, radical oxygen species (ROS) hyperproduction, oxidative stress (OS) and bacterial infections. Unfortunately, their clinical uses are limited by pharmacokinetics drawbacks and high sensitivity to environmental factors. In order to overcome these problems and to exploit the GA curative potentials, it has been linked to a biodegradable nanospherical dendrimer matrix, capable of protecting it, thus obtaining a GA-enriched nanosized dendrimer (GAD) endowed with a strong antioxidant capacity. GAD activity as an inhibitor of platelet aggregation and ROS accumulation and its antibacterial efficiency are evaluated here and compared to those of free GA, obtaining outcomes never achieved. Regarding platelet aggregation induced by thrombin and collagen, the GAD proved to be stronger by 7.1 and 7.3 times, respectively. Furthermore, the GAD showed a ROS inhibitory activity higher than that of GA by 8.1 (thrombin) and 6.9 (collagen) times. Concerning the antibacterial activities, evaluated on eleven multi-resistant Gram-positive strains of clinical relevance, the GAD is far more potent than GA, by exerting a growth inhibitory activity at MIC (µM) concentrations lower by factors in the range 12-50.

Anandamide Induces Platelet Nitric Oxide Synthase through AMP-Activated Protein Kinase.

The objective of this study was to determine whether adenosine 5' monophosphate (AMP)-activated protein kinase (AMPK) is activated by anandamide (AEA) and is involved in endothelial nitric oxide synthase (eNOS) activation. We found that AEA stimulates and activates AMPKα through a Ca2+ -dependent/Calmodulin (CaM)-dependent pathway as the specific inhibitor of the Ca2+ /Calmodulin kinase kinase ß (CaMKKß) STO-609 abolishes the AMPK phosphorylation/activation. The same inhibiting effect is shown in platelets pretreated with LY294002, an inhibitor of phosphatidylinositol 3 kinase (PI3K), or with MK2206, an inhibitor of protein kinase B (AKT), suggesting that AMPK is downstream of the PI3K/AKT pathway. Moreover, the AEA-induced eNOS activation and the consequent nitric oxide (NO) and guanosine 3'-5' cyclic monophosphate (cGMP) increase are mediated by the CaMKKß/AMPKα pathway as STO-609 significantly inhibits these parameters. In contrast, liver kinase B1 (LKB1) seems to be very poorly involved. One crucial effect of NO and cGMP elevation is the activation of protein kinase G that can phosphorylate the vasodilator-stimulated phosphoprotein (VASP). We have demonstrated that AEA stimulates VASP phosphorylation on both thr278 and ser239 that is strongly inhibited by STO-609, LY294002, and MK2206. Finally, AMPK phosphorylation/activation and VASP phosphorylation are significantly reduced by SR141716, the specific inhibitor of type 1 cannabinoid receptor (CB1). SR144528, an antagonist of type 2 cannabinoid receptor (CB2), has a less-potent effect, suggesting that the CB1 receptor is overall involved in the AEA effect. In conclusion, we show that the CaMKKß/AMPKα pathway, downstream of the PI3K/AKT pathway, is activated by AEA in human platelets and leads to increase NO levels producing beneficial effects during ischemic conditions and contributing to extend platelet survival.

Extramitochondrial energy production in platelets.

BACKGROUND INFORMATION: Energy demand in human platelets is very high, to carry out their functions. As for most human cells, the aerobic metabolism represents the primary energy source in platelets, even though mitochondria are negligibly represented. Following the hypothesis that other structures could be involved in chemical energy production, in this work, we have investigated the functional expression of an extramitochondrial aerobic metabolism in platelets. RESULTS: Oximetric and luminometric analyses showed that platelets consume large amounts of oxygen and produce ATP in the presence of common respiring substrates, such as pyruvate + malate or succinate, although morphological electron microscopy analysis showed that these contain few mitochondria. However, evaluation of the anaerobic glycolytic metabolism showed that only 13% of consumed glucose was converted to lactate. Interestingly, the highest OXPHOS activity was observed in the presence of NADH, not a readily permeant respiring substrate for mitochondria. Also, oxygen consumption and ATP synthesis fuelled by NADH were not affected by atractyloside, an inhibitor of the adenine nucleotide translocase, suggesting that these processes may not be ascribed to mitochondria. Functional data were confirmed by immunofluorescence microscopy and Western blot analyses, showing a consistent expression of the ß subunit of F1 Fo -ATP synthase and COXII, a subunit of Complex IV, but a low signal of translocase of the inner mitochondrial membrane (a protein not involved in OXPHOS metabolism). Interestingly, the NADH-stimulated oxygen consumption and ATP synthesis increased in the presence of the physiological platelets agonists, thrombin or collagen. CONCLUSIONS: Data suggest that in platelets, aerobic energy production is mainly driven by an extramitochondrial OXPHOS machinery, originated inside the megakaryocyte, and that this metabolism plays a pivotal role in platelet activation. SIGNIFICANCE: This work represents a further example of the existence of an extramitochondrial aerobic metabolism, which can contribute to the cellular energy balance.

Activation of CaMKKß/AMPKα pathway by 2-AG in human platelets.

The objective of this study was to determine whether AMPK is activated by 2-arachidonoylglycerol (2-AG) and participates to the cytoskeleton control in human platelets. We found that 2-AG stimulates the AMPKα activation through a Ca2+ /Calmodulin-dependent pathway as the specific inhibition of the CaMKKß by STO-609 inhibits the AMPKα phosphorylation/activation. Moreover, the CaMKKß/AMPKα pathway activated by 2-AG is involved in the phosphorylation of cofilin, vasodilator stimulated phosphoprotein (VASP), and myosin light chain (MLCs). These proteins participate to actin cytoskeletal remodelling during aggregation. We found that the phosphorylation/activation inhibition of these proteins is associated with a significant reduction in actin polymerization, aggregation, ATP, and α-granule secretion. Finally, AMPKα activation, Cofilin, VASP, and MLCs phosphorylation are significantly reduced by SR141716, the specific inhibitor of type 1 cannabinoid (CB1) receptor, suggesting that the CB1 receptor is involved in the 2-AG effect. In conclusion, we have shown that the CaMKKß/AMPKα pathway is activated by 2-AG in human platelets and controls the phosphorylation of key proteins involved in actin polymerization and aggregation.

The molecular mechanisms involved in lectin-induced human platelet aggregation.

We have compared the effect of three legume lectins, wheat germ agglutinin (WGA), Phaseolus vulgaris agglutinin (PHA) and Lens culinaris agglutinin (LCA), on the function of human platelets. We have found that WGA is more active than PHA in stimulating platelet activation/aggregation, while LCA has no effect. Studies on the mechanisms involved show that WGA and PHA induce phosphorylation/activation of PLCγ2 and increase [Ca2+]i. For the first time, it has been shown that Src/Syk pathway, the adapter protein SLP-76 and the exchange protein VAV, participate in the PLCγ2 activation by these lectins. Moreover WGA and PHA stimulate the PI3K/AKT pathway. PI3K, through its product phosphatidylinositol-3,4,5-trisphosphate activates Bruton's tyrosine kinase (BTK) and contributes to PLCγ2 activation. In conclusion, our findings suggest that PLCγ2 activation induced by WGA and PHA is regulated by Src/Syk and by PI3K/BTK pathways through their concerted action.

Regulation of cAMP Intracellular Levels in Human Platelets Stimulated by 2-Arachidonoylglycerol.

We demonstrated that in human platelets the endocannabinoid 2-arachidonoylglycerol (2-AG) decreased dose- and time-dependently cAMP intracellular levels. No effect on cAMP decrease induced by 2-AG was observed in the presence of the adenylate cyclase inhibitor SQ22536 as well in platelets pretreated with the thromboxane A2 receptor antagonist, SQ29548 or with aspirin, inhibitor of arachidonic acid metabolism through the cyclooxygenase pathway. An almost complete recovering of cAMP level was measured in platelets pretreated with the specific inhibitor of phosphodiesterase (PDE) 3A, milrinone. In platelets pretreated with LY294002 or MK2206, inhibitors of PI3K/AKT pathway, and with U73122, inhibitor of phospholipase C pathway, only a partial prevention was shown. cAMP intracellular level depends on synthesis by adenylate cyclase and hydrolysis by PDEs. In 2-AG-stimulated platelets adenylate cyclase activity seems to be unchanged. In contrast PDEs appear to be involved. In particular PDE3A was specifically activated, as milrinone reversed cAMP reduction by 2-AG. 2-AG enhanced PDE3A activity through its phosphorylation. The PI3K/AKT pathway and PKC participate to this PDE3A phosphorylation/activation mechanism as it was greatly inhibited by platelet pretreatment with LY294002, MK2206, U73122, or the PKC specific inhibitor GF109203X. Taken together these data suggest that 2-AG potentiates its power of platelet agonist reducing cAMP intracellular level.

Effect of 2-arachidonoylglycerol on myosin light chain phosphorylation and platelet activation: The role of phosphatidylinositol 3 kinase/AKT pathway.

The endocannabinoid 2-arachidonoylglycerol (2-AG) can be considered a true agonist as it is able to activate human platelets stimulating arachidonic acid release, thromboxane B2 formation and calcium intracellular elevation. Recently we have shown that 2-AG induces a rapid myosin light chain (MLC) phosphorylation/activation, early mediated by RhoA kinase (ROCK) signalling pathway and later by myosin light chain kinase. The aim of the present study was to investigate the role of phosphatidylinositol 3 kinase (PI3K)/AKT pathway in MLC phosphorylation and some downstream events such as actin polymerization, ATP secretion and aggregation. We demonstrated that PI3K in particular the isoforms α and ß and AKT have a role in MLC phosphorylation. The stimulation of PI3K/AKT pathway activates ROCK. ROCK is directly involved in the early phase of MLC activation stimulating thr18 phosphorylation. MLC activation is strengthened through the MLC phosphatase inhibition, that is accomplished through the phosphorylation of MYPT1, catalytic subunit of MLC phosphatase, overall mediated by ROCK. In addition we have found that the PI3Kα/ß isoforms and AKT are involved in the downstream mechanisms leading to actin polymerization, ATP secretion and aggregation of human platelets stimulated by 2-AG.