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Platelet receptors and signaling in the dynamics of thrombus formation

Platelet receptors are at the forefront of recent research and major advances have been made in understanding their molecular functions and their downstream signaling pathways. This review addresses the steps in the thrombus formation process in the arterial circulation, emphasizing our current knowledge on the role of platelet receptors and signaling. Hemostasis and pathological thrombus formation are dynamic processes that require a co-ordinated series of events involving platelet membrane receptors, bidirectional intracellular signals, and release of platelet proteins and inflammatory substances. This review aims to summarize current knowledge in the key steps in the dynamics of thrombus formation, with special emphasis on the crucial participation of platelet receptors and signaling in this process. Initial tethering and firm adhesion of platelets to the exposed subendothelium is mediated by glycoprotein (GP) Ib/IX/V complex and collagen receptors, GP VI and α2β1 integrin, in the platelet surface, and by VWF and fibrillar collagen in the vascular site. Interactions between these elements are largely influenced by flow and trigger signaling events that reinforce adhesion and promote platelet activation. Thereafter, soluble agonists, ADP, thrombin, TxA2, produced/released at the site of vascular injury act in autocrine and paracrine mode to amplify platelet activation and to recruit circulating platelets to the developing thrombus. Specific interactions of these agonists with their G-protein coupled receptors generate inside-out signaling leading to conformational activation of integrins, in particular αIIbβ3, increasing their ligand affinity. Binding of αIIbβ3 to its ligands, mainly fibrinogen, supports processes such as clot retraction and platelet aggregation. Stabilization of thrombi is supported by the late wave of signaling events promoted by close contact between aggregated platelets. The best known contact-dependent signaling is outside-in signaling through αIb β3, but new ones are being clarified such as those mediated by interaction of Eph receptors with ephrins, or by Sema 4D and Gas-6 binding to their receptors. Finally, newly identified mechanisms appear to control thrombus growth, including back-shifting of activated integrins and actuation of compensatory molecules such as ESAM or PECAM-1. The expanding knowledge of thrombotic disease is expected to translate into the development of new drugs to help management and prevention of thrombosis.

Cessation of oral anticoagulation in relation to mortality and the risk of thrombotic events in patients with atrial fibrillation

Bleeding risk (often perceived, rather than actual) is a common reason for cessation of oral anticoagulation with Vitamin K antagonists (VKA). We investigate clinical outcomes in a consecutive population of VKA naïve atrial fibrillation (AF) patients, who initiated VKA therapy in our clinic. We included consecutive VKA-naïve patients with non valvular AF, initiated on VKA therapy in our anticoagulation outpatient clinic in 2009. During follow-up, adverse events [thrombotic/vascular events (stroke, acute coronary syndrome, acute heart failure and cardiac death), major bleeding and death], and VKA cessation were recorded. At the end of the follow-up, we determined time within therapeutic range (TTR), using a linear approximation (Rosendaal method). We studied 529 patients (49% male, median age 76), median follow-up 835 days (IQR 719-954). During this period 114 patients stopped VKA treatment. 63 patients suffered a thrombotic/cardiovascular event (5.17%/year, 27 thrombotic/ischaemic strokes), 51 major bleeding (4.19%/year) and 48 died (3.94%/year). Median TTR was 54% (34-57). On multivariate analysis (adjusted by CHA₂DS₂-VASc score), VKA cessation was associated with death [Hazard Ratio (HR) 3.43; p<0.001], stroke [4.21; p=0.001] and thrombotic/cardiovascular events [2.72; p<0.001]. Independent risk factors for major bleeding were age [1.08; p<0.001], previous stroke [1.85; p=0.049], and TTR [0.97; p=0.001], but not VKA cessation. In conclusion, in AF patients AF, VKA cessation is independently associated with mortality stroke and cardiovascular events. Specifically, VKA cessation independently increased the risk of stroke, even after adjusting for CHA₂DS₂-VASc score. TTR was an independent risk factor for major bleeding following initiation of VKA therapy.

Observation versus antiplatelet therapy as primary prophylaxis for thrombosis in low-risk essential thrombocythemia

The effectiveness of antiplatelet therapy as primary prophylaxis for thrombosis in low-risk essential thrombocythemia (ET) is not proven. In this study, the incidence rates of arterial and venous thrombosis were retrospectively analyzed in 300 low-risk patients with ET treated with antiplatelet drugs as monotherapy (n = 198) or followed with careful observation (n = 102). Follow-up was 802 and 848 person-years for antiplatelet therapy and observation, respectively. Rates of thrombotic events were 21.2 and 17.7 per 1000 person-years for antiplatelet therapy and observation, respectively (P = .6). JAK2 V617F-positive patients not receiving antiplatelet medication showed an increased risk of venous thrombosis (incidence rate ratio [IRR]: 4.0; 95% CI: 1.2-12.9; P = .02). Patients with cardiovascular risk factors had increased rates of arterial thrombosis while on observation (IRR: 2.5; 95% CI: 1.02-6.1; P = .047). An increased risk of major bleeding was observed in patients with platelet count greater than 1000 x 10(9)/L under antiplatelet therapy (IRR: 5.4; 95% CI: 1.7-17.2; P = .004). In conclusion, antiplatelet therapy reduces the incidence of venous thrombosis in patients with JAK2-positive ET and the rate of arterial thrombosis in patients with associated cardiovascular risk factors. In the remaining low-risk patients, this therapy is not effective as primary prophylaxis of thrombosis, and observation may be an adequate option.

Cytotoxicity and Antiproliferative Activities of Several Phenolic Compounds Against Three Melanocytes Cell Lines: Relationship Between Structure and Activity

Abstract: Polyphenolic compounds are widely distributed in the vegetable kingdom and are therefore consumed regularly in the human diet. Epidemiological studies suggest that foods rich in polyphenolic compounds contribute to reducing the risk of cancer. The purpose of our work is to: 1) study the possible cytotoxicity and antiproliferative effects of 13 polyphenolic compounds on 3 cell lines of melanocytes, 2 of melanoma (B16F10 and SK-MEL-1), and 1 of nontransformed melanocytes (Melan-a); and 2) identify the possible relationship between the chemical structure of the tested compounds and their effect on cellular viability. The said polyphenolic compounds corresponded to 8 flavonoids with varying hydroxyl and methoxyl substituents, related structurally through the oxidation state of their flavonoid skeleton, a catechin polymer and 4 phenolic acids. The cytotoxic activity of all the studied compounds was modest or not apparent. The flavonoids luteolin, tangeretin, baicalein, quercetin, and myricetin, and gallic acid showed antiproliferative effects on the tested lines. Our results suggest that a correlation exists between the structural oxidation state and the position, number, and nature of substituents of the polyphenolic compounds studied and their antiproliferative effects.

Flavonoids inhibit platelet function through binding to the thromboxane A2 receptor.

Summary.  Background: Dietary flavonoids are known for their antiplatelet activity resulting in cardiovascular protection, although the specific mechanisms by which this inhibition occurs has not been fully established. Objective: The aim of this study was to investigate the interaction of nine flavonoids representative of various chemical classes, with platelet responses dependent on thromboxane A2 (TxA2) generation and on receptor antagonism, and to analyze the structural requirements for such effects. Methods: The effect of several types of flavonoids on platelet aggregation, serotonin release, and TxA2 generation was investigated. Competitive radioligand binding assays were used to screen for affinity of these compounds to TxA2 receptors. Results: Flavones (apigenin and luteolin) and isoflavones (genistein) abrogated arachidonic acid and collagen‐induced platelet responses, such as aggregation and secretion, with a less substantial effect on TxA2 synthesis. These compounds were identified as specific ligands of the TxA2 receptor in the µmol L−1 range, this effect accounting for antiplatelet effects related to stimulation with those agonists. Tight binding of flavonoids to the human TxA2 receptor relies on structural features such as the presence of the double bond in C2–C3, and a keto group in C4. Conclusions: The inhibition by specific flavonoids of in vitro platelet responses induced by collagen or arachidonic acid seems to be related, to a great extent, to their ability to compete for binding to the TxA2 receptor. Therefore, antagonism of this TxA2 receptor may represent an additional mechanism for the inhibitory effect of these compounds in platelet function.

The HAS-BLED Score Has Better Prediction Accuracy for Major Bleeding Than CHADS2 or CHA2DS2-VASc Scores in Anticoagulated Patients With Atrial Fibrillation

OBJECTIVES The aim of this study was to test the hypothesis that a specific bleeding risk score, HAS-BLED (hypertension, abnormal renal/liver function, stroke, bleeding history or predisposition, labile international normalized ratio, elderly, drugs/alcohol concomitantly), was better at predicting major bleeding compared with CHADS2 (congestive heart failure, hypertension, 75 years of age or older, diabetes mellitus, and previous stroke or transient ischemic attack) and CHA2DS2-VASc (congestive heart failure, hypertension, 75 years of age and older, diabetes mellitus, previous stroke or transient ischemic attack, vascular disease, 65 to 74 years of age, female) in anticoagulated atrial fibrillation (AF) patients. BACKGROUND The CHADS2 and CHA2DS2-VASc scores are well-validated stroke risk prediction scores for AF, but are also associated with increased bleeding and mortality. METHODS We recruited 1,370 consecutive AF patients (49% male; median age, 76 years) receiving oral anticoagulation therapy from our outpatient anticoagulation clinic, all of whom were receiving acenocoumarol and had an international normalized ratio between 2.0 and 3.0 during the preceding 6 months. During follow-up, major bleeding events were identified by the 2005 International Society on Thrombosis and Haemostasis criteria. Model performance was evaluated by calculating the C-statistic, and the improvement in predictive accuracy was evaluated by calculating the net reclassification improvement and integrated discrimination improvement. RESULTS After a median follow-up of 996 (range, 802 to 1,254) days, 114 patients (3.0%/year) presented with a major bleeding event; 31 of these events were intracranial hemorrhages (0.8%/year). Based on the C-statistic, HAS-BLED had a model performance superior to that of both CHADS2 and CHA2DS2-VASc (both p < 0.001). Both net reclassification improvement and integrated discrimination improvement analyses also show that HAS-BLED was more accurately associated with major bleeding compared with CHADS2 and CHA2DS2-VASc scores. CONCLUSIONS In anticoagulated AF patients, a validated specific bleeding risk score, HAS-BLED, should be used for assessing major bleeding. The practice of using CHADS2 and CHA2DS2-VASc as a measure of high bleeding risk should be discouraged, given its inferior predictive performance compared with the HAS-BLED score.

Assessment and prognostic value of the European LeukemiaNet criteria for clinicohematologic response, resistance, and intolerance to hydroxyurea in polycythemia vera

Criteria of response and definition of resistance and intolerance to hydroxyurea (HU) in polycythemia vera (PV) were proposed by the European LeukemiaNet (ELN). Such criteria were evaluated in 261 PV patients (median follow-up, 7.2 years) treated with HU for a median of 4.4 years. Complete response, partial response, and no response were observed in 24%, 66%, and 10% of patients, respectively. Achieving ELN response (complete or partial) or hematocrit response did not result in better survival or less thrombosis and bleeding. On the contrary, having no response in leukocyte count was associated with higher risk of death (HR, 2.7; 95% confidence interval [CI], 1.3%-5.4%; P = .007), whereas lack of response in platelet count involved a higher risk of thrombosis and bleeding. Resistance and intolerance to HU was registered in 11% and 13% of patients, respectively. Resistance to HU was associated with higher risk of death (HR, 5.6; 95% CI, 2.7%-11.9%; P < .001) and transformation (HR, 6.8; 95% CI, 3.0%-15.4%; P < .001). In summary, fulfilling the ELN definition for response to HU was not associated with a benefit in the clinical outcome in PV, whereas response in platelet and white blood cell counts were predictive of less thrombohemorrhagic complications and better prognosis, respectively. Resistance to HU was an adverse prognostic factor.

Plasma von Willebrand Factor Levels Are an Independent Risk Factor for Adverse Events Including Mortality and Major Bleeding in Anticoagulated Atrial Fibrillation Patients

OBJECTIVES The purpose of this study was to evaluate the prognostic value of plasma von Willebrand factor (vWF) levels and fibrin d-dimer in a large cohort of anticoagulated permanent atrial fibrillation (AF) patients. BACKGROUND In nonanticoagulated AF patients, plasma vWF levels have been related to stroke and vascular events. There are limited data on the prognostic role of biomarkers in anticoagulated AF patients in relation to adverse events (including thromboembolism), mortality, and major bleeding. METHODS We studied 829 patients (50% male; median age 76 years) with permanent AF who were stabilized (for at least 6 months) on oral anticoagulation therapy (international normalized ratio: 2.0 to 3.0). Plasma d-dimer and vWF levels were quantified by enzyme-linked immunosorbent assay. Patients were followed for 2 years, and adverse events (thrombotic and vascular events, mortality, and major bleeding) were recorded. RESULTS Patients were followed for a median of 828 days (range 18 to 1,085 days). On multivariate analysis, age 75 years and older, previous stroke, heart failure, and high plasma vWF levels (≥ 221 IU/dl) were associated with future adverse cardiovascular events (all p values <0.05). High plasma vWF levels, elderly patients, diabetes, hypercholesterolemia, and current smoking were associated with mortality (all p values <0.05). High plasma vWF levels were also an independent predictor of major bleeding (hazard ratio: 4.47, 95% confidence interval: 1.86 to 10.75; p < 0.001). High plasma vWF levels were able to refine clinical risk stratification schema for stroke (CHADS₂ [Congestive heart failure, Hypertension, Age ≥ 75, Diabetes mellitus, and prior Stroke or transient ischemic attack (doubled)], CHA₂DS₂-VASc [Congestive heart failure, Hypertension, Age ≥ 75 years, Diabetes mellitus, Stroke, Vascular disease, Age 65 to 74 years, Sex category]) and bleeding (HAS-BLED [Hypertension, Abnormal renal/liver function, Stroke, Bleeding history or predisposition, Labile International Normalized Ratio, Elderly, Drugs/alcohol concomitantly]). d-dimer did not show any significant impact on adverse events. CONCLUSIONS High plasma vWF levels (≥221 IU/dl) are an independent risk factor for adverse events in anticoagulated permanent AF patients. This biomarker may potentially be used to refine stroke and bleeding clinical risk stratification in AF.

The venous thrombosis risk factor 20210 A allele of the prothrombin gene is not a major risk factor for arterial thrombotic disease

A nucleotide change (G to A transition) at position 20210 has recently been demonstrated to be a risk factor for venous thrombosis. The relevance of this polymorphism to thrombotic disease was investigated by genotypic identification in three prospective case–control studies: 101 case patients with acute coronary heart disease (CHD), 104 patients with acute cerebrovascular disease (CVD), 82 patients with a confirmed diagnosis of deep venous thrombosis (DVT), and one control age‐ and sex‐matched for each patient. The prevalence of the genetic variation was significantly associated with the occurrence of DVT, but did not differ in patients with CHD or CVD from that in controls, suggesting that this allele should not be considered a major risk factor for arterial thrombotic disease.

Relation of the HAS-BLED bleeding risk score to major bleeding, cardiovascular events, and mortality in anticoagulated patients with atrial fibrillation.

Background— Stroke risk in atrial fibrillation (AF) using oral vitamin K antagonists is closely related to bleeding risk. The HAS-BLED (hypertension, abnormal renal/liver function, stroke, bleeding history or predisposition, labile INR [international normalized ratio], elderly, drugs/alcohol concomitantly) bleeding score has demonstrated usefulness in assessing major bleeding risk in patients with AF. However, risk factors for warfarin-associated bleeding also predict stroke risk in patients with AF. We tested the usefulness of the HAS-BLED score for predicting both major bleeding and cardiovascular events in a cohort of anticoagulated patients with AF. Methods and Results— We recruited 965 consecutive anticoagulated outpatients with permanent or paroxysmal AF who were stabilized for at least 6 months on oral anticoagulation (international normalized ratio, 2.0–3.0). Medical history and HAS-BLED score were assessed. Cox regression models were used to determine the association between clinical risk factors and bleeding episodes, adverse cardiovascular events, and mortality. The median HAS-BLED score was 2 (range, 0–6; 29% with a score ≥3 [ie, high risk]). Median follow-up was 861 days (range, 718–1016 days). Independent predictors for major bleeding were age ≥75 years (hazard ratio [HR], 1.74; 95% CI, 1.05–2.87; P=0.030), male sex (HR, 1.70; 95% CI, 1.03–2.80; P=0.036), renal impairment (HR, 2.12; 95% CI, 1.20–3.73; P=0.010), previous bleeding episode (HR, 6.00; 95% CI, 3.73–9.67; P<0.001), current alcohol consumption (HR, 2.28; 95% CI, 1.03–5.06; P=0.043), and concomitant malignant disease (HR, 2.17; 95% CI, 1.13–4.18; P=0.020). Independent predictors for adverse cardiovascular events were age >75 years (HR, 2.20; 95% CI, 1.40–3.46; P=0.001), heart failure (HR, 1.78; 95% CI, 1.20–2.86; P=0.001), and previous stroke (HR, 1.85; 95% CI, 1.20–2.86; P<0.001). The HAS-BLED score was highly predictive for major bleeding events (HR, 2.04; 95% CI, 1.68–2.49; P<0.001) and adverse cardiovascular events (HR, 1.51; 95% CI, 1.27–1.81; P<0.001). The incidence of both bleeding and adverse cardiovascular events was higher as HAS-BLED score increased, and crude bleeding rates only exceeded thrombotic events at a HAS-BLED score >3. The HAS-BLED score also predicted all-cause mortality (HR, 1.68; 95% CI, 1.40–2.01; P<0.001). Conclusions— The HAS-BLED score not only is useful in the assessment of bleeding risk, but also shows some predictive value for cardiovascular events and mortality in anticoagulated patients with AF, consistent with the relationship between thrombosis and bleeding. Nonetheless, the HAS-BLED score has been designed for predicting bleeding risk rather than thrombotic events per se, and specific risk scores for cardiovascular events and mortality should be applied for these events.