Spice active principles as the inhibitors of human platelet aggregation and thromboxane biosynthesis

Spice active principles are reported to have anti-diabetic, anti-hypercholesterolemic, antilithogenic, anti-inflammatory, anti-microbial and anti-cancer properties. In our previous report we have shown that spices and their active principles inhibit 5-lipoxygenase and also formation of leukotriene C4. In this study, we report the modulatory effect of spice active principles viz., eugenol, capsaicin, piperine, quercetin, curcumin, cinnamaldehyde and allyl sulphide on in vitro human platelet aggregation. We have demonstrated that spice active principles inhibit platelet aggregation induced by different agonists, namely ADP (50 μM), collagen (500 μg/ml), arachidonic acid (AA) (1.0 mM) and calcium ionophore A-23187 (20 μM). Spice active principles showed preferential inhibition of arachidonic acid-induced platelet aggregation compared to other agonists. Among the spice active principles tested, eugenol and capsaicin are found to be most potent inhibitors of AA-induced platelet aggregation with IC50 values of 0.5 and 14.6 μM, respectively. The order of potency of spice principles in inhibiting AA-induced platelet aggregation is eugenol>capsaicin>curcumin>cinnamaldehyde>piperine>allyl sulphide>quercetin. Eugenol is found to be 29-fold more potent than aspirin in inhibiting AA-induced human platelet aggregation. Eugenol and capsaicin inhibited thromboxane B2 (TXB2) formation in platelets in a dose-dependent manner challenged with AA apparently by the inhibition of the cyclooxygenase (COX-1). Eugenol-mediated inhibition of platelet aggregation is further confirmed by dose-dependent decrease in malondialdehyde (MDA) in platelets. Further, eugenol and capsaicin inhibited platelet aggregation induced by agonists—collagen, ADP and calcium ionophore but to a lesser degree compared to AA. These results clearly suggest that spice principles have beneficial effects in modulating human platelet aggregation.     

Potentiation of anti-aggregating activity of PGI2 by 7-ethoxycarbonyl-6,8-dimethyl-4-hydroxymethyl-1(2H)-phthalazinone (EG-626) in rabbit platelets in vitro

Anti-aggregating activity of 7-ethoxycarbonyl-6,8-dimethyl-4-hydroxymethyl-1(2H)-phthalazinone (EG-626) was tested using rabbit platelets in vitro. EG-626 alone, when added before, prevented platelet aggregation induced by ADP, as did PGI₂, papaverine and dipyridamole. Spontaneous disaggregation was also accelerated when EG-626 was added after the maximal aggregation induced by ADP. EG-626 alone also inhibited platelet aggregation induced by collagen and arachidonic acid. ID₅₀s of these agents in ADP-induced aggregation were 7–9 nM for PGI₂, 223 μM for EG-626, 266 μM for papaverine and 957 μM for dipyridamole. When EG-626 was used in combination with PGI₂, a threshold dose (50 μM) of EG-626 potentiated the anti-aggregating effect of subthreshold dose (3 nM) of PGI₂ upto 100% inhibition in collagen-induced platelet aggregation. The marked potentiating effect of EG-626 was accompanied by an accumulation of cyclic AMP in the platelets. These effects might be due to inhibition of phosphodiesterase. Papaverine and dipyridamole, other phosphodiesterase inhibitors, also potentiated the anti-aggregating activity of PGI₂. The activity of papaverine, however, was one eighth of EG-626 and that of dipyridamole was much less. The most effective combination of PGI₂ and EG-626 to induce 50% inhibition was obtained with 20% of ID₅₀ of each agent, whereas that of PGI₂ and papaverine or dipyridamole was 39 or 41%, respectively.     

Inhibition of human platelet aggregation by nitric oxide donor drugs: relative contribution of cGMP-independent mechanisms

Inhibition of platelet activation by nitric oxide (NO) is not exclusively cGMP-dependent. Here, we tested whether inhibition of platelet aggregation by structurally distinct NO donors is mediated by different mechanisms, partly determined by the site of NO release. Glyceryl trinitrate (GTN), sodium nitroprusside (SNP), S-nitrosoglutathione (GSNO), diethylamine diazeniumdiolate (DEA/NO), and a novel S-nitrosothiol, RIG200, were examined in ADP (8 microM)- and collagen (2.5 microgram/ml)-activated human platelet rich plasma. GTN was a poor inhibitor of aggregation whilst the other NO donors inhibited aggregation, irrespective of agonist. These effects were abolished by the NO scavenger, hemoglobin (Hb; 10 microM, P < 0.05, n = 6), except with high concentrations of DEA/NO, when NO concentrations exceeded the capacity of Hb. However, experiments with the soluble guanylate cyclase inhibitor, ODQ (100 microM), indicated that only SNP-mediated inhibition was exclusively cGMP-dependent. Furthermore, the cGMP-independent effects of S-nitrosothiols were distinct from those of DEA/NO, suggesting that different NO-related mediators (e.g., nitrosonium and peroxynitrite, respectively) are responsible for their actions.     

The effects of itnravenous ZK36-374, a stsable prostacyclin analogue, on normal volunteers

Prostacyclin (PGI₂) therapy has been evaluated in many vascular diseases. However, it is unstable and a potent vasodilator, able to lower blood pressure. Although such effects may be desirable in some situations, they are unwanted in others. ZK36-374 (Schering AG) is a carbacyclin derivatives with a similar action to PGI₂; however, it is chemically stable and has less of a hypotensive action.We evaluated the effects of a 4-hour I.V. infusion of ZK36-374 at a maximum dose of 2ng/Kg/min. in ten normal volunteers. Prior to the infusion and at 2 and 4 hours, blood was sampled for estimation of platelet aggregation in both platelet rich plasma and whole blood. β-thromboglobulin, 6-keto-PGF_1α and TXB₂ were measuerd by radioimmunoassay, as were other coagulation and rheological tests. The infusion was well tolerated with facial flushing, jaw trismus and some nausea at max dose. Blood pressure and pulse rate were not significantly altered. During infusion of ZK36-374, the rates of platelet aggregation to 2μm AdP and 2μg collagen in PRP were significantly decreased when compared to baseline, as was whole blood aggregation to 2μm ADP and 0.5 μg collagen. βTG also fell significantly, as did the levels of 6-keto-PGF_1α and TXB₂. Fibrinolysis, blood viscosity, and red cell deformability were unchanged.ZK36-374 is an effective anti-platelet agent without major toxic or hypotensive effects.     

Influence of short term dietary supplementation of different lipids on aggregation and arachidonic acid metabolism in rabbit platelets

Semisynthetic diets containing 8% by weight of either corn oil or butter were fed to male New Zealand rabbits for three weeks. The plasma cholesterol values were determined, the threshold concentrations for aggregation of platelet rich plasmas were measured for collagen and Na arachidonate, and the conversion of ¹⁴C arachidonic acid to thromboxane B₂ and hydroxy fatty acids (HETE and HHT) at 10, 20 and 40 μM substrate concentrations were studied. The thresholds for arachidonate induced aggregation were lower and the amplitudes of collagen induced aggregations were greater in the butter fed than in the corn oil fed rabbits. Conversions of arachidonic acid to thromboxane B₂ but not to hydroxy fatty acids were greater in the butter fed rabbits at 10 and 20 μM substrate. The observed changes were accompanied by only slight modifications of plasma cholesterol levels.     

Physiological concentrations of ADP stimulate the release of prostacyclin from bovine aortic endothelial cells

ADP (0.2−200 μN) stimulated the synthesis of prostacyclin (PGI₂), as reflected by the release of 6-keto-prostaglandin F_1α (6-K-PGF_1α), in endothelial cells cultured from bovine orta. This effect of ADP was mimicked by ATP, whereas AMP and adenosine were completely inactive. The release of 6-K-PGF_1α triggered by ADP was rapid in onset (within 5 min), transient (10 min) and followed by a period of refractoriness to a new ADP challenge. Growing and confluent cells were equally responsive to ADP. ADP stimulated the release of free arachidonic acid from the endothelial cells. ADP could be thus exert two opposite actions on platelet aggregation in vivo: a direct stimulation and an inhibition mediated by PGI₂. This last action might contribute to limit thrombus formation to areas of endothelial cell damage.     

PKC and AKT Modulate cGMP/PKG Signaling Pathway on Platelet Aggregation in Experimental Sepsis

Sepsis severity has been positively correlated with platelet dysfunction, which may be due to elevations in nitric oxide (NO) and cGMP levels. Protein kinase C, Src kinases, PI3K and AKT modulate platelet activity in physiological conditions, but no studies evaluated the role of these enzymes in platelet aggregation in sepsis. In the present study we tested the hypothesis that in sepsis these enzymes positively modulate upstream the NO-cGMP pathway resulting in platelet inhibition. Rats were injected with lipopolysaccharide (LPS, 1 mg/kg, i.p.) and blood was collected after 6 h. Platelet aggregation was induced by ADP (10 μM). Western blotting assays were carried out to analyze c-Src and AKT activation in platelets. Intraplatelet cGMP levels were determined by enzyme immunoassay kit. Phosphorylation of c-SRC at Tyr416 was the same magnitude in platelets of control and LPS group. Incubation of the non-selective Src inhibitor PP2 (10 μM) had no effect on platelet aggregation of LPS-treated rats. LPS increased intraplatelet cGMP levels by 5-fold compared with control group, which was accompanied by 76% of reduction in ADP-induced platelet aggregation. The guanylyl cyclase inhibitor ODQ (25 μM) and the PKG inhibitor Rp-8-Br-PET-cGMPS (25 μM) fully reversed the inhibitory effect of LPS on platelet aggregation. Likewise, the PKC inhibitor GF109203X (10 μM) reversed the inhibition by LPS of platelet aggregation and decreased cGMP levels in platelets. AKT phosphorylation at Thr308 was significantly higher in platelets of LPS compared with control group, which was not reduced by PI3K inhibition. The AKT inhibitor API-1 (20 μM) significantly increased aggregation and reduced cGMP levels in platelets of LPS group. However, the PI3K inhibitor wortmannin and LY29004 had no effect on platelet aggregation of LPS-treated rats. Therefore, inhibition of ADP-induced platelet aggregation after LPS injection is mediated by cGMP/PKG-dependent mechanisms, and PKC and AKT act upstream upregulating this pathway.     

Ca2+ signaling in the transformation of promastigotes to axenic amastigotes of Leishmania donovani

Three acidic phospholipases A₂ from Indian cobra (Naja naja naja) venom inhibited platelet aggregation in platelet rich plasma induced separately by ADP, collagen and epinephrine with different potencies. The order of inhibition was epinephrine > collagen > ADP. They did not inhibit platelet aggregation induced by arachidonic acid (10 M). The inhibition was dependent on concentration of the protein and the time of incubation of the phospholipases A₂ with platelet rich plasma. Parabromophenacyl bromide modified PLA₂ enzymes lost their enzymatic activity as well as platelet aggregation inhibition activity suggesting the involvement of catalytic function in platelet aggregation inhibitory activity.     

Action mechanism of the platelet aggregation inducer and inhibitor from Echis carinatus snake venom

Platelet aggregation inducer and inhibitor were isolated from Echis carinatus snake venom. The venom inducer caused aggregation of washed rabbit platelets which could be inhibited completely by heparin or hirudin. The venom inducer also inhibit both the reversibility of platelet aggregation induced by ADP and the disaggregating effect of prostaglandin E₁ on the aggregation induced by collagen in the presence of heparin. The venom inhibitor decreased the platelet aggregation induced by collagen, thrombin, ionophore A23187, arachidonate, ADP and platelet-activating factor (PAF) with an IC₅₀ of around 10 μg/ml. It did not inhibit the agglutination of formaldehyde-treated platelets induced by polylysine. In the presence of indomethacin or in ADP-refractory platelets or thrombin-degranulated platelets, the venom inhibitor further inhibited the collagen-induced aggregation. Fibrinogen antagonized competitively the inhibitory action of the venom inhibitor in collagen-induced aggregation. In chymotrypsin-treated platelets, the venom inhibitor abolished the aggregation induced by fibrinogen. It was concluded that the venom inducer caused platelet aggregation indirectly by the conversion of prothrombin to thrombin, while the venom inhibitor inhibited platelet aggregation by interfering with the interaction between fibrinogen and platelets.     

Effect of TFC-612, a 7-thia prostaglandin E1 derivative, on a peripheral arterial occlusive disease model in rats

TFC-612, methyl 6-({(1R,2S,3R)-3-hydroxy-2-{(1E,3S,5R)-3-hydroxy-5-methyl-1-nonenyl}-5-oxocyclopentyl}-thio]-hexanoate, inhibited the progression of the lesion in a lauric acid-induced peripheral arterial occlusive model at 1.0 mg/kg p.o. or 1.0 μg/rat/h s.c. in rats. Aspirin (32 mg/kg, p.o.), an anti-platelet drug, did not suppress the lesion growth. On the other hand, ketanserin (10 mg/kg, p.o.), a 5-HT₂ antagonist, also inhibited the progression of the lesion. In vitro, TFC-612 inhibited rat platelet aggregation induced by collagen and ADP with IC₅₀ values of 5.4 ng/mL and 9.5 ng/mL, respectively. Aspirin also inhibited collagen-induced aggregation with an IC₅₀ value of 6.3 μg/mL, but not ADP-induced aggregation at 180 μg/mL. Ketanserin had no effect on either aggregation at 40 μg/mL. In ex vivo experiments, aspirin inhibited platelet aggregation induced by collagen at 10 and 32 mg/kg in rats. However, TFC-612 showed significant inhibition only at 10 mglkg. TFC-612 and ketanserin increased dermal blood flow in the rat paw at 1.0 μg/kg i.v. and 100 μg/kg Lv., respectively. Aspirin had no effect on blood flow at 3.2 mg/kg i.v. These results suggest that the improvement of microcirculation, in addition to anti-platelet action by TFC-612, contributes to its inhibitory effect in a peripheral arterial occlusive model in rats.     

Mechanism of the inhibition of platelet aggregation produced by prostaglandin F2α

The inhibition of human platelet aggregation produced by PGF_2α is not specific for thromboxane A₂ mimetics. Aggregation waves induced by PAF and thrombin are also inhibited by PGF_2α (8 μM); ADP is unaffected. These effects are still seen in platelets from aspirin-treated donors and platelets desensitized to thromboxane-like agonists (e.g. 11,9-epoxymethano PGH₂). In contrast the thromboxane receptor antagonist EP 045 (up to 20 μM) had no effect on primary aggregation induced by PAF, thrombin and ADP. We have previously shown that EP 045 (IC₅₀ = 0.5 μM), displaces the specific binding of [³H] 9,11-epoxymethano PGH₂ to washed human platelets.PGF_2α produces small increases in cAMP levels, and both this effect and the anti-aggregation are diminished by the adenyl cyclase inhibitor SQ 22536. The rise in cAMP induced by PGF_2α is inhibited to a greater extent by the presence of ADP than by thrombin, PAF or a thromboxane mimetic. The ability of aggregating agents to inhibit this increase correlates inversely with their sensitivity to inhibition by PGF_2α.We suggest that the very weak effect of PGF_2α on cyclic AMP_ production is sufficient to account for its inhibitory activity, and it is unlikely to be a competitive antagonist at the platelet thromboxane receptor as suggested by others.     

Demonstration of PGl2 production by isolated perfused rat lungs with platelet aggregation test

Platelet aggregation test was used for PGl₂ measurements. The use of 6 % CO₂ in air stabilized human platelet rich plasma (PRP) so that it could be used for up to 7 hours in these measurements. The PGl₂ caused inhibition of aggregation in response to ADP was concentration dependent in the range of 0.5 ng/ml to 50 ng/ml. Isolated perfused rat lungs released spontaneously 190 ng/min PGl₂ and about 3 % of infused arachidonic acid potassium salt (equivalent to 25 μg/min arachidonic acid) was converted to PGl₂.     

Thrombin Generating Capacity and Phenotypic Association in ABO Blood Groups

Individuals with blood group O have a higher bleeding risk than non-O blood groups. This could be explained by the lower levels of FVIII and von Willebrand Factor (VWF) levels in O individuals. We investigated the relationship between blood groups, thrombin generation (TG), prothrombin activation and thrombin inactivation. Plasma levels of VWF, FVIII, antithrombin, fibrinogen, prothrombin and α₂Macroglobulin (α₂M) levels were determined. TG was measured in platelet rich (PRP) and platelet poor plasma (PPP) of 217 healthy donors and prothrombin conversion and thrombin inactivation were calculated. VWF and FVIII levels were lower (75% and 78%) and α₂M levels were higher (125%) in the O group. TG is 10% lower in the O group in PPP and PRP. Less prothrombin was converted in the O group (86%) and the thrombin decay capacity was lower as well. In the O group, α₂M plays a significantly larger role in the inhibition of thrombin (126%). In conclusion, TG is lower in the O group due to lower prothrombin conversion, and a larger contribution of α₂M to thrombin inactivation. The former is unrelated to platelet function because it is similar in PRP and PPP, but can be explained by the lower levels of FVIII.     

Role of lipoxygenase metabolism in platelet function: Effect of aspirin and salicylate

Aspirin inhibits thromboxane A₂ (TxA₂) production whereas its salicylate moiety inhibits 12-hydroxy-eiosatetraenoic acid (12-HETE) production in the platelet. The significance of the latter effect on platelet function is unclear. We examined the effects of aspirin and salicylate on (i) platelet/ collagen adhesion using ³H-adenine-labelled human platelets and collagen- coated discs, (ii) platelet aggregation induced by thrombin, collagen, ADP and arachidonic acid, and (iii) platelet TxA₂ and 12-HETE synthesis as measured by radioimmunoassay and high pressure liquid chromatography respectively. Aspirin (50 μM) decreased platelet aggregation and increased platelet adhesion. The decrease in aggregation was associated with inhibition of TxA₂ production and the increase in adhesion was associated with enhanced 12-HETE production. Salicylate had the opposite effects. Platelet aggregation was increased and platelet adhesion decreased. The increased aggregation was associated with enhanced TxA₂ production and the decrease in aggregation was associated with inhibition of 12-HETE production. These observations suggest that 12-HETE facilitates platelet adhesion which can be altered by salicylate treatment.     

In vitro effects of synthetic antioxidants and vitamin E on arachidonic acid metabolism and thromboxane formation in human platelets and on platelet aggregation

The “in vitro” effects of α-tocopherol, butylhydroxytoluene (BHT) and butylhydroxyanisole (BHA) were studied on aggregation of human platelets induced by collagen and arachidonic acid (AA), on the metabolic conversion of ¹⁴C AA through the cyclooxygenase and lipoxygenase pathways and on the formation of thromboxane B₂ (TXB₂) in washed platelets after stimulation with collagen.Vitamin E completely inhibited AA induced platelet aggregation only at high concentration (mM) and after 10 minutes of preincubation, with limited effects on AA metabolism in platelets and no effect on TXB₂ formation from endogenous substrate. BHA completely inhibited platelet aggregation in the 10⁻⁶M range, gave 50% inhibition of AA metabolism in the 10⁻⁵M range and almost complete inhibition of thromboxane formation in the 10⁻⁴M range. BHT was about 100 times less active on platelet aggregation and AA metabolism. The lipoxygenase and cyclooxygenase pathways were differentially affected at low concentrations of BHA and only at concentrations greater than 5×10⁻⁵M were both pathways depressed.     

Antiplatelet effects of clausine-D isolated from Clausena excavata

Clausine-D inhibited concentration-dependently the aggregation and release of washed rabbit platelets induced by arachidonic acid and collagen, without affecting those induced by U46619, PAF and thrombin. The IC₅₀ values of clausine-D on arachidonic acid-and collagen-induced platelet aggregation were calculated to be 9.0±1.1 and 58.9±0.9 μM, respectively. Thromboxane B₂ and prostaglandin D₂ formation in platelets caused by arachidonic acid were also suppressed. Clausine-D inhibited increased intracellular concentration of calcium in platelets caused by arachidonic acid and collagen, and also abolished the generation of inositol monophosphate caused by arachidonic acid, but not that by collagen U46619, PAF and thrombin. In human citrated platelet-rich plasma, clausine-D inhibited the secondary phase, but not the primary phase, of aggregation induced by epinephrine and ADP. These results indicate that the antiplatelet effect of clausine-D is due to inhibition of the formation of thromboxane A₂.     

Anti-platelet activity of a three-finger toxin (3FTx) from Indian monocled cobra (Naja kaouthia) venom

A low molecular weight anti-platelet peptide (6.9 kDa) has been purified from Naja kaouthia venom and was named KT-6.9. MALDI-TOF/TOF mass spectrometry analysis revealed the homology of KT-6.9 peptide sequence with many three finger toxin family members. KT-6.9 inhibited human platelet aggregation process in a dose dependent manner. It has inhibited ADP, thrombin and arachidonic acid induced platelet aggregation process in dose dependent manner, but did not inhibit collagen and ristocetin induced platelet aggregation. Strong inhibition (70%) of the ADP induced platelet aggregation by KT-6.9 suggests competition with ADP for its receptors on platelet surface. Anti-platelet activity of KT-6.9 was found to be 25 times stronger than that of anti-platelet drug clopidogrel. Binding of KT-6.9 to platelet surface was confirmed by surface plasma resonance analysis using BIAcore X100. Binding was also observed by a modified sandwich ELISA method using anti-KT-6.9 antibodies. KT-6.9 is probably the first 3FTx from Indian monocled cobra venom reported as a platelet aggregation inhibitor.     

Thrombosis Prevention by Acetylsalicylic Acid in Hyperlipemic Rats

In rats, administration of acetylsalicylic acid (ASA) by stomach tube two hours before blood removal, or addition of the drug to platelet-rich plasma in vitro, markedly inhibited platelet aggregation induced by thrombin, ADP and collagen. Addition of ASA in vitro to human platelet-rich plasma also inhibited platelet aggregation by thrombin, ADP and collagen. In hyperlipemic rats, ASA (100 to 200 mg./kg.), administered by stomach tube once or five times, markedly inhibited the production of thrombosis initiated by intravenous injection of S. typhosa endotoxin. In these experiments, thrombosis prevention by ASA was associated with both a decrease in platelet aggregation and an increase in the recalcification plasma clotting time.     

An inhibitor selective for collagen-stimulated platelet aggregation from the salivary glands of hard tick Haemaphysalis longicornis and its mechanism of action

Soluble materials of salivary glands fromHaemaphysalis longicornis were found to inhibit collagen, ADP, and thrombin-stimulated platelet aggregation. One inhibitory component was purified to salivary gland homogeneity by a combination of gel filtration, ion-exchange, and C₈ reverse phase HPLC. The purified activity, named longicomin, is a protein of molecular weight 16 000 on SDS-PAGE under both reduced and nonreduced conditions. Collagen-mediated aggegation of platelets in plasma and of washed platelets (IC₅₀ was approximately 60 nmol/L) was inhibited with the same efficacy. No inhibition of aggregation stimulated by other effectors, including ADP, arachidonic acid, thrombin, ristocetin, calcium ionophore A23187, thromboxane A2 mimetic U46619 and 12-O-phorbol-13-myristate acetate, was observed. Longiconin had no effect on platelet adhension to collagen. Not only platelet aggregation but also release reaction, and increase of intracellar ca²⁺ level of platelets in response to collagen were completely eliminated by longicomin. Increasing amounts of collagen are able to overcome the inhibition of aggregation by longicomin, indicating that longicomin probably shares with collagen a common receptor. In addition, collagen fibers did not emit fluorescence after incubation with isothocyanate-conjugated longicornin, indicating that longicomin did not bind directly to collagen fibers. The identification and isolation of longicomin demonstrates the existence of a new type of platelet inhibitor that should be useful to better undentand the mechanism of collagen stimulation of platelets. Project supported by the National Natural Science Foundation of China (Grant No. 39170591).     

Dependence of the thrombocyte aggregation reaction on plasma fibronectin and the tripeptide arginyl-glycyl-asparagilin

If was shown that the addition of fibronectin antibodies exerted the inhibition of platelet aggregation. The tripeptide RGD inhibited the platelet aggregation induced by the same agents (ADP, epinephrine, thrombin, collagen) both in blood plasma and in suspension of washed platelets.