Platelet signaling: a complex interplay between inhibitory and activatory networks

The role of platelets in hemostasis and thrombosis is dependent on a complex balance of activatory and inhibitory signaling pathways. Inhibitory signals released from the healthy vasculature suppress platelet activation in the absence of platelet receptor agonists. Activatory signals present at a site of injury initiate platelet activation and thrombus formation; subsequently, endogenous negative signaling regulators dampen activatory signals to control thrombus growth. Understanding the complex interplay between activatory and inhibitory signaling networks is an emerging challenge in the study of platelet biology and necessitates a systematic approach to utilize experimental data effectively. In this review, we will explore the key points of platelet regulation and signaling that maintain platelets in a resting state, mediate activation to elicit thrombus formation or provide negative feedback. Platelet signaling will be described in terms of key signaling molecules that are common to the pathways activated by platelet agonists and can be described as regulatory nodes for both positive and negative regulators. This article is protected by copyright. All rights reserved

GRID and docking analyses reveal a molecular basis for flavonoid inhibition of src-family kinase activity

Flavonoids reduce cardiovascular disease risk through anti-inflammatory, anti-coagulant and anti-platelet actions. One key flavonoid inhibitory mechanism is blocking kinase activity that drives these processes. Flavonoids attenuate activities of kinases including phosphoinositide-3-kinase (PI3K), Fyn, Lyn, Src, Syk, PKC, PIM1/2, ERK, JNK, and PKA. X-ray crystallographic analyses of kinase-flavonoid complexes show that flavonoid ring systems and their hydroxyl substitutions are important structural features for their binding to kinases. A clearer understanding of structural interactions of flavonoids with kinases is necessary to allow construction of more potent and selective counterparts. We examined flavonoid (quercetin, apigenin and catechin) interactions with Src-family kinases (Lyn, Fyn and Hck) applying the Sybyl docking algorithm and GRID. A homology model (Lyn) was used in our analyses to demonstrate that high quality predicted kinase structures are suitable for flavonoid computational studies. Our docking results revealed potential hydrogen bond contacts between flavonoid hydroxyls and kinase catalytic site residues. Identification of plausible contacts indicated that quercetin formed the most energetically stable interactions, apigenin lacked hydroxyl groups necessary for important contacts, and the non-planar structure of catechin could not support predicted hydrogen bonding patterns. GRID analysis using a hydroxyl functional group supported docking results. Based on these findings, we predicted that quercetin would inhibit activities of Src-family kinases with greater potency than apigenin and catechin. We validated this prediction using in vitro kinase assays. We conclude that our study can be used as a basis to construct virtual flavonoid interaction libraries to guide drug discovery using these compounds as molecular templates

Mind the gap: connexins and pannexins in platelet function

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

Tangeretin regulates platelet function through inhibition of phosphoinositide 3-Kinase and cyclic nucleotide signaling

OBJECTIVE: Dietary flavonoids have long been appreciated in reducing cardiovascular disease risk factors, but their mechanisms of action are complex in nature. In this study, the effects of tangeretin, a dietary flavonoid, were explored on platelet function, signaling, and hemostasis. APPROACH AND RESULTS: Tangeretin inhibited agonist-induced human platelet activation in a concentration-dependent manner. It inhibited agonist-induced integrin αIIbβ3 inside-out and outside-in signaling, intracellular calcium mobilization, and granule secretion. Tangeretin also inhibited human platelet adhesion and subsequent thrombus formation on collagen-coated surfaces under arterial flow conditions in vitro and reduced hemostasis in mice. Further characterization to explore the mechanism by which tangeretin inhibits platelet function revealed distinctive effects of platelet signaling. Tangeretin was found to inhibit phosphoinositide 3-kinase-mediated signaling and increase cGMP levels in platelets, although phosphodiesterase activity was unaffected. Consistent with increased cGMP levels, tangeretin increased the phosphorylation of vasodilator-stimulated phosphoprotein at S239. CONCLUSIONS: This study provides support for the ability and mechanisms of action of dietary flavonoids to modulate platelet signaling and function, which may affect the risk of thrombotic disease

A high-density immunoblotting methodology for quantification of total protein levels and phosphorylation modifications

The components of many signaling pathways have been identified and there is now a need to conduct quantitative data-rich temporal experiments for systems biology and modeling approaches to better understand pathway dynamics and regulation. Here we present a modified Western blotting method that allows the rapid and reproducible quantification and analysis of hundreds of data points per day on proteins and their phosphorylation state at individual sites. The approach is of particular use where samples show a high degree of sample-to-sample variability such as primary cells from multiple donors. We present a case study on the analysis of >800 phosphorylation data points from three phosphorylation sites in three signaling proteins over multiple time points from platelets isolated from ten donors, demonstrating the technique's potential to determine kinetic and regulatory information from limited cell numbers and to investigate signaling variation within a population. We envisage the approach being of use in the analysis of many cellular processes such as signaling pathway dynamics to identify regulatory feedback loops and the investigation of potential drug/inhibitor responses, using primary cells and tissues, to generate information about how a cell's physiological state changes over time

Priority projects for the implementation of CCS power generation with enhanced oil recovery in Mexico

In March 2014, Mexico launched its CCUS technology roadmap, outlining the actions to be taken up to 2024. One important action is the National Policy of Carbon Capture and Storage (CCS) ready and the identification of priority natural gas combined cycle (NGCC) with capture plants. This outcome could aid the creation of a technology roadmap for the design of new NGCC power plants and their operational requirements for EOR and for the reduction of CO 2 emissions. This article provides an overview of the opportunities for deploying CCS in new NGCC power plants in Mexico which were programed to begin operation throughout the period from 2016 to 2030. The attention is given to plants close to oil fields which are candidates for enhanced oil recovery (EOR), located in an inclusion zone suitable for storage. The Gulf of Mexico region, where potential EOR sites and the presence of industrial CO 2 sources are located, is within the inclusion zone for recommended sites for geological storage of CO 2 . After identifying new power plants in the inclusion zone, this article analyses which existing plants could be retrofitted and which new power plants could be designed to be ‘carbon capture ready’. In addition, the distance and the volumes of CO 2 are estimated

Severe platelet dysfunction in NHL patients receiving ibrutinib is absent in patients receiving acalabrutinib

The Bruton’s tyrosine kinase (Btk) inhibitor ibrutinib induces platelet dysfunction and causes increased risk of bleeding. Off-target inhibition of Tec is believed to contribute to platelet dysfunction and other side-effects of ibrutinib. The second generation Btk inhibitor acalabrutinib was developed with improved specificity for Btk over Tec. We investigated platelet function in patients with Non-Hodgkin Lymphoma (NHL) receiving ibrutinib or acalabrutinib by aggregometry and by measuring thrombus formation on collagen under arterial shear. Both patient groups had similarly dysfunctional aggregation responses to collagen and collagen-related peptide (CRP-XL) and comparison with mechanistic experiments in which platelets from healthy donors were treated with the Btk inhibitors suggested that both drugs inhibit platelet Btk and Tec at physiological concentrations. Only ibrutinib caused dysfunctional thrombus formation, while size and morphology of thrombi following acalabrutinib treatment were of normal size and morphology. We found that ibrutinib but not acalabrutinib inhibited SFKs and that SFKs have a critical role in platelet adhesion to collagen that is likely to underpin unstable thrombus formation observed in ibrutinib patients. We found that platelet function was enhanced by increasing levels of vWF and FVIII ex vivo by addition of intermediate purity FVIII (haemate P) to blood from patients, resulting in consistently larger thrombi. We conclude that acalabrutinib avoids major platelet dysfunction associated with ibrutinib therapy, and platelet function may be enhanced in patients with B-cell NHL by increasing plasma vWF and FVIII

Intracellular trafficking, localization, and mobilization of platelet-borne thiol isomerases

OBJECTIVE: Thiol isomerases facilitate protein folding in the endoplasmic reticulum, and several of these enzymes, including protein disulfide isomerase and ERp57, are mobilized to the surface of activated platelets, where they influence platelet aggregation, blood coagulation, and thrombus formation. In this study, we examined the synthesis and trafficking of thiol isomerases in megakaryocytes, determined their subcellular localization in platelets, and identified the cellular events responsible for their movement to the platelet surface on activation. APPROACH AND RESULTS: Immunofluorescence microscopy imaging was used to localize protein disulfide isomerase and ERp57 in murine and human megakaryocytes at various developmental stages. Immunofluorescence microscopy and subcellular fractionation analysis were used to localize these proteins in platelets to a compartment distinct from known secretory vesicles that overlaps with an inner cell-surface membrane region defined by the endoplasmic/sarcoplasmic reticulum proteins calnexin and sarco/endoplasmic reticulum calcium ATPase 3. Immunofluorescence microscopy and flow cytometry were used to monitor thiol isomerase mobilization in activated platelets in the presence and absence of actin polymerization (inhibited by latrunculin) and in the presence or absence of membrane fusion mediated by Munc13-4 (absent in platelets from Unc13dJinx mice). CONCLUSIONS: Platelet-borne thiol isomerases are trafficked independently of secretory granule contents in megakaryocytes and become concentrated in a subcellular compartment near the inner surface of the platelet outer membrane corresponding to the sarco/endoplasmic reticulum of these cells. Thiol isomerases are mobilized to the surface of activated platelets via a process that requires actin polymerization but not soluble N-ethylmaleimide-sensitive fusion protein attachment receptor/Munc13-4-dependent vesicular-plasma membrane fusion

Structural, functional and mechanistic insights uncover the fundamental role of orphan connexin-62 in platelets

Connexins (Cxs) oligomerise to form hexameric hemichannels in the plasma membrane that can further dock together on adjacent cells to form gap junctions and facilitate intercellular-trafficking of molecules. In this study, we report the expression and function of an ‘orphan’ connexin, Cx62, in human and mouse (Cx57, mouse homologue) platelets. A novel mimetic peptide (62Gap27) was developed to target the second extracellular loop of Cx62 and 3D structural models predicted its interference with gap junction and hemichannel function. The ability of 62Gap27 to regulate both gap junction and hemichannel-mediated intercellular communication was observed using FRAP analysis and flow cytometry. Cx62 inhibition by 62Gap27 suppressed a range of agonist-stimulated platelet functions and impaired thrombosis and haemostasis. This was associated with elevated PKA-dependent signalling in a cyclic adenosine monophosphate-independent manner, and was not observed in Cx57 deficient mouse platelets (in which the selectivity of 62Gap27 for this connexin was also confirmed). Notably, Cx62 hemichannels were observed to function independently of Cx37 and Cx40 hemichannels. Together, our data reveal a fundamental role for a hitherto uncharacterised connexin in the regulation of the function of circulating cells

Effect of angiotensin-converting enzyme inhibitor and angiotensin receptor blocker initiation on organ support-free days in patients hospitalized with COVID-19

IMPORTANCE Overactivation of the renin-angiotensin system (RAS) may contribute to poor clinical outcomes in patients with COVID-19. Objective To determine whether angiotensin-converting enzyme (ACE) inhibitor or angiotensin receptor blocker (ARB) initiation improves outcomes in patients hospitalized for COVID-19. DESIGN, SETTING, AND PARTICIPANTS In an ongoing, adaptive platform randomized clinical trial, 721 critically ill and 58 non–critically ill hospitalized adults were randomized to receive an RAS inhibitor or control between March 16, 2021, and February 25, 2022, at 69 sites in 7 countries (final follow-up on June 1, 2022). INTERVENTIONS Patients were randomized to receive open-label initiation of an ACE inhibitor (n = 257), ARB (n = 248), ARB in combination with DMX-200 (a chemokine receptor-2 inhibitor; n = 10), or no RAS inhibitor (control; n = 264) for up to 10 days. MAIN OUTCOMES AND MEASURES The primary outcome was organ support–free days, a composite of hospital survival and days alive without cardiovascular or respiratory organ support through 21 days. The primary analysis was a bayesian cumulative logistic model. Odds ratios (ORs) greater than 1 represent improved outcomes. RESULTS On February 25, 2022, enrollment was discontinued due to safety concerns. Among 679 critically ill patients with available primary outcome data, the median age was 56 years and 239 participants (35.2%) were women. Median (IQR) organ support–free days among critically ill patients was 10 (–1 to 16) in the ACE inhibitor group (n = 231), 8 (–1 to 17) in the ARB group (n = 217), and 12 (0 to 17) in the control group (n = 231) (median adjusted odds ratios of 0.77 [95% bayesian credible interval, 0.58-1.06] for improvement for ACE inhibitor and 0.76 [95% credible interval, 0.56-1.05] for ARB compared with control). The posterior probabilities that ACE inhibitors and ARBs worsened organ support–free days compared with control were 94.9% and 95.4%, respectively. Hospital survival occurred in 166 of 231 critically ill participants (71.9%) in the ACE inhibitor group, 152 of 217 (70.0%) in the ARB group, and 182 of 231 (78.8%) in the control group (posterior probabilities that ACE inhibitor and ARB worsened hospital survival compared with control were 95.3% and 98.1%, respectively). CONCLUSIONS AND RELEVANCE In this trial, among critically ill adults with COVID-19, initiation of an ACE inhibitor or ARB did not improve, and likely worsened, clinical outcomes. TRIAL REGISTRATION ClinicalTrials.gov Identifier: NCT0273570