Boguslawa Luzak

Platelet activation and reactivity in the convalescent phase of ischaemic stroke

The study was aimed at the evaluation of blood platelet activation and reactivity in patients in the convalescent phase of stroke (n=58) and controls matched in respect to risk factors of vascular pathology (n=55). Both groups were treated daily with acetylsalicylic acid (ASA), 150 mg/day. Using flow cytometry, the expressions of P-selectin and the active GP IIb/IIIa receptor, as well as the fraction of platelet-derived microparticles (PMPs) and total platelet aggregates (Ag), were evaluated in non-stimulated platelets and in platelets stimulated in vitro by thrombin, thrombin receptor activating peptide (TRAP) or ADP. The expression of P-selectin in non-stimulated platelets was found to be significantly (p=0.04) lower in stroke patients. In parallel, these patients manifested a significantly (p=0.0008) higher proportion of PMPs and a lowered (p=0.003) proportion of Ag, as compared to the controls. In the stroke patients the increased expressions of P-selectin and active GP IIb/IIIa in TRAP- or ADP-activated cells were less pronounced (p<0.01), while the increments in PMP fraction remained higher (p<0.05). Our results may indicate that chronic platelet activation develops in patients in the convalescent phase of stroke and the process of PMP generation prevails over blood platelet degranulation and aggregation. This shift may be particularly unfavourable due to the procoagulative and proatherosclerotic properties of PMPs, accompanied by their decreased sensitivity to the action of antiplatelet drugs.

Enhanced platelet-derived microparticle formation is associated with carotid atherosclerosis in convalescent stroke patients

Platelets participate in the development and progression of atherosclerosis. During this process they interact with endothelial cells and leukocytes. Therefore, we investigated the associations between carotid atherosclerosis and platelet reactivity markers. The platelet surface expression of P-selectin (CD62P) and the activated GPIIb/IIIa receptor (corresponding to increased binding of PAC-1), as well as the fraction of platelet-derived microparticles (PMPs) prior to and after platelet stimulation with TRAP or ADP, were determined using flow cytometry in 94 subjects in the convalescent phase of ischaemic stroke and in 76 disease controls. The mean common carotid intima-media thickness (CCAmean IMT), maximal common carotid IMT (CCAmax IMT) and maximal bifurcation IMT (BIFmax IMT) were measured bilaterally using B mode, colour Doppler ultrasonography. In stroke subjects IMT within CCA and BIF were greater than in disease controls and the percentage of PMPs prior to and after ex vivo stimulation with agonists was significantly higher than in controls. Multiple regression analysis revealed that PMPs were positively and independently correlated with both CCAmean IMT ( β = 0.23; p < 0.01) and stroke ( β = 0.21; p<0.01), while PAC-1 binding to platelets activated with ADP was negatively and independently associated with CCAmean IMT ( β = −0.29; p<0.001) and atherosclerotic carotid plaque presence ( β = −0.28, p = 0.003). We found a positive association between enhanced PMP formation and atherosclerotic thickening of carotid intima-media or carotid plaque in patients after ischaemic stroke. We demonstrated that diminished expression of active GPIIb/IIIa in the ADP-activated platelets is associated with increased carotid IMT, independently of stroke.

Pravastatin and simvastatin improves acetylsalicylic acid-mediated in vitro blood platelet inhibition.

Eur J Clin Invest 2012

Extract from Aronia melanocarpa fruits potentiates the inhibition of platelet aggregation in the presence of endothelial cells.

Introduction Some polyphenolic compounds extracted from Aronia melanocarpa fruits (AM) have been reported to be cardioprotective agents. In this study we evaluated the ability of AM extract to increase the efficacy of human umbilical vein endothelial cells (HUVECs) to inhibit platelet functions in vitro. Material and methods This study encompasses two models of monitoring platelet reactivity: optical aggregation and platelet degranulation (monitored as the surface CD62P expression) in PRP upon the stimulation with ADP. Results We observed that only at low concentrations (5 µg/ml) did AM extract significantly improve antiplatelet action of HUVECs towards ADP-activated platelets in the aggregation test. Conclusions It is concluded that the potentiating effect of AM extract on the endothelial cell-mediated inhibition of platelet aggregation clearly depends on the used concentrations of Aronia-derived active compounds. Therefore, despite these encouraging preliminary outcomes on the beneficial effects of AM extract polyphenols, more profound dose-effect studies should certainly be considered before the implementation of Aronia-originating compounds in antiplatelet therapy and the prevention of cardiovascular diseases.

The effect of a platelet cholesterol modulation on the acetylsalicylic acid-mediated blood platelet inhibition in hypercholesterolemic patients.

Aspirin (acetylsalicylic acid, ASA) is widely used in the prevention of cardiovascular disease, but its beneficial effects may be restrained in some individuals, where the reduced ability of ASA to protect against arterial thrombotic events is observed. We analyzed the influence of the treatment with atorvastatin (10mg/day) on the platelet sensitivity to ASA monitored under in vitro conditions in hypercholesterolemic patients. The associations between plasma or platelet cholesterol parameters and the ASA-mediated inhibition of platelet reactivity or the extent of platelet protein acetylation by ASA were estimated in the patients treated with atorvastatin for 1, 3, or 6 months. Out of 27 patients, in 17 individuals platelets appeared significantly more sensitive to 50 μM ASA in arachidonic acid- or collagen-induced whole blood aggregation following 1 month atorvastatin therapy (inhibition by 60.9 ± 5.6% vs. 48.8 ± 5.4%, P<0.05 for 0.5mM arachidonic acid, 40.8 ± 2.9% vs. 27.0 ± 4.1%, P<0.05 for 1 μg/ml collagen), and this effect lasted for 3 and 6 months, remaining in a weak, although significant, relation to the reduction of platelet cholesterol content (R(S)=-0.277, P<0.002 for arachidonic acid, R(S)=-0.197, P<0.02 for collagen). It was, however, not dependent upon either antiplatelet action or plasma lipid-lowering activity of atorvastatin. In addition, in about 50% of patients, we noticed that ASA (50 μM) significantly and time-dependently diminished thromboxane B(2) concentration in atorvastatin-treated patients. The ASA-induced acetylation of platelet proteins significantly increased in the course of atorvastatin therapy and was associated with reduced platelet cholesterol (R(S)=-0.598, P<0.0001). In conclusion, statin therapy may improve platelet sensitivity to ASA in some hypercholesterolemic patients. This effect may extend beyond the action of atorvastatin as merely a lipid-lowering agent. The mechanisms of resistance of some patients to such a combined ASA-statin treatment remain to be elucidated.

Comparison of cytotoxic and anti-platelet activities of polyphenolic extracts from Arnica montana flowers and Juglans regia husks.

Abstract Polyphenolic compounds of plant origin are well known to be beneficial to human health: they exert protective effects on haemostasis and have a particular influence on blood platelets. However, the anti-platelet properties of polyphenolic compounds observed so far have not been weighed against their potential cytotoxic action against platelets. The aim of this study was to demonstrate that anti-platelet and cytotoxic effects on blood platelets may interfere and therefore, may often lead to confusion when evaluating the properties of plant extracts or other agents towards blood platelets. The anti-platelet and cytotoxic in vitro effects of plant extracts obtained from the husks of walnuts (J. regia) and flowers of arnica (A. montana) on platelet reactivity and viability were examined. Platelet function was assessed using standard methods (flow cytometry: P-selectin expression, activation of GPIIbIIIa complex, vasodilator-stimulated phosphoprotein, VASP index; turbidimetric and impedance aggregometry) and newly set assays (flow cytometric monitoring of platelet cytotoxicity). The results reveal that none of the studied plant extracts demonstrated cytotoxicity towards blood platelets. The phenolic acid-rich extract of A. montana (7.5 and 15 µg/ml) significantly reduced the ADP-induced aggregation in both whole blood and PRP, and decreased the platelet reactivity index (PRI; VASP phosphorylation) in whole blood, while showing excellent antioxidant capacity. The extract of J. regia husks significantly reduced ADP-induced platelet aggregation in whole blood when applied at 7.5 µg/ml, and only slightly decreased the PRI at 15 µg/ml. Both examined extracts suppressed platelet hyper-reactivity, and such influence did not interfere with cytotoxic effects of the extracts. Thus, its high polyphenol content, excellent antioxidant capacity and distinct anti-platelet properties, in combination with its lack of toxicity, make the extract of A. montana flowers a possible candidate as an anti-platelet agent or a compounding diet supplement.

CD39/NTPDase-1 expression and activity in human umbilical vein endothelial cells are differentially regulated by leaf extracts from Rubus caesius and Rubus idaeus.

Many experimental studies have demonstrated the favorable biological activities of plants belonging to the genus Rubus, but little is known of the role of Rubus leaf extracts in the modulation of the surface membrane expression and activity of endothelial apyrase. The aim of this study was to assess the influence of 1–15 μg/ml Rubus extracts on CD39 expression and enzymatic activity, and on the activation (ICAM-1 expression) and viability of human umbilical vein endothelial cells (HUVEC). The polyphenolic contents and antioxidative capacities of extracts from dewberry (R. caesius L.) and raspberry (R. idaeus L.) leaves were also investigated. The techniques applied were flow cytometry (endothelial surface membrane expression of ICAM-1 and CD39), malachite green assay (CD39 activity), HPLC-DAD (quantitative analysis of polyphenolic extract), ABTS, DPPH and FRAP spectrometric assays (antioxidant capacity), and the MTT test (cell viability). Significantly increased CD39 expressions and significantly decreased ATPDase activities were found in the cells treated with 15 μg/ml of either extract compared to the results for the controls. Neither of the extracts affected cell proliferation, but both significantly augmented endothelial cell ICAM-1 expression. The overall antioxidant capacities of the examined extracts remained relatively high and corresponded well to the determined total polyphenol contents. Overall, the results indicate that under in vitro conditions dewberry and raspberry leaf extracts have unfavorable impact on endothelial cells.

Does grape seed extract potentiate the inhibition of platelet reactivity in the presence of endothelial cells

PURPOSE Numerous studies have suggested that grape seed extract (GSE) confers vascular protection due to the direct effect of its polyphenol content on endothelial cells. The aim of the study was to determine whether GSE confers vascular protection through the direct effect of its polyphenol content on endothelial cells. MATERIAL/METHODS After incubation with GSE-treated human umbilical vein endothelial cells (HUVECs), blood platelet reactivity was evaluated with regard to the expression of CD62P and the activated form of GPIIbIIIa in ADP-stimulated platelets. RESULTS Lower concentrations of GSE were found to enhance the antiplatelet action of HUVECs: 1 μg/ml GSE reduced platelet reactivity by about 10%. While platelet reactivity was not altered by HUVECs incubated with higher concentrations of GSE, HUVEC proliferation was significantly reduced by GSE of up to 10 μg gallic acid equivalent/ml. CONCLUSIONS The results of the study show that low doses of GSE potentiate the inhibitory action of HUVECs on platelet reactivity, which may account, at least partially, for the protective effects of grape products against cardiovascular diseases. In contrast, high concentrations of GSE significantly impair endothelial cell proliferation in vitro.

Structure, stability, and antiplatelet activity of O-acyl derivatives of salicylic acid and lipophilic esters of acetylsalicylate

The anti-thrombotic activity of acetylsalicylic acid (ASA) has been shown to be due to specific irreversible acetylation of blood platelet cyclooxygenase. The aim of our study was to investigate the associations between the antiplatelet activities of derivatives of both ASA and salicylic acid (SA), as well as the structure, stability, and molecular properties of these compounds. Homologous series of O-acyl derivatives of salicylic acid (propionyl-, butyrylsalicylic acids, PSA, BSA) and lipophilic dodecyl (C12)-, hexadecyl (C16)-, and cholesteryl acetylsalicylates were synthesized and tested for structure-activity relationships. The molecular properties (heat of formation, molecular surface area, dipole moment) of ASA and SA derivatives obtained by theoretical calculations changed with the increasing length of the acyl or alkyl residue. The inhibition of whole blood platelet aggregation and the reduction in thromboxane (TX) generation by O-acyl derivatives were concentration-dependent and decreased along with increasing the length of acyl hain. These effects correlated with the extent of platelet reactivity and P-selectin expression inhibition in collagen-activated platelets. In contrast to ASA and O-acyl derivatives of SA, none of the lipophilic ASA derivatives had a significant inhibitory effect on platelet aggregation. In conclusion, all SA and ASA derivatives studied under in vitro conditions showed much lower antiplatelet activities than ASA itself, despite their higher affinity to plasma proteins or membrane components and their equivalent ability to acetylate protein free amino groups.We suggest the significance of the carboxylic group, dipole moment, geometry, and size of these pharmaceuticals in their ability to bind to the active site of cyclooxygenase and their antiplatelet efficacy.

Xanthohumol from hop cones (Humulus lupulus L.) prevents ADP-induced platelet reactivity.

Abstract Hop cones (Humulus lupulus L.), very rich source of phenolic compounds, possessing anticancer, antioxidant and anti-inflammatory activities, are considered as beneficial diet ingredients improving human health. In this study, the antiplatelet action of xanthohumol (XN), the principal flavonoid in hop cones, was investigated. XN significantly attenuated ADP-induced blood platelet aggregation (97.2 ± 35.7 AU for 6 μg/ml of XN vs. 120.4 ± 30.1 AU for 0.17% dimethyl sulfoxide (DMSO), p < 0.001) and significantly reduced the expression of fibrinogen receptor (activated form of GPIIbIIIa) on platelets’ surface (47.6 ± 15.8 for 1.5 μg/ml XN, 44.6 ± 17.3% for 3 μg/ml XN vs. 54.5 ± 19.2% for control or 43.3 ± 18.4% for 6 μg/ml XN vs. 49.7 ± 19.4% for 0.17% DMSO, p < 0.05 or less). These findings suggest that the phenolic compounds originating from hops (XN) have a novel role as antiplatelet agents and can likely be used as dietary supplements in prophylactic approaches.