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Platelet activation plays a critical role in thrombosis and hemostasis. Several pathophysiological situations lead to hemolysis, resulting in the liberation of free ferric iron-containing hemin. Hemin has been shown to activate platelets and induce thrombo-inflammation. Classical antiplatelet therapy failed to prevent hemin-induced platelet activation. Thus, the aim of the present study was to characterize the mechanism of hemin-induced platelet death (ferroptosis). We evaluated the in vitro effect of hemin on platelet activation, signaling, oxylipins, and plasma membrane destruction using light transmission aggregometry, ex vivo thrombus formation, multiparametric flow cytometry, micro-UHPLC mass spectrometry for oxylipin profiling, and scanning ion conductance microscopy (SICM). We found that hemin induces platelet cell death indicated by increased ROS levels, phosphatidyl serine (PS) exposure, and loss of mitochondrial membrane potential (ΔΨm). Further, hemin causes lipid peroxidation and generation of distinct oxylipins, which strongly affects plasma membrane integrity leading to generation of platelet-derived microvesicles. Interestingly, hemin-dependent platelet death (ferroptosis) is specifically regulated by the subtilisin-like proprotein convertase furin. In summary, platelet undergo a non-apoptotic cell death mediated by furin. Inhibition of furin may offer a therapeutic strategy to control hemin-induced thrombosis and thrombo-inflammation at a site of hemolysis., (© 2024 The Author(s). The FASEB Journal published by Wiley Periodicals LLC on behalf of Federation of American Societies for Experimental Biology.)

Aims: The production of reactive oxygen species (ROS) by NADPH oxidase (NOX) is able to induce platelet activation, making NOX a promising target for antiplatelet therapy. In this study, we examined the effects of setanaxib, a dual NOX1/4 inhibitor, on human platelet function and ROS-related signaling pathways., Materials and Methods: In collagen-stimulated human platelets, aggregometry, assessment of ROS and Ca 2+ , immunoblotting, ELISA, flow cytometry, platelet adhesion assay, and assessment of mouse arterial thrombosis were performed in this study., Key Findings: Setanaxib inhibited both intracellular and extracellular ROS production in collagen-activated platelets. Additionally, setanaxib significantly inhibited collagen-induced platelet aggregation, P-selectin exposure from α-granule release, and ATP release from dense granules. Setanaxib blocked the specific tyrosine phosphorylation-mediated activation of Syk, LAT, Vav1, and Btk within collagen receptor signaling pathways, leading to reduced activation of PLCγ2, PKC, and Ca 2+ mobilization. Setanaxib also inhibited collagen-induced activation of integrin αIIbβ3, which is linked to increased cGMP levels and VASP phosphorylation. Furthermore, setanaxib suppressed collagen-induced p38 MAPK activation, resulting in decreased phosphorylation of cytosolic PLA 2 and reduced TXA 2 generation. Setanaxib also inhibited ERK5 activation, affecting the exposure of procoagulant phosphatidylserine. Setanaxib reduced thrombus formation under shear conditions by preventing platelet adhesion to collagen. Finally, in vivo administration of setanaxib in animal models led to the inhibition of arterial thrombosis., Significance: This study is the first to show that setanaxib suppresses ROS generation, platelet activation, and collagen-induced thrombus formation, suggesting its potential use in treating thrombotic or cardiovascular diseases., 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 Inc. All rights reserved.)

Triple-negative breast cancer (TNBC) has poor prognosis. Carboplatin (Crb) is a widely used chemotherapeutic agent, in TNBC but with serious systemic toxicity and poor tumor targeting. Bioinspired drug-loaded platelets (Plt) and Plt-coated nanocarriers evade macrophage phagocytosis by membrane proteins like CD47. The goal of this study was preparation of a novel alginate-poly (β-amino ester) (PβAE) nanoparticles (NPs) for targeted delivery of Crb to TNBC cells by developing and comparison of two bioinspired carriers of Plt membrane (PltM) coated Crb-loaded alginate-poly (β-amino ester) nanoparticles (PltM@Crb-PβAE-ALG NPs) and Plt loaded Crb (Plt@Crb). The NPs were prepared by ionic gelation and subsequently were coated by platelet membrane using ultra-sonication method. The loading efficiency, release profile, and in vitro cytotoxicity of both formulations were evaluated on HUVEC and 4 T1 cells. Additionally, the in vivo tumor targeting, therapeutic efficacy, and organ toxicity of the two formulations were assessed in a murine tumor model. Results showed both Plt@Crb and (PltM@Crb-PβAE-ALG NPs) exhibited high drug loading efficiency, sustained release, enhanced cytotoxicity against 4 T1 cells, and decreased cytotoxicity in normal cells (HUVEC) in vitro. In vivo studies revealed that although both formulations considerably improved tumor inhibition compared to free Crb, but the PltM@Crb-PβAE-ALG NPs demonstrated superior cytotoxicity and therapeutic efficacy, thanks to improved Crb's internalization efficiency, enhanced stability, and controlled release properties., 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 B.V. All rights reserved.)

Background and Aims: Glycoprotein VI (GPVI) is a platelet collagen/fibrin(ogen) receptor and an emerging pharmacological target for the treatment of thrombotic and thrombo-inflammatory diseases, notably ischaemic stroke. A first anti-human GPVI (hGPVI) antibody Fab-fragment (ACT017/glenzocimab, KD: 4.1 nM) recently passed a clinical phase 1b/2a study in patients with acute ischaemic stroke and was found to be well tolerated, safe, and potentially beneficial. In this study, a novel humanized anti-GPVI antibody Fab-fragment (EMA601; KD: 0.195 nM) was developed that inhibits hGPVI function with very high potency in vitro and in vivo., Methods: Fab-fragments of the mouse anti-hGPVI IgG Emf6.1 were tested for functional GPVI inhibition in human platelets and in hGPVI expressing (hGP6tg/tg) mouse platelets. The in vivo effect of Emf6.1Fab was assessed in a tail bleeding assay, an arterial thrombosis model and the transient middle cerebral artery occlusion (tMCAO) model of ischaemic stroke. Using complementary-determining region grafting, a humanized version of Emf6.1Fab (EMA601) was generated. Emf6.1Fab/EMA601 interaction with hGPVI was mapped in array format and kinetics and quantified by bio-layer interferometry., Results: Emf6.1Fab (KD: 0.427 nM) blocked GPVI function in human and hGP6tg/tg mouse platelets in multiple assays in vitro at concentrations ≥5 µg/mL. Emf6.1Fab (4 mg/kg)-treated hGP6tg/tg mice showed potent hGPVI inhibition ex vivo and were profoundly protected from arterial thrombosis as well as from cerebral infarct growth after tMCAO, whereas tail-bleeding times remained unaffected. Emf6.1Fab binds to a so far undescribed membrane proximal epitope in GPVI. The humanized variant EMA601 displayed further increased affinity for hGPVI (KD: 0.195 nM) and fully inhibited the receptor at 0.5 µg/mL, corresponding to a >50-fold potency compared with ACT017., Conclusions: EMA601 is a conceptually novel and promising anti-platelet agent to efficiently prevent or treat arterial thrombosis and thrombo-inflammatory pathologies in humans at risk., (© The Author(s) 2024. Published by Oxford University Press on behalf of the European Society of Cardiology.)

Understanding platelet protein functions facilitates better assessment of platelet disorders. Megakaryocyte lineage-restricted human Disabled-2 knock-in (hDAB2-KI) mice are generated to delineate the functions of hDab2, a regulator of platelet function, in the control of bleeding associated with thrombocytopenia. Here we show that hDab2-KI mice with thrombocytopenia display decreased bleeding time when compared to the control mice. hDab2 augments thromboxane A 2 (TxA 2 ) mimetic U46619- but not other agonists-stimulated granule secretion, integrin activation, and aggregation at a lower platelet concentration in vitro. Binding of hDab2 to phosphatidic acid (PA) facilitates formation of the PA-hDab2-AKT complex leading to an increase in U46619-stimulated AKT-Ser473 phosphorylation and the first wave of ADP/ATP release. Consistent with these findings, hDab2 expression in platelets from patients with immune thrombocytopenic purpura is positively correlated with U46619-stimulated ATP release, which in turn inversely correlated with their bleeding tendency. hDab2 appears crucial in regulating bleeding severity associated with thrombocytopenia by a functional interplay with ADP/ATP release underlying TxA 2 signaling., Competing Interests: Competing interests The authors declare no competing interests., (© 2024. The Author(s).)

Abstract: Platelet C-type lectin-like receptor 2 (CLEC-2) is a hem-immunoreceptor tyrosine-based activation motif-containing receptor that has a critical role in venous thrombosis but minimal involvement in hemostasis. CLEC-2 can be blocked by Btk inhibitors. Treatment with ibrutinib is associated with increased bleeding due to off-target inhibition of Src family kinases (SFKs). Patients with X-linked agammaglobulinemia (XLA) who lack Btk, however, do not bleed, suggesting selective Btk inhibition as a viable antithrombotic strategy. We assessed the effects of selective Btk inhibitors PRN1008 (rilzabrutinib) and PRN473 on platelet signaling and function mediated by CLEC-2 and glycoprotein-VI. We used healthy donors and XLA platelets to determine off-target inhibitor effects. Inferior vena cava (IVC) stenosis and Salmonella infection mouse models were used to assess antithrombotic effects of PRN473 in vivo. PRN1008 and PRN473 potently inhibited CLEC-2-mediated platelet activation to rhodocytin. No off-target inhibition of SFKs was seen. PRN1008 treatment of Btk-deficient platelets resulted in minor additional inhibition of aggregation and tyrosine phosphorylation, likely reflecting inhibition of Tec. No effect on G protein-coupled receptor-mediated platelet function was observed. PRN473 significantly reduced the number of thrombi in podoplanin-positive vessels after Salmonella infection and the presence of IVC thrombosis after vein stenosis. The potent inhibition of human platelet CLEC-2 and reduced thrombosis in in vivo models, together with the lack of off-target SFK inhibition and absence of bleeding reported in rilzabrutinib-treated patients with immune thrombocytopenia, suggest Btk inhibition as a promising antithrombotic strategy., (© 2024 by The American Society of Hematology. Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved.)

Aims: Although dual antiplatelet therapy with aspirin and a potent P2Y12 receptor inhibitor is currently recommended in patients with acute coronary syndrome (ACS), its use in elderly patients remains challenging. The aim of this trial is to evaluate the pharmacodynamic and pharmacokinetic profile of ticagrelor 60 vs. 90 mg twice daily among elderly patients (≥75 years) with ACS undergoing percutaneous coronary intervention (PCI)., Methods and Results: PLINY The ELDER (NCT04739384) was a randomized, crossover trial testing the non-inferiority of a lower vs. standard dose of ticagrelor with respect to the primary endpoint of P2Y12 inhibition as determined by pre-dose P2Y12 reaction units (PRU) using the VerifyNow-P2Y12 (Accumetrics, San Diego, CA, USA). Other pharmacodynamic tests included light transmittance aggregometry, multiple electrode aggregometry, and response to aspirin. Plasma levels of ticagrelor and its active metabolite AR-C124910XX were also evaluated. A total of 50 patients (mean age 79.6 ± 4.0 years, females 44%) were included in the trial. Ticagrelor 60 mg was non-inferior to ticagrelor 90 mg according to VerifyNow-P2Y12 results (PRU 26.4 ± 32.1 vs. 30.4 ± 39.0; least squares mean difference: -4; 95% confidence interval: -16.27 to 8.06; P for non-inferiority = 0.002). Other pharmacodynamic parameters were similar between the two ticagrelor doses and there were no differences in response to aspirin. Plasma levels of ticagrelor (398.29 ± 312.36 ng/mL vs. 579.57 ± 351.73 ng/mL, P = 0.006) and its active metabolite were significantly lower during treatment with ticagrelor 60 mg., Conclusion: Although plasma concentrations were lower, ticagrelor 60 mg twice daily provided a similar magnitude of platelet inhibition compared with ticagrelor 90 mg twice daily among elderly patients undergoing PCI., (© The Author(s) 2024. Published by Oxford University Press on behalf of the European Society of Cardiology.)

Aims: MicroRNA-126 (miR-126), one of the most abundant microRNAs in platelets, is involved in the regulation of platelet activity and the circulating miR-126 is reduced during antiplatelet therapy. However, whether intraplatelet miR-126 plays a role in thrombosis and platelet inhibition remains unclear., Methods and Results: Here, using tissue-specific knockout mice, we reported that the deficiency of miR-126 in platelets and vascular endothelial cells significantly prevented thrombosis and prolonged bleeding time. Using chimeric mice, we identified that the lack of intraplatelet miR-126 significantly prevented thrombosis. Ex vivo experiments further demonstrated that miR-126-deficient platelets displayed impaired platelet aggregation, spreading, and secretory functions. Next, miR-126 was confirmed to target phosphoinositol-3 kinase regulatory subunit 2 (PIK3R2) in platelet, which encodes a negative regulator of the phosphoinositide 3-kinase/protein kinase B pathway, enhancing platelet activation through activating the integrin αIIbβ3-mediated outside-in signalling. After undergoing myocardial infarction (MI), chimeric mice lacking intraplatelet miR-126 displayed reduced microvascular obstruction and prevented MI expansion in vivo. In contrast, overexpression of miR-126 by the administration of miR-126 agonist (agomiR-126) in wild-type mice aggravated microvascular obstruction and promoted MI expansion, which can be almost abolished by aspirin administration. In patients with cardiovascular diseases, antiplatelet therapies, either aspirin alone or combined with clopidogrel, decreased the level of intraplatelet miR-126. The reduction of intraplatelet miR-126 level was associated with the decrease in platelet activity., Conclusion: Our murine and human data reveal that (i) intraplatelet miR-126 contributes to platelet activity and promotes thrombus formation, and (ii) the reduction of intraplatelet miR-126 contributes to platelet inhibition during antiplatelet therapy., Competing Interests: Conflict of interest: none declared., (© The Author(s) 2024. Published by Oxford University Press on behalf of the European Society of Cardiology. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

Parkinson's disease (PD) is the second-most prevalent neurodegenerative disease worldwide, which worsens with advancing age. It is a common movement disorder and is often associated with several vascular diseases with decreased stroke frequency. Circulating platelets substantially regulate vascular complications, including stroke, and share striking similarities with PD neurons. Although structural alterations in platelets are well-documented in PD, their functional parameters remain unclear. This study aimed to investigate the functional abnormalities in platelets associated with PD by evaluating key functional aspects such as adhesion, activation, secretion, aggregation, and clot retraction. To achieve this, we treated human blood platelets with 6-hydroxydopamine or 6-OHDA, that selectively destroys dopaminergic neurons, thereby creating an in vitro experimental model that closely resembles the pathogenic environment in PD, and examine its impact on platelet functions. In our study, platelet adhesion was assessed and further evaluated by a microplate reader, activation and secretion by a flow cytometer, aggregation by aggregometer, and clot retraction by Sonoclot. Phase-contrast and confocal microscopic studies further verified the results from the above experiments. Our findings showed that 6-OHDA treatment significantly inhibited thrombin (a platelet agonist)-induced functions, including adhesion, activation, aggregation, secretion, and clot retraction in human-washed platelets. In summary, this research provides pioneering evidence that 6-OHDA induces abnormal platelet functions, shedding light on the previously unexplored processes by which 6-OHDA affects platelet activity., 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 International Brain Research Organization (IBRO). Published by Elsevier Inc. All rights reserved.)

Harmine (HM), a β-carboline alkaloid extracted from plants, is a crucial component of traditional Chinese medicine (TCM) known for its diverse pharmacological activities. Thrombocytopenia, a common and challenging hematological disorder, often coexists with serious illnesses. Previous research has shown a correlation between HM and thrombocytopenia, but the mechanism needs further elucidation. The aim of this study was to clarify the mechanisms underlying the effects of HM on thrombocytopenia and to develop new therapeutic strategies. Flow cytometry, Giemsa staining, and Phalloidin staining were used to assess HM's impact on Meg-01 and HEL cell differentiation and maturation in vitro. A radiation-induced thrombocytopenic mouse model was employed to evaluate HM's effect on platelet production in vivo. Network pharmacology, molecular docking, and protein blotting were utilized to investigate HM's targets and mechanisms. The results demonstrated that HM dose-dependently promoted Meg-01 and HEL cell differentiation and maturation in vitro and restored platelet levels in irradiated mice in vivo. Subsequently, HM was found to be involved in the biological process of platelet production by upregulating the expressions of Rac1, Cdc42, JNK, and 5-HTR2A. Furthermore, the targeting of HM to 5-HTR2A and its correlation with downstream Rac1/Cdc42/JNK were also confirmed. In conclusion, HM regulates megakaryocyte differentiation and thrombopoiesis through the 5-HTR2A and Rac1/Cdc42/JNK pathways, providing a potential treatment strategy for thrombocytopenia., (© 2024 John Wiley & Sons Ltd.)

Background: CpG oligonucleotides (ODNs) are synthetic single-stranded DNA sequences that act as immunostimulants. They have been increasingly used to treat several cancers; however, thrombocytopenia is a potential recognized side effect of some sequences., Objectives: We tested the ability of 2 CpG ODNs (ODN 2395 and ISIS 120704) to induce thrombocytopenia when administered to BALB/c mice and determined mechanisms associated with thrombocytopenia., Methods: BALB/c mice were prebled and then injected with titrated doses of CpG ODNs, and platelet counts were determined. The mice were treated with intravenous immunoglobulin (IVIg) or various inhibitors and antagonists of toll-like receptor 9 (TLR9) and spleen tyrosine kinase (Syk) to determine their effects on thrombocytopenia., Results: Compared with saline-treated mice or mice treated with 2'-O-methoxyethyl-modified antisense ODN, both ODN 2395 and ISIS 120704 induced acute dose-dependent thrombocytopenia within 3 and 24 hours, respectively. The thrombocytopenia was associated with significant increases in plasma monocyte chemoattractant protein 1. IVIg administration significantly rescued the CpG ODN-induced thrombocytopenia, as did treatment with either a Syk inhibitor or TLR9 antagonists. In vitro, CpG ODN could activate human platelets and this correlated significantly with enhanced IVIg- and Syk-dependent phagocytosis by THP-1 monocytes., Conclusion: These results suggest that CpG ODNs induce acute inflammatory-associated (IVIg-sensitive) thrombocytopenia that can be alleviated by Syk- or TLR9-blockade, and an IVIg- and Syk-dependent platelet clearance pathway appears primarily responsible for the thrombocytopenia., Competing Interests: Declaration of competing interests There are no competing interests to disclose., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Background:  Deep vein thrombosis (DVT) is a common condition associated with significant mortality due to pulmonary embolism. Despite advanced prevention and anticoagulation therapy, the incidence of venous thromboembolism remains unchanged. Individuals with elevated hematocrit and/or excessively high erythropoietin (EPO) serum levels are particularly susceptible to DVT formation. We investigated the influence of short-term EPO administration compared to chronic EPO overproduction on DVT development. Additionally, we examined the role of the spleen in this context and assessed its impact on thrombus composition., Methods:  We induced ligation of the caudal vena cava (VCC) in EPO-overproducing Tg(EPO) mice as well as wildtype mice treated with EPO for two weeks, both with and without splenectomy. The effect on platelet circulation time was evaluated through FACS analysis, and thrombus composition was analyzed using immunohistology., Results:  We present evidence for an elevated thrombogenic phenotype resulting from chronic EPO overproduction, achieved by combining an EPO-overexpressing mouse model with experimental DVT induction. This increased thrombotic state is largely independent of traditional contributors to DVT, such as neutrophils and platelets. Notably, the pronounced prothrombotic effect of red blood cells (RBCs) only manifests during chronic EPO overproduction and is not influenced by splenic RBC clearance, as demonstrated by splenectomy. In contrast, short-term EPO treatment does not induce thrombogenesis in mice. Consequently, our findings support the existence of a differential thrombogenic effect between chronic enhanced erythropoiesis and exogenous EPO administration., Conclusion:  Chronic EPO overproduction significantly increases the risk of DVT, while short-term EPO treatment does not. These findings underscore the importance of considering EPO-related factors in DVT risk assessment and potential therapeutic strategies., Competing Interests: None declared., (The Author(s). This is an open access article published by Thieme under the terms of the Creative Commons Attribution-NonDerivative-NonCommercial License, permitting copying and reproduction so long as the original work is given appropriate credit. Contents may not be used for commercial purposes, or adapted, remixed, transformed or built upon. (https://creativecommons.org/licenses/by-nc-nd/4.0/).)

Background: Platelet gene therapy is effective in hemophilia A (HA) mice even with inhibitors. Fludarabine (Flu), along with busulfan (Bu) or melphalan (Mel), preconditioning has been shown to be highly effective for hematopoietic stem cell transplantation in the clinic., Objectives: To evaluate the efficacy of Bu-Flu and Mel-Flu preconditioning in platelet gene therapy of HA with inhibitors., Methods: Bu-Flu and Mel-Flu were used to condition HA mice preimmunized with recombinant human factor (F)VIII. An optimal 660 centigray total body irradiation was used as a control regimen in parallel. Platelet-FVIII expression was introduced by transplantation of 2bF8 lentivirus (LV)-transduced hematopoietic stem cells. Animals were analyzed by fluorescence-activated cell sorting, quantitative polymerase chain reaction, FVIII assays, and tail bleeding tests., Results: Bu-Flu, but not Mel-Flu, enabled successful 2bF8 gene therapy. All recipients achieved >55% chimerism post hematopoietic stem cell transplantation in both Bu-Flu and 660 centigray groups, with comparable copy numbers of 2bF8 cassette and the platelet-FVIII levels. The bleeding phenotype was rescued in 2bF8LV-transduced recipients. FVIII inhibitor titers declined with time, with comparable disappearance time of inhibitors between the 2 groups. When animals were rechallenged with recombinant human FVIII after the titers dropped to undetectable levels, no inhibitors were detected in 2bF8LV-transduced recipients. In contrast, all untransduced transplanted control mice produced inhibitors. These data demonstrate that immune tolerance was established in 2bF8LV-transduced primed HA mice under Bu-Flu conditioning., Conclusion: Bu-Flu preconditioning allows for successfully introducing platelet-FVIII expression to restore hemostasis and induce immune tolerance in primed HA mice, suggesting that this approach is a promising clinically translatable strategy for gene therapy of HA with inhibitors., Competing Interests: Declaration of competing interests There are no competing interests to disclose., (Copyright © 2024 International Society on Thrombosis and Haemostasis. Published by Elsevier Inc. All rights reserved.)

Background: Platelet activation and interaction with leukocytes are crucial in inflammation. Gangliosides, sialic acid-containing glycosphingolipids, have been linked to different inflammatory conditions related to cardio- and neurodegenerative disorders. The role of gangliosides in platelet and leukocyte function, although reported, still needs further investigation., Objectives: We aimed to study the role of gangliosides in platelet activation and platelet-leukocyte interaction in vitro., Methods: Platelet activation was studied through aggregometry in platelet-rich plasma from apparently healthy human volunteers. Signaling protein phosphorylation was analyzed by immunoblotting. Platelet P-selectin expression and platelet-leukocyte aggregate formation were measured by flow cytometry., Results: The gangliosides monosialoganglioside GM1, disialoganglioside GD1a, and trisialoganglioside GT1b did not induce by themselves any platelet aggregation. Conversely, when preincubated with platelets, they potentiate platelet aggregation induced by submaximal adenosine diphosphate and collagen concentrations and increased P-selectin expression. Incubation of platelets with free sialic acid and the soluble part of monosialoganglioside GM1 induced a similar potentiating effect on platelet aggregation but not on platelet P-selectin expression. Consistently, analyzing the signaling protein phosphorylation, only the entire gangliosides activated extracellular stimuli-responsive kinase 1/2 suggesting that a complete ganglioside is crucial for its action on platelets. Both the priming effect on platelet aggregation and ERK1/2 activation were prevented by aspirin. Moreover, incubation of citrated whole blood with gangliosides induced platelet-leukocyte aggregate formation accompanied by increased expression of granulocyte and monocyte CD11b compared with untreated blood, suggesting a primary leukocyte activation., Conclusion: Gangliosides may act in vitro both on platelet and leukocyte activation and on their interaction. The observed effects might contribute to inflammatory processes in clinical conditions., Competing Interests: Declaration of competing interests There are no competing interests to disclose., (Copyright © 2024 International Society on Thrombosis and Haemostasis. Published by Elsevier Inc. All rights reserved.)

Background and Purpose: Although the amphiphilic nature of the widely used antithrombotic drug Ticagrelor is well known, it was never considered as a membranotropic agent capable of interacting with the lipid bilayer in a receptor-independent way. In this study, we investigated the influence of Ticagrelor on plasma membrane lipid order in platelets and if this modulates the potency of Ticagrelor at the P2Y 12 receptor., Experimental Approach: We combined fluorescent in situ, in vitro and in silico approaches to probe the interactions between the plasma membrane of platelets and Ticagrelor. The influence of Ticagrelor on the lipid order of the platelet plasma membrane and large unilamellar vesicles was studied using the advanced fluorescent probe NR12S. Furthermore, the properties of model lipid bilayers in the presence of Ticagrelor were characterized by molecular dynamics simulations. Finally, the influence of an increased lipid order on the dose-response of platelets to Ticagrelor was studied., Key Results: Ticagrelor incorporates spontaneously into lipid bilayers and affects the lipid order of the membranes of model vesicles and isolated platelets, in a nontrivial composition and concentration-dependent manner. We showed that higher plasma membrane lipid order in platelets leads to a lower IC 50 value for Ticagrelor. It is shown that membrane incorporation of Ticagrelor increases its potency at the P2Y 12 receptor, by increasing the order of the platelet plasma membrane., Conclusion and Implications: A novel dual mechanism of Ticagrelor action is suggested that combines direct binding to P2Y 12 receptor with simultaneous modulation of receptor-lipid microenvironment., (© 2024 British Pharmacological Society.)

Background: Snake venom botrocetin facilitates von Willebrand factor (VWF) binding to platelet GPIbα and has been widely used for the diagnosis of von Willebrand disease and GPIb-related disorders. Botrocetin is also commonly employed for the development/characterization of antithrombotics targeting the GPIb-VWF axis., Objectives: To explore the alternative receptor(s)/mechanisms that participate in botrocetin-induced platelet aggregation., Methods: The effects of botrocetin on platelet aggregation were examined using platelets from wild-type, VWF- and fibrinogen-deficient, GPIbα-deficient, IL4Rα/GPIbα-transgenic, ITGA2B and ITGB3-deficient mice, and Bernard-Soulier syndrome and healthy human samples. Platelet-fibrinogen and platelet-VWF interaction were measured using flow cytometry. GPIbα-VWF binding was evaluated utilizing enzyme-linked immunosorbent assay. Botrocetin-α IIb β 3 and botrocetin-GPIbα interactions were measured using enzyme-linked immunosorbent assay and fluorescence anisotropy assays. Heparinized whole blood from healthy donors was examined for thrombus formation and growth in a perfusion chamber., Results: Botrocetin could induce aggregation of platelets from a Bernard-Soulier syndrome patient and GPIbα-deficient mice as well as platelets lacking the N-terminal extracellular domain of GPIbα. Botrocetin could interact with α IIb β 3 and facilitated α IIb β 3 -VWF interaction independent of GPIb. Botrocetin competitively bound to the ligand-binding domain of activated rather than resting α IIb β 3 . Although botrocetin-induced platelet aggregation requires VWF, strikingly, in the absence of VWF, botrocetin blocked fibrinogen and other ligand binding to α IIb β 3 and inhibited platelet aggregation and thrombus formation. Consistently, recombinant botrocetin defective in VWF binding inhibited α IIb β 3 - and GPIb-mediated platelet aggregation, spreading, and thrombus formation., Conclusion: Our study provides insights into avoiding the misdiagnosis of GPIb-related disorders and developing botrocetin mutants as potential new antithrombotics that may simultaneously target both α IIb β 3 and GPIbα., Competing Interests: Declaration of competing interests Integrin β(3) monoclonal antibodies are patented in the United States, Canada, and Europe (United States patent number 7731966; Canadian patent number CA2628900C; European patent number EP2025685B1). GPIbα monoclonal antibodies are patented in the United States, Canada, and Europe (United States patent number 8323652; Canadian patent number CA2689726C; European patent number EP2186829B1)., (Copyright © 2024 International Society on Thrombosis and Haemostasis. Published by Elsevier Inc. All rights reserved.)

Background: EDTA-induced pseudothrombocytopenia (PTCP) during whole blood collection requires significant laboratory resources to obtain accurate results. We evaluated platelet-deaggregation function in EDTA-induced PTCP and platelet-clump flagging by the BC-6800Plus hematology analyzer using integrated digital image analysis., Methods: We prospectively collected 132 whole blood samples suspected of platelet clumping (102 in EDTA and 30 in sodium citrate) from 88 individuals. We compared platelet counts determined using the platelet count by impedance (PLT-I) function of the DxH 900 hematology analyzer and the PLT-I or optical platelet count (PLT-O) function of the BC-6800Plus. Platelet clumping was verified through manual inspection and the MC-80 digital image analyzer., Results: Among the 132 whole blood samples, 43 EDTA samples showed platelet clumping. The DxH 900 PLT-I and BC-6800Plus PLT-I results demonstrated a strong correlation (r=0.711) for the EDTA samples but only a moderate correlation with the BC-6800Plus PLT-O results (r=0.506 and 0.545, respectively). The BC-6800Plus PLT-O results were consistent with the sodium citrate platelet counts, with a median dissociation rate of 102.5% (range, 74.9%-123.1%). The DxH 900 and BC-6800Plus analyzers had sensitivity values of 0.79 and 0.72, respectively, for platelet-clump flagging. When integrating the MC-80 digital image analysis results, the sensitivity of BC-6800Plus improved to 0.89 (standard mode) or 1.0 (PLT-Pro mode)., Conclusions: BC-6800Plus PLT-O measurement results are close to the actual values obtained by platelet deaggregation with PTCP samples. Integrating the BC-6800Plus with a digital imaging analyzer effectively improved the diagnosis of PTCP and reduced the requirement for additional laboratory procedures.

Backgrounds/Objectives: Abnormal platelet functions are associated with human morbidity and mortality. Platelets have emerged as critical regulators of numerous physiological and pathological processes beyond their established roles in hemostasis and thrombosis. Maintaining physiological platelet function is essential to hemostasis and preventing platelet-associated diseases such as cardiovascular disease, cancer metastasis, immune disorders, hypertension, diabetes, sickle cell disease, inflammatory bowel disease, sepsis, rheumatoid arthritis, myeloproliferative disease, and Alzheimer's disease. Platelets become hyperactive in obesity, diabetes, a sedentary lifestyle, hypertension, pollution, and smokers. Platelets, upon activation, can trawl leukocytes and progenitor cells to the vascular sites. Platelets release various proinflammatory, anti-inflammatory, and angiogenic factors and shed microparticles in the circulation, thus promoting pathological reactions. These platelet-released factors also maintain sustained activation, further impacting these disease processes. Although the mechanisms are unknown, multiple stimuli induce platelet hyperreactivity but involve the early pathways of platelet activation. The exact mechanisms of how hyperactive platelets contribute to these diseases are still unclear, and antiplatelet strategies are inevitable for preventing these diseases. Reducing platelet function during the early stages could significantly impact these diseases. However, while this is potentially a worthwhile intervention, using antiplatelet drugs to limit platelet function in apparently healthy individuals without cardiovascular disease is not recommended due to the increased risk of internal bleeding, resistance, and other side effects. The challenge for therapeutic intervention in these diseases is identifying factors that preferentially block specific targets involved in platelets' complex contribution to these diseases while leaving their hemostatic function at least partially intact. Since antiplatelet drugs such as aspirin are not recommended as primary preventives, it is essential to use alternative safe platelet inhibitors without side effects., Methods: A systematic search of the PUBMED database from 2000 to 2023 was conducted using the selected keywords: "functional foods", "polyphenols", "fatty acids", "herbs", fruits and vegetables", "cardioprotective agents", "plant", "platelet aggregation", "platelet activation", "clinical and non-clinical trial", "randomized", and "controlled"., Results: Potent natural antiplatelet factors have been described, including omega-3 fatty acids, polyphenols, and other phytochemicals. Antiplatelet bioactive compounds in food that can prevent platelet hyperactivity and thus may prevent several platelet-mediated diseases, including cardiovascular disease., Conclusions: This narrative review describes the work during 2000-2023 in developing functional foods from natural sources with antiplatelet effects.

Background: A previous single-center study of patients with myocardial infarction (MI) showed that platelet FcγRIIa (pFCG) can distinguish patients at higher and lower risk of subsequent MI, stroke, and death., Objectives: The authors performed an 800-patient 25-center study to validate the prognostic implications of pFCG., Methods: Patients with type 1 MI (ST-segment elevation and non-ST-segment elevation) were enrolled in a prospective noninterventional trial during their index hospitalization. Enrolled patients had at least 2 of the following characteristics: age ≥65 years, multivessel coronary artery disease, previous MI, chronic kidney disease, or diabetes mellitus. Flow cytometry was used to quantify pFCG at a core laboratory. A predefined threshold was used to identify high and low pFCG. Patients were queried every 6 months by telephone with a standardized questionnaire. Events were confirmed by review of medical records., Results: Treatment with antithrombotic therapy (aspirin, P2Y 12 inhibitors, and anticoagulants) was similar in patients with high and low pFCG. The primary composite endpoint (MI, stroke, death) occurred more frequently in patients with high pFCG (HR: 2.09; 95% CI: 1.34-3.26; P = 0.001). Among individual components of the composite, both death (HR: 2.57; 95% CI: 1.50-4.40; P = 0.001) and MI (HR: 3.24; 95% CI: 1.64-6.37; P = 0.001) were more frequent in patients with high pFCG., Conclusions: Quantifying pFCG identifies patients at higher and lower risk of subsequent cardiovascular events. This prognostic information will be useful in clinical decisions regarding the intensity and duration of antiplatelet therapy. (Assessment of Individual Risk of Cardiovascular Events by Platelet FcγRIIa; NCT05175261)., Competing Interests: Funding Support and Author Disclosures This study was supported by Prolocor. Dr Schneider is named inventor on patents (U.S. 10,502,737, U.S. 11,747,335 B2) that propose the use of FcγRIIa for assaying platelet reactivity and treatment selection. Drs Schneider and DiBattiste are co-founders of Prolocor. Dr Angiolillo has received consulting fees or honoraria from Abbott, Amgen, AstraZeneca, Bayer, Biosensors, Boehringer Ingelheim, Bristol Myers Squibb, Chiesi, CSL Behring, Daiichi-Sankyo, Eli Lilly, Faraday, Haemonetics, Janssen, Merck, Novartis, Novo Nordisk, PhaseBio, PLx Pharma, Pfizer, Sanofi, and Vectura, outside of the present work; and his institution has received research grants from Amgen, AstraZeneca, Bayer, Biosensors, CeloNova, CSL Behring, Daiichi-Sankyo, Eisai, Eli Lilly, Faraday, Gilead, Janssen, Matsutani Chemical Industry Co, Merck, Novartis, Osprey Medical, Renal Guard Solutions, and the Scott R. MacKenzie Foundation. Dr Effron has received retirement income and equity from Eli Lilly. All other authors have no relationships relevant to the contents of this paper to disclose., (Copyright © 2024 American College of Cardiology Foundation. Published by Elsevier Inc. All rights reserved.)

Ethnopharmacological Relevance: Xuefu Zhuyu Decoction (XZD), a renowned traditional Chinese medicine prescription, is widely employed for the management of conditions characterized by qi-stagnation and blood stasis. Although its anti-thrombotic effect on deep vein thrombosis (DVT) patients has been clinically observed, the underlying mechanism remains largely unexplored., Aim of the Study: Our aim was to investigate the mechanisms by which XZD exerted its effect on DVT., Materials and Methods: The ultra performance liquid chromatography (UPLC) technique was employed to evaluate quality of XZD. To examine the effect of XZD on DVT, a DVT rat model with inferior vena cava (IVC) stenosis was established. The 4D-label-free proteomics approach was then utilized to uncover the possible mechanisms of XZD against DVT. Based on proteomics, citrullinated histone H3 (CitH3), along with serum levels of tumor necrosis factor-alpha (TNF-α) and interleukin-1 beta (IL-1β) were observed the inhibitory activity of XZD on neutrophil activation. Subsequently, the marker of platelet activation, specifically glycoprotein IIb (CD41) and glycoprotein IIIa (CD61), were assessed along with the secretion of von Willebrand factor (vWF) to investigate the inhibitory activity of XZD on platelet activation. Finally, we explored the impact of XZD on the sirtuin 1 (SIRT1)/nuclear factor kappa-B (NF-κB) pathway, which was associated with the activation of platelets and neutrophils., Results: Eight distinct components were identified for the quality control of XZD. XZD effectively reduced thrombus weight and length in DVT rats, without affecting the coagulation function or hematological parameters in the systemic circulation. Proteomics analysis revealed that XZD alleviated DVT by inhibiting the activation of platelets and neutrophils. The protein expression of CitH3, along with serum levels of TNF-α and IL-1β, were reduced in XZD-treated DVT rats. Similarly, protein expressions of CD41 and CD61, along with the release of vWF, were markedly down-regulated in XZD-treated DVT rats. Finally, treatment with XZD resulted in an up-regulation of SIRT1 protein expression and a down-regulation of both acetylated NF-κB/p65 and phosphorylated NF-κB/p65 protein expressions in endothelium., Conclusions: XZD alleviates DVT by inhibiting the activation of platelets and neutrophils at the injured endothelium via the regulation of SIRT1/NF-κB pathway., 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 B.V. All rights reserved.)

Introduction: An increasing number of patients attending coronary artery bypass grafting (CABG) receive preoperative antiplatelet drugs (acetylsalicylic acid, clopidogrel, prasugrel, ticagrelor). The optimal assessment of preoperative platelet function is the aim of this study for a shorter surgical timing in patients undergoing elective coronary artery bypass grafting., Methods: This study was performed on patients presenting for first-time isolated CABG on therapy with an P2Y12 receptor blockers loading dose (clopidogrel [300 mg] or prasugrel [60 mg] or ticagrelor [180 mg]) or P2Y12 receptor blockers maintenance therapy at least for five days (clopidogrel [75 mg once daily], prasugrel [10 mg once daily], ticagrelor [90 mg twice daily]). All patients received simultaneously acetylsalicylate acid (100 mg daily). Exclusion criterion was emergency CABG regardless of preoperative antiplatelet and anticoagulant therapy. All patients' data were recorded in an Excel® file and analyzed using RStudio® software., Results: Forty-eight consecutive adult patients presenting for CABG were enrolled. Preoperative thromboelastography-platelet mapping showed platelet resistance to P2Y12 blockers receptor - 25% for clopidogrel (6/24), 33% for ticagrelor (6/18), 33% for prasugrel (2/6), and this data was useful to obtain a shorter CABG waiting time in comparison with current guidelines (2.7 vs. five days for clopidogrel, 2.5 vs. five days for ticagrelor, 3.3 vs. seven days for prasugrel)., Conclusion: Preoperative thromboelastography-platelet mapping is helpful to detect harmful P2Y12 receptor blockers resistance and to minimize CABG waiting time avoiding unnecessary and life-threatening delays.

Platelets are critical mediators of hemostasis and thrombosis. Platelets circulate as discs in their resting form but change shape rapidly upon activation by vascular damage and/or soluble agonists such as thrombin. Platelet shape change is driven by a dynamic remodeling of the actin cytoskeleton. Actin filaments interact with the protein myosin, which is phosphorylated on the myosin light chain (MLC) upon platelet activation. Actin-myosin interactions trigger contraction of the actin cytoskeleton, which drives platelet spreading and contractile force generation. Filamin A (FLNA) is an actin cross-linking protein that stabilizes the attachment between subcortical actin filaments and the cell membrane. In addition, FLNA binds multiple proteins and serves as a critical intracellular signaling scaffold. Here, we used platelets from mice with a megakaryocyte/platelet-specific deletion of FLNA to investigate the role of FLNA in regulating platelet shape change. Relative to controls, FLNA-null platelets exhibited defects in stress fiber formation, contractile force generation, and MLC phosphorylation in response to thrombin stimulation. Blockade of Rho kinase (ROCK) and protein kinase C (PKC) with the inhibitors Y27632 and bisindolylmaleimide (BIM), respectively, also attenuated MLC phosphorylation; our data further indicate that ROCK and PKC promote MLC phosphorylation through independent pathways. Notably, the activity of both ROCK and PKC was diminished in the FLNA-deficient platelets. We conclude that FLNA regulates thrombin-induced MLC phosphorylation and platelet contraction, in a ROCK- and PKC-dependent manner., (© 2024 The Author(s).)

Increased platelet activity is a risk factor of thrombotic events in cardiovascular patients. We studied the relationship between platelet function, platelet size, and the content of reticulated platelets (RP) in patients with coronary heart disease (CHD, n = 55) and acute coronary syndrome (ACS, n = 95) receiving acetylsalicylic acid + clopidogrel or ticagrelor, respectively. The control group consisted of patients with risk factors for CHD, but with no CHD/ACS and free of antiplatelet drugs (n = 66). Platelet function was evaluated by the exposure of activated glycoprotein (GP) IIb-IIIa and P-selectin. In the control group, platelets were activated by TRAP (Thrombin Receptor Activating Peptide) 10 µM, and ADP 20, 5, 2.5 µM, and in the CHD/ACS groups, by TRAP 10 µM, and ADP 20 5 µM (±epinephrine 20 µM). Platelet size was assessed by the mean volume, % large forms, and forward scattering. RP were stained by thiazole orange. In the control group, activated GP IIb-IIIa and P-selectin correlated with platelet size and RP content after platelet activation by all agonists. Despite the decrease in platelet activity by antiplatelet drugs, most correlations (primarily for activated GP IIb-IIIa) were preserved in the CHD/ACS patients. In conclusion, increased platelet size and RP content are associated with increased platelet activity and the reduced efficacy of antiplatelet therapy.

Background: Genistein (Gen), a natural polyphenolic compound, has emerged as a promising candidate for lung cancer treatment. However, the potential clinical application of Gen is limited due to its poor solubility, low bioavailability, and toxic side effects. To address these challenges, a biomimetic delivery platform with cell membranes derived from natural cells as carrier material was constructed. This innovative approach aims to facilitate targeted drug delivery and solve the problem of biocompatibility of synthetic materials., Methods: First, the liposomes (LPs) loaded with Gen (LPs@Gen) was prepared using the ethanol injection method. Subsequently, PLTM-LPs@Gen was obtained through co-extrusion after mixing platelet membrane (PLTM) and LPs@Gen. Additionally, the biological and physicochemical properties of PLTM-LPs@Gen were investigated. Finally, the targeting ability, therapeutic efficacy, and safety of PLTM-LPs@Gen for lung cancer were evaluated using both a cell model and a tumor-bearing nude mouse model., Results: The optimal preparation ratio for LPs@Gen was Gen: soybean lecithin: cholesterol: DSPE-PEG2000 (3:30:5:10, mass ratio), while the ideal fusion ratio of LPs@Gen and PLTM was 1:1. The particle size of PLTM-LPs@Gen was 108.33 ± 1.06 nm, and the encapsulation efficiency and drug loading were 94.29% and 3.09% respectively. Gen was released continuously and slowly from PLTM-LPs@Gen. Moreover, PLTM-LPs@Gen exhibited good stability within one week. The results of in vitro cellular uptake and in vivo distribution experiments indicated that the carrier material, PLTM-LPs, has the immune escape ability and tumor targeting ability. Consequently, it showed better therapeutic effects than free drugs and traditional LPs in vitro and in vivo tumor models. In addition, safety experiments demonstrated that PLTM-LPs@Gen possesses good biocompatibility., Conclusion: Biomimetic nanomedicine provides a new strategy for the precision treatment of lung cancer in clinical practice., Competing Interests: The authors report no conflicts of interest in this work., (© 2024 Gao et al.)

Platelet-rich plasma (PRP) therapy holds promise for treating various clinical conditions. The activation process is crucial in releasing growth factors and cytokines from platelets, enhancing the therapeutic properties of PRP. Standard activation methods involve autologous thrombin or collagen, with variations in efficacy and growth factor release. This study explores the impact of acetylsalicylic acid (ASA), a commonly used antiplatelet drug, on PRP activation. The results indicate that non-activated PRP extracted from the whole blood of ASA-treated patients exhibits increased inflammatory cytokine concentrations, notably TNFa. After activation with autologous thrombin/CaCl 2 or collagen IV, the measured fluorescence intensities suggest varying release patterns between treated and non-treated groups. Understanding the influence of ASA on platelet activation holds implications for personalized medicine and optimizing outcomes for individual patients undergoing PRP therapy. This research sheds light on the potential challenges associated with using antiplatelet drugs, emphasizing the need for careful consideration in tailoring PRP-based regenerative therapies.

Point-of-care (POC) platelet function analysis can enable timely and precise management of bleeding and clotting in emergency rooms, operation rooms and intensive care units. However, POC platelet testing is currently not commonly performed, due to the complexity of sample preparation and limitations of existing technologies. Here, we report the development of an integrated microfluidic multiple electrode aggregometry (μMEA) sensor which uses multi-frequency impedance measurement of an embedded microelectrode array to perform platelet aggregometry directly from whole blood, sensing and measuring platelet activation in a label-free manner and without requiring any additional sample preparation. Additionally, the sensor incorporates blood flow during the assay to account for physiological flow and shear conditions. We show that the impedance signal from the sensor can be used to accurately detect and quantify platelet aggregation in a label-free manner, which was further validated by simultaneous fluorometric measurement and visualization of platelet aggregation. Further, we optimized the sensitivity and repeatability of the sensor using its frequency response and demonstrated that the sensor could be used to characterize drug dose-response in antiplatelet therapy with a frequency-tunable dynamic range. We also demonstrate that the sensor provides high sensitivity to perform platelet aggregometry under thrombocytopenic or low platelet count conditions. The μMEA sensor could thus enable POC platelet function analysis across several clinical applications.

Oxazolidinones (linezolid and tedizolid) adverse reactions include thrombocytopenia, the mechanism of which is still largely unknown. In cultured cells, oxazolidinones impair mitochondrial protein synthesis and oxidative metabolism. As mitochondrial activity is essential for megakaryocyte differentiation and maturation into platelets, we examined whether oxazolidinones impair these processes ex vivo and alter, in parallel, the activity of mitochondrial cytochrome c -oxidase (CYTOX; enzyme partly encoded by the mitochondrial genome) and cell morphology. Human CD34+ cells were isolated, incubated with cytokines (up to 14 days) and clinically relevant oxazolidinone concentrations or in control conditions, and used for (i) clonogenic assays [counting of megakaryocyte (CFU-Mk), granulocyte-monocyte (CFU-GM), burst-forming unit-erythroid (BFU-E) colonies]; (ii) the measure of the expression of megakaryocyte surface antigens (CD34 to CD41 and CD42); (iii) counting of proplatelets; (iv) the measurement of CYTOX activity; and (v) cell morphology (optic and electron microscopy). Oxazolidinones caused a significant decrease in BFU-E but not CFU-Mk or CFU-GM colonies. Yet, the megakaryocytic lineage was markedly affected, with a decreased differentiation of CD34+ into CD41+/CD42+ cells, an abolition of proplatelet formation and striking decrease in the numbers of large polylobulated nucleus megakaryocytes, with a complete loss of intracellular demarcation membrane system, disappearance of mitochondria, and suppression of CYTOX activity. These alterations were more marked in cells incubated with tedizolid than linezolid. These data suggest that oxazolidinones may induce thrombocytopenia by impairing megakaryocytic differentiation through mitochondrial dysfunction. Pharmacological interventions to prevent this toxicity might therefore be difficult as mitochondrial toxicity is most probably inherently linked to their antibacterial activity., Competing Interests: P.M.T. and F.V.B. have received speaker's and advisory boards' honoraria from Bayer and Merck (holders of marketing rights of tedizolid) and research grants from Trius Pharmaceuticals (now part of Merck) for preclinical studies of tedizolid. These companies were not involved in the design and performance of the studies presented here and did not take any part in their interpretation and/or decision to submit them to publication. The other authors have no conflict of interest to disclose in relation to the present work.

Abstract: Platelet factor XIII-A (FXIII-A) is a major cytoplasmic protein (∼3% of total), representing ∼50% of total circulating FXIII. However, mobilization of FXIII-A during platelet activation is not well defined. To determine mechanisms mediating the retention vs release of platelet FXIII-A, platelets from healthy humans and mice (F13a1-/-, Fga-/-, Plg-/-, Stim1fl/flPf4-Cre, and respective controls) were stimulated with thrombin, convulxin plus thrombin, or calcium ionophore (A23187), in the absence or presence of inhibitors of transglutaminase activity, messenger RNA (mRNA) translation, microtubule rearrangement, calpain, and Rho GTPase. Platelet releasates and pellets were separated by (ultra)centrifugation. FXIII-A was detected by immunoblotting and immunofluorescence microscopy. Even after strong dual agonist (convulxin plus thrombin) stimulation of human platelets, >80% platelet FXIII-A remained associated with the platelet pellet. In contrast, essentially all tissue factor pathway inhibitor, another cytoplasmic protein in platelets, was released to the supernatant. Pellet-associated FXIII-A was not due to de novo synthesis via platelet F13A1 mRNA. The proportion of platelet FXIII-A retained by vs released from activated platelets was partly dependent on STIM1 signaling, microtubule rearrangement, calpain, and RhoA activation but did not depend on the presence of fibrinogen or plasminogen. Immunofluorescence microscopy confirmed the presence of considerable FXIII-A within the activated platelets. Although released FXIII-A was cleaved to FXIII-A∗ and could be degraded by plasmin, platelet-associated FXIII-A remained uncleaved. Retention of substantial platelet-derived FXIII-A by activated platelets and its reduced susceptibility to thrombin- and plasmin-mediated proteolysis suggest platelet FXIII-A is a protected pool with biological role(s) that differs from plasma FXIII., (© 2024 by The American Society of Hematology. Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved.)

Platelet activation is closely related to thrombosis. Aspirin eugenol ester (AEE) is a novel medicinal compound synthesized by esterifying aspirin with eugenol using the pro-drug principle. Pharmacological and pharmacodynamic experiments showed that AEE has excellent anti-inflammatory, antioxidant, and inhibitory platelet activation effects, preventing thrombosis. However, the regulatory network and action target of AEE in inhibiting platelet activation remain unknown. This study aimed to investigate the effects of AEE on platelets of thrombosed rats to reveal its regulatory mechanism via a multi-omics approach. The platelet proteomic results showed that 348 DEPs were identified in the AEE group compared with the model group, of which 87 were up- and 261 down-regulated. The pathways in this result were different from previous results, including mTOR signaling and ADP signaling at P2Y purinoceptor 12. The metabolomics of heart and abdominal aortic tissue results showed that the differential metabolites were mainly involved in steroid biosynthesis, the citric acid cycle, phenylalanine metabolism, phenylalanine, tyrosine, and tryptophan biosynthesis, and glutathione metabolism. Molecular docking results showed that AEE had a better binding force to both the COX-1 and P2Y12 protein. AEE could effectively inhibit platelet activation by inhibiting COX-1 protein and P2Y12 protein activity, thereby inhibiting platelet aggregation. Therefore, AEE can have a positive effect on inhibiting platelet activation.

Venous thrombosis (VT) is a common vascular disease associated with reduced survival and a high recurrence rate. VT is initiated by the accumulation of platelets and neutrophils at sites of endothelial cell activation. A role for platelet αIIbβ3 in VT is not established, a task complicated by the increased bleeding risk caused by partial agonists such as tirofiban. Here, we show that m-tirofiban, a modified version of tirofiban, does not agonize αIIbβ3 based on lack of neoepitope expression and the cryo-EM structure of m-tirofiban/full-length αIIbβ3 complex. m-tirofiban abolishes agonist-induced platelet aggregation while preserving clot retraction ex vivo and, unlike tirofiban, it suppresses venous thrombogenesis in a mouse model without increasing bleeding. These findings establish a key role for αIIbβ3 in VT initiation and suggest that m-tirofiban and compounds with a similar structurally-defined mechanism of action merit consideration as potential thromboprophylaxis agents in patients at high risk for VT and hemorrhage., (© 2024. The Author(s).)

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.)

Antiplatelet and anticoagulant therapies are cornerstones of secondary prevention in high-risk cardiovascular patients. Whereas in former days the focus was set on effective antithrombotic effects, more recent trials and guidelines placed emphasis on a more balanced approach, thus including the bleeding risk for an individualized therapy. Type, strength, combination, and duration are important components to modify the individual bleeding risk. Novel antiplatelet and anticoagulant agents have shown promising results that might offer safer options in the future for high-risk cardiovascular patients. This review aims to give an overview about established drug target and pharmacologic approaches that are currently in the pipeline., Competing Interests: Disclosure None declared., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Background: We investigated whether the condition of the inner surface of hollow fibers affects the blood compatibility of hemodialyzers., Methods: We used scanning probe microscope/atomic force microscopy (SPM/AFM) to investigate the height of the swelling and flexible layers (thickness and softness) on the inner surfaces of the hollow fibers. Next, we tested the blood compatibility between dialyzers comprising a hollow fiber membrane, in which the other dialyzers, except for PVP, were additionally coated using PS membranes coated with other materials. After blood was injected into the dialyzer and plugged, dynamic stimulation was performed by slightly rotating the dialyzer for 4 h, although there was no blood circulation., Results: The vitamin E-coated polysulfone (PS) membrane showed a higher thickness and softness of the flexible layer than the asymmetric cellulose triacetate membrane without polyvinylpyrrolidone (PVP) and the PS membranes with PVP. We found that the dialyzer with vitamin E coating significantly suppressed the decrease in platelets, increase in β-TG, and increase in PF4 compared to those coated with NV polymer. Additionally, as the adsorbed protein on the inner surface, the total protein, fibronectin, and vWF levels were significantly lower in the vitamin E-coated dialyzer., Conclusion: The thickness and softness of the flexible layer of the inner surface of the hollow fiber membrane in vitro affect differences in blood coagulation performance in clinical research. Future clinical trials are required to confirm our results., Competing Interests: Declaration of conflicting interestsThe author(s) declared the following potential conflicts of interest with respect to the research, authorship, and/or publication of this article: Ryo Takatsuji, Masahide Koremoto, Yuko Saida, and Yoshihiro Hatanaka are employees of Asahi Kasei Medical Corporation. Yoko Fujimoto is an employee of Asahi Kasei Corporation.

Intracerebral hemorrhage (ICH) is a hemorrhagic disease with high mortality and disability rates. Curcumin is a promising drug for ICH treatment due to its multiple biological activities, but its application is limited by its poor watersolubility and instability. Herein, platelet membrane-coated curcumin polylactic-co-glycolic acid (PLGA) nanoparticles (PCNPs) are prepared to achieve significantly improved solubility, stability, and sustained release of curcumin. Fourier transform infrared spectra and X-ray diffraction assays indicate good encapsulation of curcumin within nanoparticles. Moreover, it is revealed for the first time that curcumin-loaded nanoparticles can not only suppress hemin-induced astrocyte proliferation but also induce astrocytes into neuron-like cells in vitro. PCNPs are used to treat rat ICH by tail vein injection, using in situ administration as control. The results show that PCNPs are more effective than curcumin-PLGA nanoparticles in concentrating on hemorrhagic lesions, inhibiting inflammation, suppressing astrogliosis, promoting neurogenesis, and improving motor functions. The treatment efficacy of intravenously administered PCNPs is comparable to that of in situ administration, indicating a good targeting effect of PCNPs on the hemorrhage site. This study provides a potent treatment for hemorrhagic injuries and a promising solution for efficient delivery of water-insoluble drugs using composite materials of macromolecules and cell membranes., (© 2024 Wiley‐VCH GmbH.)

Background and Purpose: Thromboxane A 2 (TXA 2 ) is a prostanoid produced during platelet activaton, important in enhancing platelet reactivity by activation of TP receptors. However, due to the short half-life, studying TXA 2 signalling is challenging. To enhance our understanding of TP receptor-mediated platelet biology, we therefore synthesised mono and difluorinated TXA 2 analogues and explored their pharmacology on heterologous and endogenously expressed TP receptor function., Experimental Approach: Platelet functional and signalling responses were studied using aggregometry, Ca 2+ mobilisation experiments and immunoblotting and compared with an analogue of the TXA 2 precursor prostaglandin H 2 , U46619. Gα q /Gα s receptor signalling was determined using a bioluminescence resonance energy transfer (BRET) assay in a cell line overexpression system., Key Results: BRET studies revealed that F-TXA 2 and F 2 -TXA 2 promoted receptor-stimulated TP receptor G-protein activation similarly to U46619. Unexpectedly, F 2 -TXA 2 caused reversible aggregation in platelets, whereas F-TXA 2 and U46619 induced sustained aggregation. Blocking the IP receptor switched F 2 -TXA 2 -mediated reversible aggregation into sustained aggregation. Further BRET studies confirmed F 2 -TXA 2 -mediated IP receptor activation. F 2 -TXA 2 rapidly and potently stimulated platelet TP receptor-mediated protein kinase C/P-pleckstrin, whereas IP-mediated protein kinase A/P-vasodilator-stimulated phosphoprotein was more delayed., Conclusion and Implications: F-TXA 2 is a close analogue to TXA 2 used as a selective tool for TP receptor platelet activation. In contrast, F 2 -TXA 2 acts on both TP and IP receptors differently over time, resulting in an initial wave of TP receptor-mediated platelet aggregation followed by IP receptor-induced reversibility of aggregation. This study reveals the potential difference in the temporal aspects of stimulatory and inhibitory pathways involved in platelet activation., (© 2024 The Author(s). British Journal of Pharmacology published by John Wiley & Sons Ltd on behalf of British Pharmacological Society.)

Gamma-terpinene (γ-TPN) is a cyclohexane monoterpene isolated from plant essential oils, such as tea tree (Melaleuca alternifolia), oregano (Origanum vulgare), rosemary (Rosmarinus officinalis L.), thyme (Thymus vulgaris Marchand), and eucalyptus (Eucalyptus sp.). Terpenes are widely studied molecules pharmacologically active on the cardiovascular system, hemostasis, and antioxidant actions. Herein, it was investigated the cytotoxic and antiplatelet activity of γ-TPN using different non-clinical laboratory models. For in silico evaluation, the PreADMET, SwissADME, and SwissTargetPrediction softwares were used. Molecular docking was performed using the AutoDockVina and BIOVIA Discovery Studio databases. The cytotoxicity of γ-TPN was analyzed by the MTT assay upon normal murine endothelial SVEC4-10 and fibroblast L-929 cells. Platelet aggregation was evaluated with platelet-rich (PRP) and platelet-poor (PPP) plasma from spontaneously hypertensive rats (SHR), in addition to SVEC4-10 cells pre-incubated with γ-TPN (50, 100, and 200 µM) for 24 h. SHR animals were pre-treated by gavage with γ-TPN for 7 days and divided into four groups (negative control, 25, 50, and 100 mg/kg). Blood samples were collected to measure nitrite using the Griess reagent. Gamma-TPN proved to be quite lipid-soluble (Log P = +4.50), with a qualified profile of similarity to the drug, good bioavailability, and adequate pharmacokinetics. It exhibited affinity mainly for the P2Y12 receptor (6.450 ± 0.232 Kcal/mol), moderate cytotoxicity for L-929 (CC 50 = 333.3 µM) and SVEC 4-10 (CC 50 = 366.7 µM) cells. The presence of γ-TPN in SVEC 4-10 cells was also able to reduce platelet aggregation by 51.57 and 44.20% at lower concentrations (50 and 100 µM, respectively). Then, γ-TPN has good affinity with purinergic receptors and an effect on the reversal of platelet aggregation and oxidative stress, being promising and safe for therapeutic targets and subsequent studies on the control of thromboembolic diseases., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Patient characteristics and platelet responses at romiplostim initiation according to the duration of immune thrombocytopenia (ITP) are poorly understood. Amongst romiplostim-exposed adults with ITP lasting ≥6 months during 2009-2018 in Denmark, Sweden, and Norway, we examined characteristics at romiplostim initiation, romiplostim dosage, and durable platelet response (≥75% of measurements ≥50 × 10 9 /L at 14-24 weeks) for subcohorts with newly diagnosed (duration <3 months), persistent (3-12 months), or chronic (>12 months) ITP initiating romiplostim. The 285 romiplostim initiators comprised 81 (28%) with newly diagnosed, 47 (16%) with persistent, and 157 (55%) with chronic ITP. More patients with newly diagnosed ITP than longer ITP duration, had low comorbidity levels, two or more prior ITP therapies, and previous bleeding requiring hospitalisation. The median romiplostim doses were similar across subcohorts. During treatment, median platelet counts were similar across subcohorts (75-76 × 10 9 /L), and the durable platelet response was 64.6%, 52.9%, and 52.7% for newly diagnosed, persistent, and chronic ITP, respectively. After treatment cessation, the median platelet count was 138 × 10 9 /L, 68 × 10 9 /L, and 71 × 10 9 /L, respectively. In conclusion, newly diagnosed patients, compared with romiplostim initiators with longer disease duration, had more severe ITP, higher frequency of durable platelet response, and higher median platelet count after cessation., (© 2024 The Author(s). British Journal of Haematology published by British Society for Haematology and John Wiley & Sons Ltd.)

Background and Aims: It is well known that elevated cholesterol is associated with enhanced platelet aggregation and patients suffering from familial hypercholesterolemia (FH) have a high risk of thrombotic cardiovascular events. Although decreasing cholesterol level is associated with attenuation of platelet hyperactivity, there are currently no data on the effect of convertase subtilisin/kexin type 9 monoclonal antibodies (PCSK9ab) on platelet reactivity in FH. The aim of the study was to analyse the impact of different therapies including PCSK9ab on platelet aggregation in FH., Methods: This study enrolled all 15 patients treated in the University Hospital Hradec Králové for FH. PCSK9ab have been administered in 12 of 15 patients while 8 patients were also undergoing lipid apheresis. Blood samples from all patients including pre- and post-apheresis period were tested for platelet aggregation triggered by 7 inducers, and the effect of 3 clinically used drugs (acetylsalicylic acid, ticagrelor and vorapaxar) was compared as well., Results: Although apheresis decreased the reactivity of platelets in general, platelet responses were not different between non-apheresis patients treated with PCSK9ab and apheresis patients (post-apheresis values) with the exception of ristocetin. However, when compared to age-matched healthy population, FH patients had significantly lower platelet aggregation responses to 4 out of 7 used inducers and higher profit from 2 out of 3 used antiplatelet drugs even after exclusion of FH patients regularly receiving conventional antiplatelet treatment., Conclusion: This study showed for the first time the suitability of PCSK9ab treatment for reduction of platelet reactivity in FH patients., (© 2023. The Author(s).)

Cardiac fibrosis is a prevalent pathological process observed in the progression of numerous cardiovascular diseases and is associated with an increased risk of sudden cardiac death. Although the BRD4 inhibitor JQ1 has powerful antifibrosis properties, its clinical application is extremely limited due to its side effects. There remains an unmet need for effective, safe, and low-cost treatments. Here, we present a multifunctional biomimetic nanoparticle drug delivery system (PM&EM nanoparticles) assembled by platelet membranes and erythrocyte membranes for targeted JQ1 delivery in treating cardiac fibrosis. The platelet membrane endows PM&EM nanoparticles with the ability to target cardiac myofibroblasts and collagen, while the participation of the erythrocyte membrane enhances the long-term circulation ability of the formulated nanoparticles. In addition, PM&EM nanoparticles can deliver sufficient JQ1 with controllable release, achieving excellent antifibrosis effects. Based on these advantages, it is demonstrated in both pressures overloaded induced mouse cardiac fibrosis model and MI-induced mouse cardiac fibrosis that injection of the fusion membrane biomimetic nanodrug carrier system effectively reduced fibroblast activation, collagen secretion, and improved cardiac fibrosis. Moreover, it significantly mitigated the toxic and side effects of long-term JQ1 treatment on the liver, kidney, and intestinal tract. Mechanically, bioinformatics prediction and experimental validation revealed that PM&EM/JQ1 NPs reduced liver and kidney damage via alleviated oxidative stress and mitigated cardiac fibrosis via the activation of oxidative phosphorylation activation. These results highlight the potential value of integrating native platelet and erythrocyte membranes as a multifunctional biomimetic drug delivery system for treating cardiac fibrosis and preventing drug side effects.

Purpose: Asians often face the problems of clopidogrel resistance and East Asian paradox. This study aimed to evaluate the effects of P2Y 12 inhibitors, including low-dose prasugrel 2.5 mg, on the P2Y 12 reaction unit (PRU) in the chronic phase after percutaneous coronary intervention (PCI)., Methods: A total of 348 patients were studied. PRU was measured 6-12 months after PCI and subsequently, 6 months later using a P2Y 12 assay, respectively. This study evaluated the proportion of bleeding risk (PRU ≤ 85) and ischemic risk (PRU ≥ 239) as primary endpoints, and the prediction of bleeding risk and ischemic risk using multivariable logistic regression analysis., Results: At baseline, 136 patients (39%) received prasugrel 3.75 mg, 48 patients (14%) received prasugrel 2.5 mg, and 164 patients (47%) received clopidogrel 75 mg. Clopidogrel 75 mg had a significantly higher proportion of ischemic risk within one year after PCI than the other groups, and was an independent predictor for ischemic risk with reference of prasugrel 3.75 mg. In addition, switching from clopidogrel 75 mg to prasugrel 2.5 mg significantly lowered and aggregated the PRU value. Whereas, dose reduction of prasugrel had a significantly lower proportion of bleeding risk over one year after PCI than the continuation of prasugrel 3.75 mg, and was an independent predictor for bleeding risk with reference of continuation of prasugrel 3.75 mg., Conclusions: Prasugrel 2.5 mg has a lower ischemic risk and a more stable PRU value compared with clopidogrel treatment. Prasugrel also contributes to a decline in bleeding risk with concomitant dose reduction., Trial Registration: University Hospital Medical Information Network (UMIN), ID: UMIN000029541, Date: October 16, 2017 ( https://center6.umin.ac.jp/cgi-open-bin/ctr&#95;e/ctr&#95;view.cgi?recptno=R000033395 )., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Objective: To analyze the expression of platelet membrane glycoprotein sialylation in primary immune thrombocytopenia and provide a reference for clinical diagnosis and treatment of the disease., Methods: 100 children with primary immune thrombocytopenia diagnosed and treated in the Children's Hospital of Yunhe District Central Hospital of Cangzhou City, Hebei Province from January 2020 to June 2022 were included in the study group. All children were treated with dexamethasone (DXMS) shock therapy; Another 20 healthy children who underwent routine physical examination at the same time were selected and included in the control group. The study measured the platelet membrane surface in plasma using flow cytometry in two groups: the comparative study group and the control group. The study measured the positive rates of α 2,3-sialic acid (α 2, 3-sa) and α 2,6-sialic acid (α 2, 6-sa) in both groups. The study also measured the positive rates of α 2,3-sialic acid (α 2, 3-sa) and α 2,6-sialic acid (α 2, 6-sa) before and after treatment in the comparative study group. At the same time, an enzyme-linked immunosorbent assay was used to determine the content of sialic acid and sialidase activity and content in the serum. The detection values of α2, 3-SA, and α2, 6-SA expression of children in the study group and the control group were compared, and the detection values of α2, 3-SA, and α2, 6-SA expression of children in the study group before and after treatment were compared., Results: There was no significant difference in the positive rate of α 2, 3-sa between the study group and the control group (t=0.852, P > .05); Study Group: The positive rate of α 2,6-sa was significantly lower than that of the control group (P < .05). In the study group, there was no significant difference in the positive rate of α 2,3-sa before and after treatment (P > .05). However, after treatment, the positive rate of α 2,6-sa was significantly higher than before (P < .05). The study found that the children in the study group had significantly higher levels of serum sialic acid content, sialidase activity, and content than those in the control group (P < .05). After treatment, the children in the study group showed a decrease in serum sialic acid content, sialidase activity, and content, which was statistically significant (P < .05) compared to before treatment., Conclusion: The sialylation of platelet membrane glycoprotein is abnormally expressed in primary immune thrombocytopenia. The sialylation of platelet membrane glycoprotein may be involved in the occurrence and development of the disease. It is of great practical significance to diagnose and evaluate the therapeutic effect of the disease by detecting the sialylation of platelet membrane glycoprotein.

Circulating lipoproteins may interact with platelets, increasing platelet sensitivity to aggregating agonists and their tendency towards activation and thrombus formation. In particular, patients with hypercholesterolemia exhibit a higher degree of platelet reactivity compared to normolipidemic. Moreover, accruing evidence report that lipid-lowering therapies can reduce thrombus formation, particularly in the absence of concomitant antiplatelet therapy. However, the underlying biological mechanism(s) explaining these clinical observations are not completely understood. Baseline platelet reactivity and high on-treatment platelet reactivity while on antiplatelet therapy (e.g., aspirin and clopidogrel) are associated with poor clinical outcomes. Therefore, strategies to reduce baseline platelet reactivity or improve the pharmacodynamic profile of antiplatelet therapies are an unmet clinical need. The potential use of lipid-lowering therapies for optimizing platelet reactivity provides several advantages as there is strong evidence that reducing circulating lipoproteins can improve clinical outcomes, and they may avoid the need for potent antiplatelet therapies that, although more effective, are associated with increased bleeding risk. This review will provide a systematic overview of the effects of lipid-lowering therapy on platelet reactivity in patients treated with and without antiplatelet therapy. We will focus on the potential biological mechanism(s) of action and the effect of statins, ezetimibe, proprotein convertase subtilisin/kexin 9 inhibitors, omega-3 fatty acids, and recombinant high-density lipoprotein on platelet reactivity. Ultimately, we will assess the current gaps in the literature and future perspective in the field.

Valemetostat is an EZH2/1 inhibitor that has been approved in Japan for the treatment of patients with relapsed/refractory adult T-cell leukemia/lymphoma, based mainly on results from a single-arm phase II trial. It is currently under investigation worldwide for the treatment of other non-Hodgkin lymphomas (NHLs), including peripheral T-cell lymphoma, and for solid tumors. Semi-mechanistic population pharmacokinetic modeling of total and unbound valemetostat and an analysis of the platelet time course during treatment with valemetostat were conducted using data from five clinical trials (two in patients with NHL and three in healthy volunteers). Pharmacokinetic data, including 3162 total/1871 unbound valemetostat observations from 102 patients and 72 healthy volunteers, were described by a three-compartment model with sequential zero-/first-order absorption and saturable binding in the central compartment. Alpha-1-acid glycoprotein (AAG) was the most influential covariate for total valemetostat exposure, yet had little impact on unbound exposure, meaning no dose adjustment was warranted based on AAG levels. The longitudinal platelet data from 101 patients (2313 observations) were adequately described by a modified Friberg model with two proliferation compartments, which characterized unique spontaneous recovery of platelet counts without dose modifications. A model-based simulation quantitatively assessed the proposed dose-adjustment guidance in case of platelet count decreased by comparing the probability of treatment discontinuation due to platelet count decreased with or without the dose adjustment. In summary, the models described observed total and unbound valemetostat concentrations and a unique time course of platelets during treatment, which can justify the clinical dose and provide dose-adjustment guidance., (© 2024 The Author(s). CPT: Pharmacometrics & Systems Pharmacology published by Wiley Periodicals LLC on behalf of American Society for Clinical Pharmacology and Therapeutics.)

Sweet potatoes are rich in cardioprotective phytochemicals with potential anti-platelet aggregation activity, although this benefit may vary among cultivars/genotypes. The phenolic profile [HPLC-ESI (-) -qTOF-MS 2 ], cheminformatics (ADMET properties, affinity toward platelet proteins) and anti-PA activity of phenolic-rich hydroalcoholic extracts obtained from orange (OSP) and purple (PSP) sweet potato storage roots, was evaluated. The phenolic richness [Hydroxycinnamic acids> flavonoids> benzoic acids] was PSP > OSP. Their main chlorogenic acids could interact with platelet proteins (integrins/adhesins, kinases/metalloenzymes) but their bioavailability could be poor. Just OSP exhibited a dose-dependent anti-platelet aggregation activity [inductor (IC 50, mg.ml -1 ): thrombin receptor activator peptide-6 (0.55) > Adenosine-5'-diphosphate (1.02) > collagen (1.56)] and reduced P-selectin expression (0.75-1.0 mg.ml -1 ) but not glycoprotein IIb/IIIa secretion. The explored anti-PA activity of OSP/PSP seems to be inversely related to their phenolic richness. The poor first-pass bioavailability of its chlorogenic acids (documented in silico) may represent a further obstacle for their anti-PA in vivo., 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.)

Hypertension causes platelet activation and adhesion in the brain resulting in glial activation and neuroinflammation. Further, activation of Angiotensin-Converting Enzyme 2/Angiotensin (1-7)/Mas Receptor (ACE2/Ang (1-7)/MasR) axis of central Renin-Angiotensin System (RAS), is known to reduce glial activation and neuroinflammation, thereby exhibiting anti-hypertensive and anti-neuroinflammatory properties. Therefore, in the present study, the role of ACE2/Ang (1-7)/MasR axis was studied on platelet-induced glial activation and neuroinflammation using Diminazene Aceturate (DIZE), an ACE2 activator, in astrocytes and microglial cells as well as in rat model of hypertension. We found that the ACE2 activator DIZE, independently of its BP-lowering properties, efficiently prevented hypertension-induced glial activation, neuroinflammation, and platelet CD40-CD40L signaling via upregulation of ACE2/Ang (1-7)/MasR axis. Further, DIZE decreased platelet deposition in the brain by reducing the expression of adhesion molecules on the brain endothelium. Activation of ACE2 also reduced hypertension-induced endothelial dysfunction by increasing eNOS bioavailability. Interestingly, platelets isolated from hypertensive rats or activated with ADP had significantly increased sCD40L levels and induced significantly more glial activation than platelets from DIZE treated group. Therefore, injection of DIZE pre-treated ADP-activated platelets into normotensive rats strongly reduced glial activation compared to ADP-treated platelets. Moreover, CD40L-induced glial activation, CD40 expression, and NFкB-NLRP3 inflammatory signaling are reversed by DIZE. Furthermore, the beneficial effects of ACE2 activation, DIZE was found to be significantly blocked by MLN4760 (ACE2 inhibitor) as well as A779 (MasR antagonist) treatments. Hence, our study demonstrated that ACE2 activation reduced the platelet CD40-CD40L induced glial activation and neuroinflammation, hence imparted neuroprotection., 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 B.V. All rights reserved.)

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.

Blood platelets are produced by megakaryocytes (MKs), their parent cells, which are in the bone marrow. Once mature, MK pierces through the sinusoid vessel, and the initial protrusion further elongates as proplatelet or buds to release platelets. The mechanisms controlling the decision to initiate proplatelet and platelet formation are unknown. Here, we show that the mechanical properties of the microenvironment prevent proplatelet and platelet release in the marrow stroma while allowing this process in the bloodstream. Loss of marrow confinement following myelosuppression led to inappropriate proplatelet and platelet release into the extravascular space. We further used an inert viscoelastic hydrogel to evaluate the impact of compressive stress. Transcriptional analysis showed that culture in three-dimensional gel induced upregulation of genes related to the Rho-GTPase pathway. We found higher Rho-GTPase activation, myosin light chain phosphorylation and F-actin under mechanical constraints while proplatelet formation was inhibited. The use of latrunculin-A to decrease F-actin promoted microtubule-dependent budding and proplatelet extension inside the gel. Additionally, ex vivo exposure of intact bone marrow to latrunculin-A triggered proplatelet extensions in the interstitial space. In vivo, this confinement-mediated high intracellular tension is responsible for the formation of the peripheral zone, a unique actin-rich structure. Cytoskeleton reorganization induces the disappearance of the peripheral zone upon reaching a liquid milieu to facilitate proplatelet and platelet formation. Hence, our data provide insight into the mechanisms preventing ectopic platelet release in the marrow stroma. Identifying such pathways is especially important for understanding pathologies altering marrow mechanics such as chemotherapy or myelofibrosis., Competing Interests: Competing interests statement:The authors declare no competing interest.

Oxylipins are important low abundant signaling molecules in living organisms. In platelets they play a primary role in platelet activation and aggregation in the course of thrombotic events. In vivo, they are enzymatically synthesized by cyclooxygenases, lipoxygenases, or cytochrome P450 isoenzmes, resulting in diverse polyunsaturated fatty acid (FA) metabolites including hydroxy-, epoxy-, oxo-FAs, and endoperoxides with pro-thrombotic or anti-thrombotic effects. In a recent study, it was reported that hemin induces platelet death which was accompanied by enhanced reactive oxygen species (ROS) production (measured by flow cytometry) and lipid peroxidation (as determined by proxy using flow cytometry with BODIPY-C11 as sensor). Lipidomic studies further indicated significant changes of the platelet lipidome upon ex vivo hemin treatment, amongst others oxylipins were increased. The effect could be (at least partly) reversed by riociguat/diethylamine NONOate diethylammonium salt (DEA/NO) which modulates the soluble guanylate cyclase(sGC)-cGMP-cGMP-dependent protein kinase I(cGKI) signaling axis. In the original work, oxylipins were measured by a non-enantioselective UHPLC-tandem-MS assay which may not give the full picture whether oxylipin elevation is due to ROS or by enzymatic processes. We present here the study of the stereochemical disposition of hemin-induced platelet lipidome alterations using Chiralpak IA-U column with amylose tris(3,5-dimethylphenylcarbamate) chiral selector immobilized on 1.6 µm silica particles. It was found that the major platelet oxylipins 12-HETE, 12-HEPE and 14-HDoHE (from 12-LOX) and 12-HHT (from COX-1) were present in S-configuration indicating their enzymatic formation. On the other hand, both R and S enantiomers of 9- and 13-HODE, 11- and 15-HETE were detected, possibly due to enzyme promiscuity rather than non-specific oxidation (by ROS or autoxidation), as confirmed by multi-loop based two-dimensional LC-MS using selective comprehensive mode with achiral RPLC in the 1st dimension and chiral LC in the 2nd using a multiple heart-cutting interface. For 12-HETrE, a peak at the retention time of the R-enantiomer was ruled out as isobaric interference by 2D-LC-MS. In particular, arachidonic acid derivates 12(S)-HHT, 11(R)-HETE and 15(S)-HETE were found to be sensitive to hemin and cGMP modulation., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Michael Lämmerhofer has received funding by Agilent Technologies. The authors declare no other competing financial interests, (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Ethnopharmacological Relevance: Microthrombosis is commonly seen in sepsis and COVID-19. Zixue Powder (ZXP) is a traditional Chinese herbal formula with the potential to treat microvascular and infectious diseases. However, the role and mechanism of ZXP in sepsis-associated thrombosis remain unclear., Aim of the Study: Investigating the therapeutic effectiveness and underlying mechanisms of ZXP in septic thrombosis., Materials and Methods: ZXP's compositions were examined with UPLC-QTOF-MS. The efficacy of ZXP on sepsis-induced thrombosis was assessed through various methods: liver tissue pathology was examined using hematoxylin-eosin staining, platelet count was determined by a blood cell analyzer, and an enzyme-linked immunosorbent assay (ELISA) was used to detect the levels of serum tissue factor (TF), thromboxane B2 (TXB2), D-Dimer, and plasminogen activator inhibitor-1 (PAI-1). Neutrophil extracellular traps (NETs) were localized and expressed in liver tissues by immunofluorescence, and the number of NETs in peripheral blood was evaluated by ELISA, which measured the quantity of cf-DNA and MPO-DNA in serum. Platelet P-selectin expression and platelet-neutrophil aggregation were measured by flow cytometry, and plasma P-selectin expression was measured by ELISA. Furthermore, the mechanism of the stimulator of interferon genes (STING) signaling pathway in ZXP's anti-sepsis thrombosis effect was investigated using the STING agonist, Western blot experiments, and immunoprecipitation experiments., Results: UPLC-QTOF-MS identified 40 chemical compositions of ZXP. Administration of ZXP resulted in significant improvements in liver thrombosis, platelet counts, and levels of TXB2, TF, PAI-1, and D-Dimer in septic rats. Moreover, ZXP inhibited NETs formation in both liver tissue and peripheral blood. Additionally, ZXP decreased the levels of P-selectin in both platelets and plasma, as well as the formation of platelet-neutrophil aggregates, thereby suppressing P-selectin-mediated NETs release. Immunoprecipitation and immunofluorescence staining experiments revealed that ZXP attenuated P-selectin secretion by inhibiting STING-mediated assembly of platelet soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) complex, ultimately preventing inhibition of NETs formation., Conclusion: Our study showed that ZXP effectively mitigates platelet granule secretion primarily through modulation of the STING pathway, consequently impeding NET-associated thrombosis in sepsis. These findings offer valuable insights for future research on the development and application of ZXP., Competing Interests: Declaration of competing interest The author announced that they have no interest conflicts for this work., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Sepsis is a severe inflammatory disease characterized by cytokine storm, often accompanied by disseminated intravascular coagulation (DIC). PANoptosis is a novel form of cell death triggered by cytokine storms, characterized by a cascade reaction of pyroptosis, apoptosis, and necroptosis. It exists in septic platelets and is closely associated with the onset and progression of DIC. However, there remains an unmet need for drugs targeting PANoptosis. The anti-PANoptosis effect of myricetin was predicted using network pharmacology and confirmed through molecular docking. In vitro platelet activation models demonstrated that myricetin significantly attenuated platelet particle release, integrin activation, adhesion, spreading, clot retraction, and aggregation. Moreover, in a sepsis model, myricetin reduced inflammatory infiltration in lung tissue and platelet activation while improving DIC. Additionally, whole blood sequencing samples from sepsis patients and healthy individuals were analyzed to elucidate the up-regulation of the PANoptosis targets. Our findings demonstrate the inhibitory effect of myricetin on septic platelet PANoptosis, indicating its potential as a novel anti-cellular PANoptosis candidate and therapeutic agent for septic DIC. Furthermore, our study establishes a foundation for utilizing network pharmacology in the discovery of new drugs to treat various diseases., 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. Published by Elsevier Inc.)