Platelet function measured by VerifyNow™ identifies generalized high platelet reactivity in aspirin treated patients

Selected aspirin treated patients may exhibit high platelet reactivity to agonists other than arachidonic acid. This study aimed to determine whether the VerifyNow? identifies generalized high platelet reactivity supported by correlations with other established methods that stimulate platelets with various agonists. Stable outpatients with coronary artery disease (n?=?110) were treated with aspirin in a two 3?×?3 Latin square design (81, 162 and 325?mg/day for 4 weeks each). VerifyNow? (arachidonic acid (AA) cartridge); light transmittance aggregometry; thrombelastography; PFA-100®; flow cytometry; PlateletWorks®; and urinary 11- dehydro thromboxane levels were measured. Multianalyte profiling measured fibrinogen and von Willebrand factor (vWF). Patients with ≥550 ARU by VerifyNow? had increased 5?mM AA-, 5?µM ADP-, and 2?µg/mL collagen-induced platelet aggregation compared to patients with <550 ARU (p?≤?0.001). The highest ADP- and collagen-induced aggregation was present in the upper quartile by VerifyNow? (p?<?0.05). Fibrinogen or vWF levels did not differ between VerifyNow? quartiles. Patients with high platelet reactivity to arachidonic acid identified by VerifyNow? also exhibit increased reactivity to other important platelet agonists that is independent of fibrinogen and vWF levels. These data suggest that VerifyNow? may identify a generalized high platelet reactivity phenotype.     

Platelet reactivity among Asian Indians and Caucasians

Asian Indians are reported to have higher mortality and morbidity from coronary artery disease (CAD) than other ethnic groups. This variation in events cannot be explained only by differences in conventional risk factors. Platelet activation is an important factor in the pathogenesis of CAD, however, there are limited data concerning platelet reactivity in Asian Indians. Therefore, we aimed to examine platelet reactivity in healthy Asian Indians vs. Caucasians. Thirty-five healthy, nonsmoking Asian Indians (mean age 30.1?±?3.6 years, 31.4% women) were matched for age and sex with 35 healthy, nonsmoking Caucasians (mean age 30.8?±?5 years, 31.4% women). Platelet reactivity was evaluated by measuring platelet aggregation, platelet leukocyte aggregates (PLA) formation in response to a 6-mer thrombin receptor agonist peptide (TRAP) at a final concentration of 40?µM and flow cytometry determined P-selectin expression induced by ADP, TRAP and arachidonic acid (AA). In addition, P-Selectin glycoprotein ligand-1 (PSGL-1) density on leukocytes was measured. There were no differences in platelet aggregation, basal PLA or PSGL-1 density on leukocytes between the two groups. AA-stimulated P-selectin expression was significantly higher in Asian Indians than in Caucasians (6.1?±?0.51 vs. 4.2?±?0.41 MFI, P?<?0.02). After stimulation with TRAP, platelets from Asian Indians had increased PLA formation compared with Caucasians (41.6?±?2.9% vs. 31.4?±?2.7%, P?<?0.02). AA induced P-selectin expression and TRAP stimulated PLA formation is increased in Asian Indians compared with Caucasians. These differences indicate an increase in measures of platelet reactivity among Asian Indians and may help elucidate the reported disparity in cardiovascular disease rates between the two ethnic groups.     

A platelet P-selectin test predicts adverse cardiovascular events in patients with acute coronary syndromes treated with aspirin and clopidogrel

Abstract

There is wide variation in response to antiplatelet therapy and high on-treatment platelet reactivity is associated with adverse cardiovascular events. The objective here was to determine whether the results of a novel strategy for assessing platelet reactivity (based on P-selectin measurement) are associated with clinical outcomes in patients with acute coronary syndromes (ACS). This was a prospective cohort study of 100 ACS patients taking aspirin and clopidogrel. P-selectin tests designed to assess response to P2Y₁₂ antagonists or aspirin were performed alongside light transmission aggregometry. For the P2Y₁₂ P-selectin test, an optimal cutoff for high platelet reactivity was determined by receiver operating characteristic (ROC) curve analysis. Patients were divided into two cohorts based on this value: patients with (n?=?42) or without (n?=?58) high platelet reactivity. The primary endpoint was defined as the composite of cardiovascular death, myocardial infarction and stent thrombosis. After 12 months, the primary endpoint occurred in 12 patients. ROC curve analysis determined that the P2Y₁₂ P-selectin test results were predictive of the primary endpoint (area under curve?=?0.69, p?=?0.046). The primary endpoint occurred more frequently in patients with high on-treatment platelet reactivity compared to those without (21.4% vs. 5.2%; hazard ratio (HR) 4.14; p?=?0.026). The P2Y₁₂ P-selectin test results correlated with light transmission aggregometry (Spearman p?<?0.0001). Using the Aspirin P-selectin test, only two patients demonstrated high on-treatment platelet reactivity. This study suggests that a P2Y₁₂ P-selectin test is capable of detecting high on-treatment platelet reactivity, which is associated with subsequent cardiovascular events.     

Neonatal platelets from cord blood and peripheral blood

The haemostatic system in neonates is different from that of adults, possibly contributing to an increased incidence of bleeding disorders, such as intracranial hemorrhage. In this study, we analyzed platelets from cord blood and peripheral blood, collected at three time points after delivery from 20 term and 37 preterm neonates as well as blood from 20 healthy adults. Platelet membrane glycoproteins (GP) were quantified and P-selectin expression and PAC-1 binding ability before and after stimulation with TRAP were analyzed by whole blood flow cytometry. We found no significant differences in neonatal platelets from cord blood and peripheral blood within the first 24?h of life. Platelets from infants less than 30 weeks of gestation expressed lower levels of GP (33271?±?9381 vs. 44085?±?17287 for GPIIIa, P?<?0.05) and were less reactive than platelets from term newborns (4.3?±?3.3 vs. 20.1?±?11.8% PAC-1 positive platelets after stimulation with TRAP, P?<?0.05). A significantly lower level of GPIIb/IIIa expression on platelets from peripheral blood was seen in term newborns as well as preterm infants, compared to adults. There was only a partial enhancement in the degranulation ability (α-granules) (13.4?±?12.3 vs. 50.3?±?16.1% P-selectin positive platelets, P?<?0.05) and no significant increase for PAC-1 binding (13.6?±?10.9 vs. 15.3?±?5.9% PAC-1 positive platelets, P?=?0.8) during the first 12 days of life. In conclusion, we could demonstrate that neonatal platelet reactivity increases with gestational age.     

Inverse relationships between coronary blood flow obstruction and platelet reactivity in stable angina pectoris

This study investigates relationships between platelet reactivity and coronary blood flow obstruction in stable angina pectoris. Consented were 36 patients with single-vessel disease. The subjects were divided into two groups. One group (n?=?14) had less severe (<?=?80%) and the second group (n?=?22) had severe coronary flow impairment (90%). Before elective coronary angiography platelet in vitro reactivity in venous whole blood was determined using a flow cytometry technique. A thrombin-receptor activating peptide (TRAP-6) (0.77 and 0.06?g/l) and ADP (8.5 and 1.7?µmol/l) were used to activate platelets. The number of fibrinogen positive cells (%) i.e., activated platelets after stimulation was employed as experimental parameter. Less severe flow obstruction was associated with more reactive platelets. When stimulating with 0.77?g/l TRAP-6 the number of activated platelets was 64?±?15 (SD)%. The corresponding value for the group with severe flow obstruction was 40?±?17(SD)%. The difference is significant (P?<?0.001). 0.06?g/l TRAP-6 yielded similar results (P?<?0.01). Also when using 8.5?µmol/l ADP to challenge platelets less severe flow obstruction was associated with enhanced reactivity (P?<?0.01). 1.7?µmol/l ADP generated comparable results (P?<?0.05). Thus, in stable angina pectoris coronary flow obstruction is inversely related to platelet reactivity.     

Altered plasma fibrin clot properties in essential thrombocythemia

Patients with increased thromboembolic risk tend to form denser fibrin clots which are relatively resistant to lysis. We sought to investigate whether essential thrombocythemia (ET) is associated with altered fibrin clot properties in plasma. Ex vivo plasma fibrin clot permeability coefficient (K_s), turbidimetry and clot lysis time (CLT) were measured in 43 consecutive patients with ET (platelet count from 245 to 991?×?10³/µL) and 50 control subjects matched for age, sex and comorbidities. Fibrinolysis proteins and inhibitors together with platelet activation markers were determined. Reduced K_s (?38%, p?p?p?=?0.01) and higher maximum absorbency of the turbidimetric curve (+6%, p?p?p?p?p?K_s inversely correlated with fibrinogen, PF4 and C-reactive protein. CLT positively correlated only with PAI-1. Patients with ET display prothrombotic plasma fibrin clot phenotype including impaired fibrinolysis, which represents a new prothrombotic mechanism in this disease.     

Platelet adhesion changes during storage studied with a novel method using flow cytometry and protein-coated beads

The aim of the present study was to set up and evaluate a novel method for studies of platelet adhesion and activation in blood and platelet suspensions such as platelet concentrate (PC) samples using protein-coated polystyrene beads and flow cytometry. To demonstrate its usefulness, we studied PCs during storage. PCs were prepared by aphaeresis technique (n?=?7). Metabolic variables and platelet function was measured on day 1, 5, 7 and 12 of storage. Spontaneous and TRAP-6-induced adhesion to fibrinogen- and collagen-coated beads was analyzed by flow cytometry. P-selectin and phosphatidyl serine (PS) expression was assessed on platelets bound to beads as well as on non-adherent platelets. Platelet adhesion to fibrinogen beads had increased by day 12 and adhesion to collagen beads at day 7 of storage (p?<?0.05). TRAP-6 stimulation significantly increased the platelet adhesion to fibrinogen beads (p?<?0.05) as well as the P-selectin and PS exposure on platelets bound to beads (p?<?0.01) during the first 7 days of storage, but by day 12, significant changes were no longer induced by TRAP-6 stimulation. We demonstrate that our adhesion assay using protein-coated polystyrene beads can be used to assess the adhesion properties of platelets during storage without the addition of red blood cells. Therefore it may offer a useful tool for future studies of platelet adhesive capacity in transfusion medicine and other settings.     

Comparison of aspirin and indobufen in healthy volunteers

The aim of this study is to quantify the extent and recovery of platelet inhibition after administration of indobufen and aspirin in healthy volunteers. Indobufen inhibits platelet aggregation by reversibly inhibiting the platelet cyclooxygenase enzyme, thereby suppressing thromboxane synthesis. Twenty healthy volunteers completed the study and received aspirin (200?mg/day for 2 weeks) followed by a 4-week washout period and then indobufen (200?mg twice a day for 2 weeks). The percent (%) inhibition of platelet aggregation (IPA) was assessed using arachidonic acid (0.5?mg/ml) and adenosine diphosphate (5?µM) at 4, 12, 24 and 48 hours after last dose of each drug. IPA assessed using arachidonic acid as the agonist was similar at 4 hours after the last dose of indobufen (81.07?±?9.36%) and aspirin (96.99?±?0.29%, p?=?0.10), but significantly lower at 12 hours (74.04?±?9.55% vs. 97.94?±?0.28%, p?=?0.02), 24 hours (33.39?±?11.13% vs. 97.48?±?0.32%, p?p?p?=?0.002). Indobufen (200?mg twice a day) caused equivalent initial inhibition of platelet aggregation to aspirin (200?mg daily), and the anti-aggregation effect diminished faster than after aspirin.     

Increased platelet reactivity in unstable angina patients is not related to C-reactive protein levels

Platelets are a major component of thrombi, and coronary thrombosis plays a key role in the pathogenesis of unstable angina (UA). Whether platelet aggregability is increased in UA patients however, is not known. Furthermore, no study has investigated the relationship between platelet reactivity and inflammation in UA patients In this study, venous blood samples were collected at admission in coronary care unit in 37 patients with unstable angina (Braunwald class IIIB) and in 37 sex- and age-matched patients with chronic stable angina (CSA). Patients taking thienopyridine or anticoagulant drugs were excluded from the study, as also were excluded patients with a history of acute myocardial infarction in the previous 12 months. Platelet aggregability was measured on flowing blood as time to occlude a ring coated with collagen-adenosine diphosphate (ADP), using the platelet function analyzer (PFA-100) system. By this method, the time to occlusion (closure time) is taken as a measure of platelet adhesion/aggregability, with shorter times indicating greater platelet reactivity.There were 23 men and 14 women in both groups, and age was 67.7?±?8 and 67.5?±?8 years in UA and SA, respectively (P?=?0.93). Closure time was significantly reduced in UA patients (78.8?±?14?s), compared to SA patients (93.3?±?19?s,?P?<?0.001). Among UA patients, serum C-reactive protein (CRP) levels had a median value of 5.1?mg/l (bottom and top quartile levels, 1.50–7.95). There was no significant correlation between closure time and CRP levels (r?=?0.22,?P?=?0.29). Our data show that, in patients with unstable angina there is an increase of platelet reactivity in response to ADP/collagen stimulation, which is not related to inflammation.     

In vivo platelet activation in atherothrombotic stroke is not determined by polymorphisms of human platelet glycoprotein IIIa or Ib

Platelet membrane glycoprotein polymorphisms are candidate risk factors for thrombosis, but epidemiological data are conflicting. Thus, demonstration of a genotype-dependent alteration in function is desirable to resolve these inconsistencies. We investigated in vivo platelet activation in acute thrombosis and related this to platelet genotype. Frequencies of the 1b and 2b alleles of the HPA 1a/1b and HPA 2a/2b platelet glycoprotein polymorphisms were determined in 150 (52 men/98 women, mean age 58.3 years) patients with atherothrombotic stroke, and the influence of genotype on markers of platelet activation was assessed. Platelet P-selectin (CD62P) expression and fibrinogen binding was measured using whole blood flow cytometry within 24 h of stroke and 3 months later in 77 patients who provided a repeat blood sample. Results were compared with matched controls. Neither the 1b allele [allele frequency 0.11 vs. 0.13, odds ratio (OR) confidence interval (CI) 0.8 (0.5-1.3)] nor the 2b allele [0.09 vs. 0.07, OR (CI) 1.4 (0.8-2.4)] was significantly over-represented in patients. Increased numbers of activated platelets were found following stroke (acute mean P-selectin expression 0.64% vs. control 0.35%, P < 0.001; acute mean fibrinogen binding 1.6% vs. control 0.9%, P < 0.001). Activation persisted in the convalescent phase (P < 0.001 and P = 0.005 vs. controls for P-selectin and fibrinogen respectively). Expression of P-selectin and fibrinogen was not influenced by either the HPA 1a/1b genotype (P > 0.95 for each marker, Scheffe's test) or the 2a/2b genotype (P > 0.95 for each). Although persisting platelet activation is seen in atherothrombotic stroke, it is independent of HPA 1a/1b and 2a/2b genotypes. These data suggest an underlying prothrombotic state, but do not support the polymorphisms studied as risk factors for thrombotic stroke in this population.     

Increased platelet activation in subjects chronically exposed to cadmium: A pilot study

Cadmium exposure has been reported to be associated with the risk of vascular disorders. Here, we investigated platelet activity in subjects with chronic cadmium exposure. Eighteen and 15 women participated in this study as chronically cadmium-exposed and control non-exposed subjects, respectively. Plasma P-selectin and CD40 ligand (CD40L), soluble markers of platelet activation, were measured. Platelet aggregation in whole blood, P-selectin and activated glycoprotein (aGP) IIb/IIIa expression on platelets and platelet–leukocyte aggregates were determined. The levels of plasma P-selectin and CD40L increased in subjects with chronic cadmium exposure compared with control subjects. Platelet aggregation induced by adenosine diphosphate (ADP) was higher in cadmium-exposed subjects than control subjects. Cadmium-exposed subjects had higher baseline and ADP-induced aGPIIb/IIIa expression on platelets than control subjects. Platelet–neutrophil aggregates also increased in cadmium-exposed subjects. Blood cadmium correlated with ADP-induced aggregation, aGPIIb/IIIa expression and platelet–neutrophil aggregates, while urinary cadmium correlated with soluble P-selectin. However, cadmium only at high concentration (15?µM) could potentiate ADP-induced platelet activation in vitro. In conclusion, our pilot data show that cadmium-exposed subjects have increased baseline platelet activation and reactivity.     

Impaired responsiveness of platelets to epinephrine due to α2A adrenoreceptor deficiency in Male Chinese

Epinephrine is known as a weak, but important, agonist for platelet activation. It has been reported that the responsiveness of platelets to epinephrine was markedly impaired in 6% of Caucasians and in 16% of Japanese. The purpose of this study was to screen and characterize this abnormality in healthy Taiwanese Chinese volunteers. We used aggregometry, flow cytometry and platelet function analyzer (PFA)-100 system to assess in 50 healthy male volunteers the responsiveness of platelets to epinephrine stimulation. Using α2A adrenoceptor antagonist BRL44408 maleate competition and a [³H]yohimbin binding assay, we evaluated α2A adrenoceptors on platelets. The aggregation of platelets after stimulation with 10?μM of epinephrine indicated two distinct groups of study participants: 24 (48.0%) good- and 26 (52.0%) impaired-responders to epinephrine. Flow cytometric analysis of platelets after stimulated with 1?μM epinephrine showed that glycoprotein (GP) IIb/IIIa and P-selectin expression of epinephrine good- and impaired-responders were 27.1?±?11.0% vs. 9.9?±?5.4% (p?=?0.003) and 12.2?±?6.2% vs. 3.6?±?3.5% (p?3H]yohimbine binding studies showed fewer α2A adrenoreceptors on the platelets of epinephrine-impaired-responders than on those of good-responders. The prevalence of impaired responsiveness to epinephrine was high and probably due to α2A adrenoreceptor deficiency in male Taiwanese Chinese.     

Inverse relationship between erythrocyte size and platelet reactivity in elderly

Previous work indicates that erythrocytes (RBCs) accumulate β-amyloid X-40 (Aβ₄₀) in individuals with Alzheimer disease (AD) and to a lesser extent in healthy elderly. The toxin damages RBCs and increases their mean corpuscular volume (MCV). Furthermore, AD platelets demonstrate lower reactivity. This study investigated interactions between RBCs and platelets. Older individuals with moderate hypertension (n = 57) were classified into two groups, depending on MCV in whole blood: The MCV^high group comprised individuals with higher MCV (n = 27; 97 ± 3(SD) fl) and MCV^low group had relatively lower MCV (n = 30; 90 ± 3(SD) fl). Flow cytometry was used to determine platelet reactivity, i.e., the surface binding of fibrinogen after provocation. Adenosine diphosphate (ADP) and a thrombin receptor-activating protein (TRAP-6) were used as agonists. Subsequently, blood cells were divided according to density into 17 subfractions. Intra-RBC Aβ₄₀ content was analyzed and in all platelet populations surface-bound fibrinogen was determined to estimate platelet in vivo activity. We found Aβ₄₀ inside RBCs of approximately 50% of participants, but the toxin did not affect MCV and platelet reactivity. In contrast, MCV associated inversely with platelet reactivity as judged from surface-attached fibrinogen after ADP (1.7 μmol/L) (p < 0.05) and TRAP-6 provocation (57 μmol/L (p = 0.01) and 74 μmol/L (p < 0.05)). In several density fractions (nos. 3, 4, 8, 11–13 (p < 0.05) and nos. 5–7 (p < 0.01)) MCV linked inversely with platelet-attached fibrinogen. In our community-dwelling sample, enhanced MCV associated with decreased platelet reactivity and lower in vivo platelet activity. It resembles RBCs and platelet behavior in AD-type dementia.     

Upregulation of CD40/CD40L system in rheumatic mitral stenosis with or without atrial fibrillation

Thrombelastography (TEG) analyses the status of blood coagulation including abnormalities associated with low platelet count. The aim of this study was to investigate the changes in TEG parameters in idiopathic thrombocytopenic purpura (ITP) patients. Thirty nine patients with ITP (platelet count?<?100?×?103 µl^?1) were included in the study. Age-matched 17 patients with thrombocytopenia due to chemotherapy were selected as a control group. Platelet count was positively correlated with maximum clot formation (MCF) in INTEM (r?=?0.716, p?<?0.001) and MCF in EXTEM (r?=?0.679, p?<?0.001); negatively correlated with clot formation time (CFT) in INTEM (r?=??0.755, p?<?0.001) and CFT in EXTEM (r?=??0.585, p?<?0.001) in ITP patients. Platelet count was positively correlated with MCF in INTEM (r?=?0.776, p?<?0.001) and MCF in EXTEM (r?=?0.878, p?<?0.001); negatively correlated with CFT in INTEM (r?=??0.627, p?<?0.001) in control group. Receiver operating characteristic curves to describe the critical platelet count and fibrinogen level that affect MCF revealed 31?×?103?µl^?1 and 375 mg?dl^?1 as cut-off values, respectively. In conclusion, ROTEM determines the contribution of fibrinogen and platelets to clot strength in patients with ITP. MCF appears to be the most important TEG parameter in predicting bleeding in ITP patients that makes TEG superior to other hemostatic tests.     

Reduced antiplatelet effect of aspirin during 24 hours in patients with coronary artery disease and type 2 diabetes

Reduced antiplatelet effect of aspirin has been reported in patients with type 2 diabetes, and recent studies suggest that once-daily aspirin provides insufficient platelet inhibition. We investigated if the effect of aspirin declined during the 24-hour dosing interval in patients with coronary artery disease and type 2 diabetes, and whether this correlated with increased platelet turnover. Furthermore, the intra-individual variation in platelet aggregation was determined during a 28-day period. We included 47 patients with coronary artery disease and type 2 diabetes treated with aspirin 75?mg daily. Blood samples were obtained 1 and 24 hours after aspirin intake, and this was repeated three times with a 2-week interval between each visit. Platelet aggregation was evaluated by impedance aggregometry (Multiplate® Analyzer) using arachidonic acid (1.0?mM) and collagen (3.2?µg/ml) as agonists. Markers of platelet turnover were measured by flow cytometry. Compliance was confirmed by serum thromboxane B₂. Platelet aggregation levels measured 1 and 24 hours after aspirin intake were compared using the mean of 1- and 24-hour measurements at the three study visits. The difference in platelet aggregation was 70?±?97?AU?×?min (p?<?0.0001) when using arachidonic acid as agonist and 33?±?76?AU?×?min (p?=?0.01) when using collagen. Markers of platelet turnover correlated positively, though not significantly, with residual platelet aggregation 24 hours after aspirin intake (p values 0.06 and 0.07). Median intra-individual variation of platelet aggregation was 9–16%. Patients with coronary artery disease and type 2 diabetes had increased platelet aggregation at the end of the 24-hour aspirin dosing interval. Platelet turnover did not correlate significantly with residual platelet aggregation, although a trend was observed. The intra-individual variation of platelet aggregation after aspirin intake was low.     

Low-density platelet populations demonstrate low in vivo activity in sporadic Alzheimer disease

Platelets contain a substantial quantity of amyloid-precursor protein (APP) and β-amyloid. However, despite the large importance of APP and β-amyloid to dementia, little is known about platelets in sporadic Alzheimer dementia (AD). Furthermore, platelet heterogeneity influences human pathology and has been described to affect the progression of AD. This study investigated AD platelets with respect to density diversity and in vivo activity associated with density sub-fractions. We included 39 AD patients and used, as controls, 22 elderly individuals without apparent memory disorder. A continuous Percoll? gradient covering the density span 1.04–1.09?kg/l provided the basis to divide platelets of whole blood into density fractions (n?=?16). All platelet populations were evaluated accordingly. Platelet counts were determined electronically. A flow-cytometer was put to use to measure surface-bound fibrinogen as a measure of platelet in vivo activity. Samples obtained from patients diagnosed with sporadic AD contained platelets (fractions numbers 4–16) that circulated with significantly less surface-bound fibrinogen, i.e., their platelet activation in vivo was reduced, compared with controls. In particular, highly significant differences (p?<?0.001) were obtained for the six less dense platelet populations (fractions numbers 11–16) when comparing sporadic AD with controls. In contrast, the densest AD platelets in fractions numbers 1–3 did not differ significantly from control cells with respect to in vivo platelet-bound fibrinogen. It is concluded that sporadic AD is characterized by lower density platelet populations that, while circulating, exhibited reduced activation. The clinical significance of this finding is unclear but these results suggest the importance of platelet heterogeneity in dementia as a topic for further investigation.     

Association of plasma miR-223 and platelet reactivity in patients with coronary artery disease on dual antiplatelet therapy: A preliminary report

Decreased plasma levels of microRNA-223 (miR-223), predominantly of platelet origin, were proposed as a surrogate marker of efficacy of antiplatelet therapy. However, higher on-treatment platelet reactivity was associated with lower plasma miR-223 in patients with coronary artery disease (CAD) on dual antiplatelet therapy (DAPT) including clopidogrel and aspirin. Our aim was to compare plasma miR-223 and platelet reactivity in CAD patients on DAPT with newer P2Y₁₂ antagonists vs. clopidogrel. We studied 21 men with CAD admitted to our centre owing to a non-ST-elevation acute coronary syndrome, and with an uncomplicated hospital course. From the day of admission, the patients were receiving either clopidogrel (n?=?11) or prasugrel/ticagrelor (n?=?10) in addition to aspirin. Before discharge, miR-223 expression in plasma was estimated by quantitative polymerase chain reaction using the comparative Ct method relative to miR-16 as an endogenous control. Multiple electrode aggregometry was used to assess platelet aggregation in response to adenosine diphosphate (ADP). ADP-induced platelet reactivity was decreased in the patients treated with prasugrel or ticagrelor compared with those on clopidogrel (mean?±?SD: 139?±?71 vs. 313?±?162 arbitrary units [AU]*min, p?=?0.006), due to a more potent antiplatelet activity of the novel P2Y₁₂ antagonists. Consequently, six out of seven patients in the lower tertile of the ADP-induced platelet aggregation were treated with the newer P2Y₁₂ blockers, whereas six out of seven patients in the upper tertile were on clopidogrel. Plasma miR-223 was elevated with decreasing platelet reactivity (Spearman’s rho?=?–0.52; p?=?0.015 for trend), being significantly higher in the lower tertile of the ADP-induced platelet aggregation (median [range]: 1.06 [0.25–2.31]) vs. the upper tertile (0.20 [0.13–2.30]) (p?=?0.04). In conclusion, our preliminary results argue against the notion of low plasma miR-223 as a marker of platelet responsiveness to DAPT. On the contrary, more potent platelet inhibition associated mainly with newer P2Y₁₂ antagonists appears to coincide with higher miR-223 relative to the subjects with attenuated responsiveness to DAPT.     

The effect of P2Y12 inhibition on platelet activation assessed with aggregation- and flow cytometry-based assays

Patients on P2Y₁₂ inhibitors may still develop thrombosis or bleeding complications. Tailored antiplatelet therapy, based on platelet reactivity testing, might reduce these complications. Several tests have been used, but failed to show a benefit of tailored antiplatelet therapy. This could be due to the narrowness of current platelet reactivity tests, which are limited to analysis of platelet aggregation after stimulation of the adenosine diphosphate (ADP)-pathway. However, the response to ADP does not necessarily reflect the effect of P2Y₁₂ inhibition on platelet function in vivo. Therefore, we investigated whether measuring platelet reactivity toward other physiologically relevant agonists could provide more insight in the efficacy of P2Y₁₂ inhibitors.

The effect of in vitro and in vivo P2Y₁₂ inhibition on αIIbβ3-activation, P-selectin and CD63-expression, aggregate formation, release of alpha, and dense granules content was assessed after stimulation of different platelet activation pathways. Platelet reactivity measured with flow cytometry in 72 patients on P2Y₁₂ inhibitors was compared to VerifyNow results.

P2Y₁₂ inhibitors caused strongly attenuated platelet fibrinogen binding after stimulation with peptide agonists for protease activated receptor (PAR)-1 and -4, or glycoprotein VI ligand crosslinked collagen-related peptide (CRP-xl), while aggregation was normal at high agonist concentration. P2Y₁₂ inhibitors decreased PAR-agonist and CRP-induced dense granule secretion, but not alpha granule secretion. A proportion of P2Y₁₂-inhibitor responsive patients according to VerifyNow, displayed normal fibrinogen binding assessed with flow cytometry after stimulation with PAR-agonists or CRP despite full inhibition of the response to ADP, indicating suboptimal platelet inhibition.

Concluding, measurement of platelet fibrinogen binding with flow cytometry after stimulation of thrombin- or collagen receptors in addition to ADP response identifies different patients as nonresponders to P2Y₁₂ inhibitors, compared to only ADP-induced aggregation-based assays. Future studies should investigate the value of both assays for monitoring on-treatment platelet reactivity.     

Evaluation of platelet function in thrombocytopenia

Whole blood aggregometry is a functional assay for determination of platelet function. Until now, whole blood aggregometry has not been considered feasible at low platelet counts. Hence, the objectives of the present study were to explore platelet function in thrombocytopenia using a novel index of impedance aggregometry adjusted for platelet count and evaluate the association to platelet function assessed by flow cytometry. Hirudin anticoagulated blood was collected from 20 healthy volunteers, 20 patients with primary immune thrombocytopenia (ITP), and 17 hematological cancer patients. Platelet function was analyzed by impedance aggregometry and by flow cytometry. Collagen, adenosine diphosphate, thrombin receptor agonist peptide-6, and ristocetin were used as agonists for both analyses. Thrombocytopenia in healthy whole blood was induced in vitro employing a recently published method. Platelet aggregation of thrombocytopenic patients was evaluated relative to the aggregation of healthy volunteers at the same platelet count. In flow cytometry, platelet function was described as expression of the platelet surface glycoproteins: bound fibrinogen, CD63, and P-selectin. Similar platelet counts were obtained in the patient groups (p = 0.69) (range: 13–129 × 10⁹/l). Aggregation adjusted for platelet count was significantly increased in ITP patients compared to healthy platelets across all agonists. The platelet aggregation was high in the 95% prediction interval, with 18 ITP patients above the prediction interval in at least two agonists. In contrast, the platelet aggregation was low in the prediction interval in cancer patients, and three cancer patients with platelet aggregation below the prediction interval in at least one agonist. ITP patients displayed increased expression of bound fibrinogen and CD63 following activation, compared with particularly cancer patients, but also compared with healthy platelets. This study demonstrated the feasibility of a novel approach to perform platelet function analyses in thrombocytopenia using impedance aggregometry adjusted for platelet count.     

Higher platelet reactivity and platelet-monocyte complex formation in Gram-positive sepsis compared to Gram-negative sepsis

Platelets may play a role in the high risk for vascular complications in Gram-positive sepsis. We compared the platelet reactivity of 15 patients with Gram-positive sepsis, 17 with Gram-negative sepsis and 20 healthy controls using a whole blood flow cytometry-based assay. Patients with Gram-positive sepsis had the highest median fluorescence intensity (MFI) of the platelet membrane expression of P-selectin upon stimulation with high dose adenosine diphosphate (ADP; P = 0.002 vs. Gram-negative and P = 0.005 vs. control groups) and cross-linked collagen-related peptide (CRP-XL; P = 0.02 vs. Gram-negative and P = 0.0001 vs. control groups). The Gram-positive group also demonstrated significantly higher ADP-induced fibrinogen binding (P = 0.001), as wll as platelet-monocyte complex formation (P = 0.02), compared to the Gram-negative group and had the highest plasma levels of platelet factor 4, β-thromboglobulin and soluble P-selectin. In contrast, thrombin-antithrombin complex and C-reactive protein levels were comparable in both patient groups. In conclusion, common Gram-positive pathogens induce platelet hyperreactivity, which may contribute to a higher risk for vascular complications