Residual Platelet Research Articles

Application of Raman spectroscopy using an original optical sensor to assess the laboratory efficacy of antiplatelet drugs

The main method for monitoring the laboratory effectiveness of antiplatelet drugs in modern clinical practice is aggregometry, but this method is not without limitations. In this connection, there is an objective need to develop alternative methods. One of the promising areas is the method of Raman spectroscopy (RS).Objective: development of a method to detect high residual platelet reactivity (RPR) in patients with CVD receiving acetylsalicylic acid (ASA) or clopidogrel by giant Raman spectroscopy (GRS) using an original optical biosensor.Material sand Methods. Platelet-rich plasma of patients with cardiovascular diseases (CVD) was investigated by Raman spectroscopy using an original optical biosensor. Platelet aggregation activity was investigated using a Siemens PFA-200 aggregometer with three types of cartridges – Collagen/EPI, Collagen/ADP, and P2Y. Fisher’s linear discriminant analysis was performed using Statistica 13.0 package.Results. Raman spectra analysis using different values of frequency shifts (970 cm-1 or 1590 cm-1), allows to evaluate laboratory ineffectiveness separately for ASA and clopidogrel. Thus, the number of patients with high residual platelet reactivity (RPR) was 41.7 % ± 6.3 % with ASA and 36.7 % ± 6.2 % with clopidogrel therapy; similar values using aggregometry were 43.5 % ± 10.3 % and 30.4 % ± 9.6 %.Conclusion. Application of the method of Raman spectroscopy using the original optical biosensor allows to distinguish patients with high RPR in the population of patients with CVD receiving antiaggregant therapy.

Reduction of thrombus burden by cangrelor in ticagrelor-loaded st-elevation myocardial infarction patients with high residual platelet reactivity: the imaging sub-study of the ERMIT randomized trial

Abstract Background Residual high platelet reactivity (HPR) at the time of primary percutaneous coronary intervention (PCI) for ST-elevation myocardial infarction (STEMI) is associated with higher residual thrombus burden and the subsequent suboptimal reperfusion. Purpose We sought to determine the impact of intravenous Cangrelor on the residual post-PCI thrombus burden in patients with HPR admitted for primary PCI in a prespecified intracoronary optical coherence tomography (OCT) sub-study of the ERMIT randomized trial. Methods In the ERMIT trial patients pre-treated with ticagrelor and aspirin with residual HPR (baseline platelet reactivity unit >208) were randomly assigned in a 1:1 ratio to receive cangrelor on top of standard therapy or standard therapy alone prior to PCI. The primary endpoint of the sub-study was the relative thrombus volume quantified on OCT. Other OCT quantified parameters were predefined as secondary endpoints. Results After screening of 128 patients, a total of 60 patients with HPR were enrolled and randomized. The OCT sub-study included 53 patients, 29 in the cangrelor and 24 in the control group. As reported in the table, baseline characteristics were similar between the groups. The post-PCI PRU was dramatically lower in the cangrelor as compared to the standard group (p<0.001). The primary endpoint was 4.5% [3.4;7.2] versus 6.9% [5.2;8.5], (p=0.03) in the cangrelor and control groups respectively. All other imaging parameters confirmed the significantly lower thrombus burden in patients receiving cangrelor (Table). Conclusion The imaging sub-study of the randomized ERMIT trial shows that cangrelor use is associated with a more effective platelet inhibition and lower post-PCI residual thrombus burden in ticagrelor-loaded STEMI patients with residual HPR. The clinical implications of such finding warrant larger size trials.Table

Monitoring Cell Activation and Extracellular Vesicle Generation in Platelet Concentrates for Transfusion.

Platelets play a crucial role in blood transfusions, and understanding the changes that occur during their storage is important for maintaining the quality of preparations. In this study, we examined key alternating factors, with a particular focus on platelet activation and the release of extracellular vesicles. Additionally, we compared two detection methods-imaging flow cytometry (IFC) and nanoparticle tracking analysis (NTA)-for their effectiveness in detecting particles. Platelet concentrates were prepared by pooling buffy coats from five blood group-compatible donors in an additive solution. Samples were analysed after one, three, and seven days of storage for residual white blood cells (WBCs), glucose levels, platelet activation, and extracellular vesicle concentrations. Over the storage period, the total platelet concentration decreased slightly, while the residual WBC count remained stable. Glucose levels declined, whereas platelet activation and extracellular vesicle concentration increased, with a positive correlation between the two. The particle size remained relatively unchanged throughout the storage period. Ultimately, despite controlled processing and storage conditions, platelet activation, and the release of extracellular vesicles still occurred, which may have implications for transfusion recipients. Although an optimised method is still needed, IFC has proved to be specific and potentially appropriate for detecting extracellular vesicles in transfusion preparations.

Cangrelor use in ticagrelor-loaded ST-elevation myocardial infarction patients with high residual platelet reactivity: a randomized controlled trial

Abstract Background Despite the use of potent P2Y12 inhibitor, many patients still present with high platelet reactivity (HPR) at the time of primary percutaneous coronary intervention (PCI). HPR is associated with impaired myocardial reperfusion and the subsequent poor outcome. Purpose We sought to determine the impact of Cangrelor -a rapid and potent intravenous P2Y12 inhibitor- on myocardial reperfusion in patients with HPR admitted for primary PCI in a randomized trial. Methods Patients pre-treated with ticagrelor and aspirin with residual HPR (baseline platelet reactivity unit (PRU) > 208) were randomly assigned in a 1:1 ratio to receive cangrelor (experimental group) or not (control group) just before PCI. The primary endpoint was optimal myocardial reperfusion defined as a final myocardial blush grade (MBG) 3 assessed by an independent core laboratory. Results After screening of 128 patients, a total of 60 patients were with HPR were enrolled and randomized. The ticagrelor loading dose to PRU measurement times were 83 ± 32 and 90 ± 47 minutes in the experimental and control groups, respectively. Baseline PRU were 245 ± 30 and 252 ± 35 in the experimental and standard group, respectively. A final MBG 3 was observed in 21/30 (55%) and 17/30 (45%) the experimental and standard groups (p=0.29). A final TIMI flow grade 3 was observed in 30/30 (100%) and 27/30 (90%) the experimental and standard groups (p=0.24). Peak troponin levels after PCI were 51381 ± 50047 and 51078 ± 63960 ng/L (p=0.98) in the experimental and standard group, respectively. At the end of the PCI, control PRU were 36 ± 57 and 198 ± 107 (p<0.0001) in the experimental and standard group, respectively. One patient of the experimental group presented an in-hospital major bleeding (BARC 3). Conclusion In ticagrelor-loaded STEMI patients with residual HPR at the time of PCI, cangrelor use was associated with a more effective platelet inhibition but failed to significantly improve the rates of optimal final myocardial blush grades.

Paraoxonase-1 as a Cardiovascular Biomarker in Caribbean Hispanic Patients Treated with Clopidogrel: Abundance and Functionality.

Clopidogrel, a prescription drug to reduce ischemic events in cardiovascular patients, has been extensively studied in mostly European individuals but not among Caribbean Hispanics. This study evaluated the low abundance and reduced activity of paraoxonase-1 (PON1) in clopidogrel-resistant patients as a predictive risk biomarker of poor responders and disease severity in this population. Thirty-six patients on clopidogrel (cases divided into poor and normal responders) were enrolled, along with 11 cardiovascular patients with no clopidogrel indications (positive control) and 13 healthy volunteers (negative control). Residual on-treatment platelet reactivity unit (PRU), PON1 abundance by Western blotting, and PON1 activity by enzymatic assays were measured. PON1 genotyping and computational haplotype phasing were performed on 512 DNA specimens for two genetic loci (rs662 and rs854560). No statistical differences in mean relative PON1 abundance were found among the groups (p > 0.05). However, a significantly lower enzymatic activity was found in poor responders (10.57 ± 6.79 µU/mL) when compared to controls (22.66 ± 8.30 µU/mL and 22.21 ± 9.66 µU/mL; p = 0.004). PON1 activity among carriers of the most prevalent PON1 haplotype (AA|AA) was significantly lower than in wild types (7.90 µU/mL vs. 22.03 µU/mL; p = 0.005). Our findings suggested that PON1 is a potential biomarker of cardiovascular disease severity in Caribbean Hispanics.

Antiplatelet Effect of Low-Dose Prasugrel in Elderly Patients Undergoing Percutaneous Coronary Interventions.

Low-dose prasugrel (5 mg) has been proposed for patients with Acute Coronary Syndrome (ACS) and advanced age or low body weight. However, the routine use of dose-adjusted prasugrel in this high-risk subset of patients is still debated. This study aimed to assess the prevalence and predictors of HRPR among elderly patients treated with low-dose (5 mg) prasugrel to evaluate the routine use of dose-adjusted prasugrel in this high-risk subset of patients. We included 59 elderly patients (≥75 years) treated with Dual Antiplatelet Therapy (DAPT: acetylsalicylic acid (ASA) 100-160 mg + prasugrel 5 mg) after Percutaneous Coronary Interventions (PCI) and undergoing platelet function assessment (by whole blood impedance aggregometry) 30-90 days post-discharge. At a median follow-up of 43 days (interquartile range-IQR: 32-54), high-on treatment residual platelet reactivity (HRPR) occurred in 25 patients (42.4%), who displayed a greater body mass index (BMI) (p=0.02), lower levels of vitamin D (p=0.05) and were more frequently treated with nitrates (p=0.03). After multivariate analysis, BMI was the only independent predictor of prasugrel HRPR, and a BMI >26 was the best cut-off for predicting HRPR (adjusted Odds Ratio - OR=8.6, 95%CI: 2.2-33.9, p=0.002). Among elderly patients receiving DAPT after PCI, HRPR is common with low-dose prasugrel. A greater BMI, especially for values ≥26, is the only independent predictor of HRPR with prasugrel 5 mg.

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.

Frequency of CYP2C19, P2RY12, ITGB3, ITGA2, and eNOS3 gene polymorphism in patients with myocardial infarction

The aim of the study was to assess the distribution of polymorphic variants G681A (*2) of the CYP2C19 gene, H1/H2 of the P2RY12 gene, T1565C of the ITGB3 gene, C807T of the ITGA2 gene, and T786C of the eNOS3 gene in the population of the Grodno region, and to study their associations with myocardial infarction (MI). The study of the population consists of 493 people, including 400 patients with MI aged 31 to 74 years and 93 people of the control group aged 32 to 60 years. Research data (clinical and genotyping performed by polymerase chain reaction) were analyzed using STATISTICA 10.0 software. The prevalence of carriage of genotypes associated with high residual platelet reactivity and variability in response to dual antiplatelet therapy among patients with MI was 25.2 % for the G681A polymorphic locus of the CYP2C19 gene (GA + AA), and for the H1/H2 polymorphic locus of the P2RY12 gene (H1/H2 + H2/H2) – 40.0 %, for the C807T polymorphic locus of the ITGA2 gene (CT + TT) – 65.8, for the T1565C polymorphic locus of the ITGB3 gene (TC + CC) – 25.5, for the polymorphic locus T786C of the eNOS gene (TC + CC) – 69.2 %. Among the individuals of the control group, the frequency of occurrence of these genotypes was 18.3, 46.2, 60.2, 37.6, 48.4 %, respectively. In patients with MI, compared to the control group, the TT genotype of the eNOS gene was less common (χ2 = 13.6, p = 0.0002), the CC genotype of the eNOS gene (χ2 = 5.4, p = 0.02) and the allele 786C of the eNOS gene (χ2 = 15.1, p = 0.0001) were more often detected. The carriage of the 786C allele of the eNOS gene increased the risk of MI in the studied population (OR = 2.0, 95 % CI: 1.41‒2.82, p = 0.0001). Gender differences were not found in the distribution of genotypes and alleles within the studied groups. There were no differences in carriage by the number of combinations of minor alleles between the control group and patients with MI. The most common combinations of minor alleles in both groups were comparable.

Adjunctive Cilostazol in Patients With High Residual Platelet Reactivity After Drug-Eluting Stent Implantation: A Randomized, Open-Label, Single-Center, Prospective Study (ADJUST-HPR).

Cilostazol as an adjunct to dual antiplatelet therapy (DAPT) postcoronary stenting may further reduce vascular occlusion risks. The aim of this study was to assess the impact of cilostazol on high residual platelet reactivity (HRPR) in patients undergoing drug-eluting coronary stent implantation. In a randomized, open-label, single-center, prospective study, the degree of platelet inhibition by cilostazol 100 mg twice daily was assessed on top of conventional DAPT compared with standard clopidogrel and low-dose aspirin combination in poststent patients with HRPR. HRPR was defined as P2Y12 units (PRU) > 240 as measured by the VerifyNow P2Y12 assay. In addition, the platelet activity was assessed by light transmittance aggregometry (LTA) and Multiplate electrode analyzer (MEA). The total of 148 patients were screened, and HRPR was observed in 64 (43.2%). Those were randomized for DAPT versus triple therapy (TAPT). After 30 days, TAPT group exhibited significantly lower rate of HRPR when assessed by all 3 devices (VerifyNow: 40.0 vs. 66.7% P = 0.04, LTA: 6.7 vs. 30.0% P = 0.02, MEA: 10.0 vs. 30.0% P = 0.05 L all vs. DAPT). Also, higher absolute mean difference in TAPT versus DAPT group after 30 days (VerifyNow: 71.3 ± 38.2 vs. 24.6 ± 40.2 P < 0.001, LTA: 23.9 ± 15.1 vs. 9.4 ± 11.8 P < 0.001, MEA: 9.3 ± 12.9 vs. 2.4 ± 17.3 P = 0.08) was observed. Cilostazol in addition to standard DAPT reduces the incidence of HRPR and diminishes further platelet activity in poststent patients. Whether this favorable laboratory finding will affect clinical outcomes requires an adequately powered randomized trial.

Individualized antiplatelet therapy for non-cardiogenic ischemic stroke

ObjectiveThis research aims to investigate the impact of individualized antiplatelet therapy guided by thromboelastography with platelet mapping (TEG-PM) on the clinical outcomes of patients with non-cardiogenic ischemic stroke. MethodsAmong a total of 1264 patients, 684 individuals diagnosed with non-cardiogenic ischemic stroke underwent TEG-PM testing. Based on the adjustment of antiplatelet medication, these patients were divided into individual and control groups. Within the individual group, in accordance with the TEG-PM test results, a Maximum amplitude (MA) value greater than 47mm was defined as high residual platelet reactivity (HRPR), while an MA value less than 31mm was defined as low residual platelet reactivity (LRPR). Patients with arachidonic acid (AA) less than 50% and adenosine diphosphate (ADP) less than 30% were classified as aspirin-resistant or clopidogrel-resistant. Treatment strategies for antiplatelet medication were subsequently adjusted accordingly, encompassing increment, decrement, or replacement of drugs. Meanwhile, the control group maintained their original medication regimen without alterations. ResultsThe individual group included 487 patients, while the control group had 197. In the individual group, approximately 175 patients (35.9%) were treated with increased medication dosages, 89 patients (18.3%) with reduced dosages, and 223 patients (45.8%) switched medications. The results showed that the incidence rate of ischemic events in the individual group was lower than that of the control group (5.54% vs. 12.6%, P = 0.001), but no significant difference was observed in bleeding events. Cox regression analysis revealed age (hazard ratio, 1.043; 95% CI, 1.01-1.078; P = 0.011) and coronary heart disease (hazard ratio, 1.902; 95% CI, 1.147-3.153; P = 0.013) as significant risk factors for adverse events. ConclusionIndividualized antiplatelet therapy based on TEG-PM results can reduce the risk of ischemic events in patients with non-cardiogenic ischemic stroke without increasing the risk of bleeding events or mortality. Advanced age and coronary heart disease were identified as risk factors affecting the outcomes of individualized antiplatelet therapy.

Cangrelor in contemporary patients with ST-segment elevation myocardial infarction pretreated with Ticagrelor: Pharmacodynamic data from the POMPEII study

BackgroundThere are limited data to assess pharmacodynamic (PD) profiles of patients with STEMI undergoing primary percutaneous coronary intervention (PCI) and receiving cangrelor after pretreatment with ticagrelor. MethodsThe PharmacOdynaMic effects of cangrelor in PatiEnts wIth acute or chronIc coronary syndrome undergoing percutaneous coronary intervention (POMPEII) registry (NCT04790032) is a prospective study conducted at Federico II University of Naples enrolling all patients undergoing PCI receiving cangrelor at operator’s discretion. PD assessments were performed with 3 assays: (1) the gold standard light transmittance aggregometry (LTA) (20- and 5-μM adenosine diphosphate [ADP] stimuli); (2) VerifyNow P2Y12-test; (3) Multiplate electrode aggregometry (MEA), ADP-test. ResultsWe analyzed 13 STEMI patients pretreated with ticagrelor within 1 h at the time they underwent primary PCI receiving cangrelor. All patients showed low maximal platelet aggregation at 30-minute during cangrelor infusion, as well as at 3 h and 4–6 h (corresponding to 1 h and 2–4 h after stopping cangrelor infusion) with no cases of high residual platelet reactivity. These results were consistent with all assays. ConclusionsPD data show that in contemporary real-world STEMI patients pretreated within 1 h with ticagrelor undergoing primary PCI, adding cangrelor resulted in fast and potent platelet inhibition, thus suggesting that cangrelor may bridge the gap until ticagrelor reaches its effect.

Flow-chamber device (T-TAS) to diagnose patients suspected of platelet function defects.

Patients suspected of platelet function defects represent a diagnostic challenge for the clinical laboratory, mainly due to the complexity and poor standardization of screening methods. We compared a new flow-based chip-equipped point-of-care (T-TAS) device with lumi-aggregometry and other specific tests. The study included 96 patients suspected of platelet function defects and 26 patients referred to hospital for an evaluation of residual platelet function while on antiplatelet therapy. Forty-eight of 96 patients displayed abnormal platelet function by lumi-aggregometry, and 10 of them had defective granule content and were classified as δ-storage pool disease (δ-SPD). T-TAS compared favorably with lumi-aggregometry in detecting the most severe forms of platelet function defects (i.e., δ-SPD) [test agreement (lumi-light transmission aggregometry [lumi-LTA] vs T-TAS) for the δ-SPD subgroup was 80% and K CHOEN 0.695. T-TAS was less sensitive to milder platelet function defects (i.e., primary secretion defects [PSD]). Concerning patients on antiplatelets, test agreement (lumi-LTA vs T-TAS) in detecting patients who were responders to this therapy was 54%; K CHOEN 0.150. The results indicate that T-TAS can detect the more severe forms of platelet function defects such as δ-SPD. There is limited agreement of T-TAS with lumi-aggregometry in identifying responders to antiplatelets. However, this poor agreement is commonly shared by lumi-aggregometry and other devices owing to the lack of test specificity and of prospective data from clinical trials linking platelet function with therapeutic efficacy.

Towards a greater understanding of reduced response to aspirin in children with congenital heart disease post-cardiac surgery using immature platelet fraction

ObjectiveA high platelet turnover rate may produce a population of platelets that confers an inadequate response to aspirin. We aimed to investigate the relationship between residual platelet aggregation and platelet turnover in paediatric cardiology patients on aspirin monotherapy by evaluating the fraction of immature platelets as a marker for turnover and secondly to test the predictive value of the immature platelet fraction (IPF) to classify patients as responsive or non-responsive to aspirin. MethodsSixty patients divided into two age categories (≤90 days, >90 days of age) were included in this prospective observational study. Patients were then stratified into tertiles using their IPF level. Platelet studies included thromboelastography with platelet mapping (TEGPM). ResultsThe overall incidence of ‘inadequate response to aspirin’ was 38 % in our patient cohort recently post-cardiac surgery a consequence that warrants further study. The frequency of inadequate response to aspirin was higher in the upper tertile of IPF when compared to the lower tertile, (88 %) versus (4 %) respectively (p < 0.05). The ‘cut off’ for IPF was determined to be 3.9 % with a sensitivity of 95.7 %, and a specificity of 92.9 % (area under the curve of 0.955 [CI 0.896–1.014, p < 0.05]). ConclusionThis study demonstrates that inadequate response to aspirin occurs in approximately 38 % of patients undergoing specific high-risk congenital cardiac procedures using the dosing practice of a national centre. This study supports the hypothesis that an elevated platelet turnover may result in aspirin being less effective in patients who are recently post cardiac surgery. These data are of direct translational relevance.

Impact of platelet reactivity on subclinical valve thrombosis in patients treated with TAVR

Abstract Background and introduction High residual platelet reactivity (HPR) in patients undergoing percutaneous coronary intervention with dual antiplatelet therapy (DAPT) is associated with an increased risk of thrombotic events. The relationship between HPR and the presence of subclinical valve thrombosis (SVT) following transcatheter aortic valve replacement (TAVR) is unknown. Purpose The aim of this study was to determine whether HPR is associated with the occurrence of SVT in patients undergoing TAVR treated with DAPT and to determine independent predictors of SVT. Methods This is a prospective, multicentre study. Patients with symptomatic aortic stenosis successfully treated with TAVR followed by DAPT (aspirin + clopidogrel) for 3 months were included. Patients with monotherapy, oral anticoagulation, or severe renal impairment were excluded. For the purpose of the study, platelet reactivity was measured either with Multiplate analyzer or VerifyNow point-of-care assay at baseline, at 1-2 days post, and 1-3 months post-TAVR. HPR was defined as ≥ 468AU, or ≥208PRU, respectively. The primary endpoint was the incidence of SVT measured by multi-slice CT at 3-months post TAVR in relation to HPR. Secondary objectives included the relationship between HPR following TAVR and the incidence of major adverse cardiac and cerebral clinical events (MACCE), to determine whether a pro-thrombotic inflammatory response is associated with the appearance of SVT and to evaluate factors associated with the occurrence of SVT. Results A total of 169 patients were included, the mean age was 81.5±5 and 51% were female. The overall rate of SVT was 22%. Patients with HPR demonstrated a similar incidence of SVT to those without (23,7% vs 33,6%: p=0,29). The overall bleeding rate was 4,9%. No differences in the rate of stroke at 1-year follow-up were observed (no SVT group 1.5% vs SVT group 2.6%: p=0.6). Independent predictors of SVT, were: dyslipemia OR 0,38 (0,15-0,96) p=0,039, creatinine clearance 0.98 (0.96.1.0, p=0.04), self-expandable valve OR 3,57 (1,40-9,06); p=0,006 and prosthesis size 0,78 (0,64-0,96): p=0,009. Patients with SVT expressed increased inflammatory parameters at 6 months compared to those without SVT CD14+ CD16-: 76,6±9,9 vs 70,7±13,9: p=0.02. Conclusion In contrast to coronary interventions, for the first time we have demonstrated that HPR does not appear to be associated with the development of SVT in TAVR patients treated with DAPT.

High rates of high on-aspirin platelet reactivity in patients after transcatheter aortic valve implantation

Abstract Background Guidelines recommend aspirin monotherapy in patients after transcatheter aortic valve replacement (TAVR) without an indication for oral anticoagulation. The prevalence of high on-aspirin platelet reactivity (HAPR) varies from 1% to 34% and may be associated with long-term ischemic risk or subclinical valve thrombosis which occurs in 17-40% of the patients after TAVR. Purpose To determine the prevalence of HAPR and its association with subclinical valve thrombosis in patients undergoing TAVR using methods that directly indicate the degree of platelet cyclooxygenase inhibition. Methods Data were collected in a prospective, single-center, observational cohort study between October 2020 and June 2022. Patients were enrolled after TAVR, and platelet function was assessed during the hospital stay with light-transmittance platelet aggregation (LTA) and thrombelastography (TEG) platelet mapping. Platelet aggregation was induced with arachidonic acid (AA)-induced LTA (final concentration [fc] 1.64 mM) and TEG (fc 1.0mM). The maximum percent aggregation (%aggr) was recorded. According to the literature, HAPR was defined as &amp;gt;20% aggregation by LTA or &amp;gt;50% aggregation by TEG, respectively. All patients were under maintenance of 100 mg aspirin or received a loading dose of 300-600mg aspirin before measurement. 47 of 75 patients underwent computed tomography (CT) to detect valve thrombosis (VT) 6 months after TAVR. Results Data from 75 patients under maintenance of aspirin were analyzed. The median age was 82 years (interquartile range, IQR 79-85), and 52% were male. 12 patients (16%) had a history of stroke or TIA, and 7 (9%) had suffered a previous myocardial infarction. TEG and LTA were performed during the hospital stay between days 0 and 17 post-TAVR. Median AA-induced maximum aggregation by LTA was 17 % (interquartile range, IQR 13-38) and by TEG was 29 % (IQR 5-64). When determining platelet function using LTA, 33 patients (44%) had residual platelet function (i.e., %aggrLTA &amp;gt;20%) despite aspirin treatment. According to the TEG results, 27 patients (36 %) were identified as HAPR (i.e., %aggrTEG &amp;gt;50%). The agreement (Kappa=0.06) and correlation (r=0.32, Figure 1) between the two tests were low to moderate. 13 patients had HAPR in both methods. 19 of 47 patients (40%) had VT on CT 6 months after TAVI. 12 of these 19 patients (63%) with VT responded well to aspirin in the TEG and LTA, while 7 (37%) had HAPR in both tests. HAPR was not significantly associated with HALT when measured with TEG or LTA (OR 0.90 [95% CI 0.27.2.76], p=0.86 and 0.58 [95% CI 0.17-2.04], p=0.37, respectively; Table 1). Conclusion HAPR assessed by LTA and TEG is frequent in patients undergoing TAVR. In this exploratory study, HAPR was not significantly associated with the occurrence of HALT. Further studies are required to evaluate the long-term risk of leaflet thrombosis or thromboembolic events in patients with HAPR.

In vitro and ex vivo rescue of a nonsense mutation responsible for severe coagulation factor V deficiency

BackgroundCoagulation factor V (FV) deficiency is a rare bleeding disorder that is usually managed with fresh-frozen plasma. Patients with nonsense mutations may respond to treatment with readthrough agents. ObjectivesTo investigate whether the F5 p.Arg1161Ter mutation, causing severe FV deficiency in several patients, would be amenable to readthrough therapy. MethodsF5 mRNA and protein expression were evaluated in a F5 p.Arg1161Ter-homozygous patient. Five readthrough agents with different mechanisms of action, i.e. G418, ELX-02, PTC-124, 2,6-diaminopurine (2,6-DAP), and Amlexanox, were tested in in vitro and ex vivo models of the mutation. ResultsThe F5 p.Arg1161Ter-homozygous patient showed residual F5 mRNA and functional platelet FV, indicating detectable levels of natural readthrough. COS-1 cells transfected with the FV-Arg1161Ter cDNA expressed 0.7% FV activity compared to wild-type. Treatment with 0-500 μM G418, ELX-02, and 2,6-DAP dose-dependently increased FV activity up to 7.0-fold, 3.1-fold, and 10.8-fold, respectively, whereas PTC-124 and Amlexanox (alone or in combination) were ineffective. These findings were confirmed by thrombin generation assays in FV-depleted plasma reconstituted with conditioned media of treated cells. All compounds except ELX-02 showed some degree of cytotoxicity. Ex vivo differentiated megakaryocytes of the F5 p.Arg1161Ter-homozygous patient, which were negative at FV immunostaining, turned positive after treatment with all 5 readthrough agents. Notably, they were also able to internalize mutant FV rescued with G418 or 2,6-DAP, which would be required to maintain the crucial platelet FV pool in vivo. ConclusionThese findings provide in vitro and ex vivo proof-of-principle for readthrough-mediated rescue of the F5 p.Arg1161Ter mutation.

Clopidogrel-Related High Residual Platelet Reactivity Associated with Estimated Glomerular Filtration Rate in Patients with Acute Ischemic Stroke

Introduction: There are few studies on the relationship between the occurrence of clopidogrel-related high residual platelet reactivity (HRPR) and estimated glomerular filtration rate (eGFR) at admission in patients with ischemic stroke. The aim of this study was to investigate the possible relationship between the two. Methods: Patients who were hospitalized and diagnosed with acute ischemic stroke were recruited from July 1, 2017, to June 30, 2018, at Shanghai TCM-Integrated Hospital. Renal function was measured within 24 h of enrollment and eGFR was calculated. Patients were tested for platelet reactivity using the VerifyNow system after 7 days of antiplatelet therapy with clopidogrel 75 mg/d alone, and patients with P2Y12 reaction unit values ≥230 were diagnosed with HRPR. The association between HRPR and eGFR was analyzed. Results: A total of 274 patients were enrolled in the study, of whom 91 (33.21%) had HRPR. Multivariate logistic regression analysis suggested that an increased risk of HRPR was independently associated with female sex and reduced eGFR (female sex: OR = 2.24, 95% CI: 1.26–3.99, p = 0.006; mild chronic kidney disease [CKD]: OR = 2.95, 95% CI: 1.47–5.93, p = 0.002; moderate CKD: OR = 3.07, 95% CI: 1.08–8.75, p = 0.04). Conclusion: Decreased eGFR is an independent risk factor for the occurrence of HRPR in patients with ischemic stroke.

Using the PEAR1 Polymorphisms Rs12041331 and Rs2768759 as Potential Predictive Markers of 90-Day Bleeding Events in the Context of Minor Strokes and Transient Ischemic Attack.

In this study, we explored the relationship between the platelet endothelial aggregation receptor 1 (PEAR1) polymorphisms, platelet reactivity, and clinical outcomes in patients with minor stroke or transient ischemic attack (TIA). Randomized controlled trial subgroups were assessed, wherein patients received dual antiplatelet therapy for at least 21 days. Platelet reactivity was measured at different time intervals. Genotypes were categorized as wild-type, mutant heterozygous, and mutant homozygous. Clinical outcomes were evaluated after 90 days. The rs12041331 polymorphism predominantly influenced adenosine diphosphate channel platelet activity, with the AA genotype displaying significantly lower residual platelet activity to the P2Y12 response unit (p < 0.01). This effect was more evident after 7 days of dual antiplatelet treatment (p = 0.016). Mutant A allele carriers had decreased rates of recurrent stroke and complex endpoint events but were more prone to bleeding (p = 0.015). The rs2768759 polymorphism majorly impacted arachidonic acid (AA) channel platelet activity, which was particularly noticeable in the C allele carriers. Our regression analysis demonstrated that rs12041331 AA + GA and rs2768759 CA predicted 90-day post-stroke bleeding. In conclusion, the PEAR1 polymorphisms rs12041331 and rs2768759 interfere with platelet aggregation and the performance of antiplatelet drugs. These genetic variations may contribute to bleeding events associated with minor stroke and TIA.

ДЕТЕРМИНАНТЫ ВЫСОКОЙ И НИЗКОЙ ОСТАТОЧНОЙ РЕАКТИВНОСТИ ТРОМБОЦИТОВ ПРИ ПРИЕМЕ КЛОПИДОГРЕЛА В 1-2 СУТКИ ИНФАРКТА МИОКАРДА

The aim of the study was to determine the clinical and laboratory factors that determine high and low platelet reactivity in patients taking clopidogrel on day 1-2 of myocardial infarction (MI). Material and Methods. A total of 405 patients (322 men and 83 women) with MI aged 31 to 74 years were examined. Group 1 included patients with low residual platelet reactivity (LRPR) – 89 people, group 2 consisted of patients with optimal platelet reactivity (OPR) – 223 people, group 3 was composed of patients with high residual platelet reactivity (HRPR) – 93 people. Platelet aggregation was assessed on a Multiplate impedance aggregometer (Germany) with several aggregation inducers. A blood test and a study of morphometric parameters of platelets were performed by the cytoflow method on an automated hematology analyzer Sysmex XS-500i (Japan). The study of the level of soluble CD40 ligand (sСD40L) and sP-selectin was performed in 140 patients, von Willebrand factor (VWF) and endothelin-1 were assessed in 150 people on the enzyme immunoassay analyzer Sunrise (TECAN, Austria) using Fine Biotech kits (China). All studies were performed upon admission to the hospital on day 1-2 of MI (at least 12 hours after the administration of a loading dose of clopidogrel). Results. A one-way linear regression analysis identified the following factors that independently affect the value of the ADP-test: age (β=-0.21, 95% CI: -0.42 – -0.0095; p=0.04); total cholesterol (β=1.4, 95% CI: 0.26–2.56; p=0.04); white blood cell count (β= 0.57, 95% CI: 0.003–1.14; p=0.049); hemoglobin (β=-0.16, 95% CI: -0.27 – -0.044; p=0.0067); platelet count (β=1.4, 95% CI: 0.26 – 2.56; p=0.04); PCT (β=76.8, 95% CI: 41.5 – 112.1; p=0.000024); TRAP-test (β=0.4, 95% CI: 0.33–0.46; p&lt;0.000001); CD40L (β=0.044, 95% CI: 0.013–0.074; p=0.005); sP-selectin (β=2.3, 95% CI: 0.63 – 3.97; p=0.007); VWF (β=0.06, 95% CI: 0.016 – 0.11; p=0.0085). To determine the probability of HRPR developing a binary logistic regression model was constructed based on the observational data of a sample of 398 patients, which included the following indicators: age, white blood cell count, mean platelet volume (MPV) and platelet distribution width (PDW) values, results of TRAP-test and the level of creatinine. At the probability cut-off p=0.4746, the sensitivity of the constructed model was 74.2%, specificity - 74.1%, classification accuracy – 74.1%, the area under the ROC-curve – 0.795 (CI: 0.745 – 0.844). To determine the probability of developing LRPR, a binary logistic regression model was constructed based on the observational data of 143 patients, which included the following indicators: the number of leukocytes, MPV and TRAP-test values, fibrinogen and VWF levels. With a probability cut-off p=0.5589, the sensitivity of this model was 84.6%, specificity – 77.8%, classification accuracy – 79.0%, the area under the ROC-curve – 0.826 (CI: 0.747 – 0.905). Conclusion. On days 1-2 of MI, 22% of patients with an excessive response to clopidogrel and 23% with an insufficient response to clopidogrel were identified. The determinants of HRPR are age, white blood cell count, MPV, PDW, TRAP-test and creatinine levels. The determinants of LRPR are white blood cell count, MPV values, spontaneous platelet aggregation, detected by the TRAP-test, the level of fibrinogen and VWF.

Cell Surface Platelet Tissue Factor Expression: Regulation by P2Y12 and Link to Residual Platelet Reactivity.

ADP-induced platelet activation leads to cell surface expression of several proteins, including TF (tissue factor). The role of ADP receptors in platelet TF modulation is still unknown. We aimed to assess the (1) involvement of P2Y1 and P2Y12 receptors in ADP-induced TF exposure; (2) modulation of TFpos-platelets in anti-P2Y12-treated patients with coronary artery disease. Based on the obtained results, we revisited the intracellular localization of TF in platelets. The effects of P2Y1 or P2Y12 antagonists on ADP-induced TF expression and activity were analyzed in vitro by flow cytometry and thrombin generation assay in blood from healthy subjects, P2Y12-/-, and patients with gray platelet syndrome. Ex vivo, P2Y12 inhibition of TF expression by clopidogrel/prasugrel/ticagrelor, assessed by VASP (vasodilator-stimulated phosphoprotein) platelet reactivity index, was investigated in coronary artery disease (n=238). Inhibition of open canalicular system externalization and electron microscopy (TEM) were used for TF localization. In blood from healthy subjects, stimulated in vitro by ADP, the percentage of TFpos-platelets (17.3±5.5%) was significantly reduced in a concentration-dependent manner by P2Y12 inhibition only (-81.7±9.5% with 100 nM AR-C69931MX). In coronary artery disease, inhibition of P2Y12 is paralleled by reduction of ADP-induced platelet TF expression (VASP platelet reactivity index: 17.9±11%, 20.9±11.3%, 40.3±13%; TFpos-platelets: 10.5±4.8%, 9.8±5.9%, 13.6±6.3%, in prasugrel/ticagrelor/clopidogrel-treated patients, respectively). Despite this, 15% of clopidogrel good responders had a level of TFpos-platelets similar to the poor-responder group. Indeed, a stronger P2Y12 inhibition (130-fold) is required to inhibit TF than VASP. Thus, a VASP platelet reactivity index <20% (as in prasugrel/ticagrelor-treated patients) identifies patients with TFpos-platelets <20% (92% sensitivity). Finally, colchicine impaired in vitro ADP-induced TF expression but not α-granule release, suggesting that TF is open canalicular system stored as confirmed by TEM and platelet analysis of patients with gray platelet syndrome. Data show that TF expression is regulated by P2Y12 and not P2Y1; P2Y12 antagonists downregulate the percentage of TFpos-platelets. In clopidogrel good-responder patients, assessment of TFpos-platelets highlights those with residual platelet reactivity. TF is stored in open canalicular system, and its membrane exposure upon activation is prevented by colchicine.