M. Teresa Santos

Erythrocyte Promotion of Platelet Reactivity Decreases the Effectiveness of Aspirin as an Antithrombotic Therapeutic Modality The Effect of Low-Dose Aspirin Is Less Than Optimal in Patients With Vascular Disease Due to Prothrombotic Effects of Erythrocytes on Platelet Reactivity

BACKGROUND Aspirin (acetylsalicylic acid, ASA) is widely used for secondary prevention of ischemic vascular events, although its protection only occurs in 25% of patients. We previously demonstrated that platelet reactivity is enhanced by a prothrombotic effect of erythrocytes in a thromboxane-independent manner. This diminishes the antithrombotic therapeutic potential of ASA. Recent data from our laboratory indicate that the prothrombotic effect of erythrocytes also contains an ASA-sensitive component. In accordance with this observation, intermittent treatment with high-dose ASA reduced the prothrombotic effects of erythrocytes ex vivo in healthy volunteers. In the present study, the effects of platelet-erythrocyte interactions were evaluated ex vivo in 82 patients with vascular disease: 62 patients with ischemic heart disease treated with 200 mg ASA/d and 20 patients with ischemic stroke treated with 300 mg ASA/d. METHODS AND RESULTS Platelet activation (release reaction) and platelet recruitment (fluid-phase proaggregatory activity of cell-free releasates from activated platelets) were assessed after collagen stimulation (1 microg/mL) of platelets, platelet-erythrocyte mixtures, or whole blood. Platelet thromboxane A2 synthesis was inhibited by >94% by ASA administration in all patients. Importantly, platelet recruitment followed one of three distinct patterns. In group A (n=32; 39%), platelet recruitment was blocked by ASA both in the presence and absence of erythrocytes. In group B (n=37; 45%), recruitment was abolished when platelets were evaluated alone but continued in the presence of erythrocytes, indicating a suboptimal effect of ASA on erythrocytes of this patient group. In group C (n= 13; 16%), detectable recruitment in stimulated platelets alone persisted and was markedly enhanced by the presence of erythrocytes. CONCLUSIONS In two thirds of a group of patients with vascular disease, 200 to 300 mg ASA was insufficient to block platelet reactivity in the presence of erythrocytes despite abolishing thromboxane A2 synthesis. Platelet activation in the presence of erythrocytes can induce the release reaction and generate biologically active products that recruit additional platelets into a developing thrombus. Insufficient blockade of this proaggregatory property of erythrocytes can lead to development of additional ischemic complications.

Participation of Tyrosine Phosphorylation in Cytoskeletal Reorganization, αIIbβ3 Integrin Receptor Activation, and Aspirin-Insensitive Mechanisms of Thrombin-Stimulated Human Platelets

BackgroundFibrinogen binding to the active conformation of the &agr;IIb&bgr;3 integrin receptor (glycoprotein IIb/IIIa) and cytoskeletal reorganization are important events in platelet function. Tyrosine phosphorylation of platelet proteins plays an essential role in platelet signal transduction pathways. We studied the participation of tyrosine kinases on these aspects of platelet reactivity and their importance in cyclooxygenase (COX)-1–independent mechanisms in thrombin-stimulated human platelets. Methods and ResultsUsing washed platelets from normal donors and tyrphostin-A47 and aspirin as tyrosine kinase and COX-1 inhibitors, respectively, we found that tyrphostin-A47 downregulated (1) the thrombin-activated conformational change of &agr;IIb&bgr;3, (2) actin polymerization and cytoskeletal reorganization, and (3) the quantity of tyrosine-phospho-rylated proteins associated with the reorganized cytoskeleton. The latter are important components of multimolecular signaling complexes. Concomitantly, platelet aggregation and secretion were significantly reduced. Aspirin did not affect receptor activation or tyrosine phosphorylation but did decrease the initial (30-second) burst of actin polymerization. Importantly, aspirin significantly amplified the inhibitory effect of tyrphostin-A47 on all aspects of platelet reactivity that we evaluated. ConclusionsTyrosine protein phosphorylation is a regulatory control system of the inside-out mechanism of &agr;IIb&bgr;3 activation and cytoskeletal assembly in thrombin-stimulated human platelets. Inhibition of these aspects of platelet function with tyrphostin-A47 is amplified when platelets are treated with aspirin. Therefore, tyrosine phosphorylation is a major component of early signaling events and of COX-1–independent mechanisms of thrombin-induced platelet reactivity. The study results may indicate a novel target for therapeutic intervention.

Effect of Atorvastatin on Platelet Thromboxane A2 Synthesis in Aspirin-Treated Patients With Acute Myocardial Infarction

Inhibition of platelet thromboxane A(2) (TXA(2)) by aspirin is critical in patients with acute myocardial infarction (AMI), but some patients have persistent platelet TXA(2) production within 48 hours of the onset of AMI. Statins are known to reduce TXA(2) in aspirin-free patients with hypercholesterolemia. We hypothesized that treatment with aspirin plus atorvastatin could reduce persistent TXA(2) synthesis and aspirin resistance in patients with AMI. We evaluated platelet function in 184 aspirin-treated patients within 48 hours of the onset of AMI. Patients were divided into group A (treated with aspirin alone, n = 139) and group B (treated with aspirin plus atorvastatin, n = 45). We studied collagen-induced platelet TXA(2) synthesis, serotonin ((14)C-5HT) release and recruitment, and adenosine diphosphate-, arachidonic acid-, and collagen-induced platelet aggregation. Persistent TXA(2) synthesis was detected in 25% and 9% of groups A and B, respectively (p = 0.03). TXA(2), arachidonic acid-aggregation, and collagen-induced responses were significantly reduced in patients receiving dual treatment compared to those receiving aspirin monotherapy. Atorvastatin did not modify platelet reactivity in patients with efficiently blocked TXA(2) synthesis. These results strongly suggest a direct effect of the statin on platelet eicosanoid synthesis. This was confirmed in vitro by incubating washed aspirin-free and aspirin (1 muM)-treated platelets from normal subjects with 1 to 20 microM atorvastatin. Atorvastatin in vitro significantly reduced platelet TXA(2) synthesis and collagen-induced aggregation. In conclusion, atorvastatin combined with aspirin early in the onset of the acute event significantly reduced persistent TXA(2) and TXA(2)-dependent aspirin resistance. This could contribute to the clinical benefit of atorvastatin in patients with AMI.

Composition of platelet fatty acids and their modulation by plasma fatty acids in humans: effect of age and sex

This study evaluates the influence of sex on platelet fatty acid (FA) composition, and whether sex differences are conditioned by age. Since plasma FA have a specific relationship with platelet FA their variations with age and sex are also considered. Forty-nine male-female human couples (16-75 years), where within each couple the partners were on qualitatively similar diets and of similar age, were studied. Few differences were found between the whole groups of men and women in platelet FA. A comparison of data on FA in platelet phospholipids (PL) from 3 age groups (16-40, 40-60 and over 60) showed an increase in saturated FA of middle-aged subjects, an age-dependent decrease in 20: 5 in both sexes and of 18: 2 mainly in women. The percentage of plasma phosphatidylserine plus phosphatidylinositol decreased in middle-aged subjects. With regard to the influence of FA of plasma PL on FA of platelet PL, we found a higher correlation coefficient (r) for 16:0 and 18:0 and 20:4 and a lower one for 20:5 in middle-aged men and post-menopausal women. Considering that an increase in saturated FA and 20:4 and a decrease in 20:5 in platelet PL may increase platelet function, the plasma FA influence on platelets may help to explain the higher incidence of CHD in those groups of subjects.

Effect of postprandial lipaemia on platelet function in man evaluated in whole blood.

The effect of a fatty meal (100 g of fat) on platelet function is evaluated. Two hours after the fat intake (dairy cream) there is a significant reduction in the initial stages of platelet activation by collagen (1 and 0.5 micrograms/ml) as measured by a new analytical method, the BASIC wave, and by the decrease in the beta-Thromboglobulin released by stimulated platelets. This effect is greater in platelet rich plasma (PRP) than in whole blood. Red blood cells (RBC) have a potentiating effect on platelet activation by collagen both before and after the fat intake which is indicated by an increase in the BASIC wave intensity. No significant differences were found, however, in platelet aggregation in PRP or whole blood evaluated by impedance aggregometry. These results suggest that the increase of chylomicrons after fat intake has an inhibitory effect on platelet activation but does not modify platelet aggregation. In addition, it seems that lipaemia does not modify RBC interactions with platelets in collagen stimulated samples.

Effect of Oral Contraceptives on Plasma and Platelet Lipid Composition: Influence of the length duration of time of ingestion

The action of oral contraceptives (OC) (50 μg ethi‐nylestradiol + 250 μg levonorgestrel) on plasma and platelet lipid composition was studied in two groups of women who took this OC for one year, or for a longer period of time (2–9 years). Comparison with the control group showed that the ingestion of OC modifies the composition of some fatty acids in plasma and platelets. This modification is more marked in the women who took OC for one year. In plasma a decrease in linoleic acid (18:2) was found in the cholesterol and phospholipid fractions. In platelets, an increase of stearic acid (18:0) was observed in phospholipids, triglycerides and free fatty acid fractions. The modifications observed in plasma and platelets were not parellel, which suggests a specific and different action of OC on both plasma and platelets. Finally, it is suggested that the lipid variations found in the group that took the OC for one year may be compatible with a platelet hyperfunction.

Análisis de las buenas prácticas de participación ciudadana en las unidades de gestión clínica del Servicio Andaluz de Salud

OBJECTIVE To discover good practices for inhabitant participation in the clinical management units (CMUs) of the Andalusian Health Service (AHS) (Spain) and to explore the reasons perceived by CMU and AHS professionals that may influence the presence and distribution of those good practices among the CMU. METHODS Study with mixed methodology carried out in Andalusia (Spain) in two phases (2013-2015). Firstly, an online survey was delivered to the Directors of the CMUs which had set up an inhabitant participation commission. In a second phase, a qualitative study was carried out through semi-structured interviews with professionals from the Andalusian Health Service with previous experience in inhabitant participation. A descriptive analysis of the quantitative information and a semantic content analysis of the qualitative information were carried out. RESULTS 530 CMUs took part in the survey. The inhabitant participation practices more often implemented in the CMUs are those related to the informing and consultation levels. Twelve professionals were interviewed in the second phase. Other practices with higher inhabitant involvement and delegation are secondary. The barriers which were identified by professionals are related to the beliefs and attitudes of the inhabitants, the professionals, the health system and the environment. CONCLUSION The main practices for inhabitant participation in the CMUs are related to the most basic levels of participation. The method and dynamics which facilitate inhabitant empowerment within the health system are not clearly recognised.Objective To discover good practices for inhabitant participation in the clinical management units (CMUs) of the Andalusian Health Service (AHS) (Spain) and to explore the reasons perceived by CMU and AHS professionals that may influence the presence and distribution of those good practices among the CMU.

OriginalAnálisis de las buenas prácticas de participación ciudadana en las unidades de gestión clínica del Servicio Andaluz de SaludAnalysis of good practices for inhabitant participation in the clinical management units of the Andalusian Health Service (Spain)

OBJECTIVE To discover good practices for inhabitant participation in the clinical management units (CMUs) of the Andalusian Health Service (AHS) (Spain) and to explore the reasons perceived by CMU and AHS professionals that may influence the presence and distribution of those good practices among the CMU. METHODS Study with mixed methodology carried out in Andalusia (Spain) in two phases (2013-2015). Firstly, an online survey was delivered to the Directors of the CMUs which had set up an inhabitant participation commission. In a second phase, a qualitative study was carried out through semi-structured interviews with professionals from the Andalusian Health Service with previous experience in inhabitant participation. A descriptive analysis of the quantitative information and a semantic content analysis of the qualitative information were carried out. RESULTS 530 CMUs took part in the survey. The inhabitant participation practices more often implemented in the CMUs are those related to the informing and consultation levels. Twelve professionals were interviewed in the second phase. Other practices with higher inhabitant involvement and delegation are secondary. The barriers which were identified by professionals are related to the beliefs and attitudes of the inhabitants, the professionals, the health system and the environment. CONCLUSION The main practices for inhabitant participation in the CMUs are related to the most basic levels of participation. The method and dynamics which facilitate inhabitant empowerment within the health system are not clearly recognised.Objective To discover good practices for inhabitant participation in the clinical management units (CMUs) of the Andalusian Health Service (AHS) (Spain) and to explore the reasons perceived by CMU and AHS professionals that may influence the presence and distribution of those good practices among the CMU.

C0228: Platelet Thromboxane A2 is an Extracellular Mediator of the Prothrombotic Effect of Erythrocytes

Background: The thrombotic cellular mechanisms associated with cardiovascular events remains unclear, largely because of an inability to visualize thrombus formation. In addition, the contribution of endothelial cell (EC) injuries to thrombus formation processes are unclear. Methods: We developed in vivo fluorescent imaging technique based on singleand multi-photon microscopy to revealed the multicellular processes during thrombus development. We utilized multi-color and high-speed resonance scanner systems, and applied to fluorescent protein transgenic mice. We could identify single platelet kinetics in developing thrombus in living animals. Results: We assessed dynamic cellular interplay in two thrombosis models. First, we visualized that rapidly developing thrombi composed of discoid platelets without EC disruption was triggered by reactive oxygen species by photochemically induction from moderate power laser irradiation. In this model, thrombus consisted by discoid platelet aggregations without leukocyte recruitment. The other model is, thrombus with EC disruption. High power laser induced EC erosion and extravasations of circulating leukocytes with thrombus development. Inflammatory cytokine, adhesion molecules dynamically control these two processes. As for the thrombus formation with EC disruption, chemokine expressions in endothelium and leukocyte (especially neutrophils) recruitment played a significant role in these processes. TLR4 signalling also contributed to these steps. Conclusions: In sum, using our imaging system can be a powerful tool to analyze thrombus formation and evaluate the therapeutic strategies.

Letter by Santos et al regarding article, "Pharmacodynamic effects of different aspirin dosing regimens in type 2 diabetes mellitus patients with coronary artery disease".

To the Editor: The observation of Capodanno et al1 that increasing the frequency of administration of a low dose of aspirin has a more potent platelet inhibitory effect than increasing the daily dose of aspirin was of great interest to us because we observed a similar phenomenon several years ago in 206 patients with vascular diseases.2 It is noteworthy that although they did not observe a difference between the antiplatelet effects of once-daily (OD) and twice-daily (BID) administration of 162 mg aspirin, using the whole blood VerifyNow Aspirin test, they observed significantly lower platelet aggregation in …