Variability In Platelet Function Research Articles

Characterisation of Platelet Sensitivity in Severe COVID-19

Background: Coronavirus disease 2019 (COVID-19), caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is characterised by a hyperinflammatory and prothrombotic state. In the most severe cases, patients can develop lung complications such as pneumonitis and acute respiratory distress syndrome, frequently complicated by venous and arterial thrombosis. Recent studies suggest that platelets play a role as immune mediators, and may therefore contribute to the development of hyperinflammation and thrombosis in severe COVID-19 patients. The expression and signalling of Glycoprotein VI on platelets are dependent on its association and cross-linking with the dimeric Fc receptor γ-chain, which, upon platelet activation, leads to a signalling cascade involving the Spleen Tyrosine Kinase (SYK). Fostamatinib, a potent SYK inhibitor, has been approved for treatment of Immune Thrombocytopenia (ITP), and has been implicated in abrogating thrombosis by mitigating SYK signalling in platelets. We have recently shown that SYK inhibition can abrogate the thrombosis caused by cross-linking of COVID-19 antibodies. In this study, we assessed platelet reactivity using the Platelet Phenomic Analysis (PPAnalysis), which identifies platelet sensitivity and functional response as independent measures of platelet reactivity by flow cytometry. We hypothesised that platelets in patients with severe COVID-19 are hypersensitive to platelet agonists, in particular to the collagen-related peptide agonist, which involves the SYK pathway of platelet activation. Methods: Patients with COVID-19 pneumonia, recruited to the Multi-Arm Trial of Inflammatory Signal inhibitors for COVID-19 (MATIS) clinical trial - an interventional study assessing the impact of Fostamatinib (FOS) or Ruxolitinib (RUX) against Standard of Care (SOC) to prevent escalation of COVID-19 from moderate (WHO grades 3 and 4) to severe (WHO grade 5) - were included in this study, REC reference 20/HRA/2618. Samples were collected in citrated vacutainers, taken at day 0, before starting study medication or SOC, and at days 7, 14 and 28, where possible. Samples were processed immediately to obtain platelet-rich plasma (PRP). PRP was labelled with fluorescein isothiocyanate (FITC)-conjugated anti-fibrinogen and PE-Cy5-conjugated P-Selectin antibodies, and stimulated with increasing concentrations of the platelet agonists, adenosine diphosphate (ADP; 0.03-30 µM), collagen-related peptide (CRP; 0.003-3 µg/mL), and thrombin receptor activator peptide 6 (TRAP-6; 0.05-15 µM). Changes in platelet sensitivity, measured as LogEC50, were analysed using R. Results: 40 of 182 patients recruited into MATIS were included. A total of 50 COVID-19 samples (24 at day 0, 14 at day 7, 2 at day 14, and 10 at day 28) and 59 healthy control samples were processed using PPAnalysis. The 24 samples taken at day 0 showed extensive variability in platelet sensitivities between the responses to 3 agonists, with outliers both above and below the 95% centile of controls: 0-4% (P-Selectin response) and 13-42% (fibrinogen response) of patients displayed higher platelet sensitivity (below the 95% centile for controls), and 4-42% (P-Selectin response) and 8-16% (fibrinogen response) had lower sensitivity (above the 95% centile for controls), (Figure 1). Of the 9 patients with samples also taken at day 7, no patients showed an increase in platelet sensitivity from the P-Selectin response to all agonists. 5 patients showed reduced sensitivity. 2 patients in this cohort required non-invasive ventilation (NIV). Conversely, fibrinogen response to all agonists showed increased sensitivity at day 7 in 6 patients, with 1 patient requiring NIV, and reduced sensitivity was observed in 2 patients, both requiring NIV (Figure 2). Conclusions: Patients with severe COVID-19 display a large variability in platelet function, with either higher or lower platelet sensitivity to the agonists at day 0 when compared to controls. This implicates a role for aberrant platelet reactivity in COVID-19 and its associated prothrombotic state, and could contribute to the morbidity and mortality of patients. Interpretation of day 7 results at present is limited due to small numbers. However, it does show utility of the PPAnalysis, as a useful tool in the stratification of different patient cohorts, and in studying the associations between platelet function and disease. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Haemostatic responsiveness and release of biological response modifiers following cryopreservation of platelets treated with amotosalen and ultraviolet A light.

Due to the risk of replication of contaminating pathogens, platelets have a limited storage time of 5 days, which can be prolonged to 7 days by the use of pathogen inactivation technologies. Cryopreservation (CP) may be an alternative to permit longer storage periods and increased availability. However, the preparation of platelets can result in secretion of biological response modifiers (BRM), which can cause adverse transfusion reactions in the recipient. We investigated the impact of CP on platelet function and release of BRM in untreated (conventional) and pathogen-inactivated (PI) platelet concentrates. Twelve buffy coat-derived platelet units were treated with amotosalen and ultraviolet A light to inactivate pathogens. Twelve untreated units were used as controls. The 24 units were cryopreserved and in vitro variables were analysed before and after CP. The in vitro variables investigated included platelet surface receptors and activation markers by flow cytometry, and coagulation time by viscoelastography. A panel of BRM, including cytokines, was investigated. CP of both conventional and PI platelets resulted in a significant increase of BRM with similar increases of most of the BRM after CP of conventional and PI platelet concentrates. The increase in some of the BRM correlated significantly with shortened coagulation time, increased P-selectin expression, reduced mitochondrial transmembrane potential, and reduced capacity to respond to stimulation with ADP and collagen. Cryopreservation of both conventional and PI platelets results in secretion of BRM. The increase in some of the BRM correlated with changes in platelet function variables and suggests that BRM release is affected, in part, in a similar way by CP as are changes in platelet function variables. PI with amotosalen and ultraviolet A light in combination with CP did not affect the release of immunomodulatory factors more than CP alone did.

Study of the Association of PEAR1, P2Y12, and UGT2A1 Polymorphisms with Platelet Reactivity in Response to Dual Antiplatelet Therapy in Chinese Patients

Objectives: Genetic variation is thought to contribute to considerable interindividual variability in platelet function, and there is a pressing need to identify genetic markers that can be used to predict the response to treatment. Our study investigated whether PEAR1, P2Y12, and UGT2A1 polymorphisms were associated with platelet reactivity in response to dual antiplatelet therapy in Chinese patients with acute coronary syndrome. Methods: Patients with inhibition of platelet aggregation (IPA) < 30% after treatment were classified as the high platelet reactivity (HPR) group. Patients with IPA > 30% were classified as the normal platelet reactivity (NPR) group. ADP-induced platelet aggregation was measured by thromboelastography (TEG) platelet-mapping assay. Thirteen single nucleotide polymorphisms (SNPs) of PEAR1, P2Y12 and UGT2A1 were genotyped using the Mass­ARRAY platform. Results: Seven SNPs were significantly associated with ADP-induced platelet aggregation by univariate analysis. Major allele G at rs12041331, minor allele G at rs2644592, minor allele C at rs11264580, and minor allele C at rs11249454 were significantly associated with HPR, whereas minor allele T at rs57731889, minor allele A at rs16863356, and minor allele T at rs7634096 were significantly associated with NPR. The mean IPA was significantly lower in patients suffering recurrent ischemic events than in patients without recurrent events in our study (p = 0.048). Conclusions: Our findings suggest that PEAR1, P2Y12, and UGT2A1 genetic variants may be potential biomarkers that can be used to guide clinical applications of clopidogrel and aspirin in Chinese patients.

The Effects of Andrographis paniculata on Platelet Activity in Healthy Thai Volunteers.

Background. Andrographis paniculata (AP) has been used in Thai traditional medicine to treat various infections, including the common cold and fever. Its bioactive compound, andrographolide, has shown antiplatelet activities in an in vitro study model. Since clinical studies of the effects of AP on the human platelet function have never been reported, we investigated its effect on platelet activity in ten healthy volunteers. Methods. Two grams of AP was taken 3 times within one day. The blood was withdrawn by venipuncture before and 2 and 24 hours after the AP administration to analyze the effects of AP on platelet aggregation, the expression of enzyme cyclooxygenase (COX) mRNA and protein, and TXB2, including P-selectin. Result. Even though there was no significant change in the studied parameters, this study exhibited patient-to-patient variability in platelet function. It was found that ADP-induced platelet aggregation tended to decrease after AP administration, while epinephrine-induced platelet aggregation in females tended to be higher than that in males for the entire study period. Moreover, COX-1 mRNA levels tended to decrease while P-selectin levels tended to rise after AP administration. Conclusion. These controversial results are possibly due to the multifactorial mechanisms of platelet aggregation as well as the multichemical composition of AP. Further study, probably at the molecular level, is needed to unveil the underlying mechanisms of action of AP.

Association of PEAR1 genetic variants with platelet reactivity in response to dual antiplatelet therapy with aspirin and clopidogrel in the Chinese patient population after percutaneous coronary intervention

IntroductionPlatelet Endothelial Aggregation Receptor-1 (PEAR1) is a recently reported platelet transmembrane protein which plays an important role in platelet aggregation. The aim of this study was to investigate whether PEAR1 genetic variations were associated with platelet reactivity as assessed by adenosine diphosphate(ADP)-induced platelet aggregation in Chinese patients treated with aspirin and clopidogrel. MethodsPatients with coronary heart disease (CHD) who underwent percutaneous coronary intervention (PCI) were enrolled in the study. All patients were on dual antiplatelet therapy with aspirin and clopidogrel. ADP-induced platelet aggregation was measured by thromboelastography and defined as percent inhibition of platelet aggregation (IPA). Patients (n=204) with IPA <30% were identified as high on-treatment platelet reactivity (HPR). Patients (n=201) with IPA >70% were identified as low on-treatment platelet reactivity (LPR). Sixteen single nucleotide polymorphisms (SNPs) of PEAR1 were determined by a method of improved multiple ligase detection reaction. ResultsAmong the 16 SNPs examined by univariate analysis, 5 SNPs were significantly associated with ADP-induced platelet aggregation. Minor allele C at rs11264580 (p=0.033), minor allele G at rs2644592 (p=0.048), minor allele T at rs3737224 (p=0.033) and minor allele T at rs41273215 (p=0.025) were strongly associated with HPR, whereas homozygous TT genotype at rs57731889 (p=0.009) was associated with LPR. Multivariate logistic regression analysis further revealed that the minor allele T at rs41273215 (p=0.038) was an independent predictor of HPR and the homozygous TT genotype at rs57731889 (p=0.003) was an independent predictor of LPR. ConclusionsPEAR1 genetic variations were strongly associated with ADP-induced platelet aggregation in Chinese patients with CHD treated with aspirin and clopidogrel. These genetic variations may contribute to the variability in platelet function. The utility of PEAR1 genetic variants in the assessment and prediction of cardiovascular risk warrants further investigation.

The predictive value of platelet function point-of-care tests for postoperative blood loss and transfusion in routine cardiac surgery: a systematic review.

Excessive bleeding after cardiopulmonary bypass (CPB) operations remains to be a persistent problem and weak platelet function certainly contributes to bleeding diathesis. Antiplatelet therapy (APT) is an integral component of perioperative management in patients undergoing cardiac surgery procedures, both with and without use of CPB. In addition to individual variability in platelet function, different preoperative APT administration/discontinuation management further affects platelet function, which in turn may reflect bleeding tendency. However, the impact of drug-induced platelet inhibition on early postoperative bleeding extent remains difficult to predict. Herein, we reviewed the available evidence on the association between platelet function testing values and the extent of bleeding and transfusion requirements in early perioperative period. Currently, the association between platelet function measured by ex vivo assay and the occurrence of bleeding events remains uncertain. The intent of this review is to provide comprehensive literature insight into published evidence, investigating the possibility of platelet function tests to predict bleeding extent as well as transfusion requirements in cardiac surgery patients.

Abstract 536: RNA-Seq Identifies Differential Expression of Platelet Transcripts Between People With Hyper- and Hypoaggregation to Collagen

Background: Platelet aggregation plays an important role in the pathogenesis of myocardial infarction, and high platelet aggregability is associated with increased risk of events. We hypothesized that inter-individual variability in platelet function is associated with differences in the mRNA transcripts found in platelets from hyper-aggregators and hypo-aggregators. Methods: Eight African American (discovery cohort) and four European American (validation cohort) males with family history of early onset coronary artery disease were enrolled. Individuals were divided into hyper- and hypo-aggregators based on maximal aggregation to collagen using whole blood impedance aggregometry. RNA was extracted from leukocyte-depleted platelet-rich plasma and approximately 70 million 100bp paired-end reads per sample were obtained using an Illumina HiSeq 2500. RNA-seq analysis was performed using Bowtie/TopHat/Cufflinks software pipeline. Genes with expression ≥ 1 fragments per kilobase of transcript per million reads (FPKM) were considered to be expressed. On log2 scale, ≥ 2-fold (discovery cohort) and ≥ 1-fold (replication cohort) change in gene expression were considered to be significantly differentially expressed. Results: Among the 9,373 expressed genes, the most highly expressed nonubiquitous genes included GP1BB, GP9, PPBP, PF4, &amp; TUBB1, confirming our methods. Hyper-aggregability was associated with up-regulation of 239 genes and down-regulation of 29 genes in the discovery cohort (44 and 2 replicated in validation cohort respectively). Pathway analysis found that genes related to sphingosine-1P3 (such as RHOA [ras homolog family member A], GNAZ [G protein, alpha z polypeptide], VEGFA [vascular endothelial growth factor A]) and platelet aggregation pathways (such as GP9 [platelet glycoprotein IX], TLN1 [talin 1], ITGB3 [platelet glycoprotein IIIa]) were up-regulated in hyper-aggregators. Conclusion: RNA-seq and pathway analyses found that hyper- and hypo-aggregators have significant differences in gene expression. Increased expression of sphingosine-1P3 and platelet aggregation pathway genes in hyper-aggregators highlights the relationship of signal transduction with increased platelet aggregation.

Abstract 18041: RNAseq Identifies Differential Expression of Platelet Transcripts Between Hyper and Hypo-Aggregation to Collagen

Background: Platelet aggregation plays an important role in the pathogenesis of myocardial infarction, and high platelet aggregability is associated with increased risk of events. We hypothesized that inter-individual variability in platelet function is associated with differences in the mRNA transcripts found in platelets from hyper-aggregators and hypo-aggregators. Methods: Ten individuals with family history of early onset coronary artery disease were enrolled. Individuals were divided into hyper- and hypo-aggregators based on maximal aggregation to collagen using whole blood impedance aggregometry. RNA was extracted from leukocyte-depleted platelet-rich plasma and approximately 70 million 100bp paired-end reads per sample were obtained using an Illumina HiSeq 2500. RNA-seq analysis was performed using Bowtie/TopHat/Cufflinks software pipeline. Genes with expression ≥ 0.3 fragments per kilobase of transcript per million reads (FPKM) were considered to be expressed. Genes with ≥ 2-fold change in expression on log2 scale and FDR&lt;0.1 were considered to be significantly differentially expressed. Results: Study sample consisted of 2 females and 3 males in each group. Among the 12,105 expressed genes, the most highly expressed nonubiquitous genes included GPX1, PPBP, PF4, &amp; TUBB1, confirming our methodologies. Hyper-aggregability was associated with up-regulation of 69 genes (such as MTRNR2L1 [anti-apoptotic], DEFA1 [innate immune response], and MYL4 [myosin light chain]). On the other hand, 50 genes were significantly down-regulated in hyper-aggregators (such as CCL3L1 [chemotactic for lymphocytes], CXCL5 [chemokine], and TUBB2A[beta-tubulin]). Gene ontology enrichment analysis found that genes related to innate immune response were up-regulated while genes related to B- and T-lymphocyte regulation were down-regulated in hyper-aggregators. Conclusion: RNA-seq and gene ontology analyses found that hyper- and hypo-aggregators have significant differences in gene expression. Increased expression of innate immune pathway genes in hyper-aggregators highlights the relationship of inflammation with increased platelet aggregation and suggests a role of platelets in innate immune response.

Clopidogrel Pharmacokinetics and Pharmacodynamics Vary Widely Despite Exclusion or Control of Polymorphisms (CYP2C19, ABCB1, PON1), Noncompliance, Diet, Smoking, Co-Medications (Including Proton Pump Inhibitors), and Pre-Existent Variability in Platelet Function

This study sought to determine whether known genetic, drug, dietary, compliance, and lifestyle factors affecting clopidogrel absorption and metabolism fully account for the variability in clopidogrel pharmacokinetics and pharmacodynamics. Platelet inhibition by clopidogrel is highly variable. Patients with reduced inhibition have increased risk for major adverse cardiovascular events. Identification of factors contributing to clopidogrel's variable response is needed to improve platelet inhibition and reduce risk for cardiovascular events. Healthy subjects (n = 160; ages 20 to 53 years; homozygous CYP2C19 extensive metabolizer genotype; no nicotine for 6 weeks, prescription drugs for 4 weeks, over-the-counter drugs for 2 weeks, and no caffeine or alcohol for 72 h; confined; restricted diet) received clopidogrel 75 mg/day for 9 days, at which time clopidogrel pharmacokinetic and pharmacodynamic endpoints were measured. At steady-state, clopidogrel active metabolite (clopidogrel(AM)) pharmacokinetics varied widely between subjects (coefficients of variation [CVs] 33.8% and 40.2% for clopidogrel(AM) area under the time-concentration curve and peak plasma concentration, respectively). On-treatment vasodilator stimulated phosphoprotein P2Y(12) platelet reactivity index (PRI), maximal platelet aggregation (MPA) to adenosine phosphate, and VerifyNow P2Y12 platelet response units (PRU) also varied widely (CVs 32% to 53%). All identified factors together accounted for only 18% of intersubject variation in pharmacokinetic parameters and 32% to 64% of intersubject variation in PRI, MPA, and PRU. High on-treatment platelet reactivity was present in 45% of subjects. Clopidogrel pharmacokinetics and pharmacodynamics vary widely despite rigorous exclusion or control of known disease, polymorphisms (CYP2C19, CYP3A5, ABCB1, PON1), noncompliance, co-medications, diet, smoking, alcohol, demographics, and pre-treatment platelet hyperreactivity. Thus, as yet unidentified factors contribute to high on-treatment platelet reactivity with its known increased risk of major adverse cardiovascular events. (A Study of the Effects of Multiple Doses of Dexiansoprazole, Lansoprazole, Omeprazole or Esomeprazole on the Pharmacokinetics and Pharmacodynamics of Clopidogrel in Healthy Participants: NCT00942175).

Clopidogrel Pharmacokinetics and Pharmacodynamics Vary Widely Despite Exclusion or Control of Polymorphisms (CYP2C19, ABCB1, PON1), Non-Compliance, Diet, Smoking, Co-Medications (including Proton Pump Inhibitors), and Pre-Existent Variability in Platelet Function

AbstractAbstract 4356 Background:Platelet inhibition by clopidogrel is highly variable. Patients with reduced inhibition have increased risk for major adverse cardiovascular events. The aim of this study was to determine whether known genetic, drug, dietary, compliance and lifestyle factors affecting clopidogrel absorption and metabolism fully account for the variability in clopidogrel pharmacokinetics and pharmacodynamics. Methods and Results:Healthy subjects (n=160; age 20–53; homozygous CYP2C19 extensive metabolizer genotype; no nicotine 6 weeks, prescription drugs 4 weeks, over-the-counter drugs 2 weeks, caffeine and alcohol 72 hours; confined; restricted diet) received clopidogrel 75 mg daily for 9 days. At steady-state, clopidogrel active metabolite (clopidogrelAM) pharmacokinetics varied widely between subjects (CVs 33.8% and 40.2% for clopidogrelAM AUCt and Cmax, respectively). On-treatment pharmacodynamic endpoints VASP P2Y12 platelet reactivity index (PRI), maximal platelet aggregation (MPA) to ADP, and VerifyNow P2Y12 platelet response units (PRU) also varied widely (CVs 32 – 53%). All identified factors together accounted for only 18% of inter-subject variation in pharmacokinetic parameters and 32 – 64% of inter-subject variation in PRI, MPA, and PRU. High on-treatment platelet reactivity was present in 45% of healthy, homozygous CYP2C19 extensive metabolizer subjects, free of nicotine, alcohol, and co-medications, with witnessed clopidogrel 75 mg/d treatment for 9 days. Conclusions:Clopidogrel pharmacokinetics and pharmacodynamics vary widely despite rigorous exclusion or control of known disease, polymorphisms (CYP2C19, CYP3A5, ABCB1, PON1), non-compliance, co-medications, diet, smoking, alcohol, demographics, and pre-treatment platelet hyperreactivity. Thus, as yet unidentified factors contribute to high on-treatment platelet reactivity with its known increased risk of major adverse cardiovascular events. Disclosures:Frelinger:Takeda Pharmaceuticals: Research Funding; GLSynthesis: Research Funding. Bhatt:Medscape Cardiology: Membership on an entity’s Board of Directors or advisory committees; Boston VA Research Institute: Membership on an entity’s Board of Directors or advisory committees; Society of Chest Pain Centers: Membership on an entity’s Board of Directors or advisory committees; American Heart Association Get With The Guidelines Science Subcommittee: Chair, Chair Other; American College of Cardiology: Editor, Clinical Trials, Cardiosource Other; Duke Clinical Research Institute: clinical trial steering committees, clinical trial steering committees Other; Slack Publications: Chief Medical Editor, Cardiology Today Intervention Other; WebMD: CME steering committees, CME steering committees Other; Amarin, AstraZeneca, Bristol-Myers Squibb, Eisai, Ethicon, Medtronic, Sanofi Aventis, The Medicines Company: Research Funding; PLx Pharma, Takeda: unfunded research Other. Lee:Takeda Global Research & Development Center, Inc: Employment. Mulford:Takeda Global Research & Development Center, Inc: Employment. Wu:Takeda Global Research & Development Center, Inc: Employment. Nudurupati:Takeda Global Research & Development Center, Inc: Employment. Michelson:Eli Lilly: Data monitoring committee and idependent external monitor of clinical trials, Research Funding; Takeda: Research Funding; Oxygen Biotherapeutics: Research Funding; Alexion: Research Funding; Omthera: Research Funding.

Identification of a specific intronic PEAR1 gene variant associated with greater platelet aggregability and protein expression

AbstractGenetic variation is thought to contribute to variability in platelet function; however, the specific variants and mechanisms that contribute to altered platelet function are poorly defined. With the use of a combination of fine mapping and sequencing of the platelet endothelial aggregation receptor 1 (PEAR1) gene we identified a common variant (rs12041331) in intron 1 that accounts for ≤ 15% of total phenotypic variation in platelet function. Association findings were robust in 1241 persons of European ancestry (P = 2.22 × 10−8) and were replicated down to the variant and nucleotide level in 835 persons of African ancestry (P = 2.31 × 10−27) and in an independent sample of 2755 persons of European descent (P = 1.64 × 10−5). Sequencing confirmed that variation at rs12041331 accounted most strongly (P = 2.07 × 10−6) for the relation between the PEAR1 gene and platelet function phenotype. A dose-response relation between the number of G alleles at rs12041331 and expression of PEAR1 protein in human platelets was confirmed by Western blotting and ELISA. Similarly, the G allele was associated with greater protein expression in a luciferase reporter assay. These experiments identify the precise genetic variant in PEAR1 associated with altered platelet function and provide a plausible biologic mechanism to explain the association between variation in the PEAR1 gene and platelet function phenotype.

Low platelet disaggregation predicts poor response to 150 mg clopidogrel in patients with elevated platelet reactivity

Raising the maintenance dose of clopidogrel to 150 mg enhances platelet inhibition (PI) in patients with elevated platelet reactivity (EPR); however, large differences were observed between individuals in the extent of this benefit. We aimed to find clinical and platelet function variables that might predict the response to 150 mg clopidogrel in patients with EPR. ADP 5 µM-stimulated peak aggregation (Aggmax), 6-minute late aggregation (Agglate) and 6-minute disaggregation (disAgg) were measured with light transmission aggregometry (LTA) in a consecutive cohort of 202 patients admitted for emergent or elective percutaneous coronary intervention (PCI) after receiving a 600-mg loading dose of clopidogrel. PI was assessed 12 to 18 hours after loading dose and 30 days after the intervention. EPR was defined as a Aggmax value greater than 34%. From the first day of PCI, 85 patients with EPR received 150 mg clopidogrel for 30 days, while others with normal platelet reactivity (NPR) took 75 mg clopidogrel. Baseline clinical and LTA variables were analyzed in multivariable regression models. Administration of 150 mg clopidogrel enhanced PI and decreased the prevalence of EPR at 30 days compared to the peri-interventional period (Aggmax: 46.9 ± 8.3 vs. 37.3 ± 11.5; EPR: 100% vs. 62.4%; p < 0.001 in both cases); however, the achieved level of platelet reactivity was higher than patients with NPR (Aggmax: 20.6 ± 8.1 vs. 25.3 ± 10.8, p < 0.001). Multivariable logistic regression revealed low platelet disaggregation (odds ratio (OR): 12.49; 95% CI: 2.52–62.00; P = 0.002) and acute coronary syndrome (OR: 4.83; 95% CI: 1.54–15.09; P = 0.008) as independent predictors of EPR. The area under the receiver-operating-characteristic curve was 0.72 ± 0.06 for disaggregation to predict NPR after 150 mg clopidogrel. The optimal disaggregation cut-off was 16.5% with a sensitivity of 94% and a specificity of 43%. Administering 150 mg maintenance dose of clopidogrel enhanced antiplatelet potency; however, more than half of the patients persisted with EPR. Acute coronary syndrome and low (<16.5%) platelet disaggregation are independent predictors of poor benefit from dose shift.

Antiplatelet ‘resistance’ and ‘non‐responders’: what do these terms really mean?

The term 'resistance' should be restricted to very specialized physiologic circumstances, if not abandoned altogether. The term 'non-responder' needs to be placed in the context of the question: 'Non-responder to what?' Even if we would somehow magically know what an optimal response to antiplatelet therapy was, it will still be challenging to demonstrate an 'inadequate' response to antiplatelet therapy. At present there are two alternatives--give more drug or give additional drugs. Both strategies may work in further inhibiting platelet function, but both strategies can also be associated with an increased risk of bleeding. The trick, for the future, much as with our antihypertensive and lipid-lowering armamentaria, will be to know in whom to do what with which drug, and why. Single isolated measurements are not useful--if you don't know where you started, how we would know that antiplatelet drug is producing an 'adequate' clinical effect? There is no evidence of any sort of absolute 'threshold' that must be exceeded for treatment to be effective, and in the absence of this, if we are to evaluate the effect of a given drug, we have to have baseline values (off drug), therapeutic values (on drug), and some sort of assessment of both resting (unstimulated) and agonist-provoked (stimulated) platelet function. Moreover, given all of the different things that platelets do, the ideal assessment of platelet function and drug responsiveness will need to incorporate more than one agonist and some sort of assessment of both platelet activation and platelet aggregation. No one man (or test) tells us everything; it is the totality of the information that gives us the most complete picture. And, ultimately, we need to more firmly establish how the variability in platelet function and drug-associated changes in that function correlates with long-term, hard-endpoint clinical events.

Aspirin administered to women at 100 mg every other day produces less platelet inhibition than aspirin administered at 81 mg per day: implications for interpreting the women’s health study

We aimed to determine the relative level of platelet inhibition achieved with low-dose aspirin (81 mg daily) compared with a very low-dose (100 mg every other day). The Womens Health Study (WHS) found that a dose of 100 mg every other day of aspirin provided protection against stroke as primary prophylaxis, but not myocardial infarction. In the United States, the most commonly prescribed dose of aspirin for primary prophylaxis is 81 mg per day. As a result, it is important to know whether these doses are equivalent before extrapolating the results of the WHS to women in the U.S. To achieve this goal, we have studied the effects of these two dosing regimens on platelet function in healthy women meeting the WHS inclusion criteria using a randomized design. We enrolled 49 healthy female volunteers and used a sequential, crossover design to compare the two regimens. The participants received a 17-day course of each aspirin-dosing regimen separated by a 7-day washout period. The degree of platelet inhibition was measured on days 14-17 of each dosing regimen using a point-of-care platelet function assay utilizing arachidonic acid to activate platelets (VerifyNow-Aspirin). Participants platelet response, expressed as Aspirin Response Unit (ARU) attained a significantly greater level of platelet inhibition on days 14-17 while taking aspirin 81 mg daily compared to aspirin 100 mg every other day (31.3% vs. 12.7%, P < 0.0001) with mean +/- SD ARU values of 445 +/- 50 and 570 +/- 68, P < 0.0001. Significantly more daily readings in participants were >or=550 ARU, a value correlated with clinical outcomes in several studies, with the 100 mg every other day regimen (72.0% vs. 6.4% with 81 mg daily, P < 0.0001), and this alternate-day regimen also resulted in more day-to-day variability in platelet function (P = 0.0002). We found significantly less inhibition of platelet function with the dose used in the WHS than the usual U.S. dose. We observed that the degree of platelet inhibition was significantly less with aspirin 100 mg every other day compared with aspirin 81 mg daily, suggesting that results of the Women's Health Study may have underestimated both the efficacy and toxicity of aspirin as it is commonly administered. These data need to be considered when developing recommendations about the use of aspirin in the primary prevention of cardiovascular disease in women.

Heritability, Platelet Function, and Aspirin

Platelets play a critical role in the pathophysiology of atherothrombotic disease. A pivotal event contributing to the understanding of platelet-dependent clot formation was the development of the platelet aggregometer in 1962.1 An aggregometer specifically measures the ability of platelets to adhere via glycoprotein IIb/IIIa (integrin αIIbβ3), and thousands of articles using this technique have been published, characterizing platelet function; however, the usefulness of these measurements remains unclear. Whereas the aggregometer and related techniques that measure platelet aggregation or glycoprotein expression have led to large amounts of data characterizing platelet function in various settings, the clinical importance of measurable differences in platelet function is still debated.2 The use of platelet function testing is established in rarer platelet abnormalities, such as the autosomal recessive bleeding disorder Glanzmann thrombasthenia,3 but no clear consensus has been reached on its usefulness for highly prevalent diseases caused by platelet-dependent thrombosis, such as myocardial infarction. A major factor for this discrepancy is that many of the platelet function defects that lead to bleeding are known to be caused by a single defect, whereas thrombosis in the setting of cardiovascular disease is presumed to be multifactorial. Article p 2490 The evolution of platelet function studies in various clinical settings has led to the realization that wide interindividual variability exists in the platelet activation response.4,5 What accounts for this variability? Only a few studies have systematically examined this question. Platelet function has been established as markedly dependent on the type of agonist used, the agonist concentration, and the concomitant use of antiplatelet therapy.6 In addition, in the large population-based Framingham Heart Study, O’Donnell and colleagues7 have demonstrated that heritable factors play a major role in determining platelet aggregation, as opposed to measured covariates. Less clear from the current literature is the direct …

Heritability of platelet function in families with premature coronary artery disease.

Variations in platelet function among individuals may be related to differences in platelet-related genes. The major goal of our study was to estimate the contribution of inheritance to the variability in platelet function in unaffected individuals from white and African American families with premature coronary artery disease. Platelet reactivity, in the absence of antiplatelet agents, was assessed by in vitro aggregation and the platelet function analyzer closure time. Heritability was estimated using a variance components model. Both white (n = 687) and African American (n = 321) subjects exhibited moderate to strong heritability (h(2)) for epinephrine- and adenosine diphosphate-induced aggregation (0.36-0.42 for white and >0.71 for African American subjects), but heritability for collagen-induced platelet aggregation in platelet-rich plasma was prominent only in African American subjects. Platelet lag phase after collagen stimulation was heritable in both groups (0.47-0.50). A limited genotype analysis demonstrated that the C825T polymorphism of GNB3 was associated with the platelet aggregation response to 2 muM epinephrine, but the effect differed by race. Considering the few and modest genetic effects reported to affect platelet function, our findings suggest the likely existence of undiscovered important genes that modify platelet reactivity, some of which affect multiple aspects of platelet biology.

Diagnostic Assessment of Patients with Inherited Mucocutaneous Hemorrhages (IMCH): Opening a Pandora Box?.

Diagnosis of patients with IMCH is plagued with difficulties: 1. Type 1 VWD (VWD-1) and platelet function disorders (PFD), the best characterized disorders of primary hemostasis, present inherent diagnostic difficulties. 2. Bleeding may be present in healthy individuals and may be absent in patients with diagnosed diseases. 3. An unknown proportion of bleeders do not have an identifiable haemostatic disorder. We studied prospectively 280 consecutive patients (age = 14.5±9.5, range 4–48 years) with family bleeding history who had a high probability of being pathological bleeders, based on an objective bleeding score. Only one physician interviewed the patients and the non-bleeder matched controls, (n=299, age 12.2±6.5, range 4–44 years). Exclusion criteria: age <4 and >50 years; platelet count <100.000/μL; other concurrent diseases, drug intake and C-reactive protein >1mg/dL. VWD was diagnosed when at least two tests (VWF:Ag, VWF:RCo, VWF:CB) had values <percentil 2.5 of the controls, without considering ABO type. PFD were diagnosed by PRP aggregation and 14C-5-HT secretion using pre-established criteria and reference values obtained in the 299 controls. Only 66 (23.6%) of the patients had prolonged bleeding time (BT). Frequency of diseases: 38 (13.6%) patients had VWD-1; 1 (0.35%) had type 2A VWD; 52 (18.6%) had PFD; 8 (2.9%) had concomitant VWD-1 + PFD; 7 patients (2.5%) had mild coagulation factor deficiencies (CFD, 4 with ↓FVIII:C; 2 with ↓ FIX:C; 1 with ↓ FXI:C); and 3 (1.1%) had concomitant VWD-1 + CFD; 171 bleeders (61.1%) had an unknown disorder (UD); in this UD group, 29 (17%) patients had prolonged BT, but all the other hemostatic tests were within the normal range. No distinctive bleeding pattern/severity was found among the above diagnostic categories. One patient with type 2B VWD was excluded because of thrombocytopenia and no patients with type 2N VWD were found. Patients with PFD had a secretion defect, but only 2 of them had typical storage pool disease (↓platelet 5-HT and ADP and ↓ADP/ATP ratio). No phenotypic patterns of Glanzmann or Bernard-Soulier diseases, arachidonate metabolism, or of absent ADP, collagen and TxA2 receptors were observed among the patients with PFD. Interestingly, the 170 patients with UD and the controls had almost the same mean values and distribution of VWF and platelet function variables; so we expect that very few patients with UD would qualify as VWD or PFD in repeated studies. Conclusions: 1. Prevalence of platelet secretion defects is higher than that of type 1 VWD-1 in our population. 2. Types 2 and 3 VWD, as well as paradigmatic platelet disorders (i.e., Glanzmann′s thrombasthenia, Bernard Soulier syndrome) were not detected in this study; they are of very low frequency and they are likely diagnosed at earlier age. 3. Bleeding time lacks sensitivity as a global test of primary haemostasis defects and should be omitted as a diagnostic tool. 4. After a first complete lab workup, 60% of the population with IMCH remains undiagnosed. The overwhelming majority of patients with UD probably constitute a heterogeneous group, independent of VWD or PFD, with a bleeding pattern similar to that of other disorders of primary hemostasis. Hypothetically, structural vascular defects or substances derived from the vascular wall which interfere with the normal platelet-vessel wall interaction could be involved in the pathogenesis of the hemorrhages.