P2Y12 Antagonist Research Articles

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.

Comparison of the effects of the GPIIb-IIIa antagonist Zalunfiban and the P2Y12 antagonist Selatogrel on Platelet Aggregation

Understanding the pharmacodynamic effects of platelet inhibitors is standard for developing more effective antithrombotic therapies. An example is the antithrombotic treatment of acute coronary syndrome (ACS), in particular ST-elevated myocardial infarction (STEMI) patients who are in need for rapid acting strong antithrombotic therapy despite the use of aspirin and oral P2Y12-inhibitors. In this study, we evaluated two injectable platelet inhibitors under clinical development (the P2Y12 antagonist selatogrel and the GPIIb-IIIa antagonist zalunfiban) that may be amenable to pre-hospital treatment of STEMI patients. Platelet reactivity was assessed at inhibitor concentrations that represent clinically relevant levels of platelet inhibition (IC20-50%, 1/2Cmax, and Cmax). Light transmission aggregometry (LTA), was used to evaluate the initial rate of aggregation (primary slope, PS) and maximal aggregation (MA). Both adenosine diphosphate (ADP) and thrombin receptor agonist peptide (TRAP) were used as agonists. Zalunfiban demonstrated similar inhibition of platelet aggregation when blood was collected in PPACK or TSC, whereas selatogrel demonstrated greater inhibition in PPACK. In this study, using PPACK anticoagulant, selatogrel and zalunfiban affected PS in response to ADP equivalently at all drug concentrations tested. In contrast, zalunfiban had significantly greater potency at its Cmax concentration compared to selatogrel using TRAP as agonist. Upon evaluation of MA responses at lower doses, selatogrel had greater inhibition of MA in response to ADP than zalunfiban; however, at concentrations that represent Cmax, the drugs were equivalent. Zalunfiban also had greater inhibition of MA in response to TRAP at the Cmax dose. These data suggest that zalunfiban may provide greater protection in reducing thrombus formation than selatogrel, especially since thrombin is an early, key primary agonist in the pathophysiology of thrombotic events.

Changes in process and outcome for ST elevation myocardial infarction in central China from 2011 to 2018.

Limited data are available on the changes in the quality of care for ST elevation myocardial infarction (STEMI) during China's health system reform from 2009 to 2020. This study aimed to assess the changes in care processes and outcome for STEMI patients in Henan province of central China between 2011 and 2018. We compared the data from the Henan STEMI survey conducted in 2011-2012 ( n = 1548, a cross-sectional study) and the Henan STEMI registry in 2016-2018 ( n = 4748, a multicenter, prospective observational study). Changes in care processes and in-hospital mortality were determined. Process of care measures included reperfusion therapies, aspirin, P2Y12 antagonists, β-blockers, angiotensin-converting enzyme inhibitors or angiotensin receptor blockers, and statins. Therapy use was analyzed among patients who were considered ideal candidates for treatment. STEMI patients in 2016-2018 were younger (median age: 63.1 vs . 63.8 years) with a lower proportion of women (24.4% [1156/4748] vs . 28.2% [437/1548]) than in 2011-2012. The composite use rate for guideline-recommended treatments increased significantly from 2011 to 2018 (60.9% [5424/8901] vs . 82.7% [22,439/27,129], P <0.001). The proportion of patients treated by reperfusion within 12 h increased from 44.1% (546/1237) to 78.4% (2698/3440) ( P <0.001) with a prolonged median onset-to-first medical contact time (from 144 min to 210 min, P <0.001). The use of antiplatelet agents, statins, and β-blockers increased significantly. The risk of in-hospital mortality significantly decreased over time (6.1% [95/1548] vs . 4.2% [198/4748], odds ratio [OR]: 0.67, 95% confidence interval [CI]: 0.50-0.88, P = 0.005) after adjustment. Gradual implementation of the guideline-recommended treatments in STEMI patients from 2011 to 2018 has been associated with decreased in-hospital mortality. However, gaps persist between clinical practice and guideline recommendation. Public awareness, reperfusion strategies, and construction of chest pain centers need to be further underscored in central China.

Unlocking the Conformational Changes of P2Y12: Exploring an Acridinone Compound's Effect on Receptor Activity and Conformation.

The P2Y12 receptor is an important member of the purinergic receptor family, known for its critical role in platelet activation and thrombosis. In our previously published study, the acridinone analogue NSC618159 was identified as a potent antagonist of P2Y12. In this work, we investigate the conformational changes in P2Y12 when bound to NSC618159 using molecular dynamics simulations on the receptor's active and inactive forms (4PXZ and 4NTJ, respectively). It was observed that it took the systems about 7 ns and 12 ns to stabilise when NSC618159 was in complex with the active and inactive forms of P2Y12, respectively. Additionally, the binding pocket of the crystal structure 4PXZ expanded from 172.34 Å3 to an average of 661.55 Å3 when bound to NSC618159, with a maximum pocket volume of 820.49 Å3. This expansion was attributed to the pulled away transmembrane (TM) helices and the adoption of a more open conformation by extracellular loop 2 (EL2). In contrast, 4NTJ's pocket volume was mostly consistent and had an average of 1203.82 Å3. Moreover, the RMSF profile of the NSC618159-4PXZ complex showed that residues of TM-I and TM-VII had similar fluctuations to the 4NTJ crystal structure, representing the inactive form of P2Y12. Finally, the energy components and binding affinities of NSC618159 towards the active and inactive forms of P2Y12 were predicted using the MM-PBSA approach. According to the results, the binding affinity of NSC618159 towards both active (4PXZ) and inactive (4NTJ) forms of P2Y12 was found to be almost identical, with values of -43.52 and -41.68 kcal/mol, respectively. In conclusion, our findings provide new insights into the conformational changes of P2Y12 upon binding to NSC618159 and may have implications for the development of new P2Y12 antagonists with enhanced potency and specificity.

Blocking purinergic signaling alters platelet and macrophage crosstalk and phagocytic ability in the peritoneal cavity during intra-abdominal sepsis

Intra-abdominal sepsis is a life-threatening complex syndrome caused by dysregulation of the host response to infection and is one of the major complications of intra-abdominal surgery. To date, only supportive therapies such as ventilation support, antibiotics and organ replacement are available. However, an increase in antibiotic resistance and the invasiveness of organ transplant has necessitated therapies targeting pathophysiological mechanisms. Platelets, which express the purinergic receptors P2Y1 and P2Y12, mediate macrophage polarization and secretion of pro-inflammatory markers contributing to tissue injury during sepsis. Previously, our group has shown that blocking purinergic signaling through P2Y1 and P2Y12 antagonism significantly lowered inflammatory cytokine levels and improved survival in a murine model of sepsis in a sex specific manner. We hypothesize that the inhibition of P2Y1 and P2Y12 signaling pathways improves the sex-specific outcomes of intra-abdominal sepsis by modulating the crosstalk between platelets and macrophages. Male and female mice underwent sham surgery or cecal ligation and puncture (CLP) to induce sepsis followed by intraperitoneal administration of ticagrelor (P2Y12 antagonist) or MRS2279 (P2Y1 antagonist). The peritoneal cavity fluid (PCF) was collected 4 and 24 hours post-surgery and analyzed. At 4 hours post-surgery, PCF macrophages exhibited increased bacterial phagocytosis from ticagrelor and MRS2279 treated groups in female mice only compared to CLP group. At 24 hours post-CLP surgery, platelet counts did not change but soluble p-selectin and TBX-2 levels were significantly decreased in male groups treated with either ticagrelor or MRS2279, compared to controls. In contrast, soluble p-selectin and TBX-2 levels did not change in female samples.Thus, we conclude that pharmacologically blocking P2Y1 and P2Y12 receptors can enhance the ex-vivo phagocytic activity of macrophages in a sex-related manner, and overall modulate platelet secretion and influence macrophages’ recruitment and activation in the PC during sepsis. National Institute of Health AI156627-01 to EL This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Revealing concealed cardioprotection by platelet Mfsd2b-released S1P in human and murine myocardial infarction

Antiplatelet medication is standard of care in acute myocardial infarction (AMI). However, it may have obscured beneficial properties of the activated platelet secretome. We identify platelets as major source of a sphingosine-1-phosphate (S1P) burst during AMI, and find its magnitude to favorably associate with cardiovascular mortality and infarct size in STEMI patients over 12 months. Experimentally, administration of supernatant from activated platelets reduces infarct size in murine AMI, which is blunted in platelets deficient for S1P export (Mfsd2b) or production (Sphk1) and in mice deficient for cardiomyocyte S1P receptor 1 (S1P1). Our study reveals an exploitable therapeutic window in antiplatelet therapy in AMI as the GPIIb/IIIa antagonist tirofiban preserves S1P release and cardioprotection, whereas the P2Y12 antagonist cangrelor does not. Here, we report that platelet-mediated intrinsic cardioprotection is an exciting therapeutic paradigm reaching beyond AMI, the benefits of which may need to be considered in all antiplatelet therapies.

P2X and P2Y receptor antagonists reduce inflammation in ATP-induced microglia.

P2 receptors have been implicated in the release of neurotransmitter and pro-inflammatory cytokines due to their response to neuro-excitatory substances in the microglia. The P2X4, P2X7 and P2Y12 receptors are involved in the development of pain behavior induced by peripheral nerve injury. However, it is not known if blocking P2X4, P2X7 and P2Y12 receptors is associated with the expression and the release of interleukin-1B (IL-1β), interleukin-6 (IL-6), or tumor necrosis factor-α (TNF-α) in cultured neonatal spinal cord microglia. For this reason, we examined the effects of P2X4, P2X7 and P2Y12 antagonists on the expression and the release of IL-1β, IL-6, and TNF-α in ATP-stimulated microglia. In this study, we observed the effect of A-740003, PSB-12062 and MRS 2395 (P2X4, P2X7 and P2Y12 receptors antagonist, respectively), on the expression and release of IL-1β, IL-6 and TNF-α by using real-time fluorescence quantitative polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA). ATP induced the increased expression of IL-1β, IL-6 and TNF-α at the level of messenger RNA (mRNA). ATP-evoked increase in IL-1β, IL-6 and TNF-α mRNA expression was inhibited by the P2X4 receptor antagonist A-740003 or P2X7 receptor antagonist PSB-12062, respectively. Similarly, ATP-evoked release of IL-1β, IL-6 and TNF-α was inhibited by A-740003 and PSB-12062. Furthermore, ATP-evoked increased expression of Iba-1, IL-1β, IL-6 and TNF-α mRNA, and release of IL-1β, IL-6 and TNF-α were nearly all blocked after co-administration of A-740003 plus PSB-12062. Finally, ATP-evoked increased gene expression and release of IL-1β, IL-6 and TNF-α were also inhibited by MRS 2395 (P2Y12 antagonist). These observations suggest a new clue for therapeutic strategies to treat the neuro-inflammation.

Purinergic signaling: A gatekeeper of blood-brain barrier permeation.

This review outlined evidence that purinergic signaling is involved in the modulation of blood-brain barrier (BBB) permeability. The functional and structural integrity of the BBB is critical for maintaining the homeostasis of the brain microenvironment. BBB integrity is maintained primarily by endothelial cells and basement membrane but also be regulated by pericytes, neurons, astrocytes, microglia and oligodendrocytes. In this review, we summarized the purinergic receptors and nucleotidases expressed on BBB cells and focused on the regulation of BBB permeability by purinergic signaling. The permeability of BBB is regulated by a series of purinergic receptors classified as P2Y1, P2Y4, P2Y12, P2X4, P2X7, A1, A2A, A2B, and A3, which serve as targets for endogenous ATP, ADP, or adenosine. P2Y1 and P2Y4 antagonists could attenuate BBB damage. In contrast, P2Y12-mediated chemotaxis of microglial cell processes is necessary for rapid closure of the BBB after BBB breakdown. Antagonists of P2X4 and P2X7 inhibit the activation of these receptors, reduce the release of interleukin-1 beta (IL-1β), and promote the function of BBB closure. In addition, the CD39/CD73 nucleotidase axis participates in extracellular adenosine metabolism and promotes BBB permeability through A1 and A2A on BBB cells. Furthermore, A2B and A3 receptor agonists protect BBB integrity. Thus, the regulation of the BBB by purinergic signaling is complex and affects the opening and closing of the BBB through different pathways. Appropriate selective agonists/antagonists of purinergic receptors and corresponding enzyme inhibitors could modulate the permeability of the BBB, effectively delivering therapeutic drugs/cells to the central nervous system (CNS) or limiting the entry of inflammatory immune cells into the brain and re-establishing CNS homeostasis.

Abstract WP220: Platelet Inhibition Prevents Delayed Cerebral Ischemia After Subarachnoid Hemorrhage In Mice

Subarachnoid hemorrhage (SAH) induces delayed cerebral ischemia (DCI) in about 30% of patients which is characterized by functional decline. As microthrombi is a cause of DCI, platelets may be a therapeutic target for preventing microthrombi formation and development of DCI. Clinical trials of anti-platelets have been inconclusive for their effect on outcome. However, clinical trials used different anti-platelets. Since platelets have numerous receptors which can each prevent thrombosis, there is no clear choice to target for SAH. Here, we utilize 6 platelet antagonists to identify if any receptor is a therapeutic target for DCI after SAH. Our hypothesis is that P2Y receptor inhibition will prevent microthrombi and reduce DCI after SAH. Male and female mice were randomly assigned into sham, SAH+vehicle, SAH+P2Y antagonism, SAH+PAR4 antagonism, SAH+PAF antagonism, SAH+TPβ-R antagonism, and SAH+GPIIb/IIIa antagonism. Mice were euthanized 2 (n=10/group) or 7 days (n=15/group) post-SAH. All outcomes were performed and data was analyzed by a blinded investigator. Behavior was assessed daily. Brain microthrombi were counted. Both male and female SAH mice treated with inhibitors of PAF and TPβ-R had significantly better day 1 function than vehicle-treated mice. Significantly better function on day 1 was also observed in only male mice treated the P2Y antagonists or GPIIb/IIIa antagonist. Neither sex had improved behavior for the PAR-4 inhibitor. Mice exhibiting better function had fewer brain microthrombi with the exception of the GPIIb/IIIa antagonist which reduced microthrombi in female mice but did not correspond with improved behavior. This suggests there may be another deleterious mechanism which is more prevalent/detrimental in females that is not prevented by GPIIb/IIIa inhibition, such as platelet degranulation. However, this needs to be further investigated. Additionally, inhibition of TPβ-R reduced the incidence of DCI compared to vehicle-treated mice. In conclusion, an anti-platelet drug for SAH needs to be chosen carefully as not all antagonists have a beneficial effect after SAH; of vital importance is that anti-platelet drugs may not have similar beneficial effects in both sexes.

Prasugrel — gone, but not forgotten

AbstractHuman immunodeficiency virus (HIV) infection and certain retroviral therapies are associated with an increased risk of atherosclerosis with plaque rupture resulting in acute coronary syndromes. Therefore, there is often a need to co‐prescribe antiretroviral and cardiovascular therapies, including antiplatelet agents. Antiplatelet agents, such as the P2Y12 receptor antagonists clopidogrel and prasugrel, require activation by cytochrome P450 3A4 (CYP3A4). The P2Y12 receptor antagonist ticagrelor is also metabolised by CYP3A4. Pharmacokinetic enhancers, such as ritonavir and cobicistat, are utilised to increase the systemic exposure of certain agents within multidrug HIV regimens and are potent CYP3A4 inhibitors. Using these drugs together can result in unintended and undesirable drug–drug interactions. A 55‐year‐old male with a history of HIV presented to the emergency department with central chest pain. He was found to have had a non‐ST‐elevation myocardial infarction and was commenced on dual antiplatelet therapy including ticagrelor, anticoagulation and statin therapy, in addition to his usual HIV therapy, Genvoya (elvitegravir, cobicistat, emtricitabine, tenofovir alafenamide). A drug–drug interaction between Genvoya and ticagrelor was identified by the pharmacist, and the patient was switched to prasugrel on the pharmacist's recommendation. Although prasugrel was delisted from the Pharmaceutical Benefits Scheme (PBS) in July 2020, it remains accessible as a non‐PBS medicine. Prasugrel should be considered the P2Y12 antagonist of choice for patients who are on HIV regimens containing a pharmacokinetic enhancer such as ritonavir or cobicistat.

Early Stent Patency After Endovascular Therapy for Tandem or Isolated Carotid Steno-Occlusive Disease

Early Stent Patency After Endovascular Therapy for Tandem or Isolated Carotid Steno-Occlusive Disease

QSAR, structure-based pharmacophore modelling and biological evaluation of novel platelet ADP receptor (P2Y12) antagonist.

P2Y12 has a key role in platelet aggregation and thrombus formation via an ADP-induced platelet activation mechanism. Recently, P2Y12 antagonists have become of great interest in the clinical management of antithrombotic therapy. In light of this, we explored the pharmacophoric space of P2Y12 using structure-based pharmacophore modelling. Subsequently, genetic algorithm and multiple linear regression analyses were conducted to select the best combination of physicochemical descriptors and pharmacophoric models to create useful predictive quantitative structure-activity relationship (QSAR) equation (r 2 = 0.9135, r (adj) 2 = 0.9147, r (PRESS) 2 = 0.9129, LOF = 0.3553). One pharmacophoric model emerged in the QSAR equation and was validated by analysing receiver operating characteristic (ROC) curves. The model was then used to screen 200 000 compounds from the National Cancer Institute (NCI) database. The top-ranked hits were in vitro tested, where their IC50's range between 4.20 to 35.00 μM when measured via the electrode aggregometry assay. Whilst, the VASP phosphorylation assay showed 29.70% platelet reactivity index for NSC618159, which is superior to that of ticagrelor.

Role of platelet function testing in acute coronary syndromes: a meta-analysis

ObjectiveThis meta-analysis aimed to evaluate whether using platelet function testing (PFT) in acute coronary syndromes (ACS) to personalise antiplatelet therapy including a P2Y12 antagonist offers any clinical benefits to indicate...

Endothelial biomarkers and platelet reactivity on ticagrelor versus clopidogrel in patients after acute coronary syndrome with and without concomitant type 2 diabetes: a preliminary observational study

BackgroundPleiotropic effects have been implicated in clinical benefits of ticagrelor compared to thienopyridine P2Y12 antagonists. There are conflicting data regarding effects of ticagrelor vs. thienopyridine P2Y12 blockers on endothelial function. Our aim was to compare endothelial biomarkers and their relations with platelet reactivity in real-world patients after acute coronary syndrome (ACS) on maintenance dual antiplatelet therapy (DAPT) with ticagrelor or clopidogrel stratified by diabetes status.MethodsBiochemical indices of endothelial dysfunction/activation and platelet reactivity by multiple electrode aggregometry were compared in 126 stable post-ACS subjects (mean age: 65 ± 10 years, 92 men and 34 women), including patients with (n = 61) or without (n = 65) coexistent type 2 diabetes (T2DM) on uneventful maintenance DAPT with either ticagrelor (90 mg b.d.) or clopidogrel (75 mg o.d.) in addition to low-dose aspirin. Exclusion criteria included a complicated in-hospital course, symptomatic heart failure, left ventricular ejection fraction < 40% and relevant coexistent diseases except for well-controlled diabetes, mild renal insufficiency or hypertension.ResultsClinical characteristics were similar in patients on ticagrelor (n = 62) and clopidogrel (n = 64). The adenosine diphosphate-induced platelet aggregation and circulating soluble P-selectin (sP-selectin) were decreased in ticagrelor users irrespective of T2DM status (p < 0.001 and p < 0.01 for platelet reactivity and sP-selectin, respectively). Plasma levels of soluble vascular cell adhesion molecule-1 (sVCAM-1) were lower in T2DM subjects on ticagrelor vs. clopidogrel (758 ± 162 vs. 913 ± 217 µg/L, p < 0.01). In contrast, plasma sVCAM-1 was similar in non-diabetic patients on ticagrelor and clopidogrel (872 ± 203 vs. 821 ± 210 µg/L, p > 0.7). The concentrations of sE-selectin, monocyte chemoattractant protein-1 and asymmetric dimethylarginine did not differ according to the type of P2Y12 antagonist regardless of T2DM status. Platelet reactivity was unrelated to any endothelial biomarker in subjects with or without T2DM.ConclusionsOur preliminary findings may suggest an association of ticagrelor-based maintenance DAPT with favorable endothelial effects compared to clopidogrel users in stable post-ACS patients with T2DM. If proven, this could contribute to more pronounced clinical benefits of ticagrelor in diabetic subjects.

Endothelial Extracellular Vesicles Regulate Platelet Purinergic Signaling in Sickle Cell Disease

Introduction. Acute chest syndrome (ACS) is a type of acute lung injury and the leading cause of mortality among sickle cell disease (SCD) patients. ACS is often preceded by thrombocytopenia and involves massive thrombosis across pulmonary artery branches. Although, released during hemolysis, adenosine diphosphate (ADP) is known to activate platelets by stimulating their P2Y1 and P2Y12 purinergic receptors, antagonists of P2Y12 have not shown any benefit in ACS therapy, justifying the need for better understanding of purinergic signaling in SCD. CD39 maintains ADP homeostasis by degrading excessive ADP. Though CD39 inhibits ADP-dependent platelet activation, its role in the pathophysiology of SCD is still unidentified. Methods. To evaluate SCD platelet response to ADP in vivo we used a state-of-the-art intravital lung microscopy and a novel in vivo model of ADP-triggered thrombocytopenia in transgenic humanized SCD mice. Additionally, both mouse and human SCD platelet ADP-dependent aggregation was examined using in vitro turbidimetric aggregation assay. Hemin, a major host-derived damage associated molecular pattern associated with SCD, was incubated with Human Lung Micro-Vascular Endothelial Cells (HMVEC-L). Extracellular vesicles (EVs) were obtained from mouse and human plasma samples, and cell culture medium samples using size exclusion chromatography. Obtained EVs were subjected to nanoparticle tracking analysis. EVs CD39 levels and activity were determined using ELISA, western blot and malachite green phosphate assays, respectively. Results. Intravital lung microscopy and in vivo thrombocytopenia studies revealed that intravascular administration of ADP triggered acute pulmonary thrombosis in control but not in SCD mice. In vitro aggregation study confirmed our in vivo findings and demonstrated impaired SCD mouse and human platelet response to ADP, which was, further, significantly augmented by a specific CD39 inhibitor. Hemin triggered shedding of CD39-bearing EVs by HMVEC-L. Indeed, we discovered that isolated from mouse and human plasma endothelial cell-derived SCD EVs expressed higher CD39 levels and activity in comparison to control EVs. Conclusions. Our findings suggest that CD39-bearing EVs prevent ADP-mediated platelet aggregation and pulmonary thrombosis in SCD. Current study explains why P2Y12 blockers are not effective in SCD therapy.

Mechanisms of sympathoexcitation via P2Y6 receptors.

Many drugs used in cardiovascular therapy, such as angiotensin receptor antagonists and beta-blockers, may exert at least some of their actions through effects on the sympathetic nervous system, and this also holds true for e.g., P2Y12 antagonists. A new target at the horizon of cardiovascular drugs is the P2Y6 receptor which contributes to the development of arteriosclerosis and hypertension. To learn whether P2Y6 receptors in the sympathetic nervous system might contribute to actions of respective receptor ligands, responses of sympathetic neurons to P2Y6 receptor activation were analyzed in primary cell culture. UDP in a concentration dependent manner caused membrane depolarization and enhanced numbers of action potentials fired in response to current injections. The excitatory action was antagonized by the P2Y6 receptor antagonist MRS2578, but not by the P2Y2 antagonist AR-C118925XX. UDP raised intracellular Ca2+ in the same range of concentrations as it enhanced excitability and elicited inward currents under conditions that favor Cl- conductances, and these were reduced by a blocker of Ca2+-activated Cl- channels, CaCCInh-A01. In addition, UDP inhibited currents through KV7 channels. The increase in numbers of action potentials caused by UDP was not altered by the KV7 channel blocker linopirdine, but was enhanced in low extracellular Cl- and was reduced by CaCCInh-A01 and by an inhibitor of phospholipase C. Moreover, UDP enhanced release of previously incorporated [3H] noradrenaline, and this was augmented in low extracellular Cl- and by linopirdine, but attenuated by CaCCInh-A01. Together, these results reveal sympathoexcitatory actions of P2Y6 receptor activation involving Ca2+-activated Cl- channels.

Sex-related differences in the response of anti-platelet drug therapies targeting purinergic signaling pathways in sepsis.

Sepsis, a complex clinical syndrome resulting from a serious infection, is a major healthcare problem associated with high mortality. Sex-related differences in the immune response to sepsis have been proposed but the mechanism is still unknown. Purinergic signaling is a sex-specific regulatory mechanism in immune cell physiology. Our studies have shown that blocking the ADP-receptor P2Y12 but not P2Y1 receptor was protective in male mice during sepsis, but not female. We now hypothesize that there are sex-related differences in modulating P2Y12 or P2Y1 signaling pathways during sepsis. Male and female wild-type (WT), P2Y12 knock-out (KO), and P2Y1 KO mice underwent sham surgery or cecal ligation and puncture (CLP) to induce sepsis. The P2Y12 antagonist ticagrelor or the P2Y1 antagonist MRS2279 were administered intra-peritoneally after surgery to septic male and female mice. Blood, lungs and kidneys were collected 24 hours post-surgery. Sepsis-induced changes in platelet activation, secretion and platelet interaction with immune cells were measured by flow cytometry. Neutrophil infiltration in the lung and kidney was determined by a myeloperoxidase (MPO) colorimetric assay kit. Sepsis-induced platelet activation, secretion and aggregate formation were reduced in male CLP P2Y12 KO and in female CLP P2Y1 KO mice compared with their CLP WT counterpart. Sepsis-induced MPO activity was reduced in male CLP P2Y12 KO and CLP P2Y1 KO female mice. CLP males treated with ticagrelor or MRS2279 showed a decrease in sepsis-induced MPO levels in lung and kidneys, aggregate formation, and platelet activation as compared to untreated male CLP mice. There were no differences in platelet activation, aggregate formation, and neutrophil infiltration in lung and kidney between female CLP mice and female CLP mice treated with ticagrelor or MRS2279. In human T lymphocytes, blocking P2Y1 or P2Y12 alters cell growth and secretion in vitro in a sex-dependent manner, supporting the data obtained in mice. In conclusion, targeting purinergic signaling represents a promising therapy for sepsis but drug targeting purinergic signaling is sex-specific and needs to be investigated to determine sex-related targeted therapies in sepsis.

High-Intensity Exercise Training Improves Basal Platelet Prostacyclin Sensitivity and Potentiates the Response to Dual Anti-Platelet Therapy in Postmenopausal Women.

The risk of thrombotic events dramatically increases with age and may be accelerated in women by the cessation of endogenous estrogen production at menopause. Patients at risk of thrombosis are prescribed dual anti-platelet therapy (DAPT; aspirin and a P2Y12 antagonist) and are encouraged to participate in regular physical activity, as these modalities improve nitric oxide and prostacyclin-mediated inhibition of platelet aggregation. Methods: We assessed prostacyclin sensitivity as well as basal platelet reactivity with and without in vitro DAPT before and after an 8-week high-intensity exercise training program in 13 healthy, sedentary postmenopausal women. The training intervention consisted of three 1 h sessions per week. Isolated platelets were analyzed for thromboxane A2 receptor, thromboxane A2 synthase, cyclooxygenase-1, and prostacyclin receptor protein expression. Additionally, plasma 6-keto prostaglandin F1α and thromboxane B2 levels were determined. Results: Exercise training made platelets more sensitive to the inhibitory effects of prostacyclin on thromboxane-, collagen-, and adenosine 5′-diphosphate (ADP)-induced aggregation, as well as thrombin-receptor activator peptide 6- and ADP-induced aggregation with DAPT. However, there was no change in protein expression from isolated platelets or plasma thromboxane B2 and prostacyclin levels following training. Conclusion: Together, these findings emphasize the importance of promoting physical activity as a tool for reducing thrombotic risk in postmenopausal women and suggest that training status should be considered when prescribing DAPT in this cohort.

Does the quality index of adherence to the evidence-based guidelines predict mortality in patients with myocardial infarction?

Abstract Background The SWEDEHEART quality index of hospitals' adherence to the evidence-based (EB) guidelines for myocardial infarction (MI) patients has been continuously used for several decades in Sweden. The grading protocol is based on the consensus among hospitals. The hospitals are awarded points (0, 0.5, 1) for each of the 11 indicators depending on the proportion of patients who received EB treatment and achieved treatment goals. The 11 indicators at present are reperfusion treatment in STEMI (yes/no), time to-reperfusion treatment in STEMI, time to revascularisation in NSTEMI, P2Y12 antagonists at discharge, ACE-inhibitor/ARB at discharge, the proportion of patients at follow-up, smoking cessation at one-year, participation in a physical exercise program, target LDL-cholesterol and target blood pressure at one year. Purpose To evaluate whether the SWEDEHEART quality index predicts mortality in patients with MI. Methods We used data for all MI patients reported to the SWEDEHEART registry from 72 hospitals in Sweden between 2015–2021. We calculated the difference in quality index between 2021 and 2015. The hospitals were divided into quintiles based on the difference in the score. Logistic regression with log-time offset was used to adjust for confounders (age, gender, diabetes, hypertension, hyperlipidemia, STEMI/NSTEMI, cardiac arrest before admission, occupation status, history of heart failure, prior MI, prior PCI, prior CABG, cardiogenic shock). Results We identified 98,635 patients with MI, 32,608 (33.1%) were women and 34,198 (34.7%) had STEMI. The average age was 70.8±12.2 years. The median follow-up time was 2.7 years (IQR 1.06–4.63). The crude all-cause mortality rate was 5.5% at 30-days and 22.3% after long-term follow-up. Most hospitals (72.1%) improved their quality index on average by 3.4% per year (P&amp;lt;0.001). The increase in the quality index continued during COVID-19 pandemic (2020–2021) with average increase of 8.6%, 95% CI, 0.97–1.02; P&amp;lt;0.001. The median change in SWEDEHEART quality index score among the quintiles were −1.5 (Q1), 0,5 (Q2), 2,5 (Q3), 3 (Q4), and 4 (Q5). We found no difference in mortality between the quintiles at 30-days (OR 0.99; 95% CI 0.97–1.02; p=1.02) and long-term (OR 1.01; 95% CI 0,99–1.02; p=0.850). Conclusion The SWEDEHEART quality index provides valuable descriptive information about hospitals' adherence to the guidelines. However, the index, in its current form, does not predict mortality in patients with MI. Funding Acknowledgement Type of funding sources: None.

The role of P2Y12 in cardiovascular disease beyond atherothrombosis: P2Y12 signaling promotes emergency hematopoiesis after myocardial infarction

Abstract Background Adenosine diphosphate (ADP) plays a pivotal role in platelet activation. The purinergic ADP-receptor P2Y12 has therefore been targeted in the treatment of cardiovascular disease (CVD) to prevent atherothrombosis (1). Beyond P2Y12 expression on platelets, purinergic receptors have also been described on hematopoietic stem and progenitor cells (LSK) (2). After myocardial infarction (MI), accelerated LSK proliferation launches emergency hematopoiesis as the driving force behind the inflammatory response to MI, increasing inflammatory cell production in the bone marrow (BM) and providing leukocyte resupply for local cell recruitment to the infarct (3). The inflammatory cascade after MI covers intricate multilayered interactions between the injured myocardium and the hematopoietic BM that still remain to be fully elucidated and may unearth novel therapeutic strategies. Whereas P2X receptors have recently been found to be involved in cell trafficking (4), the role of P2Y receptors in the hematopoietic BM have not yet been characterized. Purpose This study aims to characterize the influence of P2Y12 signaling on emergency hematopoiesis and cardiac remodeling after MI. Methods Permanent coronary ligation was performed for MI to assess BM activation, inflammatory cell composition, cardiac remodeling and function in murine global and platelet-specific P2Y12 knockout models and under pharmacological P2Y12 inhibition with prasugrel using flow cytometry, qPCR, immunohistochemistry and echocardiography. In vitro studies including colony forming unit (CFU) assays and flow cytometry allowed for investigation of ADP-dependent effects on LSK cells and intracellular pathway analysis. Results We identified ADP as a danger signal for the hematopoietic BM, fueling emergency hematopoiesis by promoting Akt phosphorylation and cell cycle progression. Detection of P2Y12 expression in LSK implicated a direct effect of ADP on LSK via P2Y12 signaling. P2Y12 deficiency and P2Y12 inhibition with prasugrel decelerated emergency hematopoiesis and consecutively reduced the excessive inflammatory response to MI, translating to lower numbers of hematopoietic progenitors and inflammatory cells in the blood and infarct. Ultimately, P2Y12 inhibition ameliorated chronic adverse cardiac remodeling and preserved cardiac function after MI. Conclusion ADP-dependent P2Y12-mediated activation of hematopoietic stem and progenitor cells in the BM promotes emergency hematopoiesis after MI and fuels post-ischemic inflammation, proposing a novel role of P2Y12 antagonists in CVD beyond atherothrombosis. Funding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Deutsche Forschungsgemeinschaft (DFG)