Light Transmission Aggregation Research Articles

A Novel F2RL3 Variant Predicting a Tyr157Cys Substitution in Protease-Activated Receptor 4 Is Associated with Reduced Receptor Function in Platelets

IntroductionProtease-activated receptor 4 (PAR4) is expressed widely in haemopoietic and vascular tissues and mediates thrombin-induced platelet activation primarily through Gq, protein kinase C and Ca2+ dependent pathways. Platelet PAR4 function may be modified by common non-coding variants near F2RL3 which encodes PAR4, and by epigenetic regulators which affect F2RL3 expression. However, coding sequence variants which affect PAR4 expression or function have not been reported previously. Here we characterise a novel missense variant in F2RL3associated with loss of PAR4 function in platelets from heterozygous subjects and reduced surface expression in platelets and a cell model.MethodsThe index case (P1) was identified by re-sequencing platelet G-protein coupled receptor genes in sub-groups of a collection of 2,400 adult cardiac surgery patients (GRAPHICS study; UK REC 12/SW/0286). Platelets were studied by light transmission aggregation (LTA) and by measuring P-selectin exposure, PAC-1 binding and intracellular Ca2+mobilisation in response to activating agonists. Platelet PAR4 expression was measured in platelets by flow cytometry and immunoblot and in transiently transfected HEK293 cells by flow cytometry and fluorescence microscopy.ResultsP1 was a 66 year old male with lifelong mild mucocutaneous bleeding and two gastrointestinal bleeds during aspirin treatment who harboured a heterozygous c.471A>G transition in F2RL3predicting a p.Tyr157Cys (Y157C) substitution in PAR4.Platelet phenotypeCompared to healthy controls, platelets from P1 showed reduced aggregation responses to the PAR4 agonist AYPGKF (60-600 μM; EC50 P1 266 μM vs controls 153 μM (n=11)) and to α-thrombin (5-100 mU.ml-1; EC50 P1 51 mU.ml-1 vs controls 15 mU.ml-1 (n=8)) with the greatest reduction in responses at 150 μM AYPGKF (maximum amplitude (MA) P1 1% vs controls 56%; p<0.0001) and 10 mU.ml-1 α-thrombin (MA P1 3% vs controls 67%; p<0.0001). Platelets from P1 also showed reduced P-selectin exposure, PAC-1 binding and intracellular Ca2+mobilisation in response to AYPGKF (25-1000 μM) compared to healthy controls. Functional responses of platelets from P1 to the PAR1 agonist SFLLRN (0.25-100μM) were similar to controls suggesting a selective defect in PAR4 function. Platelets from P1 showed a mean number of PAR4 receptors/platelet of 13 by flow cytometry, compared to 295 in controls (SEM 98; n=9) indicating markedly reduced surface expression. However, PAR1 surface expression in platelets from P1 was similar to controls. Immunoblots of platelet protein extracts for PAR4 from P1 and controls were similar, suggesting comparable total PAR4 expression.The c.471A>G F2RL3 variant was identified in two sons of P1, who also showed reduced platelet functional responses to AYPGKF and PAR4 surface expression that were similar to P1, indicating co-segregation of platelet phenotype and the variant F2LR3 genotype. However, these subjects did not display abnormal bleeding.Y157C PAR4 in HEK293 cellsVariant PAR4 with a C-terminal CFP tag (Y157C PAR4-CFP) expressed in HEK293 cells showed reduced surface expression by flow cytometry (mean of median fluorescence intensities 28% that of wild-type (WT) PAR4-CFP with SEM 5%; n=3). Consistent with this, fluorescence microscopy revealed predominantly intracellular expression of Y157C PAR4-CFP in the HEK293 cells with limited cell surface expression when compared to WT PAR4-CFP.ConclusionThe Y157 residue is highly conserved within transmembrane domain 3 of PAR4 that, in other G protein coupled receptors, is crucial for maintaining normal receptor structure. Therefore, the Y157C substitution identified in the index case in PAR4 is likely to adversely affect receptor function. Consistent with this, our data from multiple functional assays show reduced platelet responses to α-thrombin and to a PAR4 specific agonist, but not to a PAR1 specific agonist. This platelet phenotype co-segregated with the heterozygous Y157C substitution in the study kindred suggesting a causal association. However, this was not associated with bleeding in all kindred members. In both platelets and HEK293 cells we demonstrated the Y157C substitution was associated with loss of PAR4 function and reduced surface receptor expression.This is the first description of a missense PAR4 variant causing loss of receptor function as a heritable trait. DisclosuresNo relevant conflicts of interest to declare.

Functional and molecular characterization of inherited platelet disorders in the Iberian Peninsula: results from a collaborative study

BackgroundThe diagnostic evaluation of inherited platelet disorders (IPDs) is complicated and time-consuming, resulting in a relevant number of undiagnosed and incorrectly classified patients. In order to evaluate the spectrum of IPDs in individuals with clinical suspicion of these disorders, and to provide a diagnostic tool to centers not having access to specific platelets studies, we established the project “Functional and Molecular Characterization of Patients with Inherited Platelet Disorders” under the scientific sponsorship of the Spanish Society of Thrombosis and Haemostasis.Patients/methodsSubjects were patients from a prospective cohort of individuals referred for clinical suspicion of IPDs as well as healthy controls. Functional studies included light transmission aggregation, flow cytometry, and when indicated, Western-blot analysis of platelet glycoproteins, and clot retraction analysis. Genetic analysis was mainly performed by sequencing of coding regions and proximal regulatory regions of the genes of interest.ResultsOf the 70 cases referred for study, we functionally and molecularly characterized 12 patients with Glanzmann Thrombasthenia, 8 patients with Bernard Soulier syndrome, and 8 with other forms of IPDs. Twelve novel mutations were identified among these patients. The systematic study of patients revealed that almost one-third of patients had been previously misdiagnosed.ConclusionsOur study provides a global picture of the current limitations and access to the diagnosis of IPDs, identifies and confirms new genetic variants that cause these disorders, and emphasizes the need of creating reference centers that can help health care providers in the recognition of these defects.

Effect of intensive glycemic control on platelet reactivity in patients with long-standing uncontrolled diabetes

BackgroundIt has been previously shown that platelets of patients with diabetes are more reactive and less responsive to anti-platelet drugs compared with platelets from subjects without diabetes. Studies examining the effect of glycemic control on platelet reactivity have yielded conflicting data. Thus, in this study, we sought to explore the effect of tight glycemic control on platelet reactivity in patients with long standing uncontrolled diabetes. MethodsThe study included 30 patients with long-standing treated diabetes and a baseline HbA1c level of≥8.5%. All patients were treated with aspirin and statins. Patients were tested at baseline and after 3months of intensive glycemic and metabolic control. The treatment goal was to achieve a HbA1c level of ≤7%. Platelet reactivity was assessed by light transmission aggregation in response to 5 and 10μM ADP and to 0.5mg/ml arachidonic acid (AA). Additonally, platelet activation was assessed by plasma levels of soluble P-selectin using an enzyme-linked immunosorbent assay. ResultsThe mean duration of diabetes from the time of diagnosis was 20.46±9.31years. Baseline HbA1c was 9.4±0.8%. Following the intensive glycemic control period, the HbA1C level decreased to 8.1±0.8% (P<0.0001). Other laboratory parameters did not change significantly except for triglyceride levels, which decreased. None of the platelet aggregation studies nor P-selectin levels differed between baseline and after 3months of intensive glycemic control. ConclusionsIntensive glycemic control in patients with longstanding uncontrolled diabetes does not seem to result in a reduction in platelet reactivity.

How should we test for nonsevere heritable platelet function disorders?

Heritable platelet function disorders (HPFD) are a heterogeneous group of bleeding disorders with diverse clinical and laboratory characteristics. In contrast to the severe phenotype disorders, Glanzmann thrombasthenia and Bernard-Soulier syndrome, most nonsevere HPFD are incompletely characterized. This is a consequence of the poor standardization of diagnostic tests and of the lack of consensus about diagnostic criteria for the different subgroups of nonsevere HPFD. Distinguishing patients who have a nonsevere HPFD from those who do not is an essential first step in diagnosis which may be aided by bleeding assessment tools and screening tests such as the Platelet Function Analyser-100. However, high diagnostic accuracy can only be achieved with both light transmission aggregation (LTA) and secretion tests, for which streamlined agonist panels may be of similar utility to extended panels. Standardization of the methodology of these tests and quality assurance are essential for robust diagnosis. Identification of which platelet pathway is defective in patients with nonsevere HPFD is also usually possible with LTA and secretion tests. This strategy also sometimes enables exact diagnosis by implicating a single candidate protein and gene. Next-generation sequencing may offer a rapid approach to diagnosis of nonsevere HPFD, although rigorous strategies must be adopted to distinguish causative gene defects from bystander variations.

Functional Variations In Genes Encoding Platelet G-Protein Coupled Receptors In Unselected and Platelet Function Disorder Populations

IntroductionG-protein coupled receptors (GPCRs) are critical mediators of platelet responses to stimulatory and inhibitory agonists. In rare families with mild bleeding, it is recognised that heterozygous loss of function variations in platelet GPCR genes may diminish platelet agonist responses. However, the population prevalence of loss of function variations in these genes is unknown. We have utilised population databases and next generation sequencing from patients with inherited platelet function disorders (IPFD) to describe the extent of genetic variation in the major platelet GPCRs. We have also used predictive computation and a new consensus structure of GPCRs (Venkatakrishnan AJ et al. Nature 2013; 494) to estimate which variations confer loss of function. MethodsWe interrogated the ESP and 1000 genomes population datasets for single nucleotide (SNV) and insertion-deletion (indel) variations in the genes encoding 6 stimulatory (ADRA2A, F2R, F2RL3, P2RY1, P2RY12, TBXA2R) and 2 inhibitory (PTGER4, PTGIR) platelet GPCRs. Coding and splice region variations within the relevant Refseq transcripts were functionally annotated using the Polyphen-2, SIFT and FATHMM algorithms. Missense variations within GPCR transmembrane (TM) domains, were annotated manually by expressing the substitutions in Ballesteros-Weinstein nomenclature before comparison with the consensus GPCR structure. Missense variations in the N- and C-terminal regions (NR and CR) and the intra- and extra- cellular loops (ICL and ECL) were annotated by identifying the position of the substituted residue relative to experimentally confirmed or putative functional motifs. An identical analysis was performed using exome data from 31 unrelated patients with IPFD recruited through the UK GAPP study with clinical bleeding and abnormal platelet function by light transmission aggregation. ResultsIn 7745 individuals from the ESP and 1000 genomes cohorts, we identified 332 SNV in the target regions of the 8 GPCR genes (40.5 variations/kb) comprising 183 non-synonymous and 148 synonymous coding variants and 4 variations within intronic splice regions. There were no indel variations. Functional annotation of the non-synonymous SNVs identified 41 that potentially conferred loss of function, distributed in all the target GPCRs but with low population frequency (minor allele frequency range 1-0.008%). Five SNVs affected the NT, including Gly48Asp and Arg47His substitutions at the PAR4 receptor thrombin/trypsin cleavage site. There were 12 SNVs affecting the TM domains, of which 4 were predicted to disrupt GPCR folding, including a TPα receptor Pro305Leu substitution within the structural N/DPXXY motif and the P2Y12 receptor Met108Leu and Thr283Ile substitutions predicted to disrupt non-covalent TM network contacts. There were 14 SNVs affecting the ICL including the P2Y12 receptor Asp121Asn substitution in the E/DRY motif and prostacyclin (IP1) receptor Arg212Cys and Arg215Cys substitutions predicted to disrupt Gs coupling. Ten functional SNVs affected the CT. In 31 IPFD patients with complex laboratory phenotypes that could not be explained by loss of a single GPCR, there were 8 non-synonymous SNVs, of which 5 were predicted to confer loss of function (table).Table 1Functional GPCR variations in 31 patients with inherited platelet function disorders. +indicates loss of function predicted by the SIFT, POLYPHEN-2 and FATHMM algorithms.GPCRChangeSiteSIFTPolyphenFATHMMExperimental evidenceP2Y12Arg122HisIL2 DRY motif+++↓ Receptor functionP2Y12Thr223ArgIL3+++↓ Receptor functionP2Y12Val207AlaTM5++-↓ Receptor functionP2Y12Pro258ThrEL3++-↓ Ligand bindingIP1Arg296GlyCR+++Unknown DiscussionIn unselected populations, heterozygous loss of function GPCR gene variations which potentially affect platelet agonist responses are individually rare, but collectively numerous. Loss of function GPCR variations were also present in patients with underlying IPFD. These data illustrate that variations in platelet regulatory genes may act as modifiers of laboratory phenotype in patients with underlying IPFD and that the net phenotype may be the product of multiple gene defects. Disclosures:No relevant conflicts of interest to declare.

Platelet Activation and Inhibition iN Sickle cell disease (PAINS) study

Platelet activation/aggregation in sickle cell disease (SCD) may promote tissue ischemia, suggesting that antiplatelet therapy may be useful. However, the assessment of platelet function and the effect of antiplatelet therapy in blood from SCD patients may be confounded by hemolysis with the release of adenosine 5′-diphosphate (ADP). Here we evaluate the levels of platelet activation markers in SCD adolescents vs. normal controls and compare, by multiple methods, the effect of in vitro blockade of the platelet ADP receptor P2Y12 by prasugrel's active metabolite, R-138727. Platelet activation markers in blood from SCD adolescents (n = 15) and healthy adults (n = 10), and the effect of R-138727 (0.1–10 µM) added in vitro, were evaluated with and without ADP stimulation. The circulating levels of platelet–monocyte and platelet–neutrophil aggregates were significantly higher (p < 0.01) in SCD patients than in healthy controls. R-138727, in a concentration-dependent manner, inhibited platelet function in both SCD patients and healthy subjects as judged by ADP-stimulated light transmission aggregation, VerifyNow® P2Y12 assay, multiple electrode aggregometry, and flow cytometric analysis of platelet vasodilator-stimulated phosphoprotein, activated GPIIb-IIIa and P-selectin. The R-138727 IC50s for each assay were not significantly different in SCD vs. healthy subjects. In summary: (1) The high circulating levels of platelet–monocyte and platelet–neutrophil aggregates demonstrate in vivo platelet activation in SCD and may be useful as markers of the in vivo pharmacodynamic efficacy of antiplatelet therapy in SCD. (2) The similar in vitro R-138727 IC50s in SCD and healthy subjects suggest that the prasugrel dose-dependence for platelet inhibition in SCD patients will be similar to that previously observed in healthy subjects.

Platelet Activation and Inhibition in Sickle Cell Disease (PAINS) Study

AbstractAbstract 5147Platelet activation/aggregation in sickle cell disease (SCD) may promote tissue ischemia, suggesting antiplatelet therapy may be useful. However, assessing platelet function and the effect of antiplatelet therapy in blood from SCD patients may be confounded by hemolysis with release of ADP. Here we evaluate levels of platelet activation markers in SCD adolescents vs. normal controls and compare, by multiple methods, the effect of in vitro blockade of the platelet ADP receptor P2Y12 by prasugrel’s active metabolite, R-138727. Platelet activation markers in blood from SCD adolescents (n=15) and healthy adults (n=10), and the effect of R-138727 (0. 1 – 10 μM) added in vitro, were evaluated with and without ADP stimulation. Circulating levels of platelet-monocyte and platelet-neutrophil aggregates were significantly higher (p <0. 01) in SCD patients than in healthy controls. R-138727, in a concentration-dependent manner, inhibited platelet function in both SCD patients and healthy subjects as judged by ADP-stimulated light transmission aggregation, VerifyNow P2Y12 assay, multiple electrode aggregometry, and flow cytometric analysis of platelet vasodilator-stimulated phosphoprotein, activated GPIIb-IIIa and P-selectin. The R-138727 IC50s for each assay were not significantly different in SCD vs. healthy subjects. In summary: 1) The high circulating levels of platelet-monocyte and platelet-neutrophil aggregates demonstrate in vivo platelet activation in SCD and may be useful as markers of the in vivo pharmacodynamic efficacy of antiplatelet therapy in SCD. 2) The similar in vitro R-138727 IC50s in SCD and healthy subjects suggest that the prasugrel dose-dependence for platelet inhibition in SCD patients will be similar to that previously observed in healthy subjects. Disclosures:Frelinger:Eli Lilly: Consultancy, Research Funding; Daiichi Sankyo: Research Funding; GLSynthesis: Research Funding. Jakubowski:Eli Lilly: Employment. Heeney:Novartis: Consultancy, Research Funding; Eli Lilly and Company: Research Funding; Pfizer: Consultancy. 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.

REVERSAL OF THE ANTI-PLATELET EFFECTS OF ASPIRIN AND CLOPIDOGREL

ObjectivesGuidelines recommend stopping aspirin and clopidogrel 7–10 days before surgery to allow time for replacement of permanently inhibited platelets by newly released uninhibited platelets. The purpose of this study was...

GENDER DIFFERENCES OF ASPIRIN RESISTANCE IN PATIENTS WITH CORONARY ARTERY DISEASE

ObjectivesTo investigate the gender differences of aspirin resistance in patients with coronary artery disease.MethodsA total of 312 patients with coronary artery diseases were diagnosed by coronary angiography in Beijing Anzhen...

Relation between clopidogrel active metabolite levels and different platelet aggregation methods in patients receiving clopidogrel and aspirin

Clopidogrel is a prodrug that undergoes bioconversion via cytochrome P450 system to form an active metabolite (AM) that binds to the platelet ADP receptor. The antiplatelet effect of clopidogrel is commonly assessed by measuring the aggregatory response to 5μM ADP by light transmission aggregation (LTA) or multiple electrode aggregometry (MEA) or by the vasodilator-stimulated phosphoprotein platelet reactivity index (VASP-PRI). To determine which of these three tests of platelet ADP receptor pathway inhibition most closely correlates with clopidogrel AM levels. We analyzed blood samples from 82 patients with coronary artery disease who were randomized to receive double-dose or standard dose clopidogrel for 2weeks. We measured peak clopidogrel AM levels, platelet aggregation in response to ADP and VASP-PRI on days 1, and repeated all the measures on days 7 and 14. Linear regression analysis was used to examine the correlation between clopidogrel AM and LTA, MEA and VASP-PRI. Bland-Altman plots were used to explore the agreement between tests of the antiplatelet effects of clopidogrel. Clopidogrel AM on day 1 correlated most closely with VASP-PRI (r=-0.5767) and demonstrated weaker correlations with LTA (r=-0.4656) and MEA (r=-0.3384) (all p<0.01). Intra-class correlation (ICC) between VASP-PRI and LTA was 0.6446; VASP-PRI and MEA was 0.4720; and LTA and MEA was 0.4693. Similar results were obtained on days 7 and 14. Commonly used pharmacodynamic measures of clopidogrel response are only moderately correlated with clopidogrel AM levels and may not be suitable to measure the adequacy of clopidogrel therapy.

Genetic Polymorphisms of HO-1 and COX-1 Are Associated With Aspirin Resistance Defined by Light Transmittance Aggregation in Chinese Han Patients

Cyclooxygenase 1 (COX-1), COX-2, and HO-1 are involved in the process of aspirin's effect. The genetic susceptibility of these enzymes to aspirin resistance (AR) is unclear. A total of 431 patients took aspirin. Using arachidonic acid-induced light transmittance aggregation combined with adenosine diphosphate-induced light transmittance aggregation, 36 participants served for AR, 164 participants for semi-AR, and 231 participants for aspirin sensitivity (AS). The AR with 9 single-nucleotide polymorphism in COX-1, COX-2, and HO-1 genes was investigated. COX-1 rs1330344 (-1676A>G) is associated with AR. G-Allele carriers significantly increased the risk of AR. For patients with AS as control, P is .02 (odds ratio [OR] = 1.77, confidence interval [CI]: 1.07-2.92). For patients with semi-AR as control, P is .05. HO-1 rs2071746 (-413A>T) is associated with AR. T-Allele carriers significantly increased the risk of AR. For patients with AS as control, P is .04 (OR = 1.70, CI: 1.02-2.79). For patients with semi-AR as control, P is .05 (OR = 1.68, CI: 1.00-2.80). rs2071746 in HO-1 gene, rs1330344 in COX-1 gene contribute to AR.

Aspirin response variability after major orthopedic surgery

IntroductionVariability in platelet response to aspirin has been reported in patients undergoing cardiac surgery but has rarely been described in other operative settings and its mechanism remains uncertain. We performed a prospective cohort study to investigate the variability in platelet response to aspirin and to explore its mechanism in patients undergoing major orthopedic surgery. Materials and MethodsTwelve aspirin-treated patients undergoing elective hip or knee replacement were recruited. Once-daily aspirin was continued throughout the perioperative period. We measured platelet function using light transmission aggregation (LTA) in response to arachidonic acid (PLAA) and serum thromboxane B2 (TXB2) at baseline (before surgery) as well as on days 1, 2, 3, 4, 5, 6, and 8 after surgery. We defined aspirin low response as a PLAA>20%. ResultsSix patients exhibited aspirin low response, which typically started on post-operative days 3 or 4; the remaining 6 patients had normal response to aspirin. Compared to aspirin responders, patients with aspirin low response showed significantly higher serum TXB2 levels, a more pronounced early decrease in platelet count, and a significantly more rapid recovery of the platelet count after surgery. ConclusionAspirin response variability occurred in patients after major orthopedic surgery, with one-half of the patients in our study exhibiting post-operative aspirin low response. Increased platelet turnover might be a contributor to aspirin response variability after orthopedic surgery.

Prevalence of laboratory aspirin resistance in 431 old patients

To evaluate correlation between and agreement in light transmittance aggregation (LTA) and thromboelastography (TEG) in laboratory diagnosing aspirin resistance (AR), and to determine the prevalence of AR in old patients. Patients in the Wanshoulu District of Beijing with ischemic atherothrombotic diseases were recruited. Inclusion criteria were age ≥ 65 years, and having received regular aspirin therapy (75-100 mg daily) for at least 4 weeks. On the basis of LTA assay, the definition of AR was taken as aggregation of ≥ 20% with AA (arachidonic acid), and of ≥ 70% with ADP (adenosine diphosphate). Aspirin-sensitivity was indicated by the absence of either of these criteria; aspirinsensitivity was indicated as both criteria being met. The definition of AR by TEG is ≥ 50% via AA-induced whole blood aggregation. There were 13.69% prevalence of aspirin resistance for LTA using AA as the agonist, 30.16% prevalence of aspirin resistance for LTA using ADP as the agonist, and 23.67% prevalence of aspirin resistance for TEG using AA as the agonist. Results from these tests showed poor agreement (Kappa<0.4). However, by the method of LTA using AA and ADP as the agonists, prevalence of AR was 8.35%. By methods of AA-induced LTA and AA-induced TEG, prevalence of AR was 8.82%. Results from these two latter methods showed good agreement (Kappa = 0.793). Combined methods, as described here, have good correlation and agreement in the assays of AR, and the results with them represent a realistic measure of the prevalence of AR. Prevalence of AR of elderly patients from Wanshoulu district of Beijing is about 9%.

Reversal of the anti‐platelet effects of aspirin and clopidogrel

Guidelines recommend stopping aspirin and clopidogrel 7 to 10 days before surgery to allow time for replacement of permanently inhibited platelets by newly released uninhibited platelets. The purpose of the present study was to determine the rate of offset of the anti-platelet effects of aspirin and clopidogrel after stopping treatment and the proportion of untreated donor platelets that are required to reverse their anti-platelet effects. Cohort 1 consisted of 15 healthy subjects who received aspirin 81 mg day(-1) or clopidogrel 75 mg day(-1) for 7 days and underwent serial blood sampling until platelet function testing results normalized. Cohort 2 consisted of 36 healthy subjects who received aspirin 325 mg day(-1), clopidogrel 75 mg day(-1), aspirin 81 mg day(-1) plus clopidogrel 75 mg day(-1) or no treatment for 7 days and underwent a single blood sampling. In cohort 1, arachidonic acid (AA)-induced light transmission aggregation (LTA) returned to baseline levels in all subjects within 4 days of stopping aspirin, coinciding with the partial recovery of plasma thromboxane B(2) concentrations. ADP-induced LTA did not return to baseline levels until 10 days after stopping clopidogrel. In cohort 2, AA-induced LTA in patient treated with aspirin reached control levels after mixing with 30% untreated donor platelets whereas ADP-induced LTA in patients treated with clopidogrel reached control levels only after the addition of 90% or more donor platelets. Platelet aggregation recovers within 4 days of stopping aspirin but clopidogrel must be stopped for 10 days to achieve a normal aggregatory response.

Abstract 18022: High On-Treatment Platelet Reactivity Measured by Various Platelet Function Tests: Is the Current VASP-PRI Cutoff Too Low?

Background: High on-treatment platelet reactivity (HPR) to ADP measured by multiple methods has been linked to adverse post-PCI clinical event occurrence. The correlation of HPR cutoffs is still a matter of debate, since the predictive value and HPR group size are different depending on platelet function assays. To determine the comparable cutpoint, we correlated cutpoints for HPR determined by various assays in the same patient cohort. Methods: Platelet measures [5µM ADP-induced light transmission aggregation (LTA5μ MADP); P2Y12 reaction units by VerifyNow (PRU), and platelet reactivity index by VASP assay (PRI)] were evaluated (n=936 pairs, cohort I) in stable CAD patients during P2Y12 receptor inhibitor treatment. These assays also performed in PCI-treated patients receiving standard aspirin and clopidogrel therapy (n=584, cohort II). The HPR cutpoints were defined as LTA 5μ MADP &gt;46%, PRU &gt;235, and PRI &gt;50%. Results: In the stable CAD patients, LTA 5μ MADP showed significant correlations with PRU and PRI (r ≥0.688). In ROC curve analysis using the cutpoint of LTA 5μ MADP (&gt;46%), the matched values of PRU and PRI were &gt;234 (AUC 0.943, p&lt;0.001, sensitivity 87.3% and specificity 87.8%) and &gt;60% (Figure A). Among PCI-treated patients, the prevalence of HPR was highest based on PRI &gt;50% (69%) compared with other criteria (≤55%). The comparable values for LTA 5μ MADP &gt;46% were PRU &gt;235 (AUC 0.850, p&lt;0.001, sensitivity 85.3% and specificity 70.4%) and PRI &gt;59% (Figure B). Conclusion: The published cutoff values for HPR by LTA 5μ MADP and PRU were well correlated, whereas published PRI &gt;50% cutoff value may overestimate the risk of HPR. Updated cutoff of PRI &gt; 60% may better stratify the risk for HPR and can be used in future trials.

In Vivo Platelet Activation and Its In Vitro Inhibition by Prasugrel's Active Metabolite In Adolescents with Sickle Cell Disease

Abstract 2141 Introduction:In patients with sickle cell disease (SCD), erythrocytes contribute to microvessel occlusion resulting in tissue damage and platelet activation. Platelet activation, aggregation, local thrombus formation and platelet activation-dependent leukocyte recruitment potentially amplify tissue ischemia. Antiplatelet therapy may therefore be useful in SCD. Here we evaluate levels of platelet activation markers in adolescents with SCD vs. normal controls and the effect of in vitro blockade of the platelet ADP receptor P2Y12 by prasugrel's active metabolite, R-138727. Methods:Blood was obtained from adolescents (10 – 18 yr) with SCD and healthy adult subjects. Platelet function was evaluated by: light transmission aggregation (LTA) in platelet-rich plasma with 20 μM ADP and in whole blood by VerifyNow P2Y12; Multiple Electrode Aggregometry (MEA) with 6.5 μM ADP; vasodilator stimulated phosphoprotein (VASP) P2Y12 assay; and whole blood flow cytometric analysis of basal and in vitro ADP-stimulated levels of platelet surface activated GPIIb-IIIa (reported by monoclonal antibody PAC1) and P-selectin, platelet-monocyte aggregates (PMA) and platelet-neutrophil aggregates (PNA). These endpoints were also evaluated after in vitro incubation of whole blood with R-138727 (0.1 – 10 μM). Results:In SCD patients compared with normal subjects, circulating PMA and PNA levels were significantly higher (76.5 ± 20.3% and 55.1 ± 21.8% vs. 20.1 ± 7% and 13.9 ± 4.2% [mean ± SD], respectively, p<0.0001 for both), and in vitro ADP-stimulated platelet surface activated GPIIb-IIIa and P-selectin levels (mean fluorescence, MFI) were significantly lower (128.7 ± 66.2 and 78.1 ± 11.5 vs. 257.3 ± 50.8 and 91.6 ± 5.8, p<0.05 for both). ADP-stimulated platelet aggregation by LTA, VerifyNow and MEA, did not significantly differ between SCD and normal subjects, although whole blood platelet aggregation by MEA and VerifyNow tended to be greater in blood from SCD patients (92.5 vs. 70.4 AU, p=0.064 and 362.9 vs. 314.8 PRU, p=0.488, respectively).Treatment of whole blood in vitro with R-138727 resulted in a concentration-dependent inhibition of platelet function in both SCD patients and normal subjects. However, compared with normal subjects, the IC50 in SCD subjects was significantly lower for LTA but significantly higher for VerifyNow and VASP (Table). R-138727 inhibition of platelet function in SCD patients was similar to normal subjects as judged by MEA, whole blood flow cytometry for ADP-stimulated platelet surface P-selectin and activated GPIIb-IIIa expression, and % PMAs (Table). Sensitivity to R-138727 inhibition in SCD patient blood was greatest as measured by ADP-stimulated platelet surface P-selectin MFI, LTA, and MEA, less with ADP-stimulated platelet surface activated GPIIb-IIIa, and least with VASP, VerifyNow P2Y12 and % P-selectin-positive platelets (Table). Conclusions:1) The markedly higher circulating PMA and PNA levels in SCD patients relative to normal donors demonstrates increased in vivo platelet activation in SCD patients and suggests that PMA and PNA may be useful markers of the in vivo pharmacodynamic effects of antiplatelet therapy in SCD patients. 2) Blockade of platelet P2Y12 with R-138727 produces dose-dependent inhibition of platelet function in SCD platelets. 3) Assay-dependent differences in IC50 values between SCD patients and normal donors suggest the presence of additional variables that affect these measures of platelet function. Further studies are needed to determine the relationship between platelet inhibition measured by these assays and clinical events in SCD patients.TableComparison of R-138727 inhibition of platelet function in blood from SCD adolescents vs. healthy subjects.AssayNormal Donor, n=5 IC50 (95% CI)SCD Patient, n=10 IC50 (95% CI)PLTA, ADP 20 μM2.36 (1.90–2.93)1.59 (1.23–2.05)0.0267VASP P2Y122.14 (1.52–3.02)4.56 (3.90–5.32)0.0013VerifyNow P2Y12, % Inhibition2.09 (1.70–2.56)3.76 (3.28–4.32)<0.0001MEA, 6.5 μM ADP2.43 (1.69–3.49)1.90 (1.50–2.40)0.2479P-selectin, MFI, ADP 20 μM1.08 (0.95–1.24)1.40 (1.15–1.70)0.0503P-selectin, % positive, ADP 20 μM4.25 (3.42–5.27)3.62 (3.15–4.16)0.1891PAC1 MFI, ADP 20 μM2.50 (2.05–3.06)2.45 (2.19–2.73)0.8146% PMAs, ADP 20 μMa2.48a (2.06–3.00)3.30a (1.01–10.8)0.7389Results shown are μM R-138727.aIC50 calculated after subtracting unstimulated values. Disclosures:Frelinger:GLSynthesis: Research Funding; Lilly/Daiichi Sankyo: Consultancy, Research Funding; Takeda: Research Funding. Jakubowski:Eli Lilly and Company: Employment, Equity Ownership. Heeney:Lilly: Consultancy. Michelson:GLSynthesis: Research Funding; Lilly/Daiichi Sankyo: Data Monitoring Committee for clinical trial, Research Funding; Takeda: Research Funding.

Single nucleotide polymorphisms of HO-1 and COX-1 are associated with complete aspirin resistance defined by light transmittance aggregation in the Elderly

BackgroundAspirin remains the cornerstone of treatment for atherothrombotic diseases, but various factors associated with inflammation, platelets, the vascular system, coagulation, and metabolism may result in aspirin resistance (AR). This study...

Light Transmittance Aggregometry Induced by Different Concentrations of Adenosine Diphosphate to Monitor Clopidogrel Therapy: A Methodological Study

Light transmission aggregation (LTA) is considered the reference method to identify residual platelet reactivity (RPR) in high-risk patients with coronary artery disease on clopidogrel treatment. An international standardization of this technique is still ongoing and different concentrations of adenosine diphosphate (ADP) as the agonist for LTA have been used to evaluate the inhibitory effect of clopidogrel treatment. To evaluate RPR, LTA was performed using different ADP concentrations (2, 5, 10, and 20 μmol/L) in 466 high-risk patients with coronary artery disease on dual antiplatelet therapy who underwent percutaneous coronary intervention and in 46 healthy subjects. A VerifyNow P2Y12 assay was assessed as a point-of care system. Imprecision studies showed higher coefficients of variation for LTA by 2 and 5 μmol/L ADP (healthy subjects: 4.7% and 3.9%; patients: 6.8% and 5.2%, respectively) in comparison with those obtained determining LTA using 10 and 20 μmol/L ADP (healthy subjects: 2.2% and 2.3%; patients: 2.7% and 3.1%, respectively). In patients, a significant difference (P < 0.0001) between mean values of LTA obtained with all ADP concentrations was found, even if LTA data were significantly correlated (at least: rho = 0.88, P < 0.0001). However, data from 10 and 20 μmol/L ADP LTA were very similar and highly concordant (k = 95.9%). All agreements were significant (for all P < 0.0001), in particular the agreement between 10 and 20 μmol/L ADP LTA was very good (k = 0.86, P < 0.0001). A moderate agreement between VerifyNow and both 10 and 20 μmol/L ADP LTA was observed. LTA by 10 and 20 μmol/L ADP gave equivalent percentages of aggregation and highly concordant results in terms of RPR in patients with coronary artery disease on clopidogrel. Significant concordant results were observed between both 10 and 20 μM ADP LTA and VerifyNow. This suggests that a concentration of 10 μmol/L ADP may be considered adequate for the identification of RPR of patients on clopidogrel and should be preferred for standardization LTA.

Succinate independently stimulates full platelet activation via cAMP and phosphoinositide 3‐kinase‐β signaling

The citric cycle intermediate succinate has recently been identified as a ligand for the G-protein-coupled receptor (GPCR) SUCNR1. We have previously found that this receptor is one of the most highly expressed GPCRs in human platelets. The aim of this study was to investigate the role of SUCNR1 in platelet aggregation and to explore the signaling pathways of this receptor in platelets. Using real-time-PCR, we demonstrated that SUCNR1 is expressed in human platelets at a level corresponding to that of the P2Y(1) receptor. Light transmission aggregation experiments showed dose-dependent aggregation induced by succinate, reaching a maximum response at 0.5 mM. The effect of succinate on platelet aggregation was confirmed with flow cytometry, showing increased surface expression of activated glycoprotein IIb-IIIa and P-selectin. Intracellular SUCNR1 signaling was found to result in decreased cAMP levels, Akt phosphorylation mediated by phosphoinositide 3-kinase-β activation, and receptor desensitization. Furthermore, succinate-induced platelet aggregation was demonstrated to depend on Src, generation of thromboxane A(2), and ATP release. Platelet SUCNR1 is subject to desensitization through both homologous and heterologous mechanisms. In addition, the P2Y(12) receptor inhibitor ticagrelor completely prevented platelet aggregation induced by succinate. Our experiments show that succinate induces full aggregation of human platelets via SUCNR1. Succinate-induced platelet aggregation depends on thromboxane A(2) generation, ATP release, and P2Y(12) activation.

200 Safety of transvenous pulmonary vein isolation for the treatment of atrial fibrillation: a prospective randomized study comparing radiofrequency energy with cryoenergy

New transvenous devices using cryoenergy have been recently introduced to perform pulmonary vein isolation (PVI) for the treatment of atrial fibrillation (AF). Experimental data suggested that cryoenergy (CRYO) produced less endothelial disruption and platelet activation than radiofrequency energy (RF) offering safety benefits. We aimed to compare both systems with regards to safety in patients by measuring for the first time sensitive laboratory markers of cell damage. platelet activation and inflammation after a PVI using either one of those energies. Sixty patients with symptomatic drug-resistant AF referred for PVI (56 ± 9 years of age, 48 males. 38 with paroxysmal and 22 with persistent AF) were randomly assigned to undergo the ablation procedure using either an open irrigated tip RF catheter (Thermocool®. Biosense Webster) or a cryoballoon catheter (Arctic front®. Medtronic). Systemic markers of cell damage (procoagulant microparticles [MPs of various cellular origin], troponin T, CK and CK-MB). platelet activation (ADP-induced light transmittance aggregation [LTA], expression of the platelet surface proteins P-selectin [pSEL] and activated GPIIb/IIIa [PAC-1]) and inflammatory response (hs-CRP) were determined frequently before and 4, 24 and 48 hours after the procedure. Procedure time was significantly shorter in patients treated with the cryoballoon (177 ± 30 min versus 200 ± 46 min. p = 0.028), but there were no differences in fluoroscopic time, clinical event rate and success rate. Post-procedural increases of MPs Troponin T and hs-CRP were observed but there were no consistent differences in parameters used for comparative laboratory safety assessment of the ablation systems using either cryoenergy or radiofrequency energy. Neither systematic sensitive markers of cell damage, of platelet activation nor of inflammatory response could detect any difference in the safety profile between cryoenergy and RF energy used for transseptal PVI in patients with AF.