Ca2+ waves coordinate purinergic receptor–evoked integrin activation and polarization

The integrin GPIIb/IIIa is highly abundant on the surface of platelets and can be activated by intracellular Ca2+ signaling in an “inside-out” manner to bind to the adhesive ligand fibrinogen. Bye et al. imaged intracellular Ca2+ signaling and fibrinogen binding events in primary rat megakaryocytes activated through the ADP-stimulated receptors P2Y1 and P2Y12. The authors found that signaling by both receptors was required for full integrin activation, which depended on P2Y1-stimulated Ca2+ signaling and P2Y12-stimulated activation of the kinase PI3K. In addition, fibrinogen binding became polarized in these cells in a manner dependent on the direction of ADP-stimulated Ca2+ waves

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

Cobimetinib and trametinib inhibit platelet MEK but do not cause platelet dysfunction

The MEK inhibitors cobimetinib and trametinib are used in combination with BRAF inhibitors to treat metastatic melanoma but increase rates of hemorrhage relative to BRAF inhibitors alone. Platelets express several members of the MAPK signalling cascade including MEK1 and MEK2 and ERK1 and ERK2 but their role in platelet function and haemostasis is ambiguous as previous reports have been contradictory. It is therefore unclear if MEK inhibitors might be causing platelet dysfunction and contributing to increased hemorrhage. In the present study we performed pharmacological characterisation of cobimetinib and trametinib in vitro to investigate potential for MEK inhibitors to cause platelet dysfunction. We report that whilst both cobimetinib and trametinib are potent inhibitors of platelet MEK activity, treatment with trametinib did not alter platelet function. Treatment with cobimetinib results in inhibition of platelet aggregation, integrin activation, alpha-granule secretion and adhesion but only at suprapharmacological concentrations. We identified that the inhibitory effects of high concentrations of cobimetinib are associated with off-target inhibition on Akt and PKC. Neither inhibitor caused any alteration in thrombus formation on collagen under flow conditions in vitro. Our findings demonstrate that platelets are able to function normally when MEK activity is fully inhibited, indicating MEK activity is dispensable for normal platelet function. We conclude that the MEK inhibitors cobimetinib and trametinib do not induce platelet dysfunction and are therefore unlikely to contribute to increased incidence of bleeding reported during MEK inhibitor therapy

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

Farnesoid X Receptor and its ligands inhibit the function of platelets

Objective - While initially seemingly paradoxical due to the lack of nucleus, platelets possess a number of transcription factors that regulate their function through DNA-independent mechanisms. These include the Farnesoid X Receptor (FXR), a member of the superfamily of ligand-activated transcription factors that has been identified as a bile acid receptor. In this study, we show that FXR is present in human platelets and FXR ligands, GW4064 and 6-ECDCA, modulate platelet activation nongenomically. Approach and Results - FXR ligands inhibited the activation of platelets in response to stimulation of collagen or thrombin receptors, resulting in diminished intracellular calcium mobilization and secretion, fibrinogen binding and aggregation. Exposure to FXR ligands also reduced integrin alphaIIbbeta3 outside-in signaling and thereby reduced the ability of platelets to spread and to stimulate clot retraction. FXR function in platelets was found to be associated with the modulation of cGMP levels in platelets and associated downstream inhibitory signaling. Platelets from FXR-deficient mice were refractory to the actions of FXR agonists on platelet function and cyclic nucleotide signaling, firmly linking the non-genomic actions of these ligands to the FXR receptor. Conclusion – This study provides support for the ability of FXR ligands to modulate platelet activation. The athero-protective effects of GW4064, with its novel antiplatelet effects, indicate FXR as a potential target for prevention of athero-thrombotic disease

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

The Gene Ontology knowledgebase in 2023

The Gene Ontology (GO) knowledgebase (http://geneontology.org) is a comprehensive resource concerning the functions of genes and gene products (proteins and noncoding RNAs). GO annotations cover genes from organisms across the tree of life as well as viruses, though most gene function knowledge currently derives from experiments carried out in a relatively small number of model organisms. Here, we provide an updated overview of the GO knowledgebase, as well as the efforts of the broad, international consortium of scientists that develops, maintains, and updates the GO knowledgebase. The GO knowledgebase consists of three components: (1) the GO-a computational knowledge structure describing the functional characteristics of genes; (2) GO annotations-evidence-supported statements asserting that a specific gene product has a particular functional characteristic; and (3) GO Causal Activity Models (GO-CAMs)-mechanistic models of molecular "pathways" (GO biological processes) created by linking multiple GO annotations using defined relations. Each of these components is continually expanded, revised, and updated in response to newly published discoveries and receives extensive QA checks, reviews, and user feedback. For each of these components, we provide a description of the current contents, recent developments to keep the knowledgebase up to date with new discoveries, and guidance on how users can best make use of the data that we provide. We conclude with future directions for the project

Mutations in DONSON disrupt replication fork stability and cause microcephalic dwarfism

To ensure efficient genome duplication, cells have evolved numerous factors that promote unperturbed DNA replication and protect, repair and restart damaged forks. Here we identify downstream neighbor of SON (DONSON) as a novel fork protection factor and report biallelic DONSON mutations in 29 individuals with microcephalic dwarfism. We demonstrate that DONSON is a replisome component that stabilizes forks during genome replication. Loss of DONSON leads to severe replication-associated DNA damage arising from nucleolytic cleavage of stalled replication forks. Furthermore, ATM- and Rad3-related (ATR)-dependent signaling in response to replication stress is impaired in DONSON-deficient cells, resulting in decreased checkpoint activity and the potentiation of chromosomal instability. Hypomorphic mutations in DONSON substantially reduce DONSON protein levels and impair fork stability in cells from patients, consistent with defective DNA replication underlying the disease phenotype. In summary, we have identified mutations in DONSON as a common cause of microcephalic dwarfism and established DONSON as a critical replication fork protein required for mammalian DNA replication and genome stability

A randomized controlled trial of self-regulated modified constraint-induced movement therapy in sub-acute stroke patients

Background and purpose: Emerging research suggests the use of self-regulation (SR) for improving functional regain in patients post stroke. SR is proposed to produce an added effect to effective modified constraint-induced movement therapy (mCIMT). This study aimed to examine the effect of a self-regulated mCIMT programme (SR-mCIMT) for functional regain in patients with sub-acute stroke. Methods: Eighty-six patients completed the trial: SR-mCIMT, n = 29; mCIMT, n = 31; or conventional functional rehabilitation, n = 26. All interventions were 2-week therapist-guided training. Outcome measurements, taken by a blinded assessor, examined arm function [Action Research Arm Test (ARAT), Fugl-Meyer Assessment (FMA)], daily task performance [Lawton Instrumental Activities of Daily Living Scale (Lawton IADL)] and self-perceived arm use in functional tasks [Motor Activity Log (MAL)]. Results: Significant differences were found with the SR-mCIMT outperforming the other groups after the intervention (ARAT, P = 0.006; FMA, Lawton IADL and MAL, all Ps < 0.001). In terms of the carry-over effect, the SR-mCIMT group outperformed in the hand and coordination subscales of ARAT and FMA (P = 0.012-0.013) and the self-perceived quality of arm use (P = 0.002). Conclusion: A combination of SR and mCIMT could produce an added effect in functional regain in patients post stroke