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Platelets play a substantial role in cardiovascular disease, and for many years there has been a search for dietary componentsthat are able to inhibit platelet function and therefore decrease the risk of cardiovasculardisease. Platelets can be inhibited by alcohol, dietary fats and some antioxidants, including agroup of compounds, the polyphenols, found in fruits and vegetables. A number of these compounds have been shown to inhibit platelet function both in vitro and in vivo. In the present study the effects of the hydroxycinnamates and the flavonoid quercetin on platelet activation and cell signalling in vitro were investigated. The hydroxycinnamates inhibited platelet function, although not at levels that can be achieved in human plasma by dietary intervention. However, quercetin inhibited platelet aggregation at levels lower than those previously reported. Quercetin was also found to inhibit intracellular Ca mobilisation and whole-cell tyrosine protein phosphorylation in platelets, which are both processes essential for platelet activation. The effect of polyphenols on platelet aggregation in vivo was also investigated. Twenty subjects followed a low-polyphenol diet for 3 d before and also during supplementation. All subjects were supplemented with a polyphenol-rich meal every lunchtime for 5d. Platelet aggregation and plasma flavonols were measured at baseline and after 5d of dietary supplementation. Total plasma flavonoids increased significantly after the dietary intervention period (P = 0.001). However, no significant changes in ex vivo platelet aggregation were observed. Further investigation of the effects of individual polyphenolic compoundson platelet function, both in vitro and in vivo, is required in order to elucidate their role in the relationship between diet and the risk of cardiovascular disease. [ABSTRACT FROM AUTHOR]

Background: Extracellular vesicles (EVs) are proposed to play a role in the development of cardiovascular diseases (CVDs) and are considered emerging markers of CVDs. n-3 PUFAs are abundant in oily fish and fish oil and are reported to reduce CVD risk, but there has been little research to date examining the effects of n-3 PUFAs on the generation and function of EVs., Objectives: We aimed to investigate the effects of fish oil supplementation on the number, generation, and function of EVs in subjects with moderate risk of CVDs., Methods: A total of 40 participants with moderate risk of CVDs were supplemented with capsules containing either fish oil (1.9 g/d n-3 PUFAs) or control oil (high-oleic safflower oil) for 12 wk in a randomized, double-blind, placebo-controlled crossover intervention study. The effects of fish oil supplementation on conventional CVD and thrombogenic risk markers were measured, along with the number and fatty acid composition of circulating and platelet-derived EVs (PDEVs). PDEV proteome profiles were evaluated, and their impact on coagulation was assessed using assays including fibrin clot formation, thrombin generation, fibrinolysis, and ex vivo thrombus formation., Results: n-3 PUFAs decreased the numbers of circulating EVs by 27%, doubled their n-3 PUFA content, and reduced their capacity to support thrombin generation by >20% in subjects at moderate risk of CVDs. EVs derived from n-3 PUFA-enriched platelets in vitro also resulted in lower thrombin generation, but did not alter thrombus formation in a whole blood ex vivo assay., Conclusions: Dietary n-3 PUFAs alter the number, composition, and function of EVs, reducing their coagulatory activity. This study provides clear evidence that EVs support thrombin generation and that this EV-dependent thrombin generation is reduced by n-3 PUFAs, which has implications for prevention and treatment of thrombosis., Clinical Trial Registry: This trial was registered at clinicaltrials.gov as NCT03203512., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

The endothelial regulation of platelet activity is incompletely understood. Here we describe novel approaches to find molecular pathways implicated on the platelet-endothelium interaction. Using high-shear whole-blood microfluidics, employing coagulant or non-coagulant conditions at physiological temperature, we observed that the presence of human umbilical vein endothelial cells (HUVEC) strongly suppressed platelet adhesion and activation, via the collagen receptor glycoprotein VI (GPVI) and the PAR receptors for thrombin. Real-time monitoring of the cytosolic Ca 2+ rises in the platelets indicated no major improvement of inhibition by prostacyclin or nitric oxide. Similarly under stasis, exposure of isolated platelets to HUVEC reduced the Ca 2+ responses by collagen-related peptide (CRP-XL, GPVI agonist) and thrombin (PAR agonist). We then analyzed the label-free phosphoproteome of platelets (three donors), exposed to HUVEC, CRP-XL, and/or thrombin. High-resolution mass spectrometry gave 5463 phosphopeptides, corresponding to 1472 proteins, with good correlation between biological and technical replicates (R > .86). Stringent filtering steps revealed 26 regulatory pathways (Reactome) and 143 regulated kinase substrates (PhosphoSitePlus), giving a set of protein phosphorylation sites that was differentially (44) or similarly (110) regulated by HUVEC or agonist exposure. The differential regulation was confirmed by stable-isotope analysis of platelets from two additional donors. Substrate analysis indicated major roles of poorly studied protein kinase classes (MAPK, CDK, DYRK, STK, PKC members). Collectively, these results reveal a resetting of the protein phosphorylation profile in platelets exposed to endothelium or to conventional agonists and to endothelium-promoted activity of a multi-kinase network, beyond classical prostacyclin and nitric oxide actors, that may contribute to platelet inhibition., (© 2024 The Authors. The FASEB Journal published by Wiley Periodicals LLC on behalf of Federation of American Societies for Experimental Biology.)

Background: Inositol 1,3,4,5-tetrakisphosphate (IP 4 ) is formed from inositol 1,4,5-trisphosphate (IP 3 ) by IP 3 3-kinase (ITPK) in most cells. Its function is unknown but has been suggested to be involved in Ca 2+ entry, IP 3 regulation, and phosphoinositide 3-kinase antagonism., Objectives: To better elucidate a function for IP 4 , we tested a specific inhibitor of ITPK (GNF362) on platelets, the effects of IP 4 directly in permeabilized platelets and its effect on phosphatidylinositol 3,4,5-trisphosphate (PIP 3 ) binding to pleckstrin-homology (PH) domain-containing proteins in platelets., Methods: Human platelets were utilized in whole blood for thrombus formation, in platelet-rich plasma and washed suspensions for aggregation, and for Ca 2+ studies, or resuspended in high K + and low Na + buffers for permeabilization experiments. Phosphorylation of AKT-Ser 473 and Rap1-GTP formation were measured by Western blotting and PIP 3 binding using PIP 3 beads., Results: GNF362-enhanced platelet aggregation stimulated by low concentrations of ADP, collagen, thrombin, U46619, and thrombus formation in collagen-coated capillaries. GNF362 induced a transient elevation of Ca 2+ concentration, elevated basal levels of IP 3 , and enhanced the peak height of Ca 2+ elevated by agonists. In permeabilized platelets, IP 4 inhibited GTPγS induced formation of AKT-Ser 473 phosphorylation and platelet aggregation. IP 4 reduced GTPγS-stimulated Rap1-GTP levels and potently reduced extraction of RASA3 and BTK by PIP 3 beads., Conclusion: ITPK and IP 4 are negative regulators of platelet function. IP 4 regulation of PH domain-containing proteins may represent a pathway by which platelet activation may be controlled during thrombosis., (© 2024 The Author(s).)

Multiple myeloma (MM) and its precursor states, smoldering myeloma (SM) and monoclonal gammopathy of undetermined significance (MGUS) are associated with increased incidence of thrombosis, however the cause of this is unknown. Lenalidomide treatment of MM substantially improves patient survival, although significantly increases thrombotic risk by an unknown mechanism. This pilot study aimed to establish the impact of MM and its treatment with Lenalidomide on platelet function. We analyzed platelet function in MGUS, SM and MM compared to healthy controls. We report an increase in platelet reactivity in MGUS, SM, and MM where increases in fibrinogen binding, P-selectin exposure, altered receptor expression, elevated levels of aggregation and enhanced sensitivity to agonist stimulation were observed. We also demonstrate an increase in patient platelet reactivity post Lenalidomide treatment compared to pre-treatment. We show Lenalidomide treatment of platelets ex vivo increased reactivity that was associated with formation of larger thrombi at arterial shear rates but not venous shear rates. This study demonstrates a clear increase in platelet reactivity and prothrombotic potential in patients with MGUS, SM and MM which is elevated further upon treatment with Lenalidomide. Our observations suggest that more detailed studies are warranted to determine mechanisms of thrombotic complications to enable the development of new preventative strategies that specifically target platelets.

Previously, we and others have shown that SARS-CoV-2 spike-specific IgG antibodies play a major role in disease severity in COVID-19 by triggering macrophage hyperactivation, disrupting endothelial barrier integrity, and inducing thrombus formation. This hyperinflammation is dependent on high levels of anti-spike IgG with aberrant Fc tail glycosylation, leading to Fcγ receptor hyperactivation. For development of immune-regulatory therapeutics, drug specificity is crucial to counteract excessive inflammation whereas simultaneously minimizing the inhibition of antiviral immunity. We here developed an in vitro activation assay to screen for small molecule drugs that specifically counteract antibody-induced pathology. We identified that anti-spike-induced inflammation is specifically blocked by small molecule inhibitors against SYK and PI3K. We identified SYK inhibitor entospletinib as the most promising candidate drug, which also counteracted anti-spike-induced endothelial dysfunction and thrombus formation. Moreover, entospletinib blocked inflammation by different SARS-CoV-2 variants of concern. Combined, these data identify entospletinib as a promising treatment for severe COVID-19., (© 2023 Geyer et al.)

Proteoglycans form a heterogeneous family of proteins with covalently bound sulfated glycosaminoglycans. The extracellular matrix proteoglycan perlecan has been proposed to bind to the platelet- and megakaryocyte-specific receptor G6bB, co-regulating platelet glycoprotein VI (GPVI) signaling. The derived non-sulfate proteoglycan endorepellin was previously shown to enhance platelet adhesion via the collagen receptor, integrin α2β1. Here, we compared the roles of perlecan and other matrix proteoglycans in platelet responses and thrombus formation. We used multi-color flow cytometry to measure the degranulation and integrin αIIbβ3 activation of washed platelets in response to various proteoglycans and collagen-related peptide (CRP), the GPVI agonist. Perlecan, but not endorepellin, enhanced the CRP-induced activation of platelets in a time- and concentration-dependent manner. Similar to collagen, immobilized perlecan, but not other proteoglycans, supported static platelet adhesion and spreading. In-flowed whole-blood perlecan diminished shear-dependent platelet adhesion, while it enforced GPVI-dependent thrombus formation-to a larger extent than endorepellin-to induce more contracted aggregates of activated platelets. We concluded that the sulfated proteoglycan perlecan enhances GPVI-dependent platelet responses extending to thrombus formation, but it does so at the expense of reduced adhesion of platelets under flow.

Background: Heat shock protein 47 (HSP47) is an intracellular chaperone protein with an indispensable role in collagen biosynthesis in collagen-secreting cells. This chaperone has also been shown to be released and present on the surface of platelets. The inhibition of HSP47 in human platelets or its ablation in mouse platelets reduces platelet function in response to collagen and the glycoprotein (GP) VI collagen receptor agonist CRP-XL., Objectives: In this study, we sought, through experiments, to explore cellular distribution, trafficking, and influence on GPVI interactions to understand how HSP47 modulates collagen receptor signaling., Methods: HSP47-deficient mouse platelets and SMIH- treated human platelets were used to study the role of HSP47 in collagen mediated responses and signaling., Results: Using subcellular fractionation analysis and immunofluorescence microscopy, HSP47 was found to be localized to the platelet-dense tubular system. Following platelet stimulation, HSP47 mobilization to the cell surface was shown to be dependent on actin polymerization, a feature common to other dense tubular system resident platelet proteins that are released to the cell surface during activation. In this location, HSP47 was found to contribute to platelet adhesion to collagen or CRP-XL but not to GFOGER peptide (an integrin α2β1-binding sequence within collagens), indicating selective effects of HSP47 on GPVI function. Dimerization of GPVI on the platelet surface increases its affinity for collagen. GPVI dimerization was reduced following HSP47 inhibition, as was collagen and CRP-XL-mediated signaling., Conclusion: The present study identifies a role for cell surface-localized HSP47 in modulating platelet responses to collagen through dimerization of GPVI, thereby enhancing platelet signaling and activation., (© 2023 The Authors.)

Background: The response of platelets to activating stimuli and pharmaceutical agents varies greatly within the normal population. Current platelet function tests are used to measure end-point levels of platelet activation without taking the speed at which platelets activate into account, potentially missing vital metrics to characterize platelet reactivity., Objectives: To identify variability, to agonists and among individuals, in platelet activation kinetics and assess the impact of this on thrombus formation., Methods: We have developed a bespoke real-time flow cytometry assay and analysis package to measure the rate of platelet activation over time using 2 parameters of platelet activation, fibrinogen binding and P-selectin exposure., Results: The rate of platelet activation varied considerably within the normal population but did not correlate with maximal platelet activation, demonstrating that platelet activation rate is a separate and novel metric to describe platelet reactivity. The relative rate of platelet response between agonists was strongly correlated, suggesting that a central control mechanism regulates the rate of platelet response to all agonists., Conclusion: For the first time, we have shown that platelet response rate corresponds to thrombus size and structure, wherein faster responders form larger, more densely packed thrombi at arterial, but crucially not venous, shear. We have demonstrated that the rate of platelet activation is an important metric in stratifying individual platelet responses and will provide a novel focus for the design and development of antiplatelet therapy, targeting high-shear thrombosis without exacerbating bleeding at low shear., Competing Interests: Funding information British Heart Foundation; Grants: PG/16/36/31967 (C.I.J. and J.M.G.) RG/20/7/34866 and RG/15/2/31224 (J.M.G and C.I.J.). European Union's Horizon 2020 research and innovation program under the Marie Sklodowska-Curie grant agreement No. 766118 (IDS). Declaration of competing interests There are no competing interests to disclose., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

Zinc (Zn 2+ ) is released by platelets during a hemostatic response to injury. Extracellular zinc ([Zn 2+ ] o ) initiates platelet activation following influx into the platelet cytosol. However, the mechanisms that permit Zn 2+ influx are unknown. Fluctuations in intracellular zinc ([Zn 2+ ] i ) were measured in fluozin-3-loaded platelets using fluorometry and flow cytometry. Platelet activation was assessed using light transmission aggregometry. The detection of phosphoproteins was performed by Western blotting. [Zn 2+ ] o influx and subsequent platelet activation were abrogated by blocking the sodium/calcium exchanged, TRP channels, and ZIP7. Cation store depletion regulated Zn 2+ influx. [Zn 2+ ] o stimulation resulted in the phosphorylation of PKC substates, MLC, and β3 integrin. Platelet activation via GPVI or Zn 2+ resulted in ZIP7 phosphorylation in a casein kinase 2-dependent manner and initiated elevations of [Zn 2+ ] i that were sensitive to the inhibition of Orai1, ZIP7, or IP 3 R-mediated pathways. These data indicate that platelets detect and respond to changes in [Zn 2+ ] o via influx into the cytosol through TRP channels and the NCX exchanger. Platelet activation results in the externalization of ZIP7, which further regulates Zn 2+ influx. Increases in [Zn 2+ ] i contribute to the activation of cation-dependent enzymes. Sensitivity of Zn 2+ influx to thapsigargin indicates a store-operated pathway that we term store-operated Zn 2+ entry (SOZE). These mechanisms may affect platelet behavior during thrombosis and hemostasis.

Current in vitro thrombosis models utilise simplistic 2D surfaces coated with purified components of the subendothelial matrix. The lack of a realistic humanised model has led to greater study of thrombus formation in in vivo tests in animals. Here we aimed to develop 3D hydrogel-based replicas of the medial and adventitial layers of the human artery to produce a surface that can optimally support thrombus formation under physiological flow conditions. These tissue-engineered medial- (TEML) and adventitial-layer (TEAL) hydrogels were developed by culturing human coronary artery smooth muscle cells and human aortic adventitial fibroblasts within collagen hydrogels, both individually and in co-culture. Platelet aggregation upon these hydrogels was studied using a custom-made parallel flow chamber. When cultured in the presence of ascorbic acid, the medial-layer hydrogels were able to produce sufficient neo-collagen to support effective platelet aggregation under arterial flow conditions. Both TEML and TEAL hydrogels possessed measurable tissue factor activity and could trigger coagulation of platelet-poor plasma in a factor VII-dependent manner. Biomimetic hydrogel replicas of the subendothelial layers of the human artery are effective substrates for a humanised in vitro thrombosis model that could reduce animal experimentation by replacing current in vivo models.

Background: Factor XIII (FXIII) is an important proenzyme in the hemostatic system. The plasma-derived enzyme activated FXIII cross-links fibrin fibers within thrombi to increase their mechanical strength and cross-links fibrin to fibrinolytic inhibitors, specifically α 2 -antiplasmin, to increase resistance to fibrinolysis. We have previously shown that cellular FXIII (factor XIII-A [FXIII-A]), which is abundant in the platelet cytoplasm, is externalized onto the activated membrane and cross-links extracellular substrates. The contribution of cellular FXIII-A to platelet activation and platelet function has not been extensively studied., Objectives: This study aims to identify the role of platelet FXIII-A in platelet function., Methods: We used normal healthy platelets with a cell permeable FXIII inhibitor and platelets from FXIII-deficient patients as a FXIII-free platelet model in a range of platelet function and clotting tests., Results: Our data demonstrate that platelet FXIII-A enhances fibrinogen binding to the platelet surface upon agonist stimulation and improves the binding of platelets to fibrinogen and aggregation under flow in a whole-blood thrombus formation assay. In the absence of FXIII-A, platelets show reduced sensitivity to agonist stimulation, including decreased P-selectin exposure and fibrinogen binding. We show that FXIII-A is involved in platelet spreading where a lack of FXIII-A reduces the ability of platelets to fully spread on fibrinogen and collagen. Our data demonstrate that platelet FXIII-A is important for clot retraction where clots formed in its absence retracted to a lesser extent., Conclusion: Overall, this study shows that platelet FXIII-A functions during thrombus formation by aiding platelet activation and thrombus retraction in addition to its antifibrinolytic roles., (© 2023 The Authors.)

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

Venous thromboembolism (VTE) comprising deep venous thrombosis and pulmonary embolism is a major cause of morbidity and mortality. Short-term immobility-related conditions are a major risk factor for the development of VTE. Paradoxically, long-term immobilized free-ranging hibernating brown bears and paralyzed spinal cord injury (SCI) patients are protected from VTE. We aimed to identify mechanisms of immobility-associated VTE protection in a cross-species approach. Mass spectrometry-based proteomics revealed an antithrombotic signature in platelets of hibernating brown bears with heat shock protein 47 (HSP47) as the most substantially reduced protein. HSP47 down-regulation or ablation attenuated immune cell activation and neutrophil extracellular trap formation, contributing to thromboprotection in bears, SCI patients, and mice. This cross-species conserved platelet signature may give rise to antithrombotic therapeutics and prognostic markers beyond immobility-associated VTE.

Hepatitis is the term used to describe inflammation in the liver. It is associated with a high rate of mortality, but the underlying disease mechanisms are not completely understood and treatment options are limited. We present a mathematical model of hepatitis that captures the complex interactions between hepatocytes (liver cells), hepatic stellate cells (cells in the liver that produce hepatitis-associated fibrosis) and the immune components that mediate inflammation. The model is in the form of a system of ordinary differential equations. We use numerical techniques and bifurcation analysis to characterize and elucidate the physiological mechanisms that dominate liver injury and its outcome to a healthy or unhealthy, chronic state. This study reveals the complex interactions between the multiple cell types and mediators involved in this complex disease and highlights potential problems in targeting inflammation in the liver therapeutically., (© The Author(s) 2019. Published by Oxford University Press on behalf of the Institute of Mathematics and its Applications.)

Receptor diffusion plays an essential role in cellular signalling via the plasma membrane microenvironment and receptor interactions, but the regulation is not well understood. To aid in understanding of the key determinants of receptor diffusion and signalling, we developed agent-based models (ABMs) to explore the extent of dimerisation of the platelet- and megakaryocyte-specific receptor for collagen glycoprotein VI (GPVI). This approach assessed the importance of glycolipid enriched raft-like domains within the plasma membrane that lower receptor diffusivity. Our model simulations demonstrated that GPVI dimers preferentially concentrate in confined domains and, if diffusivity within domains is decreased relative to outside of domains, dimerisation rates are increased. While an increased amount of confined domains resulted in further dimerisation, merging of domains, which may occur upon membrane rearrangements, was without effect. Modelling of the proportion of the cell membrane which constitutes lipid rafts indicated that dimerisation levels could not be explained by these alone. Crowding of receptors by other membrane proteins was also an important determinant of GPVI dimerisation. Together, these results demonstrate the value of ABM approaches in exploring the interactions on a cell surface, guiding the experimentation for new therapeutic avenues., (© 2023. The Author(s).)

Infection by Staphylococcus aureus is the leading cause of infective endocarditis (IE). Activation of platelets by this pathogen results in their aggregation and thrombus formation which are considered to be important steps in the development and pathogenesis of IE. Here, we show that a secreted cysteine protease, staphopain A, activates human platelets and induces their aggregation. The culture supernatant of a scpA mutant deficient in staphopain A production was reduced in its ability to trigger platelet aggregation. The platelet agonist activity of purified staphopain A was inhibited by staphostatin A, a specific inhibitor, thus implicating its protease activity in the agonism. In whole blood, using concentrations of staphopain A that were otherwise insufficient to induce platelet aggregation, increased binding to collagen and thrombus formation was observed. Using antagonists specific to protease-activated receptors 1 and 4, we demonstrate their role in mediating staphopain A induced platelet activation.

Platelet and coagulation activation are highly reciprocal processes driven by multi-molecular interactions. Activated platelets secrete several coagulation factors and expose phosphatidylserine, which supports the activation of coagulation factor proteins. On the other hand, the coagulation cascade generates known ligands for platelet receptors, such as thrombin and fibrin. Coagulation factor (F)Xa, (F)XIIIa and activated protein C (APC) can also bind to platelets, but the functional consequences are unclear. Here, we investigated the effects of the activated (anti)coagulation factors on platelets, other than thrombin. Multicolor flow cytometry and aggregation experiments revealed that the 'supernatant of (hirudin-treated) coagulated plasma' (SCP) enhanced CRP-XL-induced platelet responses, i.e., integrin α IIb β 3 activation, P-selectin exposure and aggregate formation. We demonstrated that FXIIIa in combination with APC enhanced platelet activation in solution, and separately immobilized FXIIIa and APC resulted in platelet spreading. Platelet activation by FXIIIa was inhibited by molecular blockade of glycoprotein VI (GPVI) or Syk kinase. In contrast, platelet spreading on immobilized APC was inhibited by PAR1 blockade. Immobilized, but not soluble, FXIIIa and APC also enhanced in vitro adhesion and aggregation under flow. In conclusion, in coagulation, factors other than thrombin or fibrin can induce platelet activation via GPVI and PAR receptors.

Cucurbitacins are dietary compounds that have been shown to elicit a range of anti-tumour, anti-inflammatory and anti-atherosclerotic activities. Originally identified as signal transducer and activator of transcription, STAT, inhibitors, a variety of mechanisms of action have since been described, including dysregulation of the actin cytoskeleton and disruption of integrin function. Integrin outside-in signalling and cytoskeletal rearrangements are critical for the propagation of stable thrombus formation and clot retraction following platelet adhesion at the site of vessel damage. The effects of cucurbitacins on platelet function and thrombus formation are unknown. We report for the first time anti-platelet and anti-thrombotic effects of cucurbitacins B, E and I in human platelets. Treatment of platelets with cucurbitacins resulted in attenuation of platelet aggregation, secretion and fibrinogen binding following stimulation by platelet agonists. Cucurbitacins were also found to potently inhibit other integrin- and cytoskeleton-mediated events, including adhesion, spreading and clot retraction. Further investigation of cytoskeletal dynamics found treatment with cucurbitacins altered cofilin phosphorylation, enhanced activation and increased F actin polymerisation and microtubule assembly. Disruption to cytoskeletal dynamics has been previously shown to impair integrin activation, platelet spreading and clot retraction. Anti-platelet properties of cucurbitacins were found to extend to a disruption of stable thrombus formation, with an increase in thrombi instability and de-aggregation under flow. Our research identifies novel, anti-platelet and anti-thrombotic actions of cucurbitacins that appear to be linked to dysregulation of cytoskeletal dynamics and integrin function., 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/).)

The interindividual variation in the functional response of platelets to activation by agonists is heritable. Genome-wide association studies (GWASs) of quantitative measures of platelet function have identified fewer than 20 distinctly associated variants, some with unknown mechanisms. Here, we report GWASs of pathway-specific functional responses to agonism by adenosine 5'-diphosphate, a glycoprotein VI-specific collagen mimetic, and thrombin receptor-agonist peptides, each specific to 1 of the G protein-coupled receptors PAR-1 and PAR-4, in subsets of 1562 individuals. We identified an association (P = 2.75 × 10-40) between a common intronic variant, rs10886430, in the G protein-coupled receptor kinase 5 gene (GRK5) and the sensitivity of platelets to activate through PAR-1. The variant resides in a megakaryocyte-specific enhancer that is bound by the transcription factors GATA1 and MEIS1. The minor allele (G) is associated with fewer GRK5 transcripts in platelets and the greater sensitivity of platelets to activate through PAR-1. We show that thrombin-mediated activation of human platelets causes binding of GRK5 to PAR-1 and that deletion of the mouse homolog Grk5 enhances thrombin-induced platelet activation sensitivity and increases platelet accumulation at the site of vascular injury. This corroborates evidence that the human G allele of rs10886430 is associated with a greater risk for cardiovascular disease. In summary, by combining the results of pathway-specific GWASs and expression quantitative trait locus studies in humans with the results from platelet function studies in Grk5-/- mice, we obtain evidence that GRK5 regulates the human platelet response to thrombin via the PAR-1 pathway., (© 2022 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.)

Kv1.3 is a voltage-gated K + -selective channel with roles in immunity, insulin-sensitivity, neuronal excitability and olfaction. Despite being one of the largest ionic conductances of the platelet surface membrane, its contribution to platelet function is poorly understood. Here we show that Kv1.3-deficient platelets display enhanced ADP-evoked platelet aggregation and secretion, and an increased surface expression of platelet integrin α IIb . In contrast, platelet adhesion and thrombus formation in vitro under arterial shear conditions on surfaces coated with collagen were reduced for samples from Kv1.3 -/- compared to wild type mice. Use of collagen-mimetic peptides revealed a specific defect in the engagement with α 2 β 1 . Kv1.3 -/- platelets developed significantly fewer, and shorter, filopodia than wild type platelets during adhesion to collagen fibrils. Kv1.3 -/- mice displayed no significant difference in thrombus formation within cremaster muscle arterioles using a laser-induced injury model, thus other pro-thrombotic pathways compensate in vivo for the adhesion defect observed in vitro . This may include the increased platelet counts of Kv1.3 -/- mice, due in part to a prolonged lifespan. The ability of Kv1.3 to modulate integrin-dependent platelet adhesion has important implications for understanding its contribution to normal physiological platelet function in addition to its reported roles in auto-immune diseases and thromboinflammatory models of stroke.

Platelets mediate arterial thrombosis, a leading cause of myocardial infarction and stroke. During injury, platelets adhere and spread over exposed subendothelial matrix substrates of the damaged blood vessel wall. The mechanisms which govern platelet activation and their interaction with a range of substrates are therefore regularly investigated using platelet spreading assays. These assays often use differential interference contrast (DIC) microscopy to assess platelet morphology and analysis performed using manual annotation. Here, a convolutional neural network (CNN) allowed fully automated analysis of platelet spreading assays captured by DIC microscopy. The CNN was trained using 120 generalised training images. Increasing the number of training images increases the mean average precision of the CNN. The CNN performance was compared to six manual annotators. Significant variation was observed between annotators, highlighting bias when manual analysis is performed. The CNN effectively analysed platelet morphology when platelets spread over a range of substrates (CRP-XL, vWF and fibrinogen), in the presence and absence of inhibitors (dasatinib, ibrutinib and PRT-060318) and agonist (thrombin), with results consistent in quantifying spread platelet area which is comparable to published literature. The application of a CNN enables, for the first time, automated analysis of platelet spreading assays captured by DIC microscopy., (© 2022. The Author(s).)

Significance: Since protein disulfide isomerase (PDI) was first described in 1963, researchers have shown conclusively that PDI and sibling proteins are quintessential for thrombus formation. PDI, endoplasmic reticulum protein (ERp)5, ERp57, and ERp72 are released from platelets and vascular cells and interact with integrin αIIbβ3 on the outer surface of platelets. Recent Advances: At the cell surface they influence protein folding and function, propagating thrombosis and maintaining hemostasis. TMX1, which is a transmembrane thiol isomerase, is the first family member shown to negatively regulate platelets. Targets of thiol isomerases have been identified, including integrin α2β1, Von Willebrand Factor, GpIbα, nicotinamide adenine dinucleotide phosphate oxidase (Nox)-1, Nox-2, and tissue factor, all of which are pro-thrombotic, and several of which are on the cell surface. In spite of this, PDI can paradoxically catalyze the delivery of nitric oxide to platelets, which decrease thrombus formation. Critical Issues: Although the overall effect of PDI is to positively regulate platelet activation, it is still unclear how thiol isomerases function in pro-thrombotic states, such as obesity, diabetes, and cancer. In parallel, there has been a surge in the development of novel thiol isomerase inhibitors, which display selectivity, potency and modulate thrombosis and hemostasis. The availability of selective thiol isomerase inhibitors has culminated in clinical trials, with promising outcomes for the prevention of cancer-associated thrombosis. Future Directions: Altogether, thiol isomerases are perceived as an orchestrating force that regulates thrombus development. In the current review, we will explore the history of PDI in cardiovascular biology, detail known mechanisms of action, and summarize known thiol isomerase inhibitors.

Accurate and comprehensive assessment of platelet function across cohorts of donors may be key to understanding the risk of thrombotic events associated with cardiovascular disease, and, hence, to help personalize the application of antiplatelet drugs. However, platelet function tests can be difficult to perform and analyze; they also can be unreliable or uninformative and poorly standardized across studies. The Platelet Phenomic Analysis (PPAnalysis) assay and associated open-source software platform were developed in response to these challenges. PPAnalysis utilizes preprepared freeze-dried microtiter plates to provide a detailed characterization of platelet function. The automated analysis of the high-dimensional data enables the identification of subpopulations of donors with distinct platelet function phenotypes. Using this approach, we identified that the sensitivity of a donor's platelets to an agonist and their capacity to generate a functional response are distinct independent metrics of platelet reactivity. Hierarchical clustering of these metrics identified 6 subgroups with distinct platelet phenotypes within healthy cohorts, indicating that platelet reactivity does not fit into the traditional simple categories of "high" and "low" responders. These platelet phenotypes were found to exist in 2 independent cohorts of healthy donors and were stable on recall. PPAnalysis is a powerful tool for stratification of cohorts on the basis of platelet reactivity that will enable investigation of the causes and consequences of differences in platelet function and drive progress toward precision medicine., (© 2021 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.)

A subset of patients with coronavirus disease 2019 (COVID-19) become critically ill, suffering from severe respiratory problems and also increased rates of thrombosis. The causes of thrombosis in severely ill patients with COVID-19 are still emerging, but the coincidence of critical illness with the timing of the onset of adaptive immunity could implicate an excessive immune response. We hypothesized that platelets might be susceptible to activation by anti-severe acute respiratory syndrome coronavirus 2 (anti-SARS-CoV-2) antibodies and might contribute to thrombosis. We found that immune complexes containing recombinant SARS-CoV-2 spike protein and anti-spike immunoglobulin G enhanced platelet-mediated thrombosis on von Willebrand factor in vitro, but only when the glycosylation state of the Fc domain was modified to correspond with the aberrant glycosylation previously identified in patients with severe COVID-19. Furthermore, we found that activation was dependent on FcγRIIA, and we provide in vitro evidence that this pathogenic platelet activation can be counteracted by the therapeutic small molecules R406 (fostamatinib) and ibrutinib, which inhibit tyrosine kinases Syk and Btk, respectively, or by the P2Y12 antagonist cangrelor., (© 2021 by The American Society of Hematology.)

Platelets react rapidly to vascular injury and undergo activation in response to a range of stimuli to limit blood loss. Many platelet function tests measure endpoint responses after a defined time period and not the rate of platelet activation. However, the rate at which platelets convert extracellular stimuli into a functional response is an essential factor in determining how efficiently they can respond to injury, bind to a forming thrombus, and signal to recruit other platelets. This paper describes a flow cytometry-based platelet function assay that enables simultaneous data acquisition and sample stimulation and utilizes newly developed bespoke open-source software (Kinetx) to enable quantitative kinetic measurements of platelet granule release, fibrinogen binding, and intracellular calcium flux. Kinetix was developed in R so that users can alter parameters such as degree of smoothing, identification of outlying data points, or time scales. To aid users unfamiliar with the R environment, Kinetix analysis of data can be performed by a single command. Together, this allows real-time platelet activation metrics, such as rate, acceleration, time to peak-rate, time to peak-calcium, and qualitative shape changes, to be accurately and reproducibly measured and categorized. Kinetic measurements of platelet activation give a unique insight into platelets' behavior during the first stages of activation and may provide a method of predicting the recruitment of platelets into a forming thrombus.

Connexins are a family of gap junction forming proteins widely expressed by mammalian cells. They assemble into hexameric hemichannels, which can either function independently or dock with opposing hemichannels on apposite cells, forming a gap junction. Pannexins are structurally related to the connexins but extensive glycosylation of these channels prevents docking to form gap junctions and they function as membrane channels. Platelets express pannexin-1 and several connexin family members (Cx37, Cx40 and Cx62). These channels are permeable to molecules up to 1,000 Daltons in molecular mass and functional studies demonstrate their role in non-vesicular ATP release. Channel activation is regulated by (patho)physiological stimuli, such as mechanical stimulation, making them attractive potential drug targets for the management of arterial thrombosis. This review explores the structure and function of platelet pannexin-1 and connexins, the mechanisms by which they are gated and their therapeutic potential.

Background: We have previously described CxxCpep, a peptide with anti-platelet properties that inhibits peri/epicellular protein disulphide isomerase (pecPDI) by forming a mixed disulfide bond with Cys400 within the pecPDI active site., Objectives: Here we sought to determine if pecPDI targeted by CxxCpep is relevant to redox mechanisms downstream of the collagen receptor GPVI in platelets., Methods and Results: Restriction of effects of CxxCpep to the platelet surface was confirmed by LC-MS/MS following cell fractionation. Platelet aggregation was measured in platelet-rich plasma (PRP) incubated with 30 μM CxxCpep or vehicle. CxxCpep inhibited collagen-induced platelet aggregation but exerted no effect in TRAP-6-stimulated platelets. PRP was incubated with DCFDA to measure oxidative burst upon platelet adhesion to collagen. Results showed that CxxCpep decreased oxidative burst in platelets adhered to immobilized collagen while the number of adherent cells was unaffected. Furthermore, flow cytometry studies using a FITC-maleimide showed that the GPVI agonist CRP stimulated an increase in free thiols on the platelet outer membrane, which was inhibited by CxxCpep. Finally, CxxCpep inhibited platelet mitochondrial respiration upon activation with collagen, but not with thrombin., Conclusions: Our data suggest that pecPDI is a potential modulator of GPVI-mediated redox regulation mechanisms and that CxxCpep can be further exploited as a template for new antiplatelet compounds., (Copyright © 2021 Elsevier Inc. All rights reserved.)

While current oral antiplatelet therapies benefit many patients, they deregulate the hemostatic balance leaving patients at risk of systemic side-effects such as hemorrhage. Dual antiplatelet treatment is the standard approach, combining aspirin with P2Y 12 blockers. These therapies mainly target autocrine activation mechanisms (TxA 2 , ADP) and, more recently, the use of thrombin or thrombin receptor antagonists have been added to the available approaches. Recent efforts to develop new classes of anti-platelet drugs have begun to focus on primary platelet activation pathways such as through the immunoreceptor tyrosine-based activation motif (ITAM)-containing collagen receptor GPVI/FcRγ-chain complex. There are already encouraging results from targeting GPVI, with reduced aggregation and smaller arterial thrombi, without major bleeding complications, likely due to overlapping activation signaling pathways with other receptors such as the GPIb-V-IX complex. An alternative approach to reduce platelet activation could be to inhibit this signaling pathway by targeting the inhibitory pathways intrinsic to platelets. Stimulation of endogenous negative modulators could provide more specific inhibition of platelet function, but is this feasible? In this review, we explore the potential of the two major platelet immunoreceptor tyrosine-based inhibitory motif (ITIM)-containing inhibitory receptors, G6b-B and PECAM-1, as antithrombotic targets.

HSP47 (heat shock protein 47) is a collagen-specific molecular chaperone that is essential for procollagen folding and function. Previous studies have shown that HSP47 binding requires a critical Arg residue at the Y position of the (Gly-Xaa-Yaa) repeats of collagen; however, the exact binding sites of HSP47 on native collagens are not fully defined. To address this, we mapped the HSP47 binding sites on collagens through an ELISA binding assay using collagen toolkits, synthetic collagen peptides covering the entire amino acid sequences of collagen types II and III assembled in triple-helical conformation. Our results showed that HSP47 binds to only a few of the GXR motifs in collagen, with most of the HSP47 binding sites identified located near the N-terminal part of the triple-helical region. Molecular modelling and binding energy calculation indicated that residues flanking the key Arg in the collagen sequence also play an important role in defining the high-affinity HSP47 binding site of collagen. Based on this binding mode of HSP47 to collagen, virtual screening targeting both the Arg binding site and its neighboring area on the HSP47 surface, and a subsequent bioassay, we identified two novel compounds with blocking activity towards HSP47 binding of collagen. Overall, our study revealed the native HSP47 binding sites on collagen and provided novel information for the design of small-molecule inhibitors of HSP47.

Pim kinases are upregulated in several forms of cancer, contributing to cell survival and tumour development, but their role in platelet function and thrombotic disease has not been explored. We report for the first time that Pim-1 is expressed in human and mouse platelets. Genetic deletion or pharmacological inhibition of Pim kinase results in reduced thrombus formation but is not associated with impaired haemostasis. Attenuation of thrombus formation was found to be due to inhibition of the thromboxane A2 receptor as effects on platelet function was non-additive to inhibition caused by the cyclooxygenase inhibitor indomethacin or thromboxane A2 receptor antagonist GR32191. Treatment with Pim kinase inhibitors caused reduced surface expression of the thromboxane A2 receptor and resulted in reduced responses to thromboxane A2 receptor agonists, indicating a role for Pim kinase in the regulation of thromboxane A2 receptor function. Our research identifies a novel, Pim kinase dependent regulatory mechanism for the thromboxane A2 receptor and represents a new targeting strategy that is independent of COX-1 inhibition or direct antagonism of the thromboxane A2 receptor that whilst attenuating thrombosis does not increase bleeding.

Vascular complications resulting from atherosclerosis development are a major cause of death. Reactive oxygen species (ROS) are produced by platelets during activation, and have been demonstrated to positively regulate platelet activatory responses. Zn 2+ is also an important hemostatic cofactor in platelets, acting both as a platelet agonist and second messenger. Whilst the effect of Zn 2+ -dependent signaling mechanisms on ROS production in nucleated cells has been demonstrated, comparable roles in platelets have yet to be investigated. In this study we investigated the relationship between fluctuations in cytosolic Zn 2 [Zn 2+ ] i and platelet ROS production. Agonist-evoked ROS production, GSH levels and GPx activity are abrogated in platelets treated with the Zn 2+ -chelator, TPEN. Conversely, increasing platelet [Zn 2+ ] i using Zn 2+ ionophores potentiated ROS generation and decreased GSH levels and GPx activity. Zn 2+ -dependent ROS production was sensitive to pretreatment with DPI or mitoTEMPO, NADPH oxidase and mitochondria inhibitors respectively. Increasing [Zn 2+ ] i resulted in increases of Erk1/2 and JNK phosphorylation. Our data are consistent with a functional association between [Zn 2+ ] i and ROS production in platelets that could influence thrombus formation in a clinical context.

Background: Protein disulphide isomerase (PDI) and NADPH oxidase 1 (Nox-1) regulate platelet function and reactive oxygen species (ROS) generation, suggesting potentially interdependent roles. Increased platelet reactivity and ROS production have been correlated with cardiometabolic disease risk factors., Objectives: To establish whether PDI and Nox-1 cooperate to control platelet function., Methods: Immunofluorescence microscopy was utilised to determine expression and localisation of PDI and Nox-1. Platelet aggregation, fibrinogen binding, P-selectin exposure, spreading and calcium mobilization were measured as markers of platelet function. A cross-sectional population study ( n = 136) was conducted to assess the relationship between platelet PDI and Nox-1 levels and cardiometabolic risk factors., Results: PDI and Nox-1 co-localized upon activation induced by the collagen receptor GPVI. Co-inhibition of PDI and Nox-1 led to additive inhibition of GPVI-mediated platelet aggregation, activation and calcium flux. This was confirmed in murine Nox-1 -/- platelets treated with PDI inhibitor bepristat, without affecting bleeding. PDI and Nox-1 together contributed to GPVI signalling that involved the phosphorylation of p38 MAPK, p47phox, PKC and Akt. Platelet PDI and Nox-1 levels were upregulated in obesity, with platelet Nox-1 also elevated in hypertensive individuals., Conclusions: We show that PDI and Nox-1 cooperate to control platelet function and are associated with cardiometabolic risk factors.