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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.

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 β 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.

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 CaMKKβ by STO-609 inhibits the AMPKα phosphorylation/activation. Moreover, the CaMKKβ/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 CaMKKβ/AMPKα pathway is activated by 2-AG in human platelets and controls the phosphorylation of key proteins involved in actin polymerization and aggregation.

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 PLCƴ2 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., Highlights • WGA and PHA but not LCA induce ROS production in human platelets. • WGA and PHA but not LCA induce superoxide anion formation and lipid peroxidation. • WGA and PHA, but not LCA modify aerobic metabolism decreasing P/O value. • WGA and PHA decrease platelet membrane potential. • All these effects are due to increased NADPH oxidase, syk and PI3K activity.

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 β (CaMKKβ) 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 CaMKKβ/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 CaMKKβ/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.

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.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 β subunit of FData 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.This work represents a further example of the existence of an extramitochondrial aerobic metabolism, which can contribute to the cellular energy balance.

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.

The multistep preparation of the new 10-substituted 2-(1-piperazinyl)pyrimido[1,2- a ]benzimidazol-4(10 H )-ones 6a – o , and of the two isomers 10-ethyl-2-(diethylamino)pyrimido[1,2- a ]benzimidazol-4(10 H )-one 6p and 10-ethyl-4-(diethylamino)pyrimido[1,2- a ]benzimidazol-2(10 H )-one 13 , as well as the in vitro evaluation of their inhibitory activity on human platelet aggregation induced in platelet-rich plasma by ADP, collagen or the Ca 2+ ionophore A23187 were here described. Nine out of fifteen 2-(1-piperazinyl)derivatives ( 6g – o ) showed good inhibitory properties towards all the platelet aggregation agonists used. Moreover, a molecular modelling study has been performed on two of the best compounds of this series ( 6i and 6o ) to confirm in silico their interactions with the catalytic site of human platelet PDE3, using the X-ray data of the PDE3B isoform in complex with an inhibitor.

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.

In platelets of patients affected with myeloproliferative disorders, glycolytic and glycogenolytic flux have been examined. Results of studies on glucose uptake, glycogen breakdown and lactate formation have been reported. No difference in glucose uptake between controls and patients was observed, but in patients a higher lactate formation was always noted in the presence or absence of exogenous glucose, in correlation with a higher glycogen breakdown rate. Results could be explained by modified regulation mechanisms on glycogen phosphorylase.

The synthesis and in vitro antiplatelet activity significant data of coumarin derivatives 5i-x and quinolin-2(1H)-one derivatives 22a,b, as well as the corresponding structure-activity relationships are described. The recently reported 8-methyl-4-(1-piperazinyl)-7-(3-pyridylmethoxy)coumarin 5f and its potent 7-(2-morpholinoethoxy)-substituted new analogue 5u were notably more effective inhibitors of pure human platelet PDE3 than milrinone and cilostazol: these data were related, through a molecular modeling study, with the molecular interactions of the four compounds with the human PDE3A catalytic site.

The aim of this study was to examine the homocysteine effect on phospholipase Cγ2 (PLCγ2) activation and to investigate the signaling pathway involved. We found that homocysteine stimulated the tyrosine phosphorylation and activation of platelet PLCγ2. The tyrosine kinases p60src and p72syk appeared to be involved upstream. Reactive oxygen species were increased in homocysteine treated platelets. Likely oxidative stress could prime the non receptor-mediated tyrosine kinase p60src, inducing phosphorylation and activation of p72syk. The antioxidant N-acetyl-L-cysteine prevented the activation of these kinases. The phosphorylation and activation of PLCγ2 were greatly reduced by the inhibition of p72syk through piceatannol. Moreover indomethacin diminished the homocysteine effect on p60src, p72syk and PLCγ2, suggesting that thromboxane A2 could be involved. In addition the treatment of platelets with homocysteine caused intracellular calcium rise and protein kinase C activation. Finally homocysteine induced platelet aggregation, that was partially reduced by indomethacin and by N-acetyl-L-cysteine of 35% or 50% respectively, while the PLCγ2 specific inhibitor U73122 diminished platelet response to homocysteine of 70%. Altogether the data indicate that PLCγ2 plays an important role in platelet activation by homocysteine and that the stimulation of this pathway requires signals through oxygen free radicals and thromboxane A2. J. Cell. Biochem. 100: 1255–1265, 2007. © 2006 Wiley-Liss, Inc.

Background Recent evidence indicates that hyperhomocysteinaemia is an independent risk factor for atherosclerosis, thrombosis and other cardiovascular diseases. This may be secondary to impaired fibrinolysis or increased platelet reactivity. Nitric oxide (NO), a product from l-arginine by NOS and potent antiaggregating agent, plays an important role in the regulation of platelet function. Design The present study aimed to define the effect of homocysteine on the l-arginine/NO pathway in human platelets. l-arginine uptake, NO formation and Ca2+ levels were measured. Moreover the homocysteine effect on platelet activation induced by thrombin was tested. Results Homocysteine causes a concentration-dependent inhibition of l-arginine transport. Results show that homocysteine does not modify the Km parameter, but it significantly decreases the Vmax value. The nitrite and nitrate formation, strictly correlated with the l-arginine transport, also significantly decreased. In contrast, cNOS activity remained unchanged upon homocysteine treatment. In addition homocysteine in a dose dependent manner increased the intracellular Ca2+ concentration and platelet response to thrombin. Conclusions Results indicate that the l-arginine/NO pathway is one of the various targets of homocysteine in human platelets. The increased Ca2+ levels associated with reduced NO formation may generate hyperactivation and may contribute to the thrombogenic processes.

Background It has been suggested that homocysteine is implicated in the risk of atherosclerosis and thrombosis. The pathogenic mechanism has not been clarified, but oxygen-free species produced by the homocysteine metabolism and auto-oxidation could have a role. Design We have studied the effect of homocysteine on arachidonic acid release in human platelets. Two important products of arachidonic acid metabolism – thromboxane B2 (TXB2) and reactive oxygen species (ROS) – have been assayed. Results Results indicate that homocysteine induces arachidonic acid release that is partially inhibited by 5,8,11,14-eicosatetraynoic acid (ETYA). Platelet incubation with homocysteine significantly increases basal levels of TXB2 and ROS. The effect is time- and dose-dependent. The TXB2 formation is strictly correlated with the arachidonic acid release. Moreover, ROS accumulation is largely inhibited by ETYA and partially reduced by diphenyleneiodonium (DPI), suggesting the involvement both of enzymes metabolising arachidonic acid (cyclooxygenase, lipooxygenase, cytochrome P450 monooxygenase) and of NAD(P)H oxidase. Conclusion Homocysteine induces oxidative stress in human platelets in vitro. The unbalance in platelet redox-state and the increased TXB2 formation may generate hyperactivation, contributing to a thrombogenic state leading to cardiovascular diseases.

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 β 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/β isoforms and AKT are involved in the downstream mechanisms leading to actin polymerization, ATP secretion and aggregation of human platelets stimulated by 2-AG.

Treatment of human platelets with the alkylating agent N-ethylmaleimide (NEM) induces arachidonic acid release. The effect was time- and dose-dependent. NEM-stimulated arachidonic acid mobilisation could be prevented by pretreating platelets with the cytosolic phospholipase A2 (cPLA2)-specific inhibitor arachidonyltrifluoromethyl ketone. Moreover, the tyrosine kinase inhibitor genistein was able to significantly inhibit arachidonic acid mobilisation. NEM-stimulated release of arachidonic acid appears to be a Ca2+-dependent mechanism, as shown by the observation that arachidonic acid mobilisation was significantly reduced by platelet treatment with EGTA and abolished by preloading platelets with the intracellular chelator 1,2-bis (o-aminophenoxy) ethane-N,N,N′,N′-tetraacetic acid tetra (acetoxymethyl) ester (BAPTA/AM). In Fura-2-loaded platelets, NEM was able to significantly increase the intracellular Ca2+ level. The Ca2+ elevation was significantly reduced in the presence of EGTA and suppressed by cell treatment with BAPTA/AM. Arachidonic acid released by NEM produced a significant increase in reactive oxygen species (ROS) intracellular levels, which was partially inhibited by diphenyleneiodonium and almost completely suppressed by 5,8,11,14-eicosatetraynoic acid. In conclusion, the results in this study demonstrate that NEM stimulates arachidonic acid release by cPLA2 activation through intracellular Ca2+ elevation. In addition, tyrosine specific protein kinases seem to be involved in arachidonic acid release. ROS was also shown to be formed during arachidonic acid metabolisation.

A group of 29 patients with non-insulin-dependent diabetes mellitus (NIDDM) was compared with a group of 19 patients who had good glycemic control for platelet activity and hydrogen peroxide formation. NIDDM patients showed platelet hyperactivity in response to low ADP concentrations. In addition, stimulated platelets from untreated NIDDM patients produced more hydrogen peroxide than platelets of treated and normal subjects. Hydrogen peroxide accumulation was not related to modification of the enzymatic systems involved in its synthesis and break-down. The specific activities of NAD(P)H cytochrome C reductase, catalase, glutathione peroxidase and glutathione reductase were not different between patients and healthy subjects. It is likely that the platelet intracellular elevation of reactive oxygen free-radicals could play an important role in the vascular complications and thrombotic risk that is often present in NIDDM patients.

The effect on human platelets of 2-(1-piperazinyl)-4H-pyrido[1,2-a]pyrimidin-4-one (AP155) was tested in vitro by measuring cyclic adenosine monophosphate (cAMP) level, cytosolic Ca++, [125I]fibrinogen binding as well as aggregation induced by several agonists. AP155 dose-dependently inhibited aggregation both in platelet rich plasma (PRP) and in washed platelets (WP), exerting its maximal power in the presence of collagen, ADP and platelet activating factor (PAF). It specifically inhibited the activity of cAMP high affinity phosphodiesterase (PDE), resulting in a sufficient increase in cAMP levels to activate cAMP-dependent protein kinase. AP155 was able to inhibit aggregation, the increase in cytosolic Ca++ induced by thrombin, and fibrinogen binding to ADP or thrombin-stimulated platelets. Thus, this new pyridopyrimidine derivative exerts its antiplatelet activity by increasing cAMP intracellular concentration.

Free radical activity may contribute to atherosclerotic lesions which in diabetic subjects may frequently lead to vascular complications. It is known that oxidative stress is associated to diabetes. Protein glycation and glucose oxidation could be possible source of free radicals. 28 non insulin dependent diabetic subjects (NIDDM) were examined. 20 healthy subjects matched for age, sex and for the presence of hypertension and hyperlipidemia were also studied. Hydrogen peroxide, measured by intracellular levels of the fluorescent 2,7-dichloro-fluorescein (DCF), was considered as indicative parameter of free radical production. The results showed that in resting platelets the basal level of hydrogen peroxide was significantly higher in diabetic subjects than in controls. Moreover, after stimulation with thrombin, collagen, phorbol myristate acetate (PMA) and platelet activating factor (PAF), platelets of diabetic subjects generated significantly higher amounts of hydrogen peroxide than controls. Moreover, platelet aggregation induced by adenosine 5'-diphosphate (ADP) and plasma beta TG levels were higher in diabetics than in controls. In diabetic patients platelet free radical production and functional activity are increased and therefore could play a role in the elevated thrombotic risk described in diabetes.

In human platelets the endocannabinoid 2-arachidonoylglycerol (2-AG) stimulates some important pathways leading to thromboxane B2 formation, calcium intracellular elevation, ATP secretion and actin polymerisation. The aim of the present study was to examine the 2-AG effect on myosin light chain (MLC) phosphorylation and to investigate the mechanisms involved. We demonstrated that 2-AG induced a rapid MLC phosphorylation, stimulating both the RhoA kinase (ROCK) and MLC kinase (MLCK) in a dose and time-dependent manner. In addition MLC phosphorylation was strengthened through the MLC phosphatase inhibition. MLC phosphatase inhibition was accomplished through the RhoA/ROCK and protein kinase C mediated phosphorylation of MLC phosphatase inhibiting subunits MYPT1 and CPI-17. The presence of CB1 receptor in human platelets and the involvement of CB1 receptor in MLC phosphorylation and MLC phosphatase inhibition was shown.

Methylglyoxal generates a slight increase in the basal level of hydrogen peroxide in platelets. The oxidation effect of methylglyoxal significantly potentiated by thrombin, depends on both the ketoaldehyde and the agonist concentrations. A further significant increase in hydrogen peroxide accumulation was obtained in platelets pretreated with the alkylating agent N-ethylmaleimide which depletes GSH and blocks glutathione peroxidase. Resting platelets completely transform the ketoaldehyde into D(-)lactate, whereas stimulated platelets transform about 10-15 per cent of the metabolized methylglyoxal into D(-)lactate. The metabolic modifications generated by methylglyoxal such as the GSH depletion and hydrogen peroxide accumulation induce modifications in platelet function. Methylglyoxal inhibits platelet aggregation induced by several agonists and ATP release induced by thrombin.

Summary A number of 3-(dialkylamino)-1 H -pyrimido[1,2- a ]quinolin-1-ones 3 and 2-(dialkylamino)-4 H -pyrimido[2,1- a ] iso-quinolin-4-ones 4 were prepared by treating the corresponding chloro derivatives with an excess of dialkylamines. The highest in vitro antiplatelet activity was obtained when the dialkylamino substituent was 1-piperazinyl (compounds 3g and 4e ). The novel 2-(1-piperazinyl)-4 H -pyrido[1,2- a ]pyrimidin-4-one 2a was also prepared by an analogous procedure, which resulted in the most active compound towards all the platelet aggregation inducers used (ADP, collagen, A 23187). Moreover, some examples of 1-(dialkylamino)-3 H -pyrimido [1,2- a ]quinolin-3-ones 5 and 4-(dialkylamino)-2 H -pyrimido[2,1- a ]isoquinolin-2-ones 6 were also obtained (together with negligible or lower amounts of the corresponding isomers 3 and 4 , respectively) from the cyclocondensation of the appropriate ethyl N,N -dialkylmalonamate/phosphorus oxychloride reagents 13 with 2-aminoquinoline or 1-aminoisoquinoline. These latter compounds showed a rather low antiplatelet activity.

The endogenous cannabinoid 2-arachidonoylglycerol (2-AG) is described as a platelet agonist able to induce aggregation and to increase intracellular calcium. In the present report we have confirmed these data and demonstrated that the inhibitor of p38MAPK SB203580 and the inhibitor of cPLA2 metabolism ETYA affect both these parameters. Thus, we aimed to define the role of p38MAPK/cytosolic phospholipase A2 (cPLA2) pathway in 2-AG-induced human platelet activation. p38MAPK activation was assayed by phosphorylation. cPLA2 activation was assayed by phosphorylation and as arachidonic acid release and thromboxane B2 formation. It was shown that 2-AG in a dose- and time-dependent manner activates p38MAPK peaking at 10 µM after 1 min of incubation. The 2-AG effect on p38MAPK was not impaired by apyrase, indomethacin or RGDS peptide but it was significantly reduced by SR141716, specific inhibitor of type-1 cannabinoid receptor and unaffected by the specific inhibitor of type-2 cannabinoid receptor SR144528. Moreover, the incubation of platelets with 2-AG led to the phosphorylation of cPLA2 and its activation. Platelet pretreatment with SB203580, inhibitor of p38MAPK, abolished both cPLA2 phosphorylation and activation. In addition SR141716 strongly impaired cPLA2 phosphorylation, arachidonic acid release and thromboxane B2 formation, whereas SR144528 did not change these parameters. Finally platelet stimulation with 2-AG led to an increase in free oxygen radical species. In conclusion, data provide insight into the mechanisms involved in platelet activation by 2-AG, indicating that p38MAPK/cPLA2 pathway could play a relevant role in this complicated process. J. Cell. Biochem. 112: 2794–2802, 2011. © 2011 Wiley-Liss, Inc.

In this study the effect of the endocannabinoid anandamide on platelet nitric oxide (NO)/cGMP pathway was investigated. Data report that anandamide in a dose-and time-dependent manner increased NO and cGMP levels and stimulated endothelial nitric oxide synthase (eNOS) activity. These parameters were significantly reduced by LY294002, selective inhibitor of PI3K and by MK2206, specific inhibitor of AKT. Moreover anandamide stimulated both eNOSser1177 and AKTser473 phosphorylation. Finally the anandamide effect on NO and cGMP levels, eNOS and AKT phosphorylation/activation were inhibited by SR141716, specific cannabinoid receptor 1 antagonist, supporting the involvement of anandamide binding to this receptor. Overall data of this report indicate that low concentrations of anandamide, through PI3K/AKT pathway activation, stimulates eNOS activity and increases NO levels in human platelets. In such way anandamide contributes to extend platelet survival. J. Cell. Biochem. 112: 924–932, 2011. © 2010 Wiley-Liss, Inc.

1. S-D-Lactoylglutathione accumulates in human platelets activated by agonists. Among the tested inducers thrombin is the most active. 2. The effect is dose and time-dependent. S-D-Lactoylglutathione, corresponding to depleted pool of reduced glutathione, can also be detected in platelets incubated with exogenous methylglyoxal. 3. A further significant increase was observed in platelets stimulated with thrombin in the presence of methylglyoxal. 4. No change in glyoxalase activities upon platelet stimulation with thrombin was shown.

The basal platelet level of reactive oxygen species and their enhancement following stimulation by different agonists were determined in a selected group of patients with essential thrombocythaemia (ET). Activated platelets had lower levels of superoxide anion and higher intracellular concentrations of hydrogen peroxide than controls. Higher levels of lipid peroxidation induced by N-ethylmaleimide were also observed. Measurement of the most important enzymes generating and scavenging these reactive oxygen species revealed increased specific activities of NADPH-cytochrome c reductase and superoxide dismutase and a decrease in platelet catalase activity in patients with ET. Since an abnormal production of oxygen radicals seems to be implicated in various pathological conditions and aging processes, the increased amount of hydrogen peroxide found in platelets of patients could be involved in some of the platelet alterations described in ET.

Arachidonic acid can act as a second messenger regulating many cellular processes among which is nitric oxide (NO) formation. The aim of the present study was to investigate the molecular mechanisms involved in the arachidonic acid effect on platelet NO level. Thus NO, cGMP and superoxide anion level, the phosphorylation status of nitric oxide synthase, the protein kinase C (PKC), and NADPH oxidase activation were measured. Arachidonic acid dose-dependently reduced NO and cGMP level. The thromboxane A 2 mimetic U46619 behaved in a similar way. The arachidonic acid or U46619 effect on NO concentration was abolished by the inhibitor of the thromboxane A 2 receptor SQ29548 and partially reversed by the PKC inhibitor GF109203X or by the phospholipase C pathway inhibitor U73122. Moreover, it was shown that arachidonic acid activated PKC and decreased nitric oxide synthase (eNOS) activities. The phosphorylation of the inhibiting eNOSthr495 residue mediated by PKC was increased by arachidonic acid, while no changes at the activating ser1177 residue were shown. Finally, arachidonic acid induced NADPH oxidase activation and superoxide anion formation. These effects were greatly reduced by GF109203X, U73122, and apocynin. Likely arachidonic acid reducing NO bioavailability through all these mechanisms could potentiate its platelet aggregating power.

Hyperhomocysteinaemia has been associated with increased risk of thrombosis and atherosclerosis. Homocysteine produces endothelial injury and stimulates platelet aggregation. Several molecular mechanisms related to these effects have been elucidated. The study aimed to deeply investigate the homocysteine effect on nitric oxide formation in human platelets. The homocysteine-induced changes on nitric oxide, cGMP, superoxide anion levels and nitrotyrosine formation were evaluated. The enzymatic activity and the phosphorylation status of endothelial nitric oxide synthase (eNOS) at thr495 and ser1177 residues were measured. The protein kinase C (PKC), assayed by immunofluorescence confocal microscopy technique and by phosphorylation of p47pleckstrin, and NADPH oxidase activation, tested by the translocation to membrane of the two cytosolic subunits p47(phox) and p67(phox), were assayed. Results show that homocysteine reduces platelet nitric oxide and cGMP levels. The inhibition of eNOS activity and the stimulation of NADPH oxidase primed by PKC appear to be involved. PKC stimulates the eNOS phosphorylation of the negative regulatory residue thr495 and the dephosphorylation of the positive regulatory site ser1177. GF109203X and U73122, PKC and phospholipase Cgamma2 pathway inhibitors, respectively, reverse this effect. Moreover, homocysteine stimulates superoxide anion elevation and NADPH oxidase activation. These effects are significantly decreased by GF109203X and U73122, suggesting the involvement of PKC in NADPH oxidase activation. Homocysteine induces formation of the peroxynitrite biomarker nitrotyrosine. Taken together these results suggest that the homocysteine-mediated responses leading to nitric oxide impairment are mainly coupled to PKC activation. Thus homocysteine stimulates platelet aggregation and decreases nitric oxide bioavailability.

Diadenosine 5′,5‴-P1,P2-diphosphate (Ap2A) is one of the adenylic dinucleotides stored in platelet granules. Along with proaggregant ADP, it is released upon platelet activation and is known to stimulate myocyte proliferation. We have previously demonstrated synthesis of Ap2A and of two isomers thereof, called P18 and P24, from their high pressure liquid chromatography retention time, by the ADP-ribosyl cyclase CD38 in mammalian cells. Here we show that Ap2A and its isomers are present in resting human platelets and are released during thrombin-induced platelet activation. The three adenylic dinucleotides were identified by high pressure liquid chromatography through a comparison with the retention times and the absorption spectra of purified standards. Ap2A, P18, and P24 had no direct effect on platelet aggregation, but they inhibited platelet aggregation induced by physiological agonists (thrombin, ADP, and collagen), with mean IC50 values ranging between 5 and 15 μm. Moreover, the three dinucleotides did not modify the intracellular calcium concentration in resting platelets, whereas they significantly reduced the thrombin-induced intracellular calcium increase. Through binding to the purinergic receptor P2Y11, exogenously applied Ap2A, P18, and P24 increased the intracellular cAMP concentration and stimulated platelet production of nitric oxide, the most important endogenous antiaggregant. The presence of Ap2A, P18, and P24 in resting platelets and their release during thrombin-induced platelet activation at concentrations equal to or higher than the respective IC50 value on platelet aggregation suggest a role of these dinucleotides as endogenous negative modulators of aggregation.

SummaryRetinal vein occlusion (RVO) is the most common retinal vascular disorder second to diabetic retinopathy. The main risk factors in patients with RVO are hypertension, diabetes, hyperlipidemia, increased blood viscosity and glaucoma. The pathogenesis of RVO has not yet been clarified. In these events platelets could play a very important role. In the present study the platelet response to collagen was deeply investigated. Experiments were carried out on a selected group of RVO patients, which were compared to a group of healthy subjects matched for age, sex, clinical and metabolic characteristics. In resting and activated platelets of both groups of subjects p72syk phosphorylation, phospholipase Cγ 2 phosphorylation, protein kinase C activation, intracellular calcium levels and nitric oxide formation were measured. Results show that platelets of patients were more responsive to collagen or ADP than healthy subjects and that the response was significantly different (p < 0.0005) at low concentrations of these agonists. In platelets of patients stimulated with collagen increased phosphorylation of p72syk and phospholipase C γ 2 was found. Also protein kinase C was more activated in patients. In addition intracellular calcium rise induced by collagen was significantly higher in patients than in healthy subjects. RVO patients showed a lower basal level of nitric oxide both in resting and stimulated platelets compared to healthy subjects. Altogether these results suggest that the platelet hyperaggregability described in patients might be an important factor in the development of RVO contributing to the thrombogenic effects.

Introduction Type 2 diabetes mellitus shows a characteristic altered platelet function that can be due to several mechanisms such as oxidative stress. Hyperhomocysteinemia, considered as a risk factor for various arterial thrombosis, may have a role in generating oxidative damage, even if the pathogenic mechanisms are still not clear. In this report we aimed to determine the role of plasma homocysteine in inducing oxidative stress in type 2 diabetes mellitus. Materials and methods The study was performed on a group of 34 males with type 2 diabetes and 36 healthy subjects matched for sex and age. Patients and healthy subjects were undergone to laboratory evaluation for plasma homocysteine levels and other metabolic parameters. In both groups of subjects platelet reactive oxygen species, nitric oxide and guanosine 3′,5′ cyclic monophosphate levels were measured. Moreover the reduced glutathione content in platelets of patients and of healthy subjects was assayed. Results Plasma homocysteine levels were significantly increased in patients compared with healthy subjects. The basal level of reactive oxygen species was significantly higher in patients than in controls. In addition platelets of patients stimulated with thrombin produced more reactive oxygen species than healthy subjects ones. The nitric oxide, guanosine 3′,5′ cyclic monophosphate and reduced glutathione content were decreased in platelets of patients. Conclusions As homocysteine stimulates oxidative stress and inhibits nitric oxide formation, hyperhomocysteinemia measured in type 2 diabetic patients, promoting platelet hyperactivity, could have a role in the atherogenic effects described in type 2 diabetes.

Hyperhomocysteinemia is considered a risk factor in arterial and venous thrombosis. The mechanism by which homocysteine (HCy) supports atherothrombosis is still unknown and may be multifactorial. Earlier in vitro studies demonstrated that HCy induced arachidonic acid release and increased thromboxane B2 (TXB2) formation. In this work, we found that HCy stimulated the rapid and sustained phosphorylation of platelet p38 mitogen-activated protein kinase (p38 MAPK). The effect was time- and dose-dependent. The HCy effect on p38 MAPK phosphorylation was prevented by N-acetyl-L-cysteine and iloprost and was partially inhibited by nordihydroguaiaretic acid. Moreover, the incubation of platelets with HCy led to the phosphorylation of cytosolic phospholipase A2 (cPLA2). In addition HCy promoted cPLA2 activation, assessed as arachidonic acid release. The cPLA2 phosphorylation and activation were both impaired by the inhibition of p38 MAPK through SB203580. This effect was not complete, reaching at the most the 50% of the total. In FURA 2-loaded platelets, HCy induced a dose-dependent intracellular calcium rise suggesting that the calcium elevation promoted by HCy could participate in the cPLA2 activation, leading to arachidonic acid release and TXB2 formation. In conclusion, our data provide insight into the mechanisms of platelet activation induced by HCy, suggesting that the p38 MAPK/cPLA2 pathway could play a relevant role in platelet hyperactivity described in hyperhomocysteinemia.

Nitric oxide production, L-arginine transport and intracellular [Ca2+] changes in human platelets stimulated without stirring by low doses of collagen have been evaluated. Collagen decreased in a dose-dependent manner the nitric oxide formation. A reduction of about 30% of the basal level was produced by 5 microg/mL. Aspirin did not change the collagen effect. The inhibition was reversed by EGTA. Moreover collagen reduced L-arginine uptake. The exposure of platelets to 5 microg/mL collagen diminished of about 30% L-arginine transport. The specific involvement of the system y+ is suggested. In addition in FURA 2-loaded platelets collagen induced a dose-dependent slow sustained [Ca2+] rise that was almost completely cancelled by EGTA. Finally the treatment of whole platelets with collagen affected in a dose-dependent manner the maximal nitric oxide formation, suggesting a direct effect at the level of nitric oxide synthase enzyme. The phosphorylation of specific serine/threonine residues regulated by protein kinase C could be involved. In conclusion during the early phases of platelet stimulation with collagen nitric oxide formation is diminished. This reduction can be due to a lower availability of L-arginine for cytosolic nitric oxide synthase and/or to a decreased activity related to modifications of the enzyme.

Pursuing our chemical and biological studies in this field, we described the multistep preparation of the new 5-, 6-, or 7-alkoxy and 7-alkoxy-8-methyl substituted 4-(1-piperazinyl)coumarins 5d-v, as well as the in vitro evaluation of their inhibitory activity on human platelet aggregation induced in platelet-rich plasma by ADP, collagen or the Ca2+ ionophore A23187. Compounds 5h-j,p,r-u showed notably high activity towards all the platelet aggregation inducers used, and the most active one, 8-methyl-4-(1-piperazinyl)-7-(3-pyridylmethoxy)coumarin (5t), proved to be a potent in vitro antiplatelet agent.

The effect on human platelets of 8-methyl-4-(1-piperazinyl)-7-(3-pyridinylmethoxy)-2 H -1-benzopyran-2-one (RC414) was tested in vitro by measuring aggregation induced by several agonists, cAMP and cGMP levels, cAMP phosphodiesterase and PKC activities and [Ca 2+ ] i . The RC414 effect on nitric oxide production was also evaluated. RC414 in a dose-dependent manner inhibited aggregation both in platelet rich plasma and in washed platelets. It was particularly effective in platelets challenged by collagen, ADP and thrombin: ic 50 values are 0.51±0.12 μM, 0.98±0.36 μM and 1.00±0.15 μM, respectively. RC414 increased cAMP levels, through the specific inhibition of the cAMP high affinity phosphodiesterase ( ic 50 =1.73±0.35 μM). RC414 reduced [Ca 2+ ] i transients and PKC activation induced by thrombin. In addition RC414 was able to increase nitric oxide formation involving the stimulation of constitutive nitric oxide synthase enzyme. In conclusion, RC414 exerts its powerful anti-platelet activity by increasing cAMP intracellular levels and nitric oxide formation.

As a further part of our chemical and biological studies in this field, we describe the multistep preparations of the properly substituted 2-(1-piperazinyl)chromone 1b , 4-(1-piperazinyl)coumarins 5c – h , their linear benzo-fused analogues 4a , b and 8a , b , bicyclic ( 15e – g ) and tricyclic ( 15h , i ) fused derivatives of 6-(1-piperazinyl)pyrimidin-4(3 H )-one, and of the 4 H -pyrido[1,2- a ]pyrimidine derivatives 9b , c . The in vitro evaluation of their inhibitory properties towards human platelet aggregation induced in platelet-rich plasma by ADP, collagen, or the Ca 2+ ionophore A23187 showed the high activity of compounds 5d – g and 15f , g , i , among which the coumarins 5g and 5d proved to be, in that order, the most effective in vitro antiplatelet agents until now synthesized by us. Thus, in order to consider also the 4-aminocoumarin structural class, we developed a new statistically significant 3-D QSAR model, more general than the one previously obtained, through a further CoMFA study based on the antiplatelet activity data and molecular steric and electrostatic potentials of both the previously studied and herein described compounds.

Gabexate mesylate, a non-antigenic synthetic inhibitor of trypsin-like serine proteinases, is a drug used efficiently in the treatment of pancreatitis and disseminated intravascular coagulation and as a regional anticoagulant for haemodialysis. Considering the structural similarity between L-arginine and gabexate mesylate, the effect of this drug on L-arginine transport, nitric oxide (NO) formation and constitutive NO synthase activity in human platelets was investigated. Data have shown that gabexate mesylate inhibited competitively L-arginine uptake by increasing the K(m) value from 22+/-2 to 86+/-6 microM. The K(i) value was 158 microM at pH 7.4 and 37 degrees. Furthermore, gabexate mesylate decreased dose and time-dependent nitrite and nitrate formation (NO(x) release) and cGMP accumulation in whole cells. In addition, gabexate mesylate inhibited constitutive nitric oxide synthase in a cell-free extract. We concluded that gabexate mesylate could be considered an effective modulator of cellular NO synthesis.

The data presented in this report show that N-ethylmaleimide (NEM) is a powerful inhibitor of thrombin-induced platelet aggregation. NEM increased guanosine 3', 5'-cyclic monophosphate (cGMP) and adenosine 3', 5'-cyclic monophosphate (cAMP) levels in intact cells. The inhibition of cAMP high-affinity phosphodiesterase and cGMP phosphodiesterase was implicated in the elevation of the cyclic nucleotides. NEM dose dependently blocked the thrombin-stimulated, but not the phorbol myristate acetate-dependent phosphorylation of the protein kinase C substrate pleckstrin. Myosin light chain phosphorylation was also inhibited by NEM. In addition, the sulphydryl reagent inhibited Ca2+ mobilisation induced by thrombin. The data indicate that phospholipase C activation by thrombin is interrupted by NEM at the level of receptor-mediated signal transduction.

In the present study the "in vitro" influence of 3-(1-piperazinyl)-1H-pyrimido[1,2-a]quinolin-1-one (AQ11) and 2-(piperazinyl)-4H-pyrimido[2,1-a]isoquinolin-4-one (IQ3b) on human platelet aggregation, cAMP elevation, cytosolic calcium and fibrinogen binding has been investigated. Both drugs inhibited platelet aggregation in a concentration-dependent manner. In PRP AQ11 was slightly more active than IQ3b when aggregation was induced by ADP, collagen, A23187 or PMA, whereas in washed platelets challenged by thrombin, IQ3b was more effective than AQ11. Both compounds produced increase in cAMP intracellular level being the effect potentiated by the adenylate cyclase activator iloprost and IQ3b was more powerful than AQ11. Moreover IQ3b was more effective in inhibiting cAMP in high affinity phosphodiesterase (IC50 values: IQ3b 11 +/- 5 microM; AQ11 43 +/- 8 microM) and calcium elevation (IC50 values: IQ3b 9 +/- 4 microM; AQ11 32 +/- 6 microM). These compounds also inhibited fibrinogen binding in platelets challenged by ADP or thrombin. The results suggest that these new potent agents inhibit platelet phosphodiesterase activity causing an elevation in cAMP levels sufficient to inhibit calcium rise and fibrinogen binding. This mechanism can be responsible for the ability of the compounds to prevent platelet aggregation.

1. 1. Thrombin addition to human platelets stimulates l (+)lactate formation and S-D-lactoylglutathione (SDL) accumulation. 2. 2. Monoiodoacetamide decreases lactate formation and potentiates SDL accumulation through a significant increase of dihydroxyacetone phosphate and fructose 1,6bisphosphate intracellular levels both in resting and in activated platelets. 3. 3. A similar effect is produced by exogenous methylglyoxal on L(+)lactate formation and SDL accumulation. 4. 4. Resting platelets completely transform (1 hr at 37°C) the ketoaldehyde into d (-)lactate: 5. 5. When platelets are incubated in the presence of thrombin only 60% of the ketoaldehyde is found as d (-)lactate and the accumulated S- d -lactoylglutathione represents about the 0.7% of the initial substrate. 6. 6. During platelet stimulation with thrombin the hemithioacetal adduct, formed as a by-product of glycolytic pathway, can be rapidly removed for important steps of cellular activation.

The production of hydrogen peroxide was measured by following the oxidation of dichlorofluorescein (DCFH) entrapped into platelets. Resting platelets produced nanomolar quantities of DCF, which was proportional to the concentration of platelets and was steady during 1 h of incubation. A significant increase of basal DCF fluorescence was induced by stimuli namely thrombin, arachidonic acid, the Ca2+ ionophore A23187 and PMA. The effect of agonists has been also measured in the presence of 3-amino-1,2,4-triazole (AT) or N-ethylmaleimide (NEM), inhibitors of catalase and glutathione peroxidase, respectively. A further significant enhancement of DCF produced in stimulated platelets was detected only in the presence of NEM. A correlation was found between the increase in DCF and externally added hydrogen peroxide or the oxidizing species formed by xanthine oxidase plus acetaldehyde. The yield was not affected by superoxide dismutase and was higher in the presence of AT or NEM. A cooperative effect in the presence of both inhibitors was shown. Glutathione peroxidase plus glutathione diminished the level of DCF to basal levels.

The in vitro effect of 2-(diethylamino)-7-ethoxychromone (RC39XVIII) on human platelet aggregation induced by several agonists and on thromboxane B2 formation, granule release and intracellular cAMP elevation has been studied. The chromosome-derivative exerts a dose-dependent inhibitory effect on aggregation produced by U46619, arachidonic acid, thrombin, collagen and ADP. RC39XVIII inhibits aggregation, TxB2 formation and granule release in parallel. Moreover the drug potentiates cAMP accumulation induced by iloprost and forskolin. The drug also inhibits soluble cAMP phosphodiesterase in a dose-dependent manner. No effect on adenylate cyclase activity measured in platelet membranes was evident.