Variability In Platelet Response Research Articles

The Use of Clot Strength as a Predictor of Thrombosis in Peripheral Artery Disease

ObjectivesGraft/stent thrombosis is the leading cause of amputation in patients over 60, and while dual antiplatelet therapy is the standard of care, there is a significant variability in platelet response and limited guidance on measuring effectiveness. Thromboelastography with platelet mapping (TEG-PM) can objectively detail an individual’s coagulation profile, namely the strength of the clot and its response to antiplatelet medication. Although TEG-PM has been used for predicting postoperative bleeding and assessing platelet dysfunction in TBI, its application in thrombosis diseases such as peripheral artery disease (PAD) remains unexplored. The aim of this observational study was to determine if objective measures of clot strength could predict a high clinical risk of thrombosis. MethodsPatients > 60 years with peripheral artery disease (PAD) undergoing revascularization were prospectively evaluated from 2021-2023. They were clinically followed for one year to detect any thrombotic events. TEG-PM was used to objectively evaluate coagulation profiles in patients at 1, 3, 6, and 9 months. These follow-up periods were chosen based on studies showing that 1-3 month intervals in the first year after lower extremity revascularization (LER) optimize therapy and risk control. The TEG-PM data preceding a thrombotic/stenotic event in patients with thrombosis was compared to the last known well TEG-PM event in those without a thrombotic/stenotic event. We stratified the groups based on the occurrence of thrombosis/stenotic events. Descriptive statistics were applied to characterize each group and a chi-square test was conducted to assess the variance between both groups. An unpaired t-test was ran to identify differences in platelet function. ROC analysis was performed to determine the optimal TEG-PM cutoff for predicting a higher risk of thrombosis. ResultsOne hundred and fifty-eight patients were analyzed, from whom 28 (17.7%) experienced a thrombotic event. The thrombosis cohort exhibited significantly greater MAADP, MAFibrin, and MAThrombin [50.2 vs. 40.0, p<0.05], [18.19 vs. 14.64, p<0.05] and [63.8 vs. 58.5, p<0.05] respectively indicative of greater clot strength. By ROC analysis, the optimal predictor cutoff for MAADP, indicating a higher risk of thrombosis, was >42mm [p<0.05] with 82% sensitivity and 50% specificity. ConclusionsAn increase in clot strength was found to be predictive of thrombosis/stenosis within 30 days. Using a MAADP cutoff greater than 42mm might serve as an alternative approach to tailor the use of antiplatelet medication, potentially reducing the risk of thrombosis.

Perioperative platelet reactivityover time inpatients undergoing vascular surgery: An observational pilot study.

Despite Antiplatelet therapy (APT), cardiovascular patients undergoing revascularisation remain at high risk for thrombotic events. Individual response to APT varies substantially, resulting in insufficient protection from thrombotic events due to high on-treatment platelet reactivity (HTPR) in ≤40% of patients. Individual variation in platelet response impairs APT guidance on a single patient level. Unfortunately, little is known about individual platelet response to APT over time, timing for accurate residual platelet reactivity measurement, or the optimal test to monitor residual platelet reactivity. To investigate residual platelet reactivity variability over time in individual patients undergoing carotid endarterectomy (CEA) treated with clopidogrel. Platelet reactivity was determined in patients undergoing CEA in a prospective, single-centre, observational study using the VerifyNow (change in turbidity from ADP-induced binding to fibrinogen-coated beads), the VASP assay (quantification of phosphorylation of vasodilator-stimulated phosphoprotein), and a flow-cytometry-based assay (PACT) at four perioperative time points. Genotyping identified slow (CYP2C19*2 and CYP2C19*3) and fast (CYP2C19*17) metabolisers. Between December 2017 and November 2019, 50 patients undergoing CEA were included. Platelet reactivity measured with the VerifyNow (p = < .001) and VASP (p = .029) changed over time, while the PACT did not. The VerifyNow identified patients changing HTRP status after surgery. The VASP identified patients changing HTPR status after eight weeks (p = .018). CYP2C19 genotyping identified 13 slow metabolisers. In patients undergoing CEA, perioperative platelet reactivity measurements fluctuate over time with little agreement between platelet reactivity assays. Consequently, HTPR status of individual patients measured with the VerifyNow and VASP assay changed over time. Therefore, generally used perioperative platelet reactivity measurements seem unreliable for adjusting perioperative APT strategy.

Safety of Ticagrelor in Post – PCI (Percutaneous Coronary Intervention) Patients at a Tertiary Care Hospital

Background: Coronary artery disease (CAD) remains a leading cause of mortality and morbidity worldwide, despite advances in diagnosis and management. In acute coronary syndrome (ACS) patients, the combination of aspirin and clopidogrel is the standard therapy against recurrent cardiovascular events. However, clopidogrel has a delayed onset and variability in platelet response. Objective: To examine the safety profile of ticagrelor in post-PCI patients at a tertiary care hospital. Methods: This descriptive study was conducted at the Department of Cardiology, North-West General Hospital, Peshawar, from 1st April 2023 to 31st March 2024. Patients aged 30 to 80 years who underwent PCI and were started on ticagrelor were included. Baseline data such as age, gender, BMI, and cardiovascular risk factors (smoking, hypertension, diabetes mellitus) were collected. Patients received a loading dose of ticagrelor 180 mg orally followed by 90 mg twice daily. Adverse effects, including bleeding, dyspnea, and arrhythmias, were evaluated within 24 hours and at one-month follow-up. Data were analyzed using IBM SPSS version 25, with categorical variables presented as frequencies and percentages, and quantitative variables as means and standard deviations. Results: A total of 137 post-PCI patients participated in the study, with a mean age of 58 ± 10 years. The cohort included 85 men (62%) and 52 women (38%). The average BMI was 27.5 ± 3.5 kg/m². Risk factors included smoking (30%), hypertension (45%), and diabetes mellitus (25%). Within 24 hours, minor bleeding occurred in 10 patients (7.3%), dyspnea in 15 patients (10.9%), and arrhythmias in 4 patients (2.9%). At the one-month follow-up, minor bleeding was observed in 12 patients (8.8%), major bleeding in 2 patients (1.5%), dyspnea in 18 patients (13.1%), and arrhythmias in 5 patients (3.6%). Conclusion: The study supports the safety of ticagrelor as an antiplatelet therapy in post-PCI patients, with manageable adverse effects. However, careful monitoring for complications such as bleeding, dyspnea, and arrhythmias is recommended.

Navigating the Course of Dual Antiplatelet Therapy After Percutaneous Coronary Intervention: A Review of Guided Approaches.

Dual antiplatelet therapy (DAPT) is the standard approach to prevent thrombotic events in patients undergoing percutaneous coronary intervention and presenting with chronic or acute coronary syndromes. However, a sizeable proportion of patients presents with an impaired or unwarranted response to DAPT depending on genetic polymorphisms or variability in platelet response. Therefore, the concept of changing the type or dose of antiplatelet drugs based on the result of platelet function or genotype tests (ie, guided DAPT) has been introduced. The goal of guided DAPT is to intensify the antiplatelet potency in patients at high risk of thrombotic events (ie, escalation) and to decrease the antiplatelet potency in patients at high risk of bleeding (ie, de-escalation). This review aims to present an up-to-date and comprehensive overview of the latest research findings on DAPT modulation guided by either platelet function or genetic testing, discussing its current indications and future directions.

Analysis of a Naturally Occurring PAR4 Variant By Hydrogen/Deuterium Exchange Reveals Its Structural Rearrangement upon Activation By Thrombin and the Molecular Basis for Altered Reactivity

Protease activated receptors 1 and 4 (PAR1 and PAR4) are GPCRs that mediate thrombin signaling on human platelets. In this dual receptor system, PAR4 is responsible for sustained signaling required for stable platelet aggregation. In addition, common PAR4 polymorphisms affect its reactivity, which contributes to the variability in platelet response among individuals. The threonine residue 120 leads to a variant that is hyper-reactive to agonists and resistant to some antagonist compared to an alanine at this site. The molecular mechanisms by which this substitution influences PAR4 activation are not known. Further, the structural basis for PAR activation in general is also unknown. PARs are activated by enzymatic cleavage of the N-terminus exposing a tethered ligand that interacts with a binding pocket on the receptor's extracellular side. Since the ligand is attached to the receptor, there are likely substantial conformational changes that occur during PAR activation. The goal of this study was to determine the structural rearrangement of PAR4 upon activation by thrombin to test the hypothesis that polymorphisms at position 120 directly contribute to the ligand binding site and the subsequent PAR4 signaling cascades. Here, we used hydrogen-deuterium exchange mass spectrometry to map the conformational rearrangement of the hyper-reactive PAR4 variant (PAR4-120T). Full length human PAR4-120T was expressed in and purified from SF9 insect cells without additional stabilizing modifications. Our initial peptide mapping studies generated 93.5% (344/368) amino acid sequence coverage with multiple overlapping peptides. In the uncleaved (apo) state, the seven transmembrane domains of PAR4 show limited deuterium uptake as a result of being buried in detergent micelles. TM4 and TM5 displayed lower deuterium uptake than the other helices which potentially indicate the formation of dimerization interface, which is in agreement with our previous biochemical studies. Helix 8, a short α-helix between TM7 and C-terminus, showed reduced deuterium uptake. Together with other studies, this may indicate helix 8 plays a role in anchoring the receptor on lipid rafts. In comparison, the N- and C-termini displayed higher solvent accessibility based on the high deuterium uptake. Surprisingly, the tethered ligand sequence of PAR4 (GYPGQV) at the N-terminus has significantly lower solvent accessibility than neighboring regions in the apo state. This suggests it is less flexible and may be localized near the binding pocket even before thrombin cleavage. Comparison of deuterium uptake between apo and thrombin cleaved (active) states of PAR4-120T revealed reduced solvent accessibility at amino acids Cys149-Leu156 at TM3 and Ser329-Ser333 at TM7. This identifies the potential tethered ligand binding region of PAR4, which has not been previously described yet. Interestingly, in our molecular models Thr120 was located near this region spatially, which potentially explains the impact of this residue on PAR4 reactivity. We further observed an increase in deuterium uptake indicating a potential role for extracellular loop III and the connected regions of TM6 and TM7 in the receptor activation mechanism. In summary, our study on PAR4 is the first to elucidate the structural dynamics and molecular rearrangement of a protease activated receptor upon activation. Our results provide the overall molecular architecture of PAR4, uncovers the tethered ligand binding region, and further reveals the molecular basis for the inter-individual platelet reactivity based on the genotype of this key platelet receptor. Altogether, our studies may have significant clinical impact towards explaining the diversity of individual responses to existing and forthcoming therapies. DisclosuresNo relevant conflicts of interest to declare.

Adenosine Receptor Agonists Exhibit Anti-Platelet Effects and the Potential to Overcome Resistance to P2Y12 Receptor Antagonists.

Large inter-individual variation in platelet response to endogenous agonists and pharmacological agents, including resistance to antiplatelet therapy, prompts a search for novel platelet inhibitors and development new antithrombotic strategies. The present in vitro study evaluates the beneficial effects of three adenosine receptor (AR) agonists (regadenoson, LUF 5835 and NECA), different in terms of their selectivity for platelet adenosine receptors, when used alone and in combination with P2Y12 inhibitors, such as cangrelor or prasugrel metabolite. The anti-platelet effects of AR agonists were evaluated in healthy subjects (in the whole group and after stratification of individuals into high- and low-responders to P2Y12 inhibitors), using whole blood techniques, under flow (thrombus formation) and static conditions (study of platelet activation and aggregation). Compared to P2Y12 antagonists, AR agonists were much less or not effective under static conditions, but demonstrated similar antiplatelet activity in flow. In most cases, AR agonists significantly enhanced the anti-platelet effect of P2Y12 antagonists, despite possessing different selectivity profiles and antiplatelet activities. Importantly, their inhibitory effects in combination with P2Y12 antagonists were similar in high- and low-responders to P2Y12 inhibitors. In conclusion, a combination of anti-platelet agents acting via the P1 and P2 purinergic receptors represents a promising alternative to existing antithrombotic therapy.

PS1576 ADENOSINE DIPHOSPHATE RECEPTOR GENE (P2Y12) SEQUENCE VARIANTS AMONG SWEDISH, PALESTINIAN AND CONGOLESE INDIVIDUALS

Background:The P2Y12 receptor plays a central role in platelet aggregation and thrombus formation. Recently, inter‐individual variations in platelet response of healthy untreated individuals were established which was explained by genetic variations in the P2Y12 receptor gene. Several single nucleotide polymorphisms (SNPs) in the P2Y12 receptor have been associated with increased platelet reactivity and risk of cardiovascular diseases.Aims:This study aimed to evaluate the pathological H2 haplotype (using G52T as a tag‐SNP) and 18C&gt;T polymorphisms in three different ethnic groups; Palestinians, Swedish and Congolese. in addition to that, to investigate the effect of H2 haplotype pn palatelt aggregation induced by ADP/TRAP agonists.Methods:The H2 haplotype and 18C&gt;T SNPs were determined in conveniently selected healthy individuals from different ethnic groups (n = 254). The whole exon‐3 of P2Y12 was sequenced and analyzed by using ABI PRISM 310 Genetic Analyzer. The major and the minor allele frequencies of the P2Y12 SNPs were determined in the study population and the genetic differences between ethnic groups in P2Y12 were elucidated. In addition, the frequency of the genotypes were calculated among the different ethnic groups.Results:In this study, five benign single nucleotide polymorphisms (SNPs) were genotyped and identified; 18C&gt;T, 36G&gt;T, 162G&gt;T, 546C&gt;T and 989A&gt;G. The overall frequencies of each SNP in the study population (n = 254) was 21.9, 10, 0.4, 0.6 and 0.4%, respectively. The frequency of H2 haplotype was among Swedish 23.6% (n = 55), Congolese 12.0% (n = 54) and Palestinian 4.1% (n = 145). There were significant differences in frequency of H2 haplotype and 18C&gt;T among the ethnic groups (P &lt; 0.001). In regard to the pathological SNPs (767G&gt;A [p. Arg256Gln] and (793C&gt;T [Arg265Trp]) all of the study participants were negative.Summary/Conclusion:There are significant differences in the frequencies of the genetic variants of the P2Y12 exon‐3 between the studied ethnic groups. Application of these findings may explain the variable platelet response in healthy individuals and in patient under thienopyridine compounds. Further studies should be performed to study the effect of the genetic variations effect on ADP or TRAP‐ induced platelet aggregation.

Effects of genetic variation in protease activated receptor 4 after an acute coronary syndrome: Analysis from the TRACER trial

Variation in platelet response to thrombin may affect the safety and efficacy of PAR antagonism. The Thr120 variant of the common single nucleotide polymorphism (SNP) rs773902 in the protease-activated receptor (PAR) 4 gene is associated with higher platelet aggregation compared to the Ala120 variant. We investigated the relationship between the rs773902 SNP with major bleeding and ischemic events, safety, and efficacy of PAR1 inhibition in 6177 NSTE ACS patients in the TRACER trial. There was a lower rate of GUSTO moderate/severe bleeding in patients with the Thr120 variant. The difference was driven by a lower rate in the smaller homozygous group (recessive model, HR 0.13 [0.02–0.92] P = 0.042). No significant differences were observed in the ischemic outcomes. The excess in bleeding observed with PAR1 inhibition was attenuated in patients with the Thr120 variant, but the interactions were not statistically significant. In summary, lower major bleeding rates were observed in the overall TRACER cohort with the hyperreactive PAR4 Thr120 variant. The increase in bleeding with vorapaxar was attenuated with the Thr120 variant, but we could not demonstrate an interaction with PAR1 inhibition. These findings warrant further exploration, including those of African ancestry where the A allele (Thr120) frequency is ~65%.

Baseline platelet parameters for predicting early platelet response and clinical outcomes in patients with non-cardioembolic ischemic stroke treated with clopidogrel.

PurposeThe present study investigated whether routine baseline platelet parameters(BPPs) detected before clopidogrel therapy in acute non-cardioembolic ischemic stroke(NCIS) could predict early platelet response and future clinical outcomes.ResultsThe CYP2C19 polymorphisms constituted independent risk factors for LCR. The number of female patients, the incidence of diabetes mellitus (DM), the level of low-density lipoprotein(LDL) cholesterol, and the neutrophil-to-lymphocyte ratio(NLR) were significantly high in the clinical clopidogrel resistance (CCR) group. However, none of the BPPs had a significant association with laboratory clopidogrel resistance (LCR) or discriminated with the cut-off values regarding LCR or CCR. The patients were divided into two groups according to the average mean platelet volume(MPV) or platelet count(PC). We found that the HbA1c level, the number of female patients, and the CCR were higher in the groups with elevated MPV (≥ 10.6fL) and PC (≥ 235 × 109/L); the LCR, the NIHSS score at discharge, and elevated MPV and PC were risk predictors for CCR.Materials and MethodsThis study included 196 patients with acute NCIS who underwent routine blood tests upon admission, were treated with clopidogrel, and were followed up for 6 months. Early platelet response was assessed and the CYP2C19 genetic variants were screened for. All participants were categorized into either laboratory clopidogrel resistance(LCR) or clinical clopidogrel resistance (CCR) groups.ConclusionsElevated baseline MPV and PC before clopidogrel therapy, as well as CYP2C19 gene variants, should be included in a risk algorithm for NCIS. Furthermore, other nongenetic clinical risk factors should be assessed for optimal prediction of the risk for thrombotic events because of individual variability in platelet response to clopidogrel.

Platelet function testing as a biomarker for efficacy of antiplatelet drugs.

Despite the overwhelming evidence in support of the efficacy of dual antiplatelet therapy with aspirin and clopidogrel, it is also obvious that not all patients benefit from these drugs to the same extent. This interindividual variability in platelet responses may underlie clinical differences in drug efficacy, with potential for optimization of antiplatelet therapy to prevent ischemic events without excessively increasing bleeding risk. This review presents the current evidence regarding platelet function testing for monitoring of antiplatelet therapy, with emphasis on the prognostic value of platelet function testing to predict ischemic and bleeding events. The potential of platelet function testing to provide personalized antiplatelet therapy is also discussed, with an outlook toward the future of platelet function testing in high-risk individuals.

Association of the C3435T Multi-Drug Resistance Gene-1 (MDR-1) Polymorphism with Clopidogrel Resistance among Moroccan Acute Coronary Syndromes (ACS) Patients

Background: Antiplatelet drugs are recommended as one of the main medications used in Acute Coronary Syndromes (ACS) patients. However, an inter-individual variability in platelet response to Clopidogrel has been found in a substantial group of them. Genetic is known be the dominant influencing factor for individual and interethnic variations in drug responses. Polymorphisms in the genes involved in Clopidogrel absorption, biotransformation to the active metabolite, or platelet response to adenosine diphosphate (ADP) have been associated to this impaired response. Interestingly, Multi-Drug Resistance gene-1 (MDR-1) polymorphism influences oral bioavailability of clopidogrel and prognosis of ACS patients. The objectives of our study are, first, to determine the frequency of the C3435T MDR1 polymorphism among Moroccan ACS patients compared to healthy subjects; and second, to assess its effect on Clopidogrel response in a sample of Moroccan ASC patients. Methods and results: 40 ACS patients were recruited and compared to 99 healthy controls. Extracted DNA samples were genotyped by PCR-RFLP method using MboI restriction enzyme. The VerifyNow assay was used to evaluate platelet function among ACS patients. Our results showed that HTA, Smoking, Creatinine and Sex were statistically associated to Clopidogrel resistance (P=0.05; P=0.05; P=0.05 and P=0.04 respectively). 63.64% of the ST (+) patients were carrying the mutant allele, 54.5% of them having the heterozygous genotype and 36.4% the homozygous mutant one, compared to 9.1% having the homozygous wild type genotype. 62.5% of the resistant group was carrying the mutant allele (50% of them had the TT mutant genotype, 25% CT and 25% CC profiles). Among cases, 42.5% were homozygous mutant TT, 35% CC and 22.5% CT, compared to 39.4% CC, 51.5% CT and 9.1% TT among healthy controls. This polymorphism was positively correlated to ACS risk of development in the CT genotype and the additive transmission model (OR [95% CI]=0.49 [0.16-0.99], P=0.002; OR [95% CI]=2.17 [0.94-2.72], P=0.02), increasing-thus- the association of this polymorphism with the risk of pathology occurrence. Conclusion: To the best of our knowledge, our study is the first in Morocco to assess the effect of the C3435T MDR1 polymorphism on Clopidogrel response in a sample of Moroccan ACS patients; we also tried to explore the frequency of this polymorphism among Moroccan ACS patients and compare them to healthy individuals. The distribution of the mutant allele in Clopidogrel resistance groups, among ACS sub-types, and also in cases compared to controls and there correlations, suggest a potential association of this variant with Clopidogrel resistance and ACS occurrence risk in our population. Understanding the functional and clinical consequences of this MDR1 variant and others may provide a basis for treating patients more effectively. If this variability in response could be assigned to a mutation in the MDR1 gene, patients could be screened and appropriate dose adjustments could be made on the basis of their MDR1 genotype.

Genetically Determined Platelet Reactivity and Related Clinical Implications.

Many drugs are nowadays available to inhibit platelet activation and aggregation, especially in patients with acute coronary syndromes and undergoing percutaneous coronary intervention with stent implantation. Primary targets are represented by enzymes or receptors involved in platelet activation. Genetic mutations in these targets contribute to the inter-individual variability in platelet responses therefore weakening the efficacy of antiplatelet agents. High on treatment platelet reactivity is a condition characterized by low levels of platelet inhibition despite the use of antiplatelet drugs. This could be responsible for re-infarction, stent-thrombosis and strokes, affecting short and long-term prognosis after coronary revascularization. So far, to test antiplatelet resistance either the assessment of platelet function or the identification of genetic carriers of poly morphisms have been pursued. Although several methods are now available to test platelet reactivity, it is still debated whether its routine assessment gives real benefits in clinical practice. The present review aims at examining current evidences on genetic polymorphisms affecting optimal platelet inhibition.

Influence of FcγRIIA H131R Polymorphism on the Risk of Thrombosis in HIT and Variable Effect of Normal IgG on Platelet Activation

Introduction. Heparin-induced thrombocytopenia (HIT) results from an atypical immune response to platelet factor 4/heparin complexes (PF4/H), with rapid synthesis of IgG antibodies that activate platelets via FcγRIIA receptors. HIT is associated with thrombotic complications occurring in about 50% of affected patients.Moreover, the response of platelets to anti-PF4/H IgG varies from one subject to another. This variability, partly explained by gene polymorphisms affecting FcγRIIA signaling (Rollin et al, Blood 2012), might also depend on variations in the binding of HIT antibodies to FcγRIIA receptors.Objective. The influence of FcγRIIA H131R polymorphism on the risk of HIT and thrombosis was investigated in parallel with the effect of normal IgG on the platelet response to HIT antibodies.Patients and Methods. A cohort of 89 patients with definite HIT (PF4-specific ELISA and serotonin release assay both positive) and two control groups with (Abpos ; n=160) or without (Abneg ; n=179) significant levels of PF4-specific antibodies after cardiac surgery were studied. Platelet aggregation tests (PAT) were performed in 59 healthy donors using HIT plasma samples or 5B9, a humanized chimeric monoclonal antibody specific to PF4/H complexes that we have recently developed which fully mimics the effects of human HIT antibodies. The influence of normal IgG and IgG subclasses on platelet aggregation was also evaluated according to the FcγRIIA H131R polymorphism. Finally, tissue factor mRNA levels and plasma phospholipid-dependent procoagulant activity were measured after addition of HIT antibodies and heparin in the whole blood collected from healthy donors with different FCGR2A genotypes.Results. Genotypes and allele frequencies were similar in the HIT patients and both Abneg and Abpos controls. However, interestingly, homozygous FcγRIIA 131RR subjects were more frequent among the HIT patients who had developed thrombotic complications compared to those without thrombosis (34.5% vs. 8.5%, respectively, p=0.008). The FcγRIIA 131RR genotype therefore appeared to increase the risk of thrombosis in HIT significantly (OR = 5.9; 95% CI 1.7-20). This association was not found in two other cohorts of patients who had developed ischemic stroke or venous thromboembolism in a medical context other than HIT, thus indicating that the impact of FcγRIIA H131R polymorphism on the risk of thrombosis was specific to HIT. PAT performed with platelet-rich plasma (PRP) showed that the lag time measured in response to 5B9 with heparin (5B9/H) was significantly longer with FcγRIIA 131HH platelets compared to RR platelets (p=0.03). Importantly, this variable platelet response to 5B9/H related to the FcγRIIA polymorphism was no longer detectable when PAT were performed with washed platelets, when the IgG fraction had been removed from the plasma, and when collagen was used as inducer. On the other hand, the addition of human polyvalent IgG to IgG-depleted PRP inhibited the aggregation induced by 5B9/H, but lower concentrations of IgG were required to achieve this effect in FcγRIIA 131HH subjects compared to RR donors. Importantly, this variable effect of normal IgG on platelet aggregation induced by HIT antibodies was shown to depend mainly on the IgG2 subclass, which binds to the H131 isoform more effectively, whereas IgG1 exerted a similar inhibitory effect in RR and HH homozygous donors. Finally, a stronger increase in TF mRNA levels was also evidenced in homozygous FcγRIIARR donors compared to homozygous FcγRIIAHH donors after incubation of 5B9 and heparin in whole blood. Moreover, this effect was associated with a greater relative shortening of plasma clotting time indicating that the release of procoagulant phospholipid microparticles was more pronounced in RR subjects in response to HIT antibodies (p=0.04).Conclusion. These results demonstrate that HIT patients homozygous for the FcγRIIA 131R allele have a higher risk of thrombosis, probably due to an increased cell activation induced by antibodies to PF4/H with a lower inhibitory effect of endogenous IgG, especially of the IgG2subclass. DisclosuresNo relevant conflicts of interest to declare.

Pharmacology of the New P2Y12 Receptor Inhibitors: Insights on Pharmacokinetic and Pharmacodynamic Properties

The P2Y(12) receptor is a key player in platelet activation and represents an effective pharmacological target for the inhibition of platelet aggregation and prevention of atherothrombotic events. Indeed, the clinical use of the P2Y(12) receptor inhibitor clopidogrel is an effective strategy for inhibiting platelet activity in patients with acute coronary syndrome, and for preventing thrombotic events in those undergoing percutaneous coronary intervention with stenting. However, clopidogrel has several drawbacks, which include delayed onset of action, large inter-individual variability in platelet response, genetic polymorphism of the metabolizing enzyme, drug-drug interactions (DDIs), and the two-step activation process catalyzed by a series of cytochrome P450 (CYP) isoenzymes. For these reasons, new P2Y(12) receptor inhibitors have been developed in an attempt to improve on the pharmacological and clinical profile of clopidogrel. Three new P2Y(12) receptor inhibitors--prasugrel, cangrelor, and ticagrelor--have arrived, and more are coming into clinical use. Each of these antagonists has individual properties and, according to their mechanism of inhibition, can be divided into irreversible (prasugrel) and reversible inhibitors (ticagrelor, cangrelor). These agents also have different metabolic pathways: prasugrel is a prodrug that requires metabolic activation through a cytochrome-dependent pathway, while ticagrelor and cangrelor do not require metabolic conversion. However, ticagrelor is a CYP3A4 substrate/inhibitor and thus it can be involved in DDIs. Indeed, ticagrelor significantly increases the plasma levels of CYP3A4 substrates such as statins. Moreover, concomitant use with strong CYP3A4 inhibitors (such as ketoconazole, itraconazole, clarithromycin, ritonavir, telithromycin, etc.) is contraindicated, while the co-administration of ticagrelor with potent CYP3A inducers (carbamazepine, rifampicin, phenytoin, phenobarbital) is discouraged. Prasugrel and ticagrelor determine a faster, greater, and more consistent adenosine diphosphate (ADP)-receptor inhibition than clopidogrel, with a near complete inhibition of platelet aggregation between 1-2 h after administration of an oral loading dose, while cangrelor shows a rapid and potent platelet inhibitory effect with intravenous infusion. Thus, the different pharmacokinetic and pharmacodynamic characteristics of the P2Y(12) receptor inhibitors enable clinicians to personalize therapy according to patient-specific medical requirements for better prevention of atherothrombotic events. In the present review, we describe the pharmacological properties, the pharmacokinetic and pharmacodynamic differences, and the clinical efficacy of the currently available P2Y(12) receptor inhibitors.

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Variation in platelet response may affect the hemodynamic response to vasoreactivity testing in patients with secondary pulmonary hypertension due to left heart disease

Cardiologia CROATICA Objectives: Pulmonary hypertension (PH) and vascular resistance in patients with pulmonary arterial hypertension (PAH) are caused by remodeling and thrombosis of small and medium sized pulmonary arteries and arterioles, as well as vasoconstriction. These patients have increased platelet aggregation and the activated platelets are major source of thromboxane A2 which is a strong vasopressor and proaggregation molecule. PGI2 and PGE1 have opposite effects. Misbalance in eicosanoids synthesis was observed in patients with PH. The results from vasoreactivity test, in which we measure the hemodynamic response to vasodilator, particularly change in mean pulmonary pressure (mPAP), pulmonary vascular resistance (PVR) and transpulmonary gradient (TPG) in patients with PH due to the left heart disease, influence the decision on future course of treatment. The goal: to test whether the change of platelet aggregation during vasoreactivity testing is related to hemodynamic response measured as the change in mPAP, PVR and TPG. Patients and Methods: Our pilot study included 38 patients with secondary PH due to the left heart disease, 29 men (76%) and 9 women (24%). The right heart catheterization was performed in all patients and vasoreactivity testing with PGE1 in 19 patients (50%). Platelet aggregation induced by addition of AA (ASPI test), ADP (ADP test) and collagen (COL-test) was measured in blood samples from pulmonary artery with Multiplate and repeated after vasoreactivity testing. Results: Patients with the reduction of platelet aggregation in ASPI test had stronger, but insignificant reduction in mPAP (p=0.08) and PVR (p=0.15). Significant reduction was observed in the reduction of TPG (p=0.03). The reduction of platelet aggregation in ADP test was not related to hemodynamic response to PGE1. Patients with the reduction of aggregation in collagen test had stronger, though insignificant reduction in mPAP (p=0.08). However, they had significant reduction in PVR (p=0.01) and TPG (p=0.05) in comparison to those with increased aggregation. Conclusion: The data analysis in our pilot study shows insufficient size of the sample to reach a final conclusion. However, it suggest a significant potential difference in vasoreactivity among patients who respond with a decrease and those who respond with an increase in platelet aggregation after testing with PGE1. We hypothesize that variations in platelet response to PGE1 modulate its hemodynamic effect on pulmonary circulation.

Influence of genetic polymorphisms on platelet function, response to antiplatelet drugs and clinical outcomes in patients with coronary artery disease

Platelet activation and aggregation play important roles in ischemic event occurrences in patients with coronary artery disease. In the absence of a disease state and drug administration, platelet reactivity has been shown to be stable over time, indicating a high level of heritability. Interindividual variability in platelet response to agonists and in response to aspirin and clopidogrel administration along with the influence of different single nucleotide polymorphisms on platelet reactivity based on candidate gene and genome-wide association studies have been demonstrated in healthy subjects. Reduced pharmacodynamic effect and reduced clinical efficacy of clopidogrel have been well documented in high-risk coronary artery disease patients carrying a loss-of-function allele of the CYP2C19 gene. These factors are recognized by the recent American and European treatment guidelines. However, prasugrel and ticagrelor are associated with superior and predictable pharmacodynamic responses and better clinical outcomes compared with clopidogrel.

Clopidogrel

Dual antiplatelet therapy with acetylsalicylic acid (aspirin) and clopidogrel is a guideline-recommended standard of care for patients with acute coronary syndromes (ACS) and those who undergo percutaneous coronary intervention (PCI). Despite a large body of clinical evidence obtained from randomized clinical trials and patient registries supporting the efficacy and safety of aspirin plus clopidogrel therapy in these patients, questions concerning the optimal use of dual antiplatelet therapy remain. Widely debated topics pertaining to dual antiplatelet therapy in patients with ACS or undergoing PCI include (i) the appropriate clopidogrel loading dose; (ii) the optimal time to initiate the clopidogrel loading dose; (iii) the optimal duration of dual antiplatelet therapy following ACS or PCI; (iv) impact of variability of platelet response on patient outcomes; and (v) the role of other recommended and emerging P2Y₁₂ antagonists. This review discusses these ongoing controversies regarding the optimal use of dual antiplatelet therapy with aspirin and clopidogrel in patients with ACS or those undergoing PCI.

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.

Current antiplatelet options for NSTE-ACS patients

Non-ST elevation (NSTE) myocardial infarction and unstable angina are the most common clinical presentations of acute coronary syndrome (ACS). Platelet activation is central to the pathogenesis of NSTE-ACS and consensus guidelines that advocate early revascularization supported by intensive antiplatelet therapy. This review examines the drugs used concurrently with aspirin as dual antiplatelet therapy in the NSTE-ACS setting. Clopidogrel represented an important therapeutic advance. However, variations in platelet response and a relatively slow onset of action compromise outcomes with clopidogrel. Evidence reviewed in this article shows that in NSTE-ACS patients, ticagrelor and prasugrel are more effective than clopidogrel and are relatively well tolerated, with an acceptable and manageable bleeding risk. The literature suggests several differences between ticagrelor and prasugrel that should allow clinicians to better tailor treatment to the patient. Head-to-head comparisons are now needed to compare directly the risks and benefits of ticagrelor and prasugrel in NSTE-ACS. Further studies also need to address other outstanding issues such as the benefits and risks of prasugrel pre-treatment and to stratify efficacy and tolerability according to diabetes mellitus (DM) and other co-morbidities. In the meantime, the issues discussed in this review should enhance clinicians' ability to optimize and individualize NSTE-ACS treatment, thereby further reducing the morbidity and mortality associated with this common cardiovascular condition.