C. Cerletti

Aspirin resistance: position paper of the Working Group on Aspirin Resistance

A. D . MICHELSON,* M. CATTANEO, J . W . E IKELBOOM,§ P . GURBEL ,– K . KOTT KE-MARCHANT ,** T . J . K UNIC K I , F . M. PULC INELL I , C . C ERLETT I §§ and A . K . RAO,–– ON BEHALF OF THE PLATELET PHYS IOLOGY SUBC OMMI TTEE O F T HE SC I ENT IF I C AND STA NDARDI ZAT I ON COMMITT EE OF THE IN TERN AT IONA L SOC IETY ON T HROMBOS IS A ND HA EMOSTA S IS* ** *Chair, Working Group on Aspirin Resistance, International Society on Thrombosis and Haemostasis; Department of Pediatrics, Center for Platelet Function Studies, University of Massachusetts Medical School, Worcester, MA, USA; Department of Medicine, Surgery, and Dentistry, Unit of Hematology and Thrombosis, University of Milan, Ospedale San Paolo, Milan, Italy; §Department of Haematology, School of Medicine and Pharmacology, University of Western Australia, Royal Perth Hospital, Perth, Australia; –Sinai Center for Thrombosis Research, Sinai Hospital, Baltimore, MD, USA; **Hemostasis and Thrombosis, Department of Clinical Pathology, Cleveland Clinic Foundation, Cleveland, OH, USA; Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, CA, USA; Department of Experimental Medicine and Pathology, University ‘‘La Sapienza’’, Rome, Italy; §§Center for High Technology Research and Education in Biomedical Sciences, Catholic University, Campobasso, Italy; ***Chairperson, ISTH-SSC Platelet Physiology Sub-Committee; and ––Thrombosis Research Center, Temple University School of Medicine, Philadelphia, PA, USA

Human polymorphonuclear leukocytes produce and express functional tissue factor upon stimulation

Summary.  Background: Blood‐borne tissue factor (TF) plays a crucial role in thrombogenesis. Aim: To study whether polymorphonuclear leukocytes (PMN) are a source of TF. Methods and Results: Human PMN were carefully separated from other blood cells and stimulated for 3 min with purified P‐selectin or the chemotactic peptide formyl‐MetLeuPhe (fMLP): they expressed both TF procoagulant activity, as identified by specific TF MoAb and inactivated factor VIIa blockade; and TF:Ag (four to six times), as shown by flow‐cytometry and immunocytochemistry. About 40% of permeabilized PMN, both resting and stimulated, contained TF:Ag, indicating that stimulation only modifies the location of TF:Ag within PMN. By real time‐polymerase chain reaction (RT‐PCR), a very low amount of TF mRNA was detectable in resting PMN, but a 3‐ to 5‐fold increase was observed after 1‐h stimulation with P‐selectin or fMLP, respectively. Conclusions: These findings suggest that TF is not constitutively expressed in peripheral PMN, but can be up‐regulated and produced upon stimulation and specific gene transcription, as for instance during contact with activated platelets or endothelium. The stored TF is rapidly expressed in vitro as a functional molecule on the surface of activated PMN. The availability of PMN TF supports the relevance of inflammatory cells and their interaction with platelets for fibrin deposition and thrombus formation.

Effects of resveratrol and other wine polyphenols on vascular function: An update

Several epidemiologic observations show that moderate wine drinking reduces cardiovascular morbidity and mortality. Wine contains several polyphenols, and among them, resveratrol in particular has been shown to exert a number of important biologic activities on the cardiovascular system that may contribute to the protective effects of wine. The mechanisms through which resveratrol and other wine polyphenols protect from ischemic cardiovascular events are many, but protection from oxidative stress and radical oxygen species production, a facilitating activity on nitric oxide production and activity and the ability to modulate the expression of adhesive molecules by blood cells and the vascular wall seem to be the most important. In this overview, the in vitro and in vivo evidence on the activity of resveratrol on vascular function and circulating blood cells, with a special emphasis on blood platelets, is thoroughly presented.

White blood cell count, sex and age are major determinants of heterogeneity of platelet indices in an adult general population: results from the MOLI-SANI project

Background The understanding of non-genetic regulation of platelet indices - platelet count, plateletcrit, mean platelet volume, and platelet distribution width - is limited. The association of these platelet indices with a number of biochemical, environmental and clinical variables was studied in a large cohort of the general population. Design and Methods Men and women (n=18,097, 52% women, 56±12 years) were randomly recruited from various villages in Molise (Italy) in the framework of the population-based cohort study “Moli-sani”. Hemochromocytometric analyses were performed using an automatic analyzer (Beckman Coulter, IL, Milan, Italy). Associations of platelet indices with dependent variables were investigated by multivariable linear regression analysis. Results Full models including age, sex, body mass index, blood pressure, smoking, menopause, white and red blood cell counts, mean corpuscular volume, D-dimers, C-reactive protein, high-density lipoproteins, low-density lipoproteins, triglycerides, glucose, and drug use explained 16%, 21%, 1.9% and 4.7% of platelet count, plateletcrit, mean platelet volume and platelet distribution width variability, respectively; variables that appeared to be most strongly associated were white blood cell count, age, and sex. Platelet count, mean platelet volume and plateletcrit were positively associated with white blood cell count, while platelet distribution width was negatively associated with white blood cell count. Platelet count and plateletcrit were also positively associated with C-reactive protein and D-dimers (P<0.0001). Each of the other variables, although associated with platelet indices in a statistically significant manner, only explained less than 0.5% of their variability. Platelet indices varied across Molise villages, independently of any other platelet count determinant or characteristics of the villages. Conclusions The association of platelet indices with white blood cell count, C-reactive protein and D-dimers in a general population underline the relation between platelets and inflammation.

Platelet-leukocyte interactions in thrombosis

Activated platelets may adhere to leukocytes and form circulating mixed aggregates. The latter are considered a reliable marker of a prothrombotic state and are associated with several cardiovascular conditions. The molecular mechanisms responsible of this cellular interaction include a central role of platelet P-selectin and of P-selectin glycoprotein ligand-1 (PSGL-1), its counter receptor on leukocytes in a signaling cascade, resulting in the activation of the beta-2 integrin Mac-1 and in the firm adhesion between the two cell types. The interaction of P-selectin with PSGL-1 also induces upregulation of leukocyte tissue factor, biosynthesis of several cytokines and other inflammatory reactions, thereby contributing to the thrombotic progression. In this review the main determinants of mixed aggregate formation, the heritability component, the major pathological conditions associated with higher levels of mixed aggregates in the circulation will be discussed. Besides current anti-platelet or antithrombotic drugs, natural compounds, such as the polyphenols present in vegetable foods and red wine, have been tested for their inhibitory effect on mixed aggregate formation. The promising results shown by studies in vitro and in experimental animal models, remain to be carefully investigated in humans. Platelet-leukocyte aggregates provide a novel link between inflammation and thrombosis, two central processes in atherogenesis. A better understanding of the role of platelet-leukocyte interactions in athero-thrombosis will be instrumental for the progress of prevention and treatment of ischaemic cardiovascular disease.

Age- And Sex-Related Variations in Platelet Count in Italy: A Proposal of Reference Ranges Based on 40987 Subjects' Data

Background and Objectives Although several studies demonstrated that platelet count is higher in women, decreases with age, and is influenced by genetic background, most clinical laboratories still use the reference interval 150–400×109 platelets/L for all subjects. The present study was to identify age- and sex-specific reference intervals for platelet count. Methods We analysed electronic records of subjects enrolled in three population-based studies that investigated inhabitants of seven Italian areas including six geographic isolates. After exclusion of patients with malignancies, liver diseases, or inherited thrombocytopenias, which could affect platelet count, reference intervals were estimated from 40,987 subjects with the non parametric method computing the 2.5° and 97.5° percentiles. Results Platelet count was similar in men and women until the age of 14, but subsequently women had steadily more platelets than men. The number of platelets decreases quickly in childhood, stabilizes in adulthood, and further decreases in oldness. The final result of this phenomenon is that platelet count in old age was reduced by 35% in men and by 25% in women compared with early infancy. Based on these findings, we estimated reference intervals for platelet count ×109/L in children (176–452), adult men (141–362), adult women (156–405), old men (122–350) and, old women (140–379). Moreover, we calculated an “extended” reference interval that takes into account the differences in platelet count observed in different geographic areas. Conclusions The age-, sex-, and origin-related variability of platelet count is very wide, and the patient-adapted reference intervals we propose change the thresholds for diagnosing both thrombocytopenia and thrombocytosis in Italy.

Inhibition of tissue factor expression by hydroxyurea in polymorphonuclear leukocytes from patients with myeloproliferative disorders: a new effect for an old drug?

Summary.  Background: Polymorphonuclear leukocytes (PMN) from healthy subjects can produce and store tissue factor (TF), which is expressed on PMN surface upon in vitro stimulation with P‐selectin. Results: We report here that platelets and PMN from 12 patients with myeloproliferative disorders (MPD) (six with polycythemia vera, six with essential thrombocythemia) show up regulation of P‐selectin and TF, respectively, in the absence of any in vitro challenge. The number of circulating mixed platelet‐PMN aggregates was also increased. PMN TF expression as well as mixed platelet‐PMN aggregates, but not platelet P‐selectin, were significantly reduced in six MPD patients after treatment with hydroxyurea (HU). In vitro studies performed on PMN separated from healthy donors confirmed HU effects (0–1400 μm). HU prevented both P‐selectin‐induced TF expression and mixed cell aggregate formation. The inhibitory effect of HU was specific for P‐selectin‐induced PMN activation, as it did not affect formyl‐methionyl‐leucyl‐phenylalanine‐induced PMN TF expression. Conclusions: In MPD patients, platelet P‐selectin‐mediated TF expression on circulating PMN may play a role in thrombus formation and represents a novel target for the antithrombotic activity of HU.

Indomethacin prevents the long-lasting inhibitory effect of aspirin on human platelet cyclo-oxygenase activity

Aspirin and indomethacin do interact with the same site on cyclo-oxygenase. This suggestion is based on in vitro studies on ram seminal vesicles and in vivo drug interaction studies on rat platelets. The purpose of the present study was to ascertain whether the same interaction also occurred after administration of both drugs to human volunteers. Platelet aggregation induced by sodium arachidonate or by collagen, and formation of platelet MDA and TxB2 were measured before, two and 48 hours after ingestion of either indomethacin (50 mg) or aspirin (500 mg) or of both drugs (30 minutes apart). While the inhibitory effect of indomethacin on these parameters was short lasting, that of aspirin persisted for at least 48 hours. However, when both drugs were given concurrently, the long-lasting effect of aspirin was no longer detectable. Since competition at levels other than platelets was unlikely, this study indicates that indomethacin and aspirin inhibit human platelet cyclo-oxygenase by the same basic mechanism. Acetylation of the enzyme appears to be a secondary mechanism which makes the inhibitory effect of aspirin persistent.

Salicylate-aspirin interaction in the rat. Evidence that salicylate accumulating during aspirin administration may protect vascular prostacyclin from aspirin-induced inhibition.

Aspirin inhibits cyclooxygenase, thus preventing thromboxane A2 production in blood platelets and prostacyclin in vascular cells. Aspirin is rapidly hydrolyzed to salicylate in the circulation. The objectives of this study were (a) to evaluate whether administration of salicylate, though ineffective by itself, prevents the inhibitory effect of aspirin on platelet and/or vascular cyclooxygenase activity; (b) to verify whether salicylate accumulating in blood after aspirin administration interferes with the pharmacological activity of further doses of aspirin. Pretreatment of rats with sodium salicylate (25-100 mg/kg i.p.) resulted in dose-related prevention of the effect of a subsequent dose of aspirin (2.5-10 mg/kg i.v.) on both platelet and vascular cells. Sodium salicylate appeared to amplify the greater response of platelets to aspirin compared with vessel wall. Pretreatment of rats with repeated high doses of aspirin (200 mg/kg) resulted after 24 h in blood salicylate levels (150-200 microgram/ml) that significantly prevented the inhibitory effect of a subsequent dose of aspirin on newly synthesized vascular prostacyclin. Blood salicylate levels obtained after 36 or 48 h (less than 50 microgram/ml) were too low to blunt aspirins effect. The interference with aspirin of its major endogenous metabolite should be borne in mind when interpreting results obtained with high dose aspirin or during repeated administration of this drug.

Interactions of gallic acid, resveratrol, quercetin and aspirin at the platelet cyclooxygenase-1 level. Functional and modelling studies.

While resveratrol and quercetin possess antiplatelet activity, little is known on the effect of gallic acid on platelets. We studied the interactions of these three different polyphenols among themselves and with aspirin, at the level of platelet cyclooxygenase-1 (COX-1). Both functional (in vitro and in vivo) and molecular modelling approaches were used. All three polyphenols showed comparable antioxidant activity (arachidonic acid [AA]-induced intraplatelet ROS production); however, resveratrol and quercetin, but not gallic acid, inhibited AA-induced platelet aggregation. Gallic acid, similarly to salicylic acid, the major aspirin metabolite, prevented inhibition of AA-induced platelet function by aspirin but, at variance with salicylic acid, also prevented inhibition by the other two polyphenols. Molecular modelling studies, performed by in silico docking the polyphenols into the crystal structure of COX-1, suggested that all compounds form stable complexes into the COX-1 channel, with slightly different but functionally relevant interaction geometries. Experiments in mice showed that gallic acid administered before aspirin, resveratrol or quercetin fully prevented their inhibitory effect on serum TxB(2). Finally, a mixture of resveratrol, quercetin and gallic acid, at relative concentrations similar to those contained in most red wines, did not inhibit platelet aggregation, but potentiated sub-inhibitory concentrations of aspirin. Gallic acid interactions with other polyphenols or aspirin at the level of platelet COX-1 might partly explain the complex, and possibly contrasting, effects of wine and other components of the Mediterranean diet on platelets and on the pharmacologic effect of low-dose aspirin.