AA-induced Platelet Aggregation Research Articles

Abstract 3097: Platelet COX-1 Dependent And Independent Heterogeneity In Response To Low-Dose Aspirin

Background: Platelets are key mediators of thrombosis and hemostasis. While aspirin decreases cardiovascular events in high-risk populations, its pharmacological and therapeutic effect is limited by heterogeneity in response and its effect on COX-1 dependent and independent pathways. We sought to investigate the heterogeneity of response of low-dose (81mg/day) aspirin. Methods: Following an overnight fast, 39 healthy controls had 4 weekly phlebotomies and received 81 mg aspirin daily between weeks 3 and 4. At each collection, platelet activity was measured using light transmission aggregation (LTA) in response to arachidonic acid (AA [1.5 mM]; COX-1 dependent ) and epinephrine (2.0 μM), ADP (2.0 μM), and collagen (1.0 μM; COX-1 independent pathways). Results: Median age was 27 (21, 59) years, 48.7% were female, 53.9% were white, and 7.7% were Hispanic. Median platelet aggregation in response to AA (1.5 mM) was 90.9%, 91.6%, and 91.4% in weeks 1, 2, and 3, demonstrating a robust reproducible response. Baseline AA-induced platelet aggregation was modestly correlated with ADP- (r=0.28, p=0.054), collagen- (r=0.42, p=0.0036), and epinephrine-induced (r=0.32, p=0.029) platelet aggregation. Following 1 week of aspirin, AA-induced platelet aggregation decreased to 11.8% (p<0.0001). The median (interquartile range) platelet inhibition in response to AA was 87.3% (78.5%, 92.5%) demonstrating a robust COX-1 dependent response. Platelet inhibition in response to epinephrine (52.1% [46.4, 62.6]), collagen (79.5% [70.3, 87.4]), and ADP (29.8% [22.0, 36.0]), albeit statistically significant, was less robust. While platelet inhibition in response to AA was correlated to platelet inhibition in response to collagen (r=0.64 p=1.12E-5) and epinephrine (r=0.44, p=0.0052), there was no correlation in response to ADP (r=-0.04, p=0.81) induced platelet inhibition. Conclusions: Our findings demonstrate a robust COX-1 dependent aggregation response and a more heterogeneous COX-1 independent aggregation response to low-dose aspirin. The mechanism and clinical significance of these findings require further study.

Early assessment of the pharmacokinetic and pharmacodynamic effects following acetylsalicylic acid loading: toward a definition for acute therapeutic response.

Despite decades of investigations, the optimal assessment of the "therapeutic response" to early after loading dose of acetylsalicylic acid (ASA) remains unclear. Limited information is available on the relation between pharmacodynamic (PD) and pharmacokinetic (PK) measurements assessed immediately after ASA administration. Serial PD and PK analyses were performed immediately after a single 162 or 650mg dose of chewed and swallowed ASA in ten healthy adults. ASA response was defined as > 95% inhibition of serum thromboxane (Tx)B2, < 550 aspirin reaction units (ARU) by VerifyNow Aspirin (VN) test, and ≤ 20% arachidonic acid (AA)-induced platelet aggregation (PA). Correlation analyses between PK and PD measurements and receiver operating characteristic (ROC) curve analyses were performed. ASA response measured by VN test and AA-induced PA was achieved within 30min of ASA administration. A correlation was observed between ARU and AA-induced maximum PA (r = 0.69, p < 0.001), serum TxB2 (r = 0.74 and p < 0.001), and serum TxB2 inhibition (r = 0.79, p < 0.001). In ROC curve analyses, ≤ 558 ARU and ≤ 7% AA-induced PA were associated with > 95% inhibition of TxB2. 686ng/ml plasma ASA cut-off point was associated with > 95% inhibition of serum TxB2, ≤ 7% 1mM AA-induced PA, and ≤ 585 ARU. A modest ~ 50% inhibition of TxB2 inhibition was associated with marked inhibition of 1mM AA-induced platelet aggregation by LTA. Our analyses demonstrated important relationships between pharmacodynamic, and pharmacokinetic parameters measured immediately following oral ASA and cutoff values for ARU and AA-induced PA that is associated with > 95% inhibition of serum TxB2.

Abstract 13963: Redefining the Assessment of “Therapeutic” Platelet Response to Acetylsalicylic Acid

Background: Despite extensive investigations, the optimal assessment of the “therapeutic response” to acetylsalicylic acid (ASA) remains unclear. Limited information is available on the relation between serum thromboxane (Tx)B 2 ; arachidonic acid (AA)-induced platelet aggregation (PA) by light transmittance aggregometry (LTA); aspirin reaction units (ARU) by VerifyNow aspirin test (VN), and pharmacokinetics (PK). Methods: Serial PD and PK analyses were performed immediately after a single 162 or 650 mg dose of chewed and swallowed ASA in ten healthy volunteers. ASA response was defined as &gt;95% inhibition of serum TxB 2, &lt;550 ARU by VN test and ≤20% AA-induced PA. Correlation analyses between PK and PD measurements and receiver operating characteristic (ROC) curve analyses were performed. Results: ASA Response measured by VN, and AA-induced PA was achieved within 30 minutes of ASA administration. A good correlation was observed between VN ARU and 1 mM AA-induced maximum PA (r=0.80, p&lt;0.001), serum TxB 2 (r=0.76, p&lt;0.001), and inhibition of serum TxB 2 (r=0.87, p&lt;0.001). The relation between platelet aggregation and TxB 2 inhibition appears to be dichotomous, with only modest inhibition of TxB 2 (~50% inhibition) associated with marked inhibition of platelet aggregation. ROC curve analyses indicated that ≤558 ARU and ≤7% AA-induced PA were associated with &gt;95% inhibition of TxB 2 . A plasma ASA 686 ng/mL cut-off was associated with &gt;95% inhibition of serum TxB 2 , ≤7% 1 mM AA-induced PA by LTA, and ≤585 ARU. Conclusion: Our study demonstrated an important relationship between PD and PK parameters measured immediately following oral ASA and cutoff values for VN assay and LTA that is associated with &gt;95% inhibition of serum TxB 2 . We demonstrated that ~50% inhibition of TxB 2 is required to reach high levels of platelet function inhibition.

The synergistic effect of EPA and DHA with cyclooxygenase-1 inhibitors on platelet aggregation

Aim: Consumption of omega-3 fatty acids docosahexaenoic acid (DHA, 22:6ω-3) and eicosapentaenoic acid (EPA, 20:5ω-3) from a variety of foods and supplements could reduce cardiovascular events. The antiplatelet drugs aspirin (ASA) and triflusal, bind to platelet cyclooxygenase-1, thus preventing the biosynthesis of thromboxane A2 from arachidonic acid. ASA, triflusal, and their combinations with DHA or EPA were evaluated against in vitro platelet aggregation induced by the agonists AA, ADP, or TRAP-6. Materials-Methods: Platelet Rich Plasma (PRP), isolated from blood of healthy volunteers was pre-incubated with acetylsalicylic acid, triflusal, EPA, DHA, or the drug-omega-3 combinations at various concentrations for 10min at 37ºC, prior to activation by AA, TRAP-6, and ADP (0.3mM, 10μM and 6μM, respectively). Platelet aggregation was determined by Light Transmittance Aggregometry. Results: DHA significantly improves ASA and triflusal’s inhibitory effect towards AA-induced platelet aggregation, while EPA enhanced only the antiplatelet activity of ASA. Moreover, only the combination of ASA with EPA exhibited enhanced inhibitory effect against platelet aggregation induced by ADP. Furthermore, the inhibition of ASA and triflusal on TRAP-6-induced platelet aggregation was enhanced when ASA or triflusal were combined with EPA. Conclusions: Both DHA and EPA inhibit AA, ADP and TRAP-6-induced platelet aggregation in a dose dependent manner. The results of this study suggest a potentially beneficial use of EPA and DHA supplementation, in conjunction with the antiplatelet drugs ASA and triflusal.

Development of a Novel Apigenin Dosage form as a Substitute for the Modern Triple Antithrombotic Regimen

The simultaneous administration of three antiplatelet agents has been proposed as an efficient strategy for the secondary prevention of atherothrombotic events and is included in the European guidelines. However, this strategy presented an increased risk of bleeding; therefore, the identification of new antiplatelet agents, with improved efficacy and diminished side effects, is of great importance. In silico studies, UPLC/MS Q-TOF plasma stability, in vitro platelet aggregation experiments, and pharmacokinetic studies were exploited. In the present study, it has been predicted that the flavonoid apigenin could target different platelet activation pathways, including P2Y12, protease-activated receptor-1 (PAR-1), and cyclooxygenase 1 (COX-1). To enhance apigenin's potency, hybridization with docosahexaenoic acid (DHA) was performed, as fatty acids have illustrated potent efficacy against cardiovascular diseases (CVDs). The new molecular hybrid, termed 4'-DHA-apigenin, demonstrated enhanced inhibitory activity against platelet aggregation induced by thrombin receptor activator peptide-6 (TRAP-6), adenosine diphosphate (ADP), and arachidonic acid (AA), with respect to the parent apigenin. The 4'-DHA-apigenin hybrid illustrated an almost 2-fold enhanced inhibitory activity, with respect to apigenin, and an almost 3-fold enhanced inhibitory activity, with respect to DHA, for the ADP-induced platelet aggregation. Additionally, the hybrid presented a more than 12-fold enhanced inhibitory activity with respect to DHA for the TRAP-6 induced platelet aggregation. Furthermore, a 2-fold enhanced inhibitory activity was recorded for the 4'-DHA-apigenin hybrid for the AA-induced platelet aggregation with respect to apigenin. To surmount the reduced LC-MS based plasma stability, a novel dosage form in olive oil has been developed. The 4'-DHA-apigenin olive oil-based formulation presented an enhanced antiplatelet inhibitory effect in three activation pathways. To further explore the pharmacokinetic profile of 4'-DHA-apigenin in olive oil formulations, a UPLC/MS Q-TOF protocol has been established to quantify the serum levels of apigenin after oral administration to C57BL/6J wild type mice. The olive oil-based formulation of 4'-DHA-apigenin demonstrated an increase in apigenin bioavailability of 262 %. This study may offer a new therapeutic strategy tailored to improve the treatment of CVDs.

Platelet Aggregation in Apparently Healthy Elderly Populations: Reference Ranges and Influence of Hematology Parameters.

Reference intervals based on younger populations may not apply to elderly populations. The aim of the current study was to calculate reference ranges for platelet aggregation in apparently healthy Chinese elderly populations and analyze the impact of gender, age, and hematological parameters on platelet aggregation in vitro. A total of 138 males and 161 females were enrolled based on stringent inclusion and exclusion criteria. Platelet aggregation was measured with sodium citrate anticoagulation whole blood samples using a PL12 analyzer, triggered by adenosine-5'-diphosphate (ADP) and arachidonic acid (AA) agonists. Hematological parameters were measured using a Sysmex XE2100 instrument. In the total sample tested in this study, the platelet aggregation induced by AA and ADP was not dependent on age (p > 0.05) but gender. Platelet aggregation triggered by ADP and AA was more enhanced in females than males (p = 0.024 for AA, p = 0.036 for ADP). The recommended reference values of AA-induced platelet aggregation were 48.42% - 85.93% for males and 43.62% - 87.80% for females. The recommended reference values of ADP-induced platelet aggregation were 38.12% - 80.21% for males and 40.12% - 83.05% for females. Furthermore, for all agonists, a positive correlation was found between platelet aggregation and platelet count. The ADP-triggered aggregation showed a significant positive correlation with white blood cell (WBC) count and a negative correlation with red blood cell (RBC) count. Moreover, platelet aggregation showed no significant correlation with mean platelet volume or hemoglobin concentration. Combined and gender-specific reference ranges for platelet aggregation in healthy elderly populations were established in whole blood measured by a sequential platelet counting method.

Genetic Association Study and Machine Learning to Investigate Differences in Platelet Reactivity in Patients with Acute Ischemic Stroke Treated with Aspirin.

Aspirin resistance (AR) is a pressing problem in current ischemic stroke care. Although the role of genetic variations is widely considered, the data still remain controversial. Our aim was to investigate the contribution of genetic features to laboratory AR measured through platelet aggregation with arachidonic acid (AA) and adenosine diphosphate (ADP) in ischemic stroke patients. A total of 461 patients were enrolled. Platelet aggregation was measured via light transmission aggregometry. Eighteen single-nucleotide polymorphisms (SNPs) in ITGB3, GPIBA, TBXA2R, ITGA2, PLA2G7, HMOX1, PTGS1, PTGS2, ADRA2A, ABCB1 and PEAR1 genes and the intergenic 9p21.3 region were determined using low-density biochips. We found an association of rs1330344 in the PTGS1 gene with AR and AA-induced platelet aggregation. Rs4311994 in ADRA2A gene also affected AA-induced aggregation, and rs4523 in the TBXA2R gene and rs12041331 in the PEAR1 gene influenced ADP-induced aggregation. Furthermore, the effect of rs1062535 in the ITGA2 gene on NIHSS dynamics during 10 days of treatment was found. The best machine learning (ML) model for AR based on clinical and genetic factors was characterized by AUC = 0.665 and F1-score = 0.628. In conclusion, the association study showed that PTGS1, ADRA2A, TBXA2R and PEAR1 polymorphisms may affect laboratory AR. However, the ML model demonstrated the predominant influence of clinical features.

Study the Association of Nucleotide Polymorphisms in Platelet Receptor and Cytochrome P450 Genes with the Development of Resistance to Antiplatelet Drugs in Patients with Coronary Artery Disease

Aim. To study the association of nucleotide polymorphisms in platelet receptor and cytochrome P450 genes with the development of resistance to antiplatelet drugs in CHD patients.Material and Methods. The study included 243 patients diagnosed with CHD after coronary artery bypass surgery (CABG), including 140 patients in the acetylsalicylic acid (ASA) treatment group and 103 patients in the dual antiplatelet therapy (DAT) group. All patients were tested for platelet aggregation using an optical aggregometer with inducers: 5 mM ADP and 1 mM arachidonic acid (AA). DNA samples were analyzed by allele-specific PCR for the presence of polymorphisms rs2046934, rs1126643, rs5918, rs6065, rs4244285 in the platelet receptor and cytochrome P450 genes.Results. No statistically significant differences were found during comparison of the prevalence of the studied polymorphisms in the platelet receptor and cytochrome P450 genes between the groups of aspirin-sensitive and aspirin-resistant patients, as well as between the groups of clopidogrelsensitive and clopidogrel-resistant patients. No association between carriage of the minor and major alleles of the polymorphisms studied and the development of antiplatelet drug resistance was found. In the group of patients on ASA therapy, carriers of the C allele of the T1565C (rs5918) ITGB3 polymorphism had a higher rate of AA-induced platelet aggregation compared to carriers of the T allele (18,49±25,92 vs 10,43±17,34, р=0,004).Conclusion. Polymorphisms of P2RY12 (rs2046934), ITGA2 (rs1126643), ITGB3 (rs5918), GP1BA (rs6065), CYP2C19*2 (rs4244285) genes are not associated with antiplatelet drug resistance in both patients on ASC therapy and on DAT. The presence of minor alleles of the rs2046934, rs1126643, rs6065, rs4244285 polymorphisms are not associated with increased platelet aggregation activity before CABG.However, in the group of patients on ASA therapy C-allele carriers of the rs5918 polymorphism of the ITGB3 gene had a higher rate of AA-induced platelet aggregation compared to T-allele carriers.

Exploring the Q-markers of Angelica sinensis (Oliv.) Diels of anti-platelet aggregation activity based on spectrum-effect relationships.

The radix of Angelica sinensis (Oliv.) Diels (RAS) is widely used in medicinal and dietary applications in China, and has the function for replenishing and invigorating the blood, stopping pain and moistening the intestines. In this study, RAS from the main geoherb regions showed better efficacy in inhibiting Adenosine diphosphate- or arachidonic acid-induced platelet aggregation than those from non-geoherb regions. In addition, the HPLC fingerprints of 30 batches of RAS, as part of the comprehensive evaluation of RAS, were established and used for spectral efficiency to screen the quality markers for anti-platelet aggregation activities. Five compounds in RAS-senkyunolide I, uridine, guanine, ferulic acid and adenosine-were demonstrated to contribute significantly to the anti-platelet aggregation activity. These bioactive compounds, especially senkyunolide I and ferulic acid with stronger activities, could be used as quality markers of RAS for quality control of RAS.

Effects of ticagrelor monotherapy vs. clopidogrel monotherapy on platelet reactivity: a randomized, crossover clinical study (SINGLE study)

Increasing clinical trials demonstrated that the discontinuation of aspirin while maintaining a P2Y12 inhibitor monotherapy could decrease the risk of bleeding without losing the antithrombotic effect. However, no data are available on the platelet reactivity of patients undergoing ticagrelor monotherapy vs. clopidogrel. Therefore, we performed this study to observe the efficacy of ticagrelor monotherapy vs. clopidogrel in Chinese patients with chronic coronary syndrome. This randomized, single-blinded, crossover trial enrolled 50 patients who were administered with ticagrelor (90 mg twice daily for 2 weeks) or clopidogrel (75 mg once daily for 2 weeks). Followed by a 2-week washout period, the two groups of patients underwent a crossover trial. Light transmission aggregometry (LTA) and thromboelastography (TEG) assays were used to test platelet reactivity. The platelet aggregation rate (PAgR) of ADP and AA was significantly lower with ticagrelor than clopidogrel (PAgR of ADP, 27.30% (7.30%-42.635%) vs. 35.55% (12.03%-69.25%), P = .0254; PAgR of AA, 77.80% (21.60%-86.43%) vs. 83.10% (67.13%-87.20%), P = .0400). There was no significant difference in PAgR of collagen and epinephrine between the two groups. The TEG assay showed that ADP and AA, which induced the inhibition of platelet aggregation, were significantly higher in the ticagrelor group than those in the clopidogrel group [ADP%, 69.00% (59.68%–88.95%) vs. 60.55% (35.98%–78.35%), P = .0020; AA%, 53.65% (22.75%–79.28%) vs. 15.15% (5.75%–70.25%), P = .0127]. High on-treatment platelet reactivity (HTPR) on ADP was 2.17% with ticagrelor and 19.57% with clopidogrel. HTPR on AA was 50.00% with ticagrelor and 69.57% with clopidogrel. Ticagrelor and clopidogrel caused the inhibition of ADP and AA-induced platelet aggregation. Moreover, ticagrelor monotherapy had a stronger inhibitory effect than clopidogrel monotherapy (except on collagen and epinephrine).

S1R agonist modulates rat platelet eicosanoid synthesis and aggregation

Sigma-1 receptor (S1R) is detected in different cell types and can regulate intracellular signaling pathways. S1R plays a role in the pathomechanism of diseases and the regulation of neurotransmitters. Fluvoxamine can bind to S1R and reduce the serotonin uptake of neurons and platelets. We therefore hypothesized that platelets express S1R, which can modify platelet function. The expression of the SIGMAR1 gene in rat platelets was examined with a reverse transcription polymerase chain reaction and a quantitative polymerase chain reaction. The receptor was also visualized by immunostaining and confocal laser scanning microscopy. The effect of S1R agonist PRE-084 on the eicosanoid synthesis of isolated rat platelets and ADP- and AA-induced platelet aggregation was examined. S1R was detected in rat platelets both at gene and protein levels. Pretreatment with PRE-084 of resting platelets induced elevation of eicosanoid synthesis. The rate of elevation in thromboxane B2 and prostaglandin D2 synthesis was similar, but the production of prostaglandin E2 was higher. The concentration-response curve showed a sigmoidal form. The most effective concentration of the agonist was 2 µM. PRE-084 increased the quantity of cyclooxygenase-1 as detected by ELISA. PRE-084 also elevated the ADP- and AA-induced platelet aggregation. S1R of platelets might regulate physiological or pathological functions.

Increased symmetric dimethyl-arginine is a predictor factor of decreased platelet reactivity and increased bleeding risk in patients with acute coronary syndrome

Abstract Background One of the promising biomarkers in CVD are asymmetric dimethylarginine (ADMA) and symmetric dimethylarginine (SDMA), which are products of L-arginine methylation and are both involved in endothelial dysfunction. ADMA, SDMA and L-homoarginine, have emerged as biomarkers linked to cardiovascular outcomes [1]. Purpose To investigate the association of SDMA with platelet reactivity and bleeding risk in patients with acute coronary syndrome (ACS) treated with potent P2Y12 inhibitors prasugrel and ticagrelor. Methods Our prospective observational study enrolled 292 patients with ACS undergoing percuteneus coronary intervention [2]. Plasma concentrations of SDMA were measured during the hospitalization for ACS. Impedance aggregometry was used. The primary study endpoint was the concentration of metabolites and platelet reactivity. The primary clinical outcome endpoint was the incidence of Thrombolysis in Myocardial Infarction (TIMI) bleeding events (major, minor and minimal). The efficacy endpoint was the composite of major adverse cardiac events (MACE: stent thrombosis, myocardial infarction, stroke and cardiac death). Results There was an inverse correlation between SDMA serum levels and platelet reactivity (r=−0.25; p&amp;lt;0.000). The ADP+PGE1-induced platelet reactivity was 33% lower among patients with the highest SDMA quartile (4th) as compared to those with the 1–3rd SDMA quartile (8 [0–29] vs 12 [0–126] U; p&amp;lt;0.001). The AA-induced platelet reactivity was 56% lower among patients with the highest SDMA quartile (4th) as compared to those with the 1–3rd SDMA quartile (4 [0–48] vs 9 [0–133]; p&amp;lt;0.001). In a multivariate model, the highest SDMA (4th) quartile was found to be an independent predictor of the lowest ADP+PGE1 and AA induced platelet aggregation (OR: 2.666, 95% CI [1.184–5.999], p=0.018). Conclusions Our study shows that high plasma concentration of SDMA, but not ADMA, is independently associated with low platelet reactivity to ADP and AA and is associated with major and minor bleeding events in patients with ACS on potent antiplatelet therapies. Therefore, SDMA might have a potential to be further evaluated as a blood biomarker for individualization of duration and potency of antiplatelet therapies in an ACS population at high risk of bleeding complications. Acknowledgment I-COMET research team Funding Acknowledgement Type of funding sources: None. Figure 1Table 1

The influence of metabolic activity of platelets on sensitivity to Acetylsalicylic acid in patients with coronary heart disease

Abstract Background Acetylsalicylic acid (ASA) is used to reduce the risk of shunt occlusion after coronary artery bypass grafting (CABG). From 5% to 60% of CHD patients do not respond to ASA. This phenomenon was defined as ASA resistance. The functional activity of platelets is largely determined by the state of their metabolism. Methods The venous blood samples were acquired from 66 patients with CHD before CABG, on the first day after surgery, and on the 8–10th day after surgery. The aggregometry was carried out for all participants by an optical aggregometer with 1 mM of arachidonic acid (AA) and 5 mM Adenosinediphosphate (ADP). Resistance to ASA was determined at the level of platelet aggregation with AA over 20% on ASA therapy or over 20% after platelet incubation with ASA in vitro before CABG. Patients were divided into ASA sensitive (sASA) and ASA resistant (rASA) groups. The level of synthesis of primary and secondary reactive oxygen species (ROS) by platelets was determined using chemiluminescent analysis. We investigated the overall level of radical synthesis from the values of Imax and S (area under the chemiluminescence curve), which, respectively, characterizes the maximum synthesis per unit time and the total amount of radical. The kinetics of ROS synthesis was characterized by Tmax (time to reach the maximum for the chemiluminescent curve). The activity of NAD- and NADP-dependent dehydrogenases in platelets was determined by the bioluminescent method. Results It was found that the aggregation activity of platelets depended on the sensitivity of CHD patients to ASA and decreased during postoperative ASA therapy. The most pronounced differences in metabolic parameters of platelets in sASA and rASA patients were detected by Nox2 activity. Platelet aggregation activity was correlated with platelet Nox2 activity only in sASA patients and only before CABG. The level of AA-induced platelet aggregation with the addition of ASA in sASA patients before CABG was negatively correlated with the Imax of ADP-induced lucigenin-enhanced (r=−0.27, p=0.046) and S of spontaneous luminol-enhanced (r=−0.31, p=0.022) platelet chemiluminescence. Patients with rASA before CABG had positive correlations of AA-induced aggregation with S of spontaneous (r=0.63, p=0.029) as well as Imax (r=0.85, p&amp;lt;0.001) and S (r=0.87, p&amp;lt;0.001) of ADP-induced luminol-enhanced chemiluminescence of platelets. Therefore, the absence of correlations between platelet aggregation activity and Nox2 activity was determined by ASA resistance and postoperative ASA therapy. The synthesis of secondary ROS (Table) by platelets of CHD patients did not depend on the sensitivity of patients to ASA but increased during postoperative treatment with ASA. The activity of NAD(P)-dependent dehydrogenases in platelets did not differ in sASA and rASA patients with CHD. Conclusions Metabolic Activity of Platelets could influence on resistance to ASA in Patients with CHD. Funding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): The reported study was funded by Russian Foundation for Basic Research, Government of Krasno-yarsk Territory, Krasnoyarsk Regional Fund of Science to the research project: “Antiplatelet therapy personification in patients with coronary heart disease (CHD) depending on the level of P-selectin gene expression, the intensity of intercellular interaction and inflammation”

The Synergistic Effect of Ginkgo biloba Extract 50 and Aspirin Against Platelet Aggregation.

PurposeWe aimed to investigate potential synergistic antiplatelet effects of Ginkgo biloba extract (GBE50) in combination with aspirin using in vitro models.MethodsArachidonic acid (AA), platelet activating factor (PAF), adenosine 5ʹ-diphosphate (ADP) and collagen were used as inducers. The antiplatelet effects of GBE50, aspirin and 1:1 combination of GBE50 and aspirin were detected by microplate method using rabbit platelets. Synergy finder 2.0 was used to analyze the synergistic antiplatelet effect. The compounds in GBE50 were identified by UPLC-Q/TOF-MS analysis and the candidate compounds were screened by TCMSP database. The targets of candidate compounds and aspirin were obtained in TCMSP, CCGs, Swiss target prediction database and drugbank. Targets involving platelet aggregation were obtained from GenCLiP database. Compound-target network was constructed and GO and KEGG enrichment analyses were performed to identify the critical biological processes and signaling pathways. The levels of thromboxane B2 (TXB2), cyclic adenosine monophosphate (cAMP) and PAF receptor (PAFR) were detected by ELISA to determine the effects of GBE50, aspirin and their combination on these pathways.ResultsGBE50 combined with aspirin inhibited platelet aggregation more effectively. The combination displayed synergistic antiplatelet effects in AA-induced platelet aggregation, and additive antiplatelet effects occurred in PAF, ADP and collagen induced platelet aggregation. Seven compounds were identified as candidate compounds in GBE50. Enrichment analyses revealed that GBE50 could interfere with platelet aggregation via cAMP pathway, AA metabolism and calcium signaling pathway, and aspirin could regulate platelet aggregation through AA metabolism and platelet activation. ELISA experiments showed that GBE50 combined with aspirin could increase cAMP levels in resting platelets, and decreased the levels of TXB2 and PAFR.ConclusionOur study indicated that GBE50 combined with aspirin could enhance the antiplatelet effects. They exerted both synergistic and additive effects in restraining platelet aggregation. The study highlighted the potential application of GBE50 as a supplementary therapy to treat thrombosis-related diseases.

Prevalence and Clinical Predictors of Aspirin Resistance in Heart Transplant Recipients

<h3>Purpose</h3> Enhanced platelet activation is strongly associated with the development and progression of transplant vasculopathy. Aspirin is thought to be associated with improved graft survival and reduced cardiac allograft vasculopathy in heart transplant (HT) recipients. The assessment of aspirin resistance by laboratory methods is potentially of great therapeutic importance, as it would enable identification of patients at risk for clinical events and allow intervention to prevent subsequent morbidity or mortality. We aimed to determine the prevalence and clinical predictors of aspirin resistance following HT. <h3>Methods</h3> We evaluated 67 stable HT recipients receiving antiplatelet monotherapy with aspirin (100 mg/day≥1 month). Platelets were stimulated with adenosine diphosphate (ADP) and arachidonic acid (AA), and aggregation was assessed using light-transmitted aggregometry. Aggregation was expressed as the maximal percent change in light transmittance from baseline. Aspirin resistance was defined as AA-induced platelet aggregation of ≥20%. <h3>Results</h3> Mean AA-induced aggregation was 29.6 ±24.8%. Of the total cohort, 40 patients (60%) were aspirin resistant and 27 (40%) were not. Aspirin-resistant patients had higher HbA1c values (7.7%±1.5 vs 5.3%±0.9, p<0.001) and higher prevalence of de-novo donor specific antibodies (DSA) (43 vs 11%, p=0.013). There were no differences in aspirin resistance by baseline donor and recipient characteristics (i.e., age, gender, etiology, BMI, hypertension, hemodynamics, immunosuppression). In a multivariable analysis (Figure) HbA1c and DSA were independently associated with a significantly higher risk of aspirin resistance. <h3>Conclusion</h3> Our study suggests a high rate of aspirin resistance in HT patients, which was associated with higher DSA and HbA1c levels, both known risk factors for transplant vasculopathy and survival. Further studies are needed to correlate aspirin resistance with clinical outcomes and hence to tailor therapeutic interventions.

Arachidonic acid-induced platelet aggregation and acetylsalicylic acid treatment during pregnancy in women with recurrent miscarriage, a post hoc study

In this post hoc study, arachidonic acid (AA)-induced platelet aggregation during pregnancy with and without acetylsalicylic acid (ASA) treatment was studied in 323 women with unexplained recurrent first-trimester miscarriage and in 59 healthy women with normal pregnancies. All women had normal AA-induced platelet aggregation in the non-pregnant state. Women with recurrent miscarriage were treated with 75 mg ASA or placebo daily. AA-induced platelet aggregation was measured with multiple electrode impedance aggregometry and presented in units (U), where 1 U = 10 aggregation units x minutes. There were no significant differences in platelet aggregation between placebo-treated women with recurrent miscarriage and healthy women. The mean differences were -0.7 (95%CI; −7.0; 5.6) U in the non-pregnant state, 3.8 (95%CI; −4.6; 12.2) U during the late first trimester and 1.7 (95%CI; −6.7; 10.3) U and 4.1 (95%CI; −3.9; 12.0) U during the early and late third trimester, respectively. ASA reduced platelet aggregation by median −84.0% (Q1; Q3; −89.8; −76.3), −79.9% (−84.7; −69.2) and −75.7% (−83.5; −49.5), respectively, during pregnancy. The degree of inhibition by ASA decreased during the third trimester (p < .0001). There were two (1.9%) complete non-responders to ASA and 32.1% with a partial response. The rate of subsequent miscarriage was not affected by ASA, which did not seem to influence the rate of early miscarriage if treatment was initiated when a viable pregnancy was detectable by ultrasound.

The Effect of Enhanced External Counterpulsation on Platelet Aggregation in Patients with Coronary Heart Disease.

Resistance to antiplatelet therapy, especially aspirin or clopidogrel, triggers other therapies for patients with coronary heart disease (CHD). Enhanced external counterpulsation (EECP) is a noninvasive, pneumatic technique that provides beneficial effects for patients with CHD. However, the physiological effects of EECP have not been fully studied, and the role of EECP on platelet function remains poorly understood. A total of 168 patients with CHD were finally selected from the Second Xiangya Hospital and randomly assigned to either a control group or EECP group. The control group accepted only standard medical treatment, while the EECP group accepted standard medical treatment and EECP treatment. Blood samples were collected from patients at baseline and after EECP, and platelet aggregation was assessed. Changes in platelet aggregation were compared before and after treatment. There was no difference in the basal levels of arachidonic acid (AA) induced platelet maximum aggregation ratio (MAR) between the two groups. The AA-induced platelet MAR was significantly decreased after EECP therapy. The logistic analysis showed that low HDL-C was not favorable for the decrease in platelet aggregation. EECP therapy is favorable for lowering platelet aggregation in patients with CHD, especially the AA-induced platelet aggregation ratio.

Conversion and outcome of dual antiplatelet therapy after bleeding in patients with acute coronary syndrome

Abstract Objective To explore the conversion and outcome of antiplatelet therapy in patients with acute coronary syndrome (ACS) who have suffered bleeding after receiving aspirin combined with ticagrelor. Methods Patients who diagnosed with ACS and given aspirin 100 mg once daily combined with ticagrelor 90 mg twice daily for dual antiplatelet therapy (DAPT) were selected. The patients were divided into bleeding group and non-bleeding group according to whether bleeding events occurred within one year after DAPT. The bleeding group was divided into the next four groups according to the converted antiplatelet treatment strategy decided by the doctor and the patient. Patients in group A to D were treated with ticagrelor 90mg twice a day plus aspirin 0–50mgonce a day, ticagrelor 60mg twice a day plus aspirin 100mgonce a day, clopidogrel 75mgonce a day plus aspirin 100mg once a day and ticagrelor 90mg twice a day plus aspirin 100mg once a day, respectively. All patients were followed up for 1 year after DAPT. The primary outcome is change of bleeding degree by BARC. The secondary outcomes includes BARC bleeding, all-cause mortality and MACE. Results A total of 752 cases were enrolled, including 250 in the bleeding group and 502 in the non-bleeding group. The bleeding group was divided into four treatment groups, which were 63, 43, 38 and 95 cases among A, B, C and D group. There was a significant difference in the improvement of bleeding among the four groups. To be noted, patients in group A has the highest improvement rate of hemorrhage (65.1% vs 62.8% vs 47.4% vs 24.2%, P&amp;lt;0.01). There were no significant differences in the incidence of MACE and all-cause mortality (P&amp;gt;0.05). The incidence of major MACE in patients in the bleeding group was 14.4%, which was higher than that in the non-bleeding group (9.2%, P=0.03).The result of multivariate logistic regression analysis showed that female (OR=5.18, 95% CI: 1.61–16.65, P=0.006), low body weight (OR=7.73, 95% CI: 2.46–24.49, P&amp;lt;0.001) and low AA-induced maximum platelet aggregation rate (OR=2.72, 95% CI:1.09–6.84, P=0.033) were independent risk factors for hemorrhage. Conclusion Patients with acute coronary syndrome who have suffered bleeding after receiving aspirin combined with ticagrelor were at a higher risk of MACE than those who did not. Low-dose aspirin (0–50mg/d) combined with ticagrelor or even ticagrelor alone has the best effect on reducing bleeding without increasing the risk of ischemia. Female, low body mass and low AA-induced maximum platelet aggregation rate are independent risk factors for bleeding. Funding Acknowledgement Type of funding source: None

Prevalence and Clinical Predictors of Aspirin Resistance in Heart Transplant Recipients

Enhanced platelet activation is strongly associated with the development and progression of transplant vasculopathy. Aspirin is thought to be associated with improved graft survival and reduced cardiac allograft vasculopathy in heart transplant (HT) recipients. The assessment of aspirin resistance by laboratory methods is potentially of great therapeutic importance, as it would enable identification of patients at risk for clinical events and allow intervention to prevent subsequent morbidity or mortality. We aimed to determine the prevalence and clinical predictors of aspirin resistance following HT. We evaluated 67 stable HT recipients receiving antiplatelet monotherapy with aspirin (100 mg/day≥1 month). Platelets were stimulated with adenosine diphosphate (ADP) and arachidonic acid (AA), and aggregation was assessed using light-transmitted aggregometry. Aggregation was expressed as the maximal percent change in light transmittance from baseline. Aspirin resistance was defined as AA-induced platelet aggregation of ≥20%. Mean AA-induced aggregation was 29.6 ±24.8%. Of the total cohort, 40 patients (60%) were aspirin resistant and 27 (40%) were not. Aspirin-resistant patients had higher HbA1c values (7.7%±1.5 vs 5.3%±0.9, p<0.001) and higher prevalence of de novo donor-specific antibodies (DSA) (43 vs 11%, p=0.013). There were no differences in aspirin resistance by baseline donor and recipient characteristics (i.e., age, gender, etiology, BMI, hypertension, hemodynamics, immunosuppression). In a multivariable analysis (Figure) HbA1c and DSA were independently associated with a significantly higher risk of aspirin resistance. Our study suggests a high rate of aspirin resistance in HT patients, which was associated with higher DSA and HbA1c levels, both known risk factors for transplant vasculopathy and survival. Further studies are needed to correlate aspirin resistance with clinical outcomes and hence to tailor therapeutic interventions.

Ticagrelor Inhibits Toll-Like and Protease-Activated Receptor Mediated Platelet Activation in Acute Coronary Syndromes

PurposeSince ticagrelor inhibits the cellular uptake of adenosine, thereby increasing extracellular adenosine concentration and biological activity, we hypothesized that ticagrelor has adenosine-dependent antiplatelet properties. In the current study, we compared the effects of ticagrelor and prasugrel on platelet activation in acute coronary syndrome (ACS).MethodsPlatelet surface expression of P-selectin and activated glycoprotein (GP) IIb/IIIa in response to adenosine diphosphate (ADP), the toll-like receptor (TLR)-1/2 agonist Pam3CSK4, the TLR-4 agonist lipopolysaccharide (LPS), the protease-activated receptor (PAR)-1 agonist SFLLRN, and the PAR-4 agonist AYPGKF were measured by flow cytometry in blood from 80 ticagrelor- and 80 prasugrel-treated ACS patients on day 3 after percutaneous coronary intervention. Residual platelet aggregation to arachidonic acid (AA) and ADP were assessed by multiple electrode aggregometry and light transmission aggregometry.ResultsADP-induced platelet activation and aggregation, and AA-induced platelet aggregation were similar in patients on ticagrelor and prasugrel, respectively (all p ≥ 0.3). Further, LPS-induced platelet surface expression of P-selectin and activated GPIIb/IIIa did not differ significantly between ticagrelor- and prasugrel-treated patients (both p > 0.4). In contrast, Pam3CSK4-induced platelet surface expression of P-selectin and activated GPIIb/IIIa were significantly lower in ticagrelor-treated patients (both p ≤ 0.005). Moreover, SFLLRN-induced platelet surface expression of P-selectin and activated GPIIb/IIIa were significantly less pronounced in patients on ticagrelor therapy compared to prasugrel-treated patients (both p < 0.03). Finally, PAR-4 mediated platelet activation as assessed by platelet surface expression of activated GPIIb/IIIa following stimulation with AYPGKF was significantly lower in patients receiving ticagrelor (p = 0.02).ConclusionTicagrelor inhibits TLR-1/2 and PAR mediated platelet activation in ACS patients more strongly than prasugrel.