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Disorders of hemostasis resulting in bleeding or thrombosis are leading cause of mortality in the world. Blood platelets are main players in hemostasis, providing the primary response to the vessel wall injury. In this case, they rapidly switch to the activated state in reaction to the exposed chemical substances such as ADP, collagen and thrombin. Molecular mechanisms of platelet activation are known, and detailed computational models are available. However, they are too complicated for large-scale problems (e.g. simulation of the thrombus growth) where less detailed models are required, which still should take into account the variation of agonist concentration and heterogeneity of platelets. In this paper, we present a simple model of the platelet population response to a spatially inhomogeneous stimulus. First, computational nodes modeling platelets are placed randomly in space. Each platelet is assigned the specific threshold for agonist, which determines whether it becomes activated at a given time. The distribution of the threshold value in a population is assumed to be log-normal. The model was validated against experimental data in a specially designed system, where the photorelease of ADP was caused by localized laser stimulus. In this system, a concentration of ADP obeys 2-dimensional Gaussian distribution which broadens due to the diffusion. The response of platelets to the point-like source of ADP is successfully described by the presented model. Our results advance the understanding of platelet function during hemostatic response. The simulation approach can be incorporated into larger computational models of thrombus formation., Competing Interests: NO authors have competing interests, (Copyright: © 2024 Starodubtseva et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

The omega-3 polyunsaturated fatty acids (PUFAs) Docosahexaenoic acid (DHA) and Eicosapentaenoic acid (EPA) exert multiple cardioprotective effects, influencing inflammation, platelet activation, endothelial function and lipid metabolism, besides their well-established triglyceride lowering properties. It is not uncommon for omega-3 PUFAs to be prescribed for hypertriglyceridemia, alongside antiplatelet therapy in cardiovascular disease (CVD) patients. In this regard, we studied the effect of EPA and DHA, in combination with antiplatelet drugs, in platelet aggregation and P-selectin and α IIb β 3 membrane expression. The antiplatelet drugs aspirin and triflusal, inhibitors of cyclooxygenase-1 (COX-1); ticagrelor, an inhibitor of the receptor P2Y 12 ; vorapaxar, an inhibitor of the PAR-1 receptor, were combined with DHA or EPA and evaluated against in vitro platelet aggregation induced by agonists arachidonic acid (AA), adenosine diphosphate (ADP) and TRAP-6. We further investigated procaspase-activating compound 1 (PAC-1) binding and P-selectin membrane expression in platelets stimulated with ADP and TRAP-6. Both DHA and EPA displayed a dose-dependent inhibitory effect on platelet aggregation induced by AA, ADP and TRAP-6. In platelet aggregation induced by AA, DHA significantly improved acetylsalicylic acid (ASA) and triflusal's inhibitory activity, while EPA enhanced the inhibitory effect of ASA. In combination with EPA, ASA and ticagrelor expressed an increased inhibitory effect towards ADP-induced platelet activation. Both fatty acids could not improve the inhibitory effect of vorapaxar on AA- and ADP-induced platelet aggregation. In the presence of EPA, all antiplatelet drugs displayed a stronger inhibitory effect towards TRAP-6-induced platelet activation. Both omega-3 PUFAs inhibited the membrane expression of α IIb β 3 , though they had no effect on P-selectin expression induced by ADP or TRAP-6. The antiplatelet drugs exhibited heterogeneity regarding their effect on P-selectin and α IIb β 3 membrane expression, while both omega-3 PUFAs inhibited the membrane expression of α IIb β 3 , though had no effect on P-selectin expression induced by ADP or TRAP-6. The combinatory effect of DHA and EPA with the antiplatelet drugs did not result in enhanced inhibitory activity compared to the sum of the individual effects of each component.

Background: Leukocyte-platelet aggregates comprise a pathogenic link between hemostasis and immunity, but the prerequisites and mechanisms of their formation remain not understood., Aims: To quantify the formation, composition, and morphology of leukocyte-platelet aggregates in vitro under the influence of various cellular activators., Methods: Phorbol-12-myristate-13-acetate (PMA), lipopolysaccharide (LPS), thrombin receptor-activating peptide (TRAP-6), and adenosine diphosphate (ADP) were used as cellular activators. Flow cytometry was utilized to identify and quantify aggregates in whole human blood and platelet-rich plasma. Cell types and cellular aggregates were identified using fluorescently labeled antibodies against the appropriate cellular markers, and cell activation was assessed by the expression of appropriate surface markers. For confocal fluorescent microscopy, cell membranes and nuclei were labeled. Neutrophil-platelet aggregates were studied using scanning electron microscopy., Results: In the presence of PMA, ADP or TRAP-6, about 17-38 % of neutrophils and 61-77 % of monocytes formed aggregates with platelets in whole blood, whereas LPS did not induce platelet aggregation with either neutrophils or monocytes due the inability to activate platelets. Similar results were obtained when isolated neutrophils were added to platelet-rich plasma. All the cell types involved in the heterotypic aggregation expressed molecular markers of activation. Fluorescent and electron microscopy of the aggregates showed that the predominant platelet/leukocyte ratios were 1:1 and 2:1., Conclusions: Formation of leukocyte-platelet aggregates depends on the nature of the cellular activator and the spectrum of its cell-activating ability. An indispensable condition for formation of leukocyte-platelet aggregates is activation of all cell types including platelets, which is the restrictive step., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Acetylcholine is the main neurotransmitter at the vertebrate neuromuscular junctions (NMJs). ACh exocytosis is precisely modulated by co-transmitter ATP and its metabolites. It is assumed that ATP/ADP effects on ACh release rely on activation of presynaptic G i protein-coupled P 2 Y 13 receptors. However, downstream signaling mechanism of ATP/ADP-mediated modulation of neuromuscular transmission remains elusive. Using microelectrode recording and fluorescent indicators, the mechanism underlying purinergic regulation was studied in the mouse diaphragm NMJs. Pharmacological stimulation of purinoceptors with ADP decreased synaptic vesicle exocytosis evoked by both low and higher frequency stimulation. This inhibitory action was suppressed by antagonists of P 2 Y 13 receptors (MRS 2211), Ca 2+ mobilization (TMB8), protein kinase C (chelerythrine) and NADPH oxidase (VAS2870) as well as antioxidants. This suggests the participation of Ca 2+ and reactive oxygen species (ROS) in the ADP-triggered signaling. Indeed, ADP caused an increase in cytosolic Ca 2+ with subsequent elevation of ROS levels. The elevation of [Ca 2+ ] in was blocked by MRS 2211 and TMB8, whereas upregulation of ROS was prevented by pertussis toxin (inhibitor of G i protein) and VAS2870. Targeting the main components of lipid rafts, cholesterol and sphingomyelin, suppressed P 2 Y 13 receptor-dependent attenuation of exocytosis and ADP-induced enhancement of ROS production. Inhibition of P 2 Y 13 receptors decreased ROS production and increased the rate of exocytosis during intense activity. Thus, suppression of neuromuscular transmission by exogenous ADP or endogenous ATP can rely on P 2 Y 13 receptor/G i protein/Ca 2+ /protein kinase C/NADPH oxidase/ROS signaling, which is coordinated in a lipid raft-dependent manner., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Introduction: Previous literature suggests that sphingolipids may impact systemic coagulation and platelet aggregation, thus modulating the risks of thrombotic events. The goal of this investigation was to evaluate the role of serum sphingolipids on intrinsic platelet function to assess whether pharmacologic manipulation of sphingolipid metabolites would impact platelet aggregability., Methods: C57BL/6J mice were injected with either normal saline, 1 mg/kg FTY720 (synthetic sphingosine-1-phosphate [S1P] receptor analog), or 5 mg/kg SLM6031434 (sphingosine kinase two inhibitor). Mice were sacrificed at 6 h and whole blood (WB) was collected for impedance aggregometry assessing platelet responsiveness to arachidonic acid or adenosine diphosphate. Ex vivo studies utilized WB or platelet-rich plasma that was pretreated with S1P, FTY720, amitriptyline, or d-sphingosine then analyzed by aggregability and flow cytometry for platelet and platelet-derived microvesicle characteristics., Results: FTY720 and SLM6031434 pretreated induced similar arachidonic acid and adenosine diphosphate-mediated platelet aggregation as controls. Ex vivo WB and platelet-rich plasma treatment with S1P, FTY720, amitriptyline and d-sphingosine did not impact platelet aggregation. The percentages of CD41+, CD62P+ and CD41+/ceramide+, CD62P+/ceramide + platelets, and platelet-derived microvesicle were not significantly different between amitriptyline-treated and normal saline-treated cohorts., Conclusions: Sphingolipid modulating agents, such as FTY720, SLM6031434, S1P, amitriptyline, ceramide, and d-sphingosine do not appear to independently impact platelet aggregation in murine models., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Introduction: Platelets play an important role in cardiovascular disease mainly in the development of acute thrombotic events. Elevated platelet indices have been proposed as a risk factor for acute coronary syndrome (ACS). It remains uncertain whether increased platelet indices are the result or the cause of ACS., Aim and Objective: This study aimed to correlate mean platelet volume (MPV) and platelet aggregation response to know the functional status of platelets based on their size., Materials and Methods: A total of 50 patients with ST-segment elevation ACS (STE-ACS) or non-ST-segment elevation ACS (NSTE-ACS) were included and their MPV was measured and platelet aggregometry was performed. Patients were divided into two groups, patients with MPV ≤9.1 fl as group 1 and those with MPV >9.1 fl as group 2. The mean maximum platelet aggregation response (MMPAR) with ADP, Collagen, and Epinephrine, of both the groups, were compared. MMPAR to ADP, Collagen, and Epinephrine in group 1 was 74.47%, 66.13%, and 72.9%, respectively, and in group 2, 72.94%, 59.97%, and 72.43%, respectively. There was no statistically significant difference in the MMPAR to ADP, Collagen, and Epinephrine among the two groups., Conclusion: Increased MPV does not indicate the platelets are hyperreactive. An increase in MPV may be because of the increased release of immature platelets from bone marrow as there is increased consumption of platelets at the site of thrombus formation in ACS., (Copyright © 2023 Copyright: © 2023 Indian Journal of Pathology and Microbiology.)

Arterial thrombosis manifesting as heart attack and stroke is the leading cause of death worldwide. Platelets are central mediators of thrombosis that can be activated through multiple activation pathways. Platelet-derived extracellular vesicles (pEVs), also known as platelet-derived microparticles, are granular mixtures of membrane structures produced by platelets in response to various activating stimuli. Initial studies have attracted interest on how platelet agonists influence the composition of the pEV proteome. In the current study, we used physiological platelet agonists of varying potencies which reflect the microenvironments that platelets experience during thrombus formation: adenosine diphosphate, collagen, thrombin as well as a combination of thrombin/collagen to induce platelet activation and pEV generation. Proteomic profiling revealed that pEVs have an agonist-dependent altered proteome in comparison to their cells of origin, activated platelets. Furthermore, we found that various protein classes including those related to coagulation and complement (prothrombin, antithrombin, and plasminogen) and platelet activation (fibrinogen) are attributed to platelet EVs following agonist stimulation. This agonist-dependent altered proteome suggests that protein packaging is an active process that appears to occur without de novo protein synthesis. This study provides new information on the influence of physiological agonist stimuli on the biogenesis and proteome landscape of pEVs., (© 2024 The Authors. PROTEOMICS published by Wiley‐VCH GmbH.)

Background: Protease-activated receptor 4 (PAR4) mediates thrombin signaling on platelets and other cells. Our recent structural studies demonstrated that a single nucleotide polymorphism in extracellular loop 3 and PAR4-P310L (rs2227376) leads to a hyporeactive receptor., Objectives: The goal of this study was to determine how the hyporeactive PAR4 variant in extracellular loop 3 impacts platelet function in vivo using a novel knock-in mouse model (PAR4-322L)., Methods: A point mutation was introduced into the PAR4 gene F2rl3 via CRISPR/Cas9 to create PAR4-P322L, the mouse homolog to human PAR4-P310L. Platelet response to PAR4 activation peptide (AYPGKF), thrombin, ADP, and convulxin was monitored by αIIbβ3 integrin activation and P-selectin translocation using flow cytometry or platelet aggregation. In vivo responses were determined by the tail bleeding assay and the ferric chloride-induced carotid artery injury model., Results: PAR4-P/L and PAR4-L/L platelets had a reduced response to AYPGKF and thrombin measured by P-selectin translocation or αIIbβ3 activation. The response to ADP and convulxin was unchanged among genotypes. In addition, both PAR4-P/L and PAR4-L/L platelets showed a reduced response to thrombin in aggregation studies. There was an increase in the tail bleeding time for PAR4-L/L mice. The PAR4-P/L and PAR4-L/L mice both showed an extended time to arterial thrombosis., Conclusion: PAR4-322L significantly reduced platelet responsiveness to AYPGKF and thrombin, which is in agreement with our previous structural and cell signaling studies. In addition, PAR4-322L had prolonged arterial thrombosis time. Our mouse model provides a foundation to further evaluate the role of PAR4 in other pathophysiological contexts., Competing Interests: Declaration of competing interests There are no competing interests to disclose, (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Peripheral mononuclear cells (PBMCs) exhibit robust changes in mitochondrial respiratory capacity in response to health and disease. While these changes do not always reflect what occurs in other tissues, such as skeletal muscle, these cells are an accessible and valuable source of viable mitochondria from human subjects. PBMCs are exposed to systemic signals that impact their bioenergetic state. Thus, expanding our tools to interrogate mitochondrial metabolism in this population will elucidate mechanisms related to disease progression. Functional assays of mitochondria are often limited to using respiratory outputs following maximal substrate, inhibitor, and uncoupler concentrations to determine the full range of respiratory capacity, which may not be achievable in vivo. The conversion of adenosine diphosphate (ADP) to adenosine triphosphate (ATP) by ATP-synthase results in a decrease in mitochondrial membrane potential (mMP) and an increase in oxygen consumption. To provide a more integrated analysis of mitochondrial dynamics, this article describes the use of high-resolution fluorespirometry to measure the simultaneous response of oxygen consumption and mitochondrial membrane potential (mMP) to physiologically relevant concentrations of ADP. This technique uses tetramethylrhodamine methylester (TMRM) to measure mMP polarization in response to ADP titrations following maximal hyperpolarization with complex I and II substrates. This technique can be used to quantify how changes in health status, such as aging and metabolic disease, affect the sensitivity of mitochondrial response to energy demand in PBMCs, T-cells, and monocytes from human subjects.

Platelets play essential roles in the formation of blood clots by clumping with coagulation factors at the site of vascular injury to stop bleeding; therefore, a reduction in the platelet number or disorder in their function causes bleeding risk. In our research, we developed a method to assess platelet aggregation using an optical approach within a microfluidic chip's channel by evaluating the size of laser speckles. These speckles, associated with slowed blood flow in the microfluidic channel, had a baseline size of 28.54 ± 0.72 µm in whole blood. Removing platelets from the sample led to a notable decrease in speckle size to 27.04 ± 1.23 µm. Moreover, the addition of an ADP-containing agonist, which activates platelets, resulted in an increased speckle size of 32.89 ± 1.69 µm. This finding may provide a simple optical method via microfluidics that could be utilized to assess platelet functionality in diagnosing bleeding disorders and potentially in monitoring therapies that target platelets.

Background: This study aims to investigate the relevance of platelet aggregation markers, specifically arachidonic acid (AA) and adenosine diphosphate (ADP), in relation to the prognosis of sepsis patients., Methods: A cohort of 40 sepsis patients was included and stratified, based on their 28-day post-treatment prognosis, into two groups: a survival group (n = 31) and a severe sepsis group (n = 9. Then, their various clinical parameters, including patient demographics, platelet counts (PLT), inflammatory markers, and platelet aggregation rates (PAR) induced by AA and ADP between the two groups, were compared. Long-term health implications of sepsis were assessed using the Acute Physiologic Assessment and Chronic Health Evaluation II (APACHE II) score, and logistic regression analysis was conducted to evaluate the prognostic significance of PAR in sepsis patients., Results: Patients with severe sepsis exhibited significantly elevated levels of procalcitonin (PCT), platelet adhesion rates, and PAR induced by ADP (P < 0.05), but having lower PLT (P < 0.05), compared to those in the survival group. Logistic regression analysis demonstrated that PAR induced by ADP was a protective factor in predicting prognosis in sepsis patients (P < 0.01)., Conclusions: Activation of platelets in sepsis intensifies inflammatory response. Patients with sepsis whose ADP-induced PAR was < 60% displayed significant impairment in platelet aggregation function, and had higher mortality rate. Monitoring ADP-induced PAR is crucial for management of sepsis., Competing Interests: The authors stated that there were no conflicts of interest to declare.

In in vitro model of short-term therapeutic inhalation of Xe/O 2 mixture, xenon in millimolar concentrations led to a pronounced decrease in induced platelet aggregation in the platelet-enriched blood plasma. The maximum and statistically significant decrease occurred in response to induction by collagen (by ≈30%, p≤0.01) and ADP (by ≈25%, p≤0.01). A slightly weaker but statistically significant reduction in aggregation appeared in response to ristocetin (by ≈12%, p≤0.01) and epinephrine (by ≈9%, p≤0.01). It should be noted that the spontaneous aggregation exceeded the reference values in the control group. Nevertheless, even at minimal absolute values, spontaneous platelet aggregation decreased by 2 times in response to xenon (p≤0.01). The reasons for the decrease of spontaneous and induced aggregation are xenon accumulation in the lipid bilayer of the membrane with subsequent nonspecific (mechanical) disassociation of membrane platelet structures and specific block of its distinct from neuronal NMDA receptor., (© 2024. Springer Science+Business Media, LLC, part of Springer Nature.)

Objectives: Flow cytometry with adenosine diphosphate (ADP) allows to characterize molecular changes of platelet function caused by this physiologically important activation, but the methodology has not been thoroughly investigated, standardized and characterized yet. We analyzed the influence of several major variables and chose optimal conditions for platelet function assessment., Methods: For activation, 2.5 μM CaCl 2 , 5 μM ADP and antibodies were added to diluted blood and incubated for 15 min. We analyzed kinetics of antibody binding and effects of their addition sequence, agonist concentration, blood dilution, exogenous calcium addition and platelet fixation., Results: We tested our protocol on 11 healthy children, 22 healthy adult volunteers, 9 patients after a month on dual antiplatelet therapy after percutaneous coronary intervention (PCI), 7 adult patients and 14 children with immune thrombocytopenia (ITP). We found that our protocol is highly sensitive to ADP stimulation with low percentage of aggregates formation. The assay is also sensitive to platelet function inhibition in post-PCI patients. Finally, platelet preactivation with ITP plasma was stronger and caused increase in activation response to ADP stimulation compared to preactivation with low dose of ADP., Conclusions: Our assay is sensitive to antiplatelet therapy and platelet preactivation in ITP patients under physiological conditions with minimal percentage of aggregates formation., (© 2023 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Background and Aims:  Glycoprotein VI (GPVI) is the major platelet-specific collagen receptor. GPVI shedding with generation of soluble GPVI (sGPVI) is an endogenous feedback mechanism preventing platelet overstimulation. sGPVI has not been investigated in patients with chronic coronary syndrome (CCS) undergoing percutaneous coronary intervention (PCI), especially regarding its potential value as a predictor of ischemic and bleeding risk., Methods:  Baseline plasma sGPVI levels were available in 318 patients with CCS undergoing PCI. Platelet function was assessed by measuring both adenosine diphosphate (ADP) and collagen-induced platelet aggregation. Co-primary endpoints were a composite of death or myocardial injury at 48 hours after PCI, and Bleeding Academic Research Consortium (BARC) type 1 to 5 bleeding at 30 days., Results:  There was no significant correlation between sGPVI and platelet function at baseline or at 48 hours after PCI and loading with antiplatelet drugs. Baseline plasma sGPVI levels were not associated with the ischemic risk: the incidence of the ischemic endpoint was 25.0% in the lower, 22.9% in the middle, and 26.7% in the upper sGPVI tertile ( p  = 0.82). There was a significant nonlinear relationship between sGPVI and the risk of bleeding: the incidence of the bleeding endpoint was 11.8% in the lower, 12.6% in the middle, and 26.4% in the upper sGPVI tertile ( p  = 0.006)., Conclusion:  In patients with CCS undergoing PCI, plasma levels of sGPVI did not correlate with ADP- or collagen-induced platelet aggregation. Patients with higher baseline levels of sGPVI may carry an increased risk of bleeding at 30 days after PCI but no excess risk of ischemic events., Competing Interests: R.H.-R. reported grants from German Center for Cardiovascular Research, Deutsches Herzzentrum München, the Federal Ministry of Education and Research, and advanceCOR GmbH during the conduct of the study. I.B. reported personal fees from Sysmex Europe GmbH outside the submitted work. S.S. reported grants from Else Kröner-Fresenius-Stiftung (Else Kröner-Memorial grant) during the conduct of the study and DZHK (German Center for Cardiovascular Research) for the Intracoronary Stenting and Antithrombotic Regimen: Lesion Platelet Adhesion as Selective Target of Endovenous Revacept in Patients with Chronic Coronary Syndromes Undergoing Percutaneous Coronary Intervention trial and personal fees from Bayer Vital GmbH, Daiichi Sankyo, and Biopas Laboratories outside the submitted work. S.H. has received research grants and honoraria from Roche Diagnostics, Bristol Myers Squibb, Merck KGaA, Sysmex Inostics, and Volition SPRL outside the submitted work. M.G. is cofounder of advanceCor, the manufacturer of Revacept. H.S. reports honoraria from AstraZeneca, Bayer Vital, MSD Sharp & Dohme, Novartis, Servier, Sanofi-Aventis, Boehringer Ingelheim, Daiichi Sankyo, Amgen, Pfizer and consulting fees from AstraZeneca, Amgen, MSD Sharp & Dohme, not related to the current work. G.M. is the founder of advanceCOR GmbH, Martinsried, Germany. K.A. is employee of advanceCOR GmbH, Martinsried, Germany., (Thieme. All rights reserved.)

Platelet functional activity was assessed in healthy volunteers (HV, n=92), patients with stable angina pectoris (SA, n=42) and acute coronary syndrome (ACS, n=73), treated with acetylsalicylic acid (ASA) + clopidogrel and ASA + ticagrelor, respectively. In all HV and patients we have compared parameters of platelet aggregation (maximum light transmission and velocity, Tmax and Vmax) and parameters, characterizing exposure of platelet activation markers, evaluated by flow cytometry. HV platelets were activated by 10 μM, 1 μM TRAP, and 20 μM, 5 μM, 2.5 μM ADP; patient platelets were activated by 10 μM TRAP and by 20 μM and 5 μM ADP. Strong and significant correlations between the aggregation and flow cytometry parameters (the r correlation coefficient from 0.4 up to >0.6) most frequently were registered in HV platelet during activation by 1 μM TRAP and in SA patients during platelet activation by 20 μM and 5 μM ADP. However, in many other cases these correlations were rather weak (r < 0.3) and sometimes statistically insignificant. In HV the differences in PAC-1 binding parameters between platelets activated by 10 μM TRAP (the strongest agonist) and all ADP concentrations were negligible (≤ 10%), while CD62P binding (at all ADP concentrations) and LTA parameters for (5 μM and 2.5 μM ADP) were significantly lower (by 40-60%). Antiplatelet therapy in patients decreased all parameters as compared to HV, but to varying extents. For 10 μM TRAP the MFI index for PAC-1 binding (40-50% decrease) and for both ADP concentrations the Tmax values (60-85% decrease) appeared to be the most sensitive in comparison with the other parameters that decreased to a lesser extent. The data obtained indicate a possibility of inconsistency between different LTA and flow cytometry parameters in assessing platelet activity and efficacy of antiplatelet drugs.

Objectives: The aim of the current study was to assess the relationship among thrombin receptor activator peptide 6 (TRAP test), adenosine-5'-diphosphate (ADP test), arachidonic acid (ASPI test), and stroke/transient ischemic attack (TIA), using the multiple electrode aggregometry (Multiplate) in patients undergoing carotid thromboendarterectomy (CEA)., Design: A retrospective study., Setting: Vascular surgery operating rooms of a university hospital., Participants: One hundred thirty-one out of 474 patients undergoing CEA between November 2020 and October 2022., Interventions: None., Measurements and Main Results: A preoperative blood sample of all enrolled patients was analyzed using the Multiplate analyzer. Receiver operating characteristics curves, were generated to test the ability of TRAP, ADP, and ASPI in discriminating perioperative thromboembolic stroke/TIA. A logistic LASSO regression model was used to identify factors independently associated with stroke/TIA. Eight patients experienced a perioperative stroke/TIA. Although all the platelet functional assays showed excellent predictive performance, an ADP value exceeding 72 U showed the highest specificity (87%) and sensitivity (68%) in discriminating patients who had a perioperative thromboembolic stroke/TIA, with a negative predictive value of 99% and a positive predictive value of 15%. After LASSO regression, an ADP >72 U and the need for a shunt during CEA were the only 2 variables independently associated with perioperative stroke/TIA., Conclusion: Because the ADP test was independently associated with perioperative stroke/TIA, the assessment of platelet reactivity using Multiplate may offer potential utility in monitoring patients undergoing CEA., Competing Interests: Declaration of competing interest None, (Copyright © 2023 Elsevier Inc. All rights reserved.)

Abstract: Sildenafil, a common over-the-counter pill often self-administered at high doses for erectile dysfunction, has been reported to rarely cause prothrombotic events and sudden cardiac death in a few case reports. Therefore, we investigated the in vitro and in vivo effect of sildenafil treatment and dosage on platelet activation and mitogen-activated protein kinase (MAPK) phosphorylation. BALB/C mice were segregated into four groups, each having four mice each (control, low [3.25 mg/kg], medium [6.5 mg/kg], and high [13 mg/kg] sildenafil), and after the treatment, blood was drawn from each mouse and washed platelets prepared. Washed platelets were incubated with CD41 PE-Cy7 and Phospho-p38 MAPK PE antibodies and analyzed using a flow cytometer for platelet activation and adenosine 5'- diphosphate (ADP)/collagen-induced MAPK phosphorylation. Washed platelets obtained from the venous blood of 18 human volunteers, were incubated with PAC-1 FITC and Phospho-p38 MAPK PE antibodies, and platelet activation (ADP and collagen), followed by flow cytometry analysis. There was a significant increase in both platelet activation as well as MAPK phosphorylation in the presence of collagen in the high-dose (13 mg/kg) sildenafil group (P = 0.000774). Further, increased platelet activation was observed in samples that were treated with high-dose sildenafil as compared to the untreated samples (P < 0.00001). These studies show the risk of prothrombotic episodes in patients on high-dose sildenafil (100 mg), in those with even mild endothelial dysfunction due to ADP, and collagen-induced platelet activation through MAPK phosphorylation, which was not seen in the low-and intermediate-dose cohorts., (Copyright © 2024 Copyright: © 2024 Indian Journal of Pharmacology.)

Background and Purpose: Platelet function during inflammation is dependent on activation by endogenous nucleotides. Non-canonical signalling via the P2Y 1 receptor is important for these non-thrombotic functions of platelets. However, apart from ADP, the role of other endogenous nucleotides acting as agonists at P2Y 1 receptors is unknown. This study compared the effects of ADP, Ap3A, NAD + , ADP-ribose, and Up4A on platelet functions contributing to inflammation or haemostasis., Experimental Approach: Platelets obtained from healthy human volunteers were incubated with ADP, Ap3A, NAD + , ADP-ribose, or Up4A, with aggregation and fibrinogen binding measured (examples of function during haemostasis) or before exposure to fMLP to measure platelet chemotaxis (an inflammatory function). In silico molecular docking of these nucleotides to the binding pocket of P2Y 1 receptors was then assessed., Key Results: Platelet aggregation and binding to fibrinogen induced by ADP was not mimicked by NAD + , ADP-ribose, and Up4A. However, these endogenous nucleotides induced P2Y 1 -dependent platelet chemotaxis, an effect that required RhoA and Rac-1 activity, but not canonical PLC activity. Analysis of molecular docking of the P2Y 1 receptor revealed distinct differences of amino acid interactions and depth of fit within the binding pocket for Ap3A, NAD + , ADP-ribose, or Up4A compared with ADP., Conclusion and Implications: Platelet function (aggregation vs motility) can be differentially modulated by biased-agonist activation of P2Y 1 receptors. This may be due to the character of the ligand-binding pocket interaction. This has implications for future therapeutic strategies aimed to suppress platelet activation during inflammation without affecting haemostasis as is the requirement of current ant-platelet drugs., Linked Articles: This article is part of a themed issue on Platelet purinergic receptor and non-thrombotic disease. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v181.4/issuetoc., (© 2023 The Authors. British Journal of Pharmacology published by John Wiley & Sons Ltd on behalf of British Pharmacological Society.)

Adenosine diphosphate (ADP) is a nucleotide that is structurally very similar to ATP but lacks one of the two high-energy bonds due to hydrolysis. In muscle studies, ADP is usually considered exclusively as a product formed during myosin cross-bridge cycling and is not otherwise involved in this process. In our study, we question the widely held view of ADP as a final product formed during muscle contraction. Using biophysical and biochemical methods, we managed to show that ADP can act as a substrate for myosins in at least three types of muscles: smooth and striated adductor muscles of bivalves (Mytilidae and Pectinidae), and also vertebrate skeletal muscles. According to our data, the differences in the effect of ATP and ADP on the optical, biochemical, and structural properties of actomyosins are exclusively quantitative. We explain the previous ideas about ADP as a compound capable of inhibiting the ATPase activity of actomyosin by the ability of ATP and ADP to depolymerize the polymeric myosin when the concentration in the medium reaches more than 0.3 mM., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Vascular endothelial cells serve as barriers between blood components and subendothelial tissue and regulate the blood coagulation-fibrinolytic system. Ionizing radiation is a common physical stimulant that induces a bystander effect whereby irradiated cells influence neighboring cells through signalings, including purinergic receptor signaling, activated by adenosine 5'-triphosphate (ATP), adenosine 5'-diphosphate (ADP), and adenosine as secondary soluble factors. Human vascular endothelial EA.hy926 cells were cultured and irradiated with γ-rays or treated with ATP, ADP, or adenosine under non-toxic conditions. RNA-seq, gene ontology, and hierarchical clustering analyses were performed. The transcriptome analysis of differentially expressed genes in vascular endothelial cells after γ-ray irradiations suggests that the change of gene expression by γ-irradiation is mediated by ATP and ADP. In addition, the expression and activity of the proteins related to blood coagulation and fibrinolysis systems appear to be secondarily regulated by ATP and ADP in vascular endothelial cells after exposure to γ-irradiation. Although it is unclear whether the changes of the gene expression related to blood coagulation and fibrinolysis systems by γ-irradiation affected the increased hemorrhagic tendency through the exposure to γ-irradiation or the negative feedback to the activated blood coagulation system, the present data indicate that toxicity associated with γ-irradiation involves the dysfunction of vascular endothelial cells related to the blood coagulation-fibrinolytic system, which is mediated by the signalings, including purinergic receptor signaling, activated by ATP and ADP.

Background and Purpose: Ticagrelor is labelled as a reversible, direct-acting platelet P2Y 12 receptor (P2Y 12 R) antagonist that is indicated clinically for the prevention of thrombotic events in patients with acute coronary syndrome (ACS). As with many antiplatelet drugs, ticagrelor therapy increases bleeding risk in patients, which may require platelet transfusion in emergency situations. The aim of this study was to further examine the reversibility of ticagrelor at the P2Y 12 R., Experimental Approach: Studies were performed in human platelets, with P2Y 12 R-stimulated GTPase activity and platelet aggregation assessed. Cell-based bioluminescence resonance energy transfer (BRET) assays were undertaken to assess G protein-subunit activation downstream of P2Y 12 R activation., Key Results: Initial studies revealed that a range of P2Y 12 R ligands, including ticagrelor, displayed inverse agonist activity at P2Y 12 R. Only ticagrelor was resistant to washout and, in human platelet and cell-based assays, washing failed to reverse ticagrelor-dependent inhibition of ADP-stimulated P2Y 12 R function. The P2Y 12 R agonist 2MeSADP, which was also resistant to washout, was able to effectively compete with ticagrelor. In silico docking revealed that ticagrelor and 2MeSADP penetrated more deeply into the orthosteric binding pocket of the P2Y 12 R than other P2Y 12 R ligands., Conclusion and Implications: Ticagrelor binding to P2Y 12 R is prolonged and more akin to that of an irreversible antagonist, especially versus the endogenous P2Y 12 R agonist ADP. This study highlights the potential clinical need for novel ticagrelor reversal strategies in patients with spontaneous major bleeding, and for bleeding associated with urgent invasive procedures., (© 2023 The Authors. British Journal of Pharmacology published by John Wiley & Sons Ltd on behalf of British Pharmacological Society.)

Background: Recently the US Food and Drug Administration has granted variances to select blood centers to supply cold-stored platelet components (CSP). In hemorrhage resuscitation warming of blood components with approved fluid warming devices is common., Study Design and Methods: Pathogen-reduced apheresis platelet units were collected and stored in one of two ways: (1) CSP-I, (2) CSP-D. CSP-I were collected and immediately stored at 1-6°C until used. CSP-D were collected and stored at 20-24°C for 5 days and transferred to storage at 1-6°C until use. Aggregometry using arachidonic acid (AA), adenosine diphosphate (ADP) and collagen as agonists was performed on the unit samples before and after the units were infused through a Ranger blood-warming device., Results: CSP-I, 23 units, had very high aggregation responses to all agonists (all ≥47.6 ± 20.7). There was a statistically significant reduction in ADP-induced aggregometry results from 55.1 ± 23.2 before compared to 33.5 ± 14.6 following infusion of the PLT through the blood warmer (p < .001). There were no differences in AA and collagen aggregometry results before and after the infusion of the platelets through the blood warmer. CSP-D had 5 of the 15 units with visible clotting in the bag. The 10 CSP-Ds studied had lower aggregation than all agonists before and after infusion through the blood-warming device (all ≤49.9 ± 35.9)., Conclusion: We detected a statistically significant reduction in ADP-induced aggregometry in CSP-I run through a Ranger blood-warming device with no change with AA or collagen agonist aggregometry., (© 2023 AABB.)

Background: Several conventional studies focused on platelet pinocytosis for possible utilization as drug delivery systems. Although platelet pinocytosis is important in such utilization, the impact of the shear rate on pinocytosis is unclear., Objective: Our objective was to investigate the relationship between shear rate and platelet pinocytosis in vitro. In addition, this study addressed the change in platelet aggregation reactivity with adenosine diphosphate (ADP) stimulation after pinocytosis., Method: Porcine platelet-rich plasma was mixed with fluorescein isothiocyanate (FITC)-conjugated dextran and incubated for 15 min under shear conditions of 0, 500, and 1500 s-1. After incubation, confocal microscopic scanning and three-dimensional rendering were performed to confirm the internalization of FITC-dextran into platelets. The amount of FITC-dextran accumulated via platelet pinocytosis was compared using flow cytometry at each shear rate. In addition, light transmission aggregometry by ADP stimulation was applied to platelets after pinocytosis., Results: The amount of intracellular FITC-dextran increased with higher shear rates. Platelets with increased amounts of intracellular FITC-dextran did not show changes in the aggregation reactivity to ADP., Conclusions: A higher shear rate promotes platelet pinocytosis, but enhanced pinocytosis does not affect aggregation sensitivity, which is stimulated by ADP.

Growth differentiation factor-15 (GDF-15) is proposed to be strongly associated with several cardiovascular diseases, such as heart failure and atherosclerosis. Moreover, some recent studies have reported an association between GDF-15 and platelet activation. In this study, we isolated peripheral blood platelets from healthy volunteers and evaluated the effect of GDF-15 on adenosine diphosphate (ADP)-induced platelet activation using the platelet aggregation assay. Subsequently, we detected the expression of GDF-15-related receptors on platelets, including the epidermal growth factor receptor (EGFR), human epidermal growth factor receptor 2 (HER2), human epidermal growth factor receptor 3 (HER3), transforming growth factor-beta receptor I (TGF-βRI), transforming growth factor-beta receptor II (TGF-βRII), glial-cell-line-derived neurotrophic factor family receptor α-like (GFRAL), and those rearranged during transfection (RET). Then, we screened for GDF-15 receptors using the GDF-15-related receptor microarray comprising these recombinant proteins. We also performed the immunoprecipitation assay to investigate the interaction between GDF-15 and the receptors on platelets. For the further exploration of signaling pathways, we investigated the effects of GDF-15 on the extracellular signal-regulated kinase (ERK), protein kinase B (AKT), and Janus kinase 2 (JAK2) pathways. We also investigated the effects of GDF-15 on the ERK and AKT pathways and platelet aggregation in the presence or absence of RET agonists or inhibition. Our study revealed that GDF-15 can dose-independently inhibit ADP-induced human platelet aggregation and that the binding partner of GDF-15 on platelets is GFRAL. We also found that GDF-15 inhibits ADP-induced AKT and ERK activation in platelets. Meanwhile, our results revealed that the inhibitory effects of GDF-15 can be mediated by the GFRAL/RET complex. These findings reveal the novel inhibitory mechanism of ADP-induced platelet activation by GDF-15.

Background: Antiplatelet therapy is the cornerstone of treatment for patients presenting with acute coronary syndrome (ACS) treated with percutaneous coronary intervention (PCI). Some patients may not respond to such therapy adequately, which is associated with a greater risk of ischemic events. Reticulated platelets are the youngest, largest, and most active platelet subtype. They have been initially shown to be associated with an increased risk of cardiovascular (CV) events and increased platelet activity., Objectives: The aim of the presented study was to evaluate whether the immature platelet fraction (IPF) reflects the response to antiplatelet treatment in invasively managed ACS patients., Material and Methods: This prospective study enrolled ACS patients treated with PCI and dual antiplatelet therapy (DAPT) comprising acetylsalicylic acid (ASA) and clopidogrel or ticagrelor. In all patients, venous blood was collected within 24 h after the procedure. Platelet parameters were measured, including IPF using the Sysmex hematological analyzer and adenosine diphosphate (ADP)-induced platelet reactivity using the Multiplate® Analyzer., Results: A total of 108 patients were enrolled, including 62 with ST-segment elevation ACS (STE-ACS) and 46 with non-ST-segment elevation ACS (NSTE-ACS). Of them, 20.4% had diabetes mellitus, 26.9% had a history of MI and 59.2% of smoking. Spearman's correlation analysis demonstrated that higher IPF and immature platelet count (IPC) values are associated with increased ADP-induced platelet reactivity (respectively: rho = 0.387, 95% confidence interval (95% CI): 0.101-0.615, p = 0.008; and rho = 0.458, 95% CI: 0.185-0.666, p = 0.001) in NSTE-ACS but not in STE-ACS patients., Conclusion: Immature platelet count and IPF may be valuable markers of platelet activity in patients with NSTE-ACS treated invasively and receiving DAPT (ClinicalTrials.gov No. NCT06177587).

Purpose: The incidence of acute myocardial infarctions (AMI) shows circadian variation typically peaking during morning hours with a decline at night. However, this variation does not occur in patients with diabetes mellitus (DM). The night's decline of AMI may be partially explained by melatonin-related platelet inhibition. Whether this effect is absent in diabetic patients is unknown. The aim was to study the effect of melatonin on in-vitro platelet aggregation in healthy individuals and patients with type 2 DM., Methods: Platelet aggregation was measured in blood samples from healthy individuals (n = 15) and type 2 DM patients (n = 15) using multiple electrode aggregometry. Adenosine diphosphate (ADP), arachidonic acid (ASPI) and thrombin (TRAP) were used as agonists. Aggregability for each subject was tested after adding melatonin in two concentrations., Results: In healthy individuals, melatonin inhibited platelet aggregation in both higher (10-5 M) and lower concentrations (10-9 M) induced by ADP, ASPI, and TRAP (p < 0.001, p = 0.002, p = 0.029, respectively). In DM patients, melatonin did not affect platelet aggregation in both concentrations induced by ADP, ASPI, and TRAP. Melatonin decreased platelet aggregation induced by ADP, ASPI, and TRAP significantly more in healthy individuals compared to patients with DM. (p = 0.005, p = 0.045 and p = 0.048, respectively)., Conclusion: Platelet aggregation was inhibited by melatonin in healthy individuals. In-vitro antiplatelet effect of melatonin in type 2 DM patients is significantly attenuated., (© 2023. The Author(s).)

Activated platelets possess procoagulant activity expressing on their surface phosphatidylserine (PS), a substrate for assembling coagulation complexes. We examined the effects of platelets activated by different agonists on fibrin formation and thrombin generation and compared these effects with each other and with PS expression. Modified plasma recalcification assay was developed to assess platelet effects on fibrin formation. Washed human platelets were left intact or activated by A23187 ionophore, collagen, arachidonic acid, ADP or TRAP (Thrombin Receptor Activating Peptide) and spun down in 96-well plates. Plasma was then added, recalcified, and fibrin formation was monitored by light absorbance. Platelets prepared in the same way were tested for their effect on thrombin generation. PS expression was evaluated by flow cytometry using annexin V staining. Platelets significantly accelerated fibrin formation and thrombin generation. They shortened lag phase and increased maximum rate of plasma clotting, and increased peak and maximum rate of thrombin generation. In both tests platelets were presumably activated by endogenous thrombin formed in plasma after triggering coagulation reactions. However, pretreatment with exogenous agonists additionally increased platelet procoagulant activity. It reached the maximum after incubation with A23187, being lower with collagen and arachidonic acid and minimum with ADP and TRAP (the latter might be ineffective due to competition with endogenous thrombin). The effects of platelets activated by different agonists on fibrin formation and thrombin generation correlate with each other and correspond to PS expression on their surface.

Background: The Platelet function analyzer-200 (PFA-200) can determine the effect of clopidogrel in cats, but analysis traditionally must be performed at point-of-care (POC). The ability to ship samples of blood to a laboratory would allow widespread access., Objectives: We aimed to validate the shipping of blood samples for PFA-200 analysis in cats to determine the effect of clopidogrel., Methods: Twenty healthy cats and 10 cats receiving clopidogrel were recruited. Blood was collected from cats and aliquoted into two samples, one was analyzed at POC within 2 hours using the PFA-200, and the other was packaged and transported to a location 4 km away, stored, and transported back to the lab for analysis the following day., Results: Median closure times (CTs) with the collagen/adenosine diphosphate (COL/ADP) cartridge in healthy cats were 51.5 seconds (POC) and 78.8 seconds (shipped), which were significantly different (P < 0.001), and for cats on clopidogrel, median CTs were 147.5 seconds (POC) and 190 seconds (shipped), which were not significantly different (P = 0.131). Median CTs with the P2Y cartridge in healthy cats were 50.5 seconds (POC) and 64.9 seconds (shipped), which were significantly different (P = 0.03), and in cats receiving clopidogrel, median CTs were 300 seconds (POC) and 300 seconds (shipped) which were not significantly different (P = 1.000). Reference intervals for CTs differed for COL/ADP at POC (19.8-89.7 seconds) and shipped (50.9-161.6 seconds) and for P2Y at POC (35.5-118.8 seconds) and shipped (35.1-108.9 seconds). Receiver operating characteristics showed similar areas under the curve (AUCROCs) regarding the effect of clopidogrel for COL/ADP at POC (0.994 seconds) and shipped (0.932) and for P2Y at POC (0.904 seconds) and shipped (0.975 seconds). When classifying for the presence of clopidogrel effects, Cohen's Kappa was 0.62 for COL/ADP and 1.00 for P2Y., Conclusions: Shipping blood samples for PFA analysis are feasible with similar performance to POC analyses for determining the effect of clopidogrel in cats., (© 2023 The Authors. Veterinary Clinical Pathology published by Wiley Periodicals LLC on behalf of the American Society for Veterinary Clinical Pathology.)

Exercise training (ET) can lower platelet reactivity in patients with cardiovascular risk factors. However, the effects of ET on platelet reactivity in higher-risk patients is unknown. The aim of this study was to evaluate the effects of ET on platelet reactivity in patients with recent myocardial infarction (MI). Ninety patients were randomly assigned 1 month post-MI to the intervention (patients submitted to a supervised ET program) or control group. All patients were on dual antiplatelet therapy (DAPT). Platelet reactivity by VerifyNow-P2Y 12 (measured by P2Y 12 reaction units - PRUs) test was determined at baseline and at the end of 14 ± 2 weeks of follow-up at rest (primary endpoint), and multiplate electrode aggregometry (MEA) adenosine diphosphate (ADP) and aspirin (ASPI) tests were performed immediately before and after the maximal cardiopulmonary exercise test (CPET) at the same time points (secondary endpoints). Sixty-five patients (mean age 58.9 ± 10 years; 73.8% men; 60% ST elevation MI) completed follow-up (control group, n = 31; intervention group, n = 34). At the end of the follow-up, the mean platelet reactivity was 172.8 ± 68.9 PRUs and 166.9 ± 65.1 PRUs for the control and intervention groups, respectively (p = .72). Platelet reactivity was significantly increased after the CPET compared to rest at the beginning and at the end of the 14-week follow-up (among the intervention groups) by the MEA-ADP and MEA-ASPI tests (p < .01 for all analyses). In post-MI patients on DAPT, 14 weeks of supervised ET did not reduce platelet reactivity. Moreover, platelet reactivity was increased after high-intensity exercise (ClinicalTrials.gov: NCT02958657; https://clinicaltrials.gov/ct2/show/NCT02958657).

Pancreatic beta cells secrete insulin in response to plasma glucose. The ATP-sensitive potassium channel (K ATP ) links glucose metabolism to islet electrical activity in these cells by responding to increased cytosolic [ATP]/[ADP]. It was recently proposed that pyruvate kinase (PK) in close proximity to beta cell K ATP locally produces the ATP that inhibits K ATP activity. This proposal was largely based on the observation that applying phosphoenolpyruvate (PEP) and ADP to the cytoplasmic side of excised inside-out patches inhibited K ATP . To test the relative contributions of local vs. mitochondrial ATP production, we recorded K ATP activity using mouse beta cells and INS-1 832/13 cells. In contrast to prior reports, we could not replicate inhibition of K ATP activity by PEP + ADP. However, when the pH of the PEP solutions was not corrected for the addition of PEP, strong channel inhibition was observed as a result of the well-known action of protons to inhibit K ATP . In cell-attached recordings, perifusing either a PK activator or an inhibitor had little or no effect on K ATP channel closure by glucose, further suggesting that PK is not an important regulator of K ATP . In contrast, addition of mitochondrial inhibitors robustly increased K ATP activity. Finally, by measuring the [ATP]/[ADP] responses to imposed calcium oscillations in mouse beta cells, we found that oxidative phosphorylation could raise [ATP]/[ADP] even when ADP was at its nadir during the burst silent phase, in agreement with our mathematical model. These results indicate that ATP produced by mitochondrial oxidative phosphorylation is the primary controller of K ATP in pancreatic beta cells. KEY POINTS: Phosphoenolpyruvate (PEP) plus adenosine diphosphate does not inhibit K ATP activity in excised patches. PEP solutions only inhibit K ATP activity if the pH is unbalanced. Modulating pyruvate kinase has minimal effects on K ATP activity. Mitochondrial inhibition, in contrast, robustly potentiates K ATP activity in cell-attached patches. Although the ADP level falls during the silent phase of calcium oscillations, mitochondria can still produce enough ATP via oxidative phosphorylation to close K ATP . Mitochondrial oxidative phosphorylation is therefore the main source of the ATP that inhibits the K ATP activity of pancreatic beta cells., (© 2023 The Authors. The Journal of Physiology published by John Wiley & Sons Ltd on behalf of The Physiological Society.)

Aging muscle experiences functional decline in part mediated by impaired mitochondrial ADP sensitivity. Elamipretide (ELAM) rapidly improves physiological and mitochondrial function in aging and binds directly to the mitochondrial ADP transporter ANT. We hypothesized that ELAM improves ADP sensitivity in aging leading to rescued physiological function. We measured the response to ADP stimulation in young and old muscle mitochondria with ELAM treatment, in vivo heart and muscle function, and compared protein abundance, phosphorylation, and S-glutathionylation of ADP/ATP pathway proteins. ELAM treatment increased ADP sensitivity in old muscle mitochondria by increasing uptake of ADP through the ANT and rescued muscle force and heart systolic function. Protein abundance in the ADP/ATP transport and synthesis pathway was unchanged, but ELAM treatment decreased protein s-glutathionylation incuding of ANT. Mitochondrial ADP sensitivity is rapidly modifiable. This research supports the hypothesis that ELAM improves ANT function in aging and links mitochondrial ADP sensitivity to physiological function. ELAM binds directly to ANT and ATP synthase and ELAM treatment improves ADP sensitivity, increases ATP production, and improves physiological function in old muscles. ADP (adenosine diphosphate), ATP (adenosine triphosphate), VDAC (voltage-dependent anion channel), ANT (adenine nucleotide translocator), H + (proton), ROS (reactive oxygen species), NADH (nicotinamide adenine dinucleotide), FADH 2 (flavin adenine dinucleotide), O 2 (oxygen), ELAM (elamipretide), -SH (free thiol), -SSG (glutathionylated protein)., (© 2023. The Author(s).)

Background: Diabetic patients tend to have increased platelet reactivity after coronary artery bypass grafting (CABG). The aim of this study was to determine the association between hemoglobin A1c (HbA1c) values and platelet reactivity and to evaluate the consequent impact on clinical outcomes in patients undergoing CABG., Methods: This prospective observational trial consecutively enrolled 225 diabetic patients undergoing CABG, between February 2014 and October 2018. HbA1c levels and platelet function (multiple electrode aggregometry [MEA]) were analyzed the day before surgery and on postoperative day 4 (POD 4). Patients were divided into two groups according to the HbA1c value: HBA1c < 7% and HbA1c ≥ 7%., Results: Significantly higher postoperative ASPI (platelet function test based on arachidonic acid) and ADP (platelet function test based on adenosine diphosphate) test values were observed at POD 4 compared with preoperative values (ASPI test: p  < 0.001; ADP test: p  < 0.001). The prevalence of preoperative aspirin resistance (AR) was 46.4% relative to 57.2% after surgery showing consistent increase in postoperative AR by approximately 10%. In addition, the prevalence of AR in the HbA1c < 7% group was higher by 10% compared with the HbA1c ≥ 7% group, both before and after surgery. We did not demonstrate differences in clinical outcomes between the HbA1c groups., Conclusion: Perioperative assessment of platelet reactivity in diabetic patients detects those with AR who may be at increased risk of adverse ischemic events. A personalized approach guided by MEA and administration of early and more potent antiaggregation therapy after CABG can be beneficial in this group of patients., Competing Interests: None declared., (Thieme. All rights reserved.)

Background: Light transmission aggregation (LTA) is used widely by the clinical and research communities. Although it is a gold standard, there is a lack of interlaboratory harmonization., Objectives: The primary objective was to assess whether sources of activators (mainly adenosine diphosphate [ADP], collagen, arachidonic acid, epinephrine, and thrombin receptor activating peptide6) and ristocetin contribute to poor LTA reproducibility. The secondary objective was to evaluate interindividual variability of results to appreciate the distribution of normal values and consequently better interpret pathologic results., Methods: An international multicenter study involving 28 laboratories in which we compared LTA results obtained with center-specific activators and a comparator that we supplied., Results: We report variability in the potency (P) of activators in comparison with the comparator. Thrombin receptor activating peptide 6 (P, 1.32-2.68), arachidonic acid (P, 0.87-1.43), and epinephrine (P, 0.97-1.34) showed the greatest variability. ADP (P, 1.04-1.20) and ristocetin (P, 0.98-1.07) were the most consistent. The data highlighted clear interindividual variability, notably for ADP and epinephrine. Four profiles of responses were observed with ADP from high-responders, intermediate-responders, and low-responders. A fifth profile corresponding to nonresponders (5% of the individuals) was observed with epinephrine., Conclusion: Based on these data, the establishment and adoption of simple standardization principles should mitigate variability due to activator sources. The observation of huge interindividual variability for certain concentrations of activators should lead to a cautious interpretation before reporting a result as abnormal. Confidence can be taken from the fact that difference between sources is not exacerbated in patients treated with antiplatelet agents., Competing Interests: Declaration of competing interests M.L. has received speaker fees from Bayer, participated in industry-funded trials from Idorsia, served on advisory boards for Servier and JAMP/Orimed Pharma, and received in-kind support for investigator-initiated grants from Fujimori Kogyo. M.C.A. has received fees from Novo Nordisk, participated in industry-funded trials from Agrobio, participated in PIA-funded project in collaboration with Stago and Agrobio, and served on advisory boards for Novo Nordisk. U.J.S. has received research grants from Octapharma; consulting fees from Bayer, SOBI, CSL Behring, and Pfizer; and travel support from Bayer, SOBI, CSL Behring, Biotest, Takeda, and Leo Pharma. D.F. has received honorarium and travel support from Viatris. S.V. has received travel support from Stago. F.M. has received speaker fees from Fresenius, Technoclone, and Werfen. P.F. has received travel support from SOBI and Novo Nordisk. A.G. reports personal fees from Aspen, Bayer Vital, Chromatec, Instrumentation Laboratory, Portola, Sanofi-Aventis, Roche, and GTH e.V.; grants from Ergomed, Boehringer Ingelheim, Rovi, Sagent, Biokit, Fa. Blau Farmaceutics, Prosensa/Biomarin, DRK-BSD Baden-Würtemberg/Hessen, Deutsche Forschungsgemeinschaft, Robert-Koch-Institut, Dilaflor, and GIZ Else-Körner-Stiftung; grants and personal fees from Macopharma; grants and other fee from DRK-BSD NSTOB; and nonfinancial support from Veralox, Vakzine Projekt Management GmbH, AstraZeneca, and Janssen Vaccines & Prevention B.V. outside the submitted work. In addition, A.G. has a patent—screening methods for transfusion-related acute lung injury (TRALI)—with royalties paid to EP2321644, 18.05.2011. M.C. has received research grant from Agios Pharmaceuticals, Genentech Inc, and Novartis Inc; consulting fees from Novo Nordisk; and honoraria for board participation with Novo Nordisk, Takeda Inc, Genentech Inc, BPL Inc, CSL Behring Inc, and Genzyme Corp Agios Pharmaceuticals. C.P. has received research grant from Stago. I.D. has received travel support from Sobi, Takeda, and Novonordisk and speaker fees from Novonordisk. A.B. has received fees from Aguettant, Alexion, and Viatris. J.R. has received support from Instituto de Salud Carlos (PI20/00926 [ISCIII&Feder], PMP21/00052 [ISCIII&NG EU], and CB15/00055) and Sociedad Española de Trombosis y Hemostasia (Ayuda a Grupo Español de Alteraciones Plaquetarias Congénitas). P.G. has received speaker’s fees from Sanofi and Roche, and honoraria for board participation with Viatris. M.R.C., M.C., C.L.-B., M.I.K., L.G., B.H., A.S., C.C., A.N.S., M.B., M.F., K.D., W.H., M.F.H., A.N.M., J.P.F., P.S., F.P., A.C., N.B., J.R., and D.L. have no conflict of interest to declare., (Copyright © 2023 International Society on Thrombosis and Haemostasis. Published by Elsevier Inc. All rights reserved.)

Purpose: Obese patients exhibit an overall increased platelet reactivity and a reduced sensitivity to antiplatelet therapy. The aim of this study is to evaluate the platelet reactivity measured by impedance aggregometry in overweight and obese patients and chronic coronary syndrome (CCS) that were treated with dual antiplatelet therapy (DAPT)., Methods: Platelet aggregation was assessed by impedance aggregometry in patients with CCS receiving DAPT (aspirin plus clopidogrel). We compared the platelet reactivity in patients with a normal weight versus overweight or obese patients. Furthermore, the correlation between the body mass index (BMI) and adenosine diphosphate- (ADP-) or thrombin receptor-activating peptide- (TRAP-) dependent platelet aggregation was analyzed., Results: 64 patients were included in the study of which 35.9% were patients with normal weight. A higher ADP- and TRAP-dependent platelet reactivity was observed in overweight and obese patients (ADP: median 27 units (U) [IQR 13-39.5] vs. 7 U [6-15], p < 0.001 and TRAP: 97 U [73-118.5] vs. 85 U [36-103], p = 0.035). Significant positive correlations were observed between agonist-induced platelet reactivity and BMI., Conclusion: Despite the use of DAPT, a higher platelet reactivity was found in overweight and obese patients with CCS. If these patients will benefit from treatment with more potent platelet inhibitors, it needs to be evaluated in future clinical trials., (© 2022. The Author(s).)

Background: Fibrinogen γ-chain peptide-coated, adenosine 5'-diphosphate (ADP)-encapsulated liposomes (H12-ADP-liposomes) are potent hemostatic adjuvants that promote platelet thrombi formation at bleeding sites. Although we have reported the efficacy of these liposomes in a rabbit model of cardiopulmonary bypass coagulopathy, we are yet to address the possibility of their hypercoagulative potential, especially in human beings., Objectives: Considering its future clinical applications, we herein investigated the safety of using H12-ADP-liposomes in vitro using blood samples from patients who had received platelet transfusion after cardiopulmonary bypass surgeries., Methods: Ten patients receiving platelet transfusions after cardiopulmonary bypass surgery were enrolled. Blood samples were collected at the following 3 points: at the time of incision, at the end of the cardiopulmonary bypass, and immediately after platelet transfusion. After incubating the samples with H12-ADP-liposomes or phosphate-buffered saline (PBS, as a control), blood coagulation, platelet activation, and platelet-leukocyte aggregate formation were evaluated., Results: Patients' blood incubated with H12-ADP-liposomes did not differ from that incubated with PBS in coagulation ability, degree of platelet activation, and platelet-leukocyte aggregation at any of the time points., Conclusion: H12-ADP-liposomes did not cause abnormal coagulation, platelet activation, or platelet-leukocyte aggregation in the blood of patients who received platelet transfusion after a cardiopulmonary bypass. These results suggest that H12-ADP-liposomes could likely be safely used in these patients, providing hemostasis at the bleeding sites without causing considerable adverse reactions. Future studies are needed to ensure robust safety in human beings., (Copyright © 2023 International Society on Thrombosis and Haemostasis. Published by Elsevier Inc. All rights reserved.)

The process of platelet aggregation is often influenced by several factors including sex and age. A literature review confirmed the existence of sex-related differences in platelet aggregation. Although 68 out of 78 papers found such differences, there are still some controversies regarding these differences, which can be due to multiple factors (age, trigger, concomitant disease, sample handling, etc.). These outcomes are discussed in line with novel results obtained from a local study, in which blood samples from a total of 53 overall healthy women and men with ages ranging from 20 to 66 years were collected. Aggregation was induced with seven different triggers (ristocetin, thrombin receptor activating peptide 6 [TRAP-6], arachidonic acid [AA], platelet-activating factor 16 [PAF-16], ADP, collagen, or thromboxane A 2 analog U-46619) ex vivo. In addition, three FDA-approved antiplatelet drugs (vorapaxar, ticagrelor, or acetylsalicylic acid [ASA]) were also tested. In general, women had higher aggregation responses to some agonists (ADP, TRAP), as well as lower benefit from inhibitors (ASA, vorapaxar). The aggregatory responses to AA and TRAP decreased with age in both sexes, while responses to ADP, U-46619, and PAF were affected by age only in women. In conclusion, more studies are needed to decipher the biological importance of sex-related differences in platelet aggregation in part to enable personalized antiplatelet treatment., Competing Interests: None declared., (Thieme. All rights reserved.)

Background: The hemostatic plug formation at sites of vascular injury is strongly dependent on rapid platelet activation and integrin-mediated adhesion and aggregation. However, to prevent thrombotic complications, platelet aggregate formation must be a self-limiting process. The second-wave mediator adenosine diphosphate (ADP) activates platelets via Gq-coupled P2Y 1 and Gi-coupled P2Y 12 receptors. After ADP exposure, the P2Y 1 receptor undergoes rapid phosphorylation-induced desensitization, a negative feedback mechanism believed to be critical for limiting thrombus growth., Objective: The objective of this study was to examine the role of rapid P2Y 1  receptor desensitization on platelet function and thrombus formation in vivo., Methods: We analyzed a novel knock-in mouse strain expressing a P2Y 1 receptor variant that cannot be phosphorylated beyond residue 340 (P2Y 1 340-0P ), thereby preventing the desensitization of the receptor., Results: P2Y 1 340-0P mice followed a Mendelian inheritance pattern, and peripheral platelet counts were comparable between P2Y 1 340-0P/340-0P and control mice. In vitro, P2Y 1 340-0P/340-0P platelets were hyperreactive to ADP, showed a robust activation response to the P2Y 1 receptor-selective agonist, MRS2365, and did not desensitize in response to repeated ADP challenge. We observed increased calcium mobilization, protein kinase C substrate phosphorylation, alpha granule release, activation of the small GTPase Rap1, and integrin inside-out activation/aggregation. This hyperreactivity, however, did not lead to increased platelet adhesion or excessive plug formation under physiological shear conditions., Conclusion: Our studies demonstrate that receptor phosphorylation at the C-terminus is critical for P2Y 1 receptor desensitization in platelets and that impaired desensitization leads to increased P2Y 1 receptor signaling in vitro. Surprisingly, desensitization of the P2Y 1 receptor is not required for limiting platelet adhesion/aggregation at sites of vascular injury, likely because ADP is degraded quickly or washed away in the bloodstream., Competing Interests: Declaration of competing interests All authors have no conflicts of interest to declare., (Copyright © 2023 International Society on Thrombosis and Haemostasis. Published by Elsevier Inc. All rights reserved.)

In the cerebral cortex, glutamate activates NMDA receptors (NMDARs), localized in noradrenergic neurons, inducing noradrenaline release that may have a permissive effect on glutamatergic transmission, and therefore, on the modulation of long-term plasticity. ATP is co-released with noradrenaline, and with its metabolites (ADP and adenosine) is involved in the purinergic modulation of electrically-evoked noradrenaline release. However, it is not known if noradrenaline release evoked by activation of NMDARs is also under purinergic modulation. The present study aimed to investigate and to characterize the purinergic modulation of noradrenaline release evoked by NMDARs. Stimulation of rat cortical slices with 30 µM NMDA increased noradrenaline release, which was inhibited by ATP upon metabolization into ADP and adenosine and by the selective agonists of A 1 and A 2A receptors, CPA and CGS2680, respectively. It was also inhibited by UTP and UDP, which are mainly released under pathophysiological situations. Characterization of the effects mediated by these compounds indicated the involvement of P2Y 1 , P2Y 6 , A 1 and A 2A receptors. It is concluded that, in the rat brain cortex, NMDA-evoked noradrenaline release is modulated by several purinergic receptors that may represent a relevant mechanism to regulate the permissive effect of noradrenaline on NMDA-induced neuroplasticity.

Background: Platelet dysfunctions are shared by cardiovascular diseases and a wide range of inflammatory diseases., Aims: To determine the ability of a new whole tomato-based food supplement (WTBFS) containing carotenoid and olive polyphenols to inhibit platelet aggregation., Methods: Aggregation was evaluated in platelet-rich plasma using microtiter plates and a plate reader., Results: Platelets treated with WTBFS showed a >70% reduction of 5 µM adenosine diphosphate (ADP)-induced platelet aggregation; at 10 µM of ADP, the inhibitory effect of WTBFS was reduced of about 50%. Similarly, 78% and 48% reduction were obtained using 5 µg/mL and 10 µg /mL of collagen as an agonist., Conclusion: Since the compounds in WTBFS share the ability to inhibit STAT3, the inhibition of its signaling pathway may represent the mechanism underlying the antiplatelet activities. The activity of a lipophilic solution prepared from WTBS was in vitro tested on the platelet aggregation in response to ADP agonists and Collagen.

We studied the properties of N6-chloroadenosine phosphates (ATP, ADP, and AMP chloramines) as compounds with potentially increased antiplatelet efficacy determined by their binding to the plasma membrane of platelets. Chloramine derivatives of ATP, ADP, and AMP do not differ in their optical absorption characteristics: their absorption spectra are in the range of 220-340 nm with a maximum at 264 nm. Chloramines of adenosine phosphates are characterized by high reactivity with respect to thiol compounds. In particular, the rate constants of the reaction of N6-chloroadenosine-5'-diphosphate with N-acetylcysteine, reduced glutathione, dithiothreitol, and cysteine reach 59,000, 250,000, 340,000, and 1,250,000 M -1 ×sec -1 , respectively, and only 1.10±0.02 M -1 ×sec -1 with methionine. It has been found that N6-chloradenosine-5'-triphosphate is a strong inhibitor of platelet functions: it effectively suppresses ADP-induced cell aggregation (IC 50 in the whole blood is 5 μM) and inhibits aggregation of preactivated platelets and induces dissociation of their aggregates., (© 2023. Springer Science+Business Media, LLC, part of Springer Nature.)

Background: Especially in disease conditions, platelets can encounter activating agents in circulation., Objectives: To investigate the extent to which previously activated platelets can be reactivated and whether in-and reactivation applies to different aspects of platelet activation and thrombus formation., Methods: Short-and long-term effects of glycoprotein VI (GPVI) and G protein-coupled receptor (GPCR) stimulation on platelet activation and aggregation potential were compared via flow cytometry and plate-based aggregation. Using fluorescence and electron microscopy, we assessed platelet morphology and content, as well as thrombus formation., Results: After 30 minutes of stimulation with thrombin receptor activator peptide 6 (TRAP6) or adenosine diphosphate (ADP), platelets secondarily decreased in PAC-1 binding and were less able to aggregate. The reversibility of platelets after thrombin stimulation was concentration dependent. Reactivation was possible via another receptor. In contrast, cross-linked collagen-related peptide (CRP-XL) or high thrombin stimulation evoked persistent effects in α IIb β 3 activation and platelet aggregation. However, after 60 minutes of CRP-XL or high thrombin stimulation, when α IIb β 3 activation slightly decreased, restimulation with ADP or CRP-XL, respectively, increased integrin activation again. Compatible with decreased integrin activation, platelet morphology was reversed. Interestingly, reactivation of reversed platelets again resulted in shape change and if not fully degranulated, additional secretion. Moreover, platelets that were previously activated with TRAP6 or ADP regained their potential to contribute to thrombus formation under flow. On the contrary, prior platelet triggering with CRP-XL was accompanied by prolonged platelet activity, leading to a decreased secondary platelet adhesion under flow., Conclusion: This work emphasizes that prior platelet activation can be reversed, whereafter platelets can be reactivated through a different receptor. Reversed, previously activated platelets can contribute to thrombus formation., (Crown Copyright © 2023. Published by Elsevier Inc. All rights reserved.)

During trauma and surgery, bleeding is a major concern. One of the crucial strategies for hemostasis is the use of biological hemostatic material. Herein, we reported an amino acid-based hydrogel FmocF-ADP hydrogel, which consisted of N-[(9H-fluoren-9-ylmethoxy) carbonyl]-3-phenyl-L-alanine (FmocF) and adenosine diphosphate (ADP) sodium solution. The hydrogel was created by FmocF self-assembling to nanofiber in ADP sodium solution and then cross-linking to hydrogel. FmocF-ADP hydrogel showed good in vitro coagulation activity as measured by whole blood clotting assays, platelet clotting assays, platelet activation assays, and platelet adhesion assays. Further, it was noted to reveal an exceptional in vivo hemostatic effect in a mouse liver bleeding model. Together with the previous report of the good biocompatibility and antimicrobial activity of FmocF hydrogel, our study would extend the biomedical application of FmocF hydrogel. In conclusion, the present study would provide a constructive strategy for the development of new antimicrobial and hemostatic materials or develop a potential hemostatic material., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Austria, part of Springer Nature.)

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.

Background: Platelet activation and aggregation are critical to the initiation of hemostasis after trauma with hemorrhage. Platelet dysfunction is a well-recognized phenomenon contributing to trauma-induced coagulopathy. The goal of this study was to evaluate the timing and severity of platelet dysfunction in massively transfused, traumatically injured patients during the first 72 hours after injury and its association with 30-day survival., Methods: A retrospective secondary cohort study of platelet count and function was performed using samples from the Pragmatic Randomized Optimal Platelet and Plasma Ratios trial. Platelet characteristics were measured at 8 timepoints during the first 72 hours of hospitalization and compared between 30-day survivors and nonsurvivors. Platelet counts were assessed via flow cytometry. Platelet function was analyzed with the use of serial thrombelastography and impedance aggregometry with agonists arachidonic acid, adenosine diphosphate, collagen, thrombin receptor activating peptide, and ristocetin., Results: In total, 680 patients were included for analysis. Platelet counts were significantly lower from baseline to 72 hours after hospital admission with further 1.3 to 2-fold reductions noted in nonsurvivors compared to survivor patients. Platelet aggregation via adenosine diphosphate, arachidonic acid, collagen, thrombin receptor activating peptide, and ristocetin was significantly lower in nonsurvivors at all time points. The nadir of platelet aggregation was 2 to 6 hours after admission with significant improvements in viscoelastic maximum clot formation and agonist-induced aggregation by 12 hours without concomitant improvement in platelet count., Conclusion: Platelet aggregability recovers 12 hours after injury independent of worsening thrombocytopenia. Failure of platelet function to recover portends a poor prognosis., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Objective: Premature adrenarche (PA) has been associated with an increase in adrenal androgens, and the hyperandrogenic hormonal environment is known to lead to increased platelet (PLT) aggregation. Here, we evaluated the effects of PA on PLT aggregation in PLT-rich plasma samples from female patients., Methods: The study included 40 female patients diagnosed with PA between February, 2014 and June, 2018 and 30 healthy female individuals as a control group. Adenosine diphosphate (ADP) and collagen-induced PLT aggregation were studied via the photometric aggregometry method., Results: There were no significant differences in the PLT count or volume values between those participants with PA and the control group. Additionally, the ADP-induced maximum aggregation time, value, and slope values did not significantly differ between the patient and control groups (p>0.05). However, the collagen-induced maximum aggregation time, value, and slope values were significantly higher in the studygroup (p<0.001)., Conclusion: Increased collagen-induced PLT aggregation was detected in female patients with PA. As PA is associated with a higher risk of cardiovascular events later in life, close follow-up of PA in this respect may be beneficial., (©Copyright 2023 by Turkish Society for Pediatric Endocrinology and Diabetes | The Journal of Clinical Research in Pediatric Endocrinology published by Galenos Publishing House.)

Background: Temporomandibular joint osteoarthritis (TMJ-OA) causes cartilage degeneration, bone cavitation, and fibrosis of the TMJ. However, the mechanisms underlying the fibroblast-like synoviocyte (FLS)-mediated inflammatory activity in TMJ-OA remain unclear., Methods and Results: Reverse transcription-quantitative polymerase chain reaction analysis revealed that the P2Y 1 , P2Y 12 , and P2Y 13 purinergic receptor agonist adenosine 5'-diphosphate (ADP) significantly induces monocyte chemotactic protein 1 (MCP-1)/ C-C motif chemokine ligand 2 (CCL2) expression in the FLS1 synovial cell line. In contrast, the uracil nucleotide UTP, which is a P2Y 2 and P2Y 4 agonist, has no significant effect on MCP-1/CCL2 production in FLS1 cells. In addition, the P2Y 13 antagonist MRS 2211 considerably decreases the expression of ADP-induced MCP-1/CCL2, whereas ADP stimulation enhances extracellular signal-regulated kinase (ERK) phosphorylation. Moreover, it was found that the mitogen-activated protein kinase/ERK kinase (MEK) inhibitor U0126 reduces ADP-induced MCP-1/CCL2 expression., Conclusion: ADP enhances MCP-1/CCL2 expression in TMJ FLSs via P2Y 13 receptors in an MEK/ERK-dependent manner, thus resulting in inflammatory cell infiltration in the TMJ. Collectively, the findings of this study contribute to a partial clarification of the signaling pathway underlying the development of inflammation in TMJ-OA and can help identify potential therapeutic targets for suppressing ADP-mediated purinergic signaling in this disease., (© 2022. The Author(s).)

Objectives: Methods for assessing platelet function in patients with neurovascular disease remain controversial and poorly studied. This study aimed to assess associations between thromboelastography 6s (TEG6s) measurements and postoperative ischemic complications in patients with unruptured intracranial aneurysms (UIAs) treated by coil embolization., Methods: Eighty-four patients with UIAs taking a combined aspirin and clopidogrel protocol were retrospectively reviewed from January 2021 to May 2022. Blood samples were obtained for TEG6s to assess platelet function on the day of coil embolization. To identify acute ischemic complications, diffusion-weighted imaging (DWI) was performed within 24 h after coil embolization. Multivariate logistic regression analysis was conducted to identify potential risk factors for postoperative positive DWI (DWI (+)) lesions., Results: Forty-three of the 84 patients (51%) with DWI (+) lesions were identified. Compared with patients without DWI (+) lesions, Adenosine diphosphate (ADP)-induced platelet-fibrin clot strength (MA ADP ) was significantly higher (53.6 mm [Interquartile range (IQR): 48.3-58.3 mm] vs 46.7 mm [IQR: 36.8-52.2 mm]; p=0.001) and ADP inhibition rate (ADP%) was significantly lower (19% [IQR: 11-31%] vs 31% [IQR: 21-44%]; p=0.001) in DWI (+) patients. Multivariate analysis identified MA ADP , ADP%, and procedure time as significant independent predictors of subsequent DWI (+) lesions (odds ratios: 1.07, 0.96, and 1.02, respectively). Based on receiver operating characteristic curve analysis, MA ADP >50.9 mm and ADP% <28.8% were associated with postoperative DWI (+) lesions in patients undergoing coil embolization for UIAs., Conclusions: MA ADP and ADP% as assessed by TEG6s can offer reliable parameters to predict postoperative ischemic complications after coil embolization of UIAs. Lower MA ADP values and higher ADP% may decrease the risk of postoperative ischemic complications., Competing Interests: Declaration of Competing Interest This research has not obtained particular funding from any public, commercial, or non-commercial funding institutions., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Background: 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., Methods: 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., Results: 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., Conclusions: 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.

Platelet hyperactivation could lead to various cardiovascular and cerebrovascular diseases, while epidemiological analyses have found that long-term tea drinking could prevent and restrain cardiovascular diseases. Existing studies have shown that catechins, especially epigallocatechin gallate (EGCG), are the main functional factors of tea in alleviating thrombosis, which could inhibit arterial thrombosis and platelet aggregation induced by a variety of agonists. However, their structure-activity relationship and the underlying mechanisms are still unclear. Based on the above background, this study took six typical catechins as research objects, constructed platelet activation models with different inducers, and explored the inhibitory effects and potential mechanisms of catechins with different structures on platelet aggregation through flow cytometry, immunoblotting, cell spreading, and other experiments. It was found that ester catechins could inhibit platelet aggregation induced by adenosine diphosphate (ADP), while epigallocatechin (EGC) with three hydroxyls on the B ring in non-ester catechins was also able to effectively inhibit platelet aggregation. Our data suggested that gallic acyl on the C ring and three hydroxyls on the B ring were the main functional groups affecting the antithrombotic effect of catechins, and the effect of gallic acyl on platelets was significantly stronger than that of the hydroxyl.