Influence of renal function and platelet turnover on the antiplatelet effect of aspirin

Introduction

Kidney disease predisposes to cardiovascular events. This study investigated the influence of renal function and platelet turnover on the antiplatelet effect of aspirin in patients with coronary artery disease.

Materials and Methods

We included 124 aspirin-treated patients with coronary artery disease and normal to moderately reduced renal function. All tests were performed one hour after aspirin ingestion. Renal function was assessed using creatinine, estimated glomerular filtration rate (eGFR), and cystatin C. The antiplatelet effect of aspirin was evaluated using the VerifyNow® Aspirin assay and multiple electrode aggregometry (MEA, Multiplate®) induced by collagen (1.0 μg/mL) and arachidonic acid (1.0 mmol/L). Von Willebrand factor was measured as a marker of endothelial dysfunction. Platelet turnover was evaluated by measurements of immature, reticulated platelets.

Results

Renal function did not influence the antiplatelet effect of aspirin evaluated by MEA (r = − 0.2-0.09, p = 0.03-0.77) or the VerifyNow® (r = − 0.12-0.11, all p-values > 0.1). In contrast, renal function correlated inversely with von Willebrand factor levels (r_creatinine = 0.48, p < 0.0001; r_eGFR = − 0.46, p < 0.001; r_{cystatin C} = 0.54, p < 0.0001). The number of immature platelets correlated with platelet aggregation according to MEA (r = 0.20-0.39, all p-values < 0.03), but not according to VerifyNow® (r = − 0.07, p = 0.50).

Conclusions

A reduced antiplatelet effect of aspirin may be explained by an increased number of immature platelets. Moderately impaired renal function was associated with high levels of von Willebrand factor, but not with a reduced antiplatelet effect of aspirin.     

Evaluation of platelet function and pharmacological platelet inhibition in patients with myeloproliferative disorders using multiple electrode aggregometry

Background

The aim of this study was to describe platelet aggregation characteristics by multiple electrode aggregometry (MEA) and to evaluate MEA for its potential to detect platelet dysfunction and response to anti-aggregatory drugs in patients with myeloproliferative disorders (MPD).

Methods

We compared the platelet response to arachidonic acid (ASPI test), adenosine diphosphate (ADP test) and thrombin receptor activating peptide (TRAP test) in hirudin-anticoagulated blood of 55 patients with polycythaemia vera and essential thrombocythaemia and 75 controls.

Results

Comparing MPD patients and controls no statistically significant difference indicative of platelet dysfunction was found in MPD patients. Analysis of covariance revealed platelet- and leukocyte count as a significant influencing factor on MEA function. Furthermore we could demonstrate that ASA and clopidogrel treatment results in a statistically significant lower ASPI (Controls: p < 0.0001, MPD: p < 0.0001) and ADPtest value (MPD: p = 0.00125) compared to untreated patients thereby validating the method for monitoring of anti-aggregatory therapy.

Conclusion

In this study MEA was confirmed as a valid method for monitoring of ASA and clopidogrel treatment in patients with MPD and normal control subjects. The platelet and leukocyte count were identified as major influencing factors on MEA aggregation tests both in MPD patients and controls. No functional platelet abnormalities were detected in MPD patients.     

Reduced platelet response to aspirin in patients with coronary artery disease and type 2 diabetes mellitus

Introduction

Diabetes mellitus is complicated by accelerated atherosclerosis, resulting in an increased risk of coronary artery disease (CAD) and thrombosis. Despite the proven benefits of aspirin, previous studies indicate a reduced cardiovascular protection from aspirin in diabetic patients. We aimed to investigate whether diabetes mellitus influenced the platelet response to aspirin in patients with CAD.

Materials and Methods

Platelet aggregation and activation were evaluated during aspirin treatment in 85 diabetic and 92 non-diabetic patients with CAD. Adherence to aspirin was carefully controlled. All patients had CAD verified by coronary angiography and were taking 75 mg non-enteric coated aspirin daily.

Results

Diabetic patients showed significantly higher levels of platelet aggregation compared to non-diabetic patients evaluated by VerifyNow^® Aspirin (p = 0.03) and Multiplate^® aggregometry using arachidonic acid (AA) 0.5 mM (p = 0.005) and 1.0 mM (p = 0.009). In addition, platelet activation determined by soluble P-selectin was significantly higher in diabetics compared to non-diabetics (p = 0.005). The higher AA-induced aggregation was associated with higher levels of HbA_1c. Compliance was confirmed by low levels of serum thromboxane B₂ (below 7.2 ng/mL). Diabetics had significantly higher levels of serum thromboxane B₂ (p < 0.0001).

Conclusions

Diabetic patients with CAD had significantly higher levels of both platelet aggregation and activation compared to non-diabetic patients with CAD despite treatment with the same dosage of aspirin. These findings may partly explain the reduced cardiovascular protection from aspirin in diabetic patients.     

Similarities in Thromboelastometric (ROTEM®) Findings between Humans and Baboons

Introduction

Interest in visco-elastic testing in different clinical scenarios has increased but few data are available on thromboelastometric findings in primates.

Materials and Methods

Blood cell count (hemoglobin, hematocrit, platelet count), coagulation parameters (prothrombin time, International Normalized Ratio, fibrinogen), and ROTEM® (Tem International GmbH, Munich, Germany) variables were analyzed using blood from 25 anesthetized male baboons and 21 non-anesthetized healthy volunteers. The platelet component of the clot was calculated as the difference in maximum clot elasticity (MCE) between the whole blood clot (EXTEM test) and the fibrin-based clot (FIBTEM test). In subgroups of each species, 10 μg abciximab was added to the regular FIBTEM reagent (cytochalasin D) for additional platelet inhibition.

Results

Blood cell count was comparable between humans and primates. Both fibrinogen concentration (p < 0.0001) and maximum clot firmness (MCF) in FIBTEM assays were significantly lower in baboons (p > 0.0001, and p = 0.006, respectively). PT, INR, and clotting time in NATEM assays were significantly prolonged in humans compared with baboons. MCF in NATEM, EXTEM and INTEM assays was not different between baboons and humans. Clot lysis in NATEM, EXTEM and INTEM assays was significantly higher in humans (p < 0.0001). In contrast FIBTEM clot lysis was significantly higher in baboons (p = 0.01). Addition of abciximab into the FIBTEM assay resulted in a significant reduction in MCF and MCE (p < 0.001) and, consequently, the platelet component increased similar in both humans and baboons (p < 0.001).

Conclusion

Activated ROTEM® tests revealed broad similarities between humans and baboons. ROTEM® assays developed for use in humans can also be used in baboons.     

Genetic determinants of response to aspirin: appraisal of 4 candidate genes

Introduction

Intersubject variability in platelet response to aspirin could be related to genetic factors that regulate platelet enzymes or receptors. This study evaluates the impact of the selected polymorphisms in the COX-1 gene, the CYP5A1 gene, the P2RY1 receptor gene, and the GPIIbIIIa receptor gene on platelet response to aspirin and risk of suffering from major adverse cardiovascular and cerebrovascular events (MACCE).

Materials and methods

192 Caucasian patients with stable coronary artery disease treated with daily aspirin were recruited and followed for 3 years. Platelet aggregation was measured by light transmission aggregometry with arachidonic acid (1.6 mM) and adenosine diphosphate (5, 10 or 20 μM) used as agonists. Genotyping was performed by standard PCR methods.

Results

Arachidonic acid-induced platelet aggregation was unaffected by the COX-1 22C/T and by the Pl^A1/A2 polymorphisms. However, carriers of the 1622 G/G genotype of the P2RY1 gene had significantly higher levels of arachidonic acid-induced platelet aggregation compared with non-carriers (AA 2.0%, AG 2.0% vs. GG 9.0%, p = 0.047). Carrying the 1622 G/G genotype increased the risk of inadequate platelet response to aspirin, defined as arachidonic acid-induced aggregation ≥ 20%, by a factor of 8.5 (1.4 - 53.3, p = 0.022) and the risk of 3-year MACCE by a factor of 7 (1.4 - 34.7, p = 0.017).

Conclusion

The 1622A/G mutation of the P2RY1 gene could contribute to inadequate platelet response to aspirin and is associated with an increased risk of suffering from MACCE.     

Aspirin response evaluated by the VerifyNow Aspirin System and light transmission aggregometry

Introduction

Patients with inadequate platelet inhibition by aspirin, referred to as aspirin resistance, might have an increased risk of suffering cardiovascular events. Therefore, identification of these patients by measuring platelet function is of great interest. Our objectives were to evaluate performance parameters of VerifyNow™ and to determine the agreement between VerifyNow™ and light transmission aggregometry (LTA) ad modum Born.

Materials and Methods

We included 21 healthy volunteers and 40 patients with stable coronary artery disease. Duplicate measurements of platelet aggregation were performed using VerifyNow™ and LTA (arachidonic acid 1.0 mM) in healthy volunteers before aspirin and in all participants on four consecutive days during treatment with non-enteric-coated aspirin 75 mg daily. VerifyNow™ test results were expressed in Aspirin Reaction Units (ARU) and LTA test results in percent of maximal aggregation. The cut-off for determination of aspirin resistance was ≥ 550 ARU and ≥ 20%, respectively.

Results

All participants were compliant, confirmed by complete suppression of serum-thromboxane B₂. VerifyNow™ was highly repeatable with a coefficient of variance of 0.5% at baseline and 3.0% during aspirin treatment. No individuals were identified as aspirin resistant with VerifyNow™, whereas seven (12%) individuals were identified with LTA. ROC analysis using LTA as the gold standard showed poor sensitivity and good specificity with a cut-off at 550 ARU.

Conclusion

VerifyNow™ was highly repeatable, but further studies are needed to investigate the relevance of the cut-off level at 550 ARU for detecting aspirin resistance.     

Sustained enhancement of residual platelet reactivity after coronary stenting in patients with myocardial infarction compared to elective patients

Introduction

Elevated platelet reactivity despite antiplatelet therapy is associated with an increased cardiovascular risk after percutaneous coronary interventions. Current guidelines recommend uniform antiplatelet maintenance regimen after percutaneous coronary interventions for patients with myocardial infarction and elective patients. We sought to demonstrate that there is a persistent enhancement of residual platelet reactivity after myocardial infarction, requiring an intensified antiplatelet maintenance therapy.

Materials and Methods

A total of 66 patients after coronary stenting for myocardial infarction (n = 36) or elective coronary stenting (n = 30) were included in this prospective, controlled study. Platelet reactivity to adenosine-5-diphosphate and arachidonic acid under treatment with clopidogrel (75 mg) and acetyl salicylic acid (100 mg) were assessed 48 hours and 30 days after coronary stenting using light transmission aggregometry and multiple electrode platelet aggregometry (Multiplate analyzer) simultaneously.

Results

Fourty-eight hours after coronary stenting all measures of residual platelet reactivity were significantly elevated in the infarction group. After a mean follow up of 37 days, residual platelet reactivity to adenosine-5-diphosphate was still consistently elevated, albeit statistically not significant. Contrarily, residual platelet reactivity to arachidonic acid significantly decreased and returned to normal by the time of follow up. Regression analyses revealed myocardial infarction, C-reactive protein and fibrinogen as predictors of enhanced platelet reactivity 48 hours after coronary stenting.

Conclusions

Patients undergoing coronary stenting for acute myocardial infarction exhibit an enhancement of residual platelet reactivity sustaining for at least 48 hours following coronary stenting. This finding provides a rationale for a continued intensified antiplatelet therapy after myocardial infarction.     

Reference intervals for platelet aggregation assessed by multiple electrode platelet aggregometry

Introduction

Analyses of platelet aggregation in hirudin whole blood using Multiplate® was validated. Reference intervals for the most commonly used agonists were established, and the association between platelet aggregation, age, gender and haematological values was analysed.

Material and methods

We included 121 healthy individuals to establish reference intervals and six healthy individuals for evaluation of the day-to-day variation. Platelet aggregation was evaluated on hirudin whole blood employing Multiplate® induced by arachidonic acid, ADP, collagen and ristocetin (RISTOlow and RISTOhigh). Measurements of haematological values were performed employing Sysmex K-4500.

Results

We found no association between platelet aggregation and age (p > 0.57 for all agonists, except RISTOlow: p = 0.05). Platelet aggregation was significantly higher in women compared to men for all agonists (p < 0.0003), except RISTOlow (p = 0.05). A reference interval is presented as 95% confidence interval suitable for any age and both sex. Day-to-day variation was < 11% for all agonists except for RISTOlow. No association was found between platelet aggregation and haematocrit or red blood cell count after adjusting for age and gender except for RISTOhigh. A positive significant association was found between platelet count and platelet aggregation (p < 0.04). Finally, a significant positive association was found between platelet aggregation and white blood cell count for all agonists (p < 0.05) except RISTOlow and RISTOhigh (p > 0.05).

Conclusion

Reference intervals for platelet aggregation in healthy individuals (age: 17 to 66 years) were established in hirudin whole blood measured by Multiplate® employing the most commonly used agonists.     

Aspirin resistance following pediatric cardiac surgery

Introduction

Aspirin is often used to prevent thrombosis in pediatric cardiac surgery. The primary study aim was to assess aspirin resistance in this context. Secondary aims were to evaluate (1) the relationship between elevated inflammatory markers and thrombosis and (2) aspirin's effect on these levels.

Materials and Methods

This was a prospective observational study of children undergoing cardiac surgery managed with and without aspirin. Aspirin response was assessed using the VerifyNow™ system and urinary 11-dehydrothromboxane B₂ (uTxB₂) measurements. Laboratory studies of inflammation were also obtained.

Results

101 subjects were studied; 50 received aspirin. Six subjects (5.9%), 5 aspirin-treated, experienced symptomatic thrombosis. When measured by VerifyNow™ resistance was 43% after aspirin suppositories and 14% after additional days of oral aspirin. There was no correlation with thrombosis. Upper quartile post-operative day (POD) #5 uTxB₂ was correlated with thrombosis in aspirin treated subjects (p < 0.01). High risk aspirin-treated subjects who experienced thrombosis had higher POD#5 uTxB_2. This finding did not reach statistical significance (p = 0.07). Elevated pre-operative C-reactive protein (CRP) was independently associated with thrombosis (p < 0.02) in all subjects and in high risk subjects (p = 0.01). Inflammatory markers were not affected by aspirin.

Conclusions

Aspirin inhibited ex-vivo platelet function with a low incidence of resistance. Elevated POD#5 uTxB₂ and pre-operative CRP were correlated with thrombosis in aspirin treated subjects. Further studies are needed to determine whether children with high levels of uTxB₂ despite aspirin therapy and/or those with elevated preoperative CRP are at increased risk for thrombosis.     

Platelet aggregometry in the presence of PGE(1) provides a reliable method for cilostazol monitoring

Introduction

Cilostazol has been shown to be effective for prevention and treatment of cerebral infarction. However, there appears to be no widely accepted method appropriate for monitoring cilostazol. We attempted to establish an assay system for cilostazol monitoring, using platelet aggregation induced by arachidonic acid (AA) in the presence of PGE₁ which upregulates intracellular cyclic AMP.

Methods

Blood was drawn from stroke patients before and after cilostazol intake. AA-induced platelet aggregation after pretreatment with 0 ~ 30 nM PGE₁ for 2 minutes was measured by light transmittance aggregometry.

Results

AA-induced platelet aggregation was 73.1 ± 2.2% in the absence of PGE₁, and pretreatment with 30 nM PGE₁ had virtually no inhibitory effect on platelet aggregation prior to cilostazol intake. In contrast, after cilostazol intake, 30 nM PGE₁ significantly inhibited platelet aggregation to 12.7 ± 4.5% (p = 7.8 × 10(− 11)) , while in the absence of PGE₁ platelet aggregation remained similar to that of prior-to-cilostazol value (70.6 ± 3.5%). The plasma concentration of cilostazol ranged from 0.55 to 3.51 μM. In the presence of 30 nM PGE₁, all the patients with cilostazol concentrations exceeding 1 μM had their platelet aggregation inhibited almost completely. ROC analysis suggests that AA-induced platelet aggregation in the presence of 30 nM PGE₁ had the excellent sensitivity (90.5%) and specificity (88.4%) for monitoring cilostazol.

Conclusions

AA-induced platelet aggregation in the presence of 30 nM PGE₁ could give good estimate on plasma concentrations of cilostazol. It is suggested that this system is a good tool for monitoring cilostazol.     

Platelet protease-activated receptor 1 and membrane expression of P-selectin in pulmonary arterial hypertension

Introduction

Pulmonary arterial hypertension (PAH) is frequently associated with thrombotic events, particularly involving the pulmonary microcirculation at sites of vascular injury. We therefore decided to analyse protease-activated receptor 1 (PAR1), a key element in the activation of human platelets by thrombin, in PAH patients in stable clinical condition.

Methods

Using flow cytometry, we analyzed platelet PAR1 density, PAR1-mediated exposure of P-selectin and the formation of platelet-leukocyte aggregates in 30 PAH patients aged 11 to 78 years (median 50.5 years). The control group consisted of 25 healthy subjects with the same age range as patients.

Results

In patients, total platelet PAR1 density and uncleaved PAR1 density correlated negatively with platelet count (r² = 0.33 and r² = 0.34 respectively, p < 0.0015). In patients with a low platelet count (< 150 × 10⁹ platelets/L), both densities were increased relative to controls (82% and 33% respectively, p < 0.05). Thrombin peptide-induced platelet exposure of P-selectin was directly related to total and uncleaved PAR1 density (respectively, r² = 0.33 and r² = 0.29, p < 0.0025) and increased in subjects with low platelet count (46% versus those with normal platelet count, p < 0.05). Patients with low platelet count had decreased in vitro thrombin-induced formation of platelet-leukocyte aggregates (57% decrease versus controls, p < 0.05).

Conclusions

There seems to be a subpopulation of PAH patients with increased propensity to thrombotic events as suggested by increased platelet PAR1 expression and PAR-mediated surface exposure of P-selectin associated with decreased platelet count.     

Post interventional cardiology urinary thromboxane correlates with PlateletMapping® detected aspirin resistance

Introduction

We have previously defined aspirin resistance detected by TEG PlateletMapping using arachidonic acid (AA). This aspirin resistance is observed as platelet activation (> 20%) by AA in whole blood, even though the isolated platelets are inhibited by aspirin. This platelet activation in whole blood is due to a transcellular pathway mediated by platelets and leukocytes.

Methods

To determine if this PlateletMapping assay of aspirin resistance on pre-procedure blood samples correlated with an in vivo response we assayed the first voided urine samples collected 2-8 hours post interventional cardiology procedures for 11-dehydro thromboxane B2.

Results and Conclusions

We detected 27 aspirin resistant patients out of a total of 81 (33%), in agreement with our previous study. All of these patients were on aspirin therapy, confirmed by a < 20% aggregation response to AA by light transmission platelet aggregometry using isolated platelet rich plasma. Aspirin resistant patients urine samples (14 out of a total of 60 patients analyzed) contained significantly (P = 0.008) higher 11-dehydro thromboxane B2 levels than the other 46 aspirin sensitive patients urine samples. Since our previous study implicated 12- and 15-lipoxygenases in this pathway, we also assayed for polymorphisms to determine any correlation with aspirin resistance. A correlation was found in a polymorphism affecting the lipoxygenase domain of platelet 12-lipoxygenase. This result indicates that aspirin resistance detected in whole blood by the TEG PlateletMapping assay correlates with a physiological consequence in terms of thromboxane formation. This is the first report of such a correlation.     

Utility of whole blood impedance aggregometry for the assessment of clopidogrel action using the novel Multiplate analyzer--comparison with two flow cytometric methods

Background

Non-responsiveness to anti-platelet therapy has been reported and has been linked to the occurrence of adverse events. No standard method to monitor clopidogrel efficacy is available at present. We aimed at comparing the utility of whole blood impedance aggregometry for the assessment of clopidogrel action using the novel Multiplate® analyzer with two flow cytometric methods.

Methods

Platelet function was determined before and after the initiation of clopidogrel therapy (300 mg loading dose, followed by 75 mg qd) in 40 patients (observational study). Furthermore, 77 patients and 77 referents with and without clopidogrel treatment (75 mg qd) were evaluated (case control study). Platelet function was assessed by Multiplate® ADP and ADP + PG tests, by P-selectin (CD62P) expression, and by vasodilator stimulated phosphoprotein (VASP) phosphorylation status.

Results

The observational study revealed that platelet reactivity decreased significantly after clopidogrel administration with all 4 methods (p < 0.001 for each). In the case control study the median values of all 4 tests were significantly higher in the referents without clopidogrel treatment than in the patients on clopidogrel therapy (p < 0.001 for each). Applying test specific lower reference limits as criterion for the differentiation between responders and non-responders to clopidogrel treatment, 57% of the patients on clopidogrel therapy were classified as non-responders with the Multiplate® ADP test, 38% with the Multiplate® ADP + PG test, 55% with the P-selectin assay, 9% with the PLT VASP/P2Y12 assay.

Conclusions

The VASP phosphorylation assay appeared to be advantageous for the assessment of clopidogrel action compared to the Multiplate® ADP + PG test, the P-selectin assay, and the Multiplate® ADP test (listed in descending order). However, our method comparison study underscores the critical nature of the dependence of results on the techniques used in specific studies, and it remains to be elucidated which method correlates best with the occurrence of adverse events.     

Influence of cytochrome 2C19 allelic variants on on-treatment platelet reactivity evaluated by five different platelet function tests

Background

The antiplatelet effect of clopidogrel has been linked to cytochrome P450 2C19 (CYP2C19) carrier status. The presence of loss of function and gain of function variants were found to have a gene-dose effect on clopidogrel metabolism. However, genotyping is only one aspect of predicting response to clopidogrel and several platelet function tests are available to measure platelet response.Patients and methodsWe studied the influence of CYP2C19 allelic variants on on-treatment platelet reactivity as assessed by light transmission aggregometry (LTA), the VerifyNow P2Y12 assay, the VASP assay, multiple electrode aggregometry (MEA), and the Impact-R in 288 patients after stenting for cardiovascular disease. Allelic variants of CYP2C19 were determined with the Infiniti® CYP450 2C19 + assay and categorized into four metabolizer states (ultrarapid, extensive, intermediate, poor).

Results

Platelet reactivity increased linearly from ultrarapid to poor metabolizers using the VerifyNow P2Y12 assay (P = 0.04), the VASP assay (P = 0.02) and the Impact-R (P = 0.04). The proportion of patients with high on-treatment residual platelet reactivity (HRPR) identified by LTA, the VerifyNow P2Y12 assay and the VASP assay increased when the metabolizer status decreased, while no such relationship could be identified for results of MEA and Impact-R. The presence of loss of function variants (*2/*2, *2-8*/wt, *2/*17) was an independent predictor of HRPR in LTA and the VASP assay while it did not reach statistical significance in the VerifyNow P2Y12 assay, MEA, and the Impact-R.

Conclusion

Depending on the type of platelet function test differences in the association of on-treatment platelet reactivity with CYP2C19 carrier status are observed.     

Antiplatelet effects of antidepressant treatment: A randomized comparison between escitalopram and nortriptyline

Introduction

Depressive disorders have been identified as independent risk factors for coronary heart disease. The present study (i) compared platelet function of depressed patients with that of healthy controls, (ii) analysed possible aggregability changes during 3 months of treatment with antidepressants, and (iii) sought to assess different effects of escitalopram and nortriptyline on platelet aggregation.

Methods

Blood samples of 91 major depressed patients and 91 healthy controls were analysed with whole blood aggregometry in a case-control setting. Depressed patients were randomized to two groups treated either with escitalopram (n = 47) or nortriptyline (n = 44). Platelet aggregation was studied on days 0, 1, 3, 7, 14, 21, 84 of continuing medication and was determined in response to adenosine diphosphate (ADP) and collagen.

Results

Platelet aggregation induced by ADP was increased among depressive patients compared with that of healthy controls (26%, p = 0.006). With antidepressant treatment, changes in platelet aggregation remained comparable in both groups at early time points (d1 to 21). In contrast, at day 84, patients with antidepressive response revealed significant differences in both medication groups: Patients receiving escitalopram showed a 23% decrease of ADP induced aggregation (p = 0.03) and a 15% decrease of collagen induced aggregation (p = 0.03). With nortriptyline the increase in impedance was reduced by 29% after ADP induction (p = 0.046).

Conclusion

Depressed patients have higher ex vivo platelet aggregation that may contribute to increased cardiovascular morbidity. After three months of antidepressant treatment with either escitalopram or nortriptyline, platelet aggregation was significantly reduced in antidepressant responders, irrespective of the antidepressant medication type.     

Aspirin insensitive thromboxane generation is associated with oxidative stress in type 2 diabetes mellitus

Introduction

Aspirin (ASA) irreversibly inhibits platelet cyclooxygenase-1 (COX-1) leading to decreased thromboxane-mediated platelet activation. The effect of ASA ingestion on platelet activation, thromboxane generation, oxidative stress and anti-oxidant biomarkers was studied in type 2 diabetes mellitus (DM).

Material and methods

Baseline and post-ASA samples (100/325 mg x 7 days) were obtained from 75 DM patients and 86 healthy controls for urinary 11-dehydro-thromboxane B2 (11dhTxB2), 8-iso-prostaglandin-F2α (8-isoPGF2α) and serum sP-Selectin, nitrite (NO₂^-), nitrate (NO₃^-) and paraoxonase 1 (PON1) activity.

Results

Compared to baseline controls, baseline DM had higher mean levels of 11dhTxB2 (3,665 ± 2,465 vs 2,450 ± 1,572 pg/mg creatinine, p = 0.002), 8-isoPGF2α (1,457 ± 543 vs 1,009 ± 412 pg/mg creatinine, p < 0.0001), NO₂^- (11.8 ± 7.3 vs 4.8 ± 5.3 μM, p < 0.0001), NO₃^- (50.4 ± 39.3 vs 20.9 ± 16.7 μM, p < 0.0001) and sP-Selectin (120.8 ± 56.7 vs 93.0 ± 26.1 ng/mL, p = 0.02), and the same held for post-ASA levels (p < 0.0001). ASA demonstrated no effect on 8-isoPGF2α, NO₂^-, NO₃^-, sP-Selectin or PON1 activity in either DM or controls. Post ASA inhibition of urinary 11dhTxB2 was 71.5% in DM and 75.1% in controls. There were twice as many ASA poor responders in DM than in controls (14.8% and 8.4%) based on systemic thromboxane reduction. Urinary 8-isoPGF2α excretion was greater in DM ASA poor responders than good responders (p < 0.009).

Conclusions

This suggests that oxidative stress may maintain platelet function irrespective of COX-1 pathway inhibition and/or increase systemic generation of thromboxane from non-platelet sources.     

P2Y12 platelet reactivity after thrombolytic therapy for ST-segment elevation myocardial infarction

Introduction

Thrombolysis, as reperfusion therapy for ST segment elevation myocardial infarction (STEMI), induces a pro-thrombotic status with enhanced platelet activity; this study aims to evaluate P2Y12 platelet reactivity and response to clopidogrel in the post-thrombolysis scenario.

Materials and Methods

Observational, prospective study, including consecutive patients with elective angiography after thrombolytic therapy for STEMI. Every patient received antiplatelet therapy with loading doses of 250 mg aspirin and 300 mg clopidogrel on admission followed by 100 mg aspirin and 75 mg clopidogrel daily. P2Y12-dependent platelet reactivity (expressed in P2Y12-Reaction Units, PRU) was assessed with VerifyNow® device on admission, daily after thrombolysis and pre-angiography.

Results

41 patients fulfilled the inclusion criteria. Median time between thrombolysis and angiography was 2,5 days (IQR 1,8-4,1). Post-treatment platelet reactivity (PPR) showed poor correlation with time on clopidogrel treatment (r2 = 0.04) and reached a maximum value of 274 ± 84 PRU during the first 24 h after thrombolysis (Day + 1 determination). After this, values showed a progressive reduction until the point of angiography (249 ± 82 PRU), without significant differences between consecutive time-points (p = 0,549).Inhibition of platelet aggregation (IPA) assessed as a percentage of P2Y12 receptor blockage was poor, increasing gradually from 0 ± 4% on admission to 11 ± 6% the day of the angiography (p = 0,001). 71,4% of patients showed PPR ≥ 208 PRU during angiography.

Conclusions

Platelet reactivity, as assessed by post-treatment P2Y12 mediated reactivity, is heightened after thrombolytic therapy during STEMI management. In this scenario, standard doses of clopidogrel did not achieve significant inhibition of ADP-mediated platelet reactivity.     

The effect of clopidogrel besylate and clopidogrel hydrogensulfate on platelet aggregation in patients with coronary artery disease: a retrospective study

Background

Recently several alternative forms of the original clopidogrel hydrogensulfate (CHS) were spread worldwide. A large amount of such drugs turned out to be clopidogrel besylate (CB). Only three studies, involving healthy volunteers, investigated the antiplatelet effect of CB, whereas its attribute remained unexplored in the case of patients with cardiovascular diseases. This retrospective study aimed to evaluate the difference between the antiplatelet effects of two clopidogrel formulas, CHS and CB, on patients with coronary artery diseases.

Methods

Data of 150 patients with previous CHS treatment were investigated. According to the documentations, the CHS therapy was shifted to CB. 94 patients of the selected population received dual antiplatelet therapy, clopidogrel and aspirin. The antiplatelet effects of CHS and CB were compared by ADP induced platelet aggregation measurements using light transmission aggregometry.

Results

Irrespective of the therapeutic combinations the performed statistical investigations failed to show significant difference (p = 0.30) between the effect of CB (AGGmax_CB: 27.6 ± 13.7%) or CHS (AGGmax_CHS: 29.0 ± 15.3%) on the ADP induced platelet aggregation. Insignificant deviations were found in both forms of clopidogrel salts, either in the lack (AGGmax_CB : 32.5 ± 14,2%; AGGmax_CHS: 34,0 ± 16,1%; p = 0,29) or in the presence of aspirin (AGGmax_CB: 24.7 ± 12,5%; AGGmax_CHS: 26,0 ± 14,1%; p = 0,31).

Conclusion

Our results indicated that both CB and CHS had an identical inhibitory effect on ADP induced platelet aggregation in patients with cardiovascular diseases. Moreover their efficiency showed no overall significant difference in the case of dual antiplatelet therapy with aspirin as well. However there might be an inter- and intraindividual variability between the two clopidogrel formulas.     

Baseline platelet size is increased in patients with acute coronary syndromes developing early stent thrombosis and predicts future residual platelet reactivity. A case-control study

Introduction

Pre-procedural predictors of early stent thrombosis (ST) and future response to platelet inhibitors are in demand. We sought to evaluate the impact of baseline platelet indices on the occurrence of early ST and future residual platelet reactivity.

Materials and methods

Hundred and eight patients with acute coronary syndromes (ACS) in whom stents were implanted were included: 36 consecutive ST cases and 72 matched controls. Platelet indices assessed with flow cytometry before stent implantation were retrieved from the department's data base. Residual platelet reactivity specific to aspirin (aspirin reaction units-ARU) and clopidogrel (P2Y12 reaction units-PRU) was assessed prospectively with VerifyNow^® under dual antiplatelet treatment.

Results

Platelet size reported as mean platelet volume (MPV) or proportion of large platelets (LPLT) was significantly higher in ST cases compared with controls (10.4, 95% confidence intervals [CI], 10.1-10.8 vs. 9.7, CI, 9.5-9.9, P = 0.0004 and 35.8, CI, 34.2-37.3 vs. 33.3, CI, 32.2-34.3, P = 0.007, respectively). Dual aspirin and clopidogrel poor-responsiveness was diagnosed significantly more often in ST cases than in controls (19.6% vs. 1.4%, P = 0.004), whereas no difference was observed for single aspirin or clopidogrel poor-responsiveness. A strong correlation was found between MPV and both, ARU (r = 0.66, P < 0.0001) and PRU (r = 0.55, P < 0.0001). Similarly, higher LPLT was associated with higher ARU (r = 0.47, P < 0.0001) and PRU (r = 0.38, P = 0.0001).

Conclusions

Baseline platelet size is increased in patients with ACS developing early ST and correlates with future residual platelet reactivity under aspirin and clopidogrel therapy. Dual but not isolated aspirin or clopidogrel poor-responsiveness appears to be associated with early ST.     

The Cone-and-Plate(let) analyzer is not suitable to monitor clopidogrel therapy: A comparison with the flowcytometric VASP assay and optical aggregometry

Introduction

High on-clopidogrel platelet reactivity has been associated with an increased risk for atherothrombotic events. A new player on the horizon is the IMPACT-R ADP-test using ADP pre-stimulation. We here report the results of a thorough evaluation of this new device.

Materials and methods

The IMPACT-R ADP-test was evaluated in different categories of subjects. First, normal range values were determined in healthy subjects (n = 46). Second, the effect of 600 mg of clopidogrel was evaluated with the IMPACT-R ADP-test and two other well-validated methods (flowcytometric VASP-analysis and optical aggregometry) in 21 patients. Third, a head-to-head comparison between the IMPACT-R ADP-test and optical aggregometry was performed in a large cohort of patients on dual antiplatelet therapy.

Results

The results of the IMPACT-R ADP-test were highly variable throughout healthy subjects. The administration of a high clopidogrel loading dose resulted in a small but significant increase in surface coverage but 61.9% of the patients were still identified as clopidogrel nonresponder. In contrast, optical aggregometry and VASP-analysis identified 24% and 33% of these patients as a clopidogrel nonresponder, respectively. Head-to-head comparison with optical aggregometry in 451 patients showed only a modest correlation between both methods (r ∼ 0.20, p < 0.0001).

Conclusions

The IMPACT-R ADP-test is relatively insensitive to the effects of clopidogrel and cannot substitute for methods such as flowcytometric VASP-analysis and optical aggregometry. Further studies are required to establish the clinical usefulness of IMPACT-R ADP-test to accurately predict the occurrence of major adverse cardiovascular events in patients with high on-clopidogrel platelet reactivity before it can be implemented in clinical practice.