Storage temperature determines platelet GPVI levels and function in mice and humans.
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ABSTRACT:
SUBMITTER: Miles J
PROVIDER: S-EPMC8679671 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:BackgroundContinuous agitation during storage slows down the platelet storage lesions. However, in special circumstances, manual-mixing can be alternatively used to store products for short time periods without compromising platelet quality. Based on this finding, and given the role of shear stress in modulating receptor expression, we were interested in comparing the levels of platelet adhesion receptor, GPVI and platelet adhesion capacity under each storage condition.MethodsPlatelet concentrates (PCs) were divided into three groups: continuously-agitated PCs (CAG-PCs) with or without PP2 (Src kinase inhibitor) and manually-mixed PCs (MM-PCs). Platelet count/MPV, swirling, GPVI and P-selectin expression, GPVI shedding, platelet adhesion/spreading to collagen were examined during 5 days of storage.ResultsWhile MM- and CAG-PCs showed similar levels of P-selectin expression, GPVI expression was significantly elevated in MM-PCs with lower GPVI shedding/expression ratios, enhanced platelet adhesion/spreading and swirling in manually-mixed PCs. Of note, CAG-PCs treated with PP2 also demonstrated lower P-selectin expression and GPVI shedding, higher GPVI expression and attenuated swirling and spreading capability.ConclusionGiven the comparable platelet activation state in MM and CAG-PCs as indicated by P-selectin expression, enhanced platelet adhesion/spreading in MM-PCs, along with relatively higher GPVI expression here, supports previous studies demonstrating a role for biomechanical forces in modulating GPVI-dependent function. Thus, lower GPVI expression in CAG-PCs may be due to shear forces induced by agitation, which keeps this receptor down-regulated while also attenuating platelet adhesion/spreading capacities during storage. Low platelet function in PP2-CAG-PCs also highlights the importance of Src-kinases threshold activity in maintaining platelets quality.
| S-EPMC8756730 | biostudies-literature
Project description:BackgroundDue to mTOR (mammalian/mechanistic target of rapamycin) gene-loss mice die during embryonic development, the role of mTOR in platelets has not been evaluated using gene knockout technology.MethodsA mouse model with megakaryocyte/platelet-specific deletion of mTOR was established, and be used to evaluate the role of mTOR in platelet activation and thrombus formation.ResultsmTOR-/- platelets were deficient in thrombus formation when grown on low-concentration collagen-coated surfaces; however, no deficiency in thrombus formation was observed when mTOR-/- platelets were perfused on higher concentration collagen-coated surfaces. In FeCl3-induced mouse mesenteric arteriole thrombosis models, wild-type (WT) and mTOR-/- mice displayed significantly different responses to low-extent injury with respect to the ratio of occluded mice, especially within the first 40 min. Additionally, mTOR-/- platelets displayed reduced aggregation and dense granule secretion (ATP release) in response to low doses of the glycoprotein VI (GPVI) agonist collagen related peptide (CRP) and the protease-activated receptor-4 (PAR4) agonist GYPGKF-NH2; these deficiencies were overcame by stimulation with higher concentration agonists, suggesting dose dependence of the response. At low doses of GPVI or PAR agonist, the activation of αIIbβ3 in mTOR-/- platelets was reduced. Moreover, stimulation of mTOR-/- platelets with low-dose CRP attenuated the phosphorylation of S6K1, S6 and Akt Ser473, and increased the phosphorylation of PKCδ Thr505 and PKCε Ser729. Using isoform-specific inhibitors of PKCs (δ, ɛ, and α/β), we established that PKCδ/ɛ, and especially PKCδ but not PKCα/β or PKCθ, may be involved in low-dose GPVI-mediated/mTOR-dependent signaling.ConclusionThese observations indicate that mTOR plays an important role in GPVI-dependent platelet activation and thrombus formation.
| S-EPMC8111939 | biostudies-literature
Project description:Reports from both adult and paediatric populations indicate that approximately two-thirds of platelet transfusions are used prophylactically to prevent bleeding, while the remaining one-third are used therapeutically to manage active bleeding. These two indications, prophylactic and therapeutic, serve two very distinct purposes and therefore will have two different functional requirements. In addition, disease aetiology in a given patient may require platelets with different functional characteristics. These characteristics can be derived from the various manufacturing methods used in platelet product production, including collection methods, processing methods, and storage options. The iterative combinations of manufacturing methods can result in a number of unique platelet products with different efficacy and safety profiles, which could potentially be used to benefit patient populations by meeting diverse clinical needs. In particular, cold storage of platelet products causes many biochemical and functional changes, of which the most notable characterised to date include increased haemostatic activity and altered expression of molecules inherent to platelet:leucocyte interactions. The in vivo consequences, both short- and long-term, of these molecular and cellular cold-storage-induced changes have yet to be clearly defined. Elucidation of these mechanisms would potentially reveal unique biologies that could be harnessed to provide more targeted therapies. To this end, in this new era of personalised medicine, perhaps there is an opportunity to provide individual patients with platelet products that are tailored to their clinical condition and the specific indication for transfusion.
| S-EPMC6683864 | biostudies-literature
Project description:AbstractGlycoprotein VI (GPVI) plays a key role in collagen-induced platelet aggregation. Affimers are engineered binding protein alternatives to antibodies. We screened and characterized GPVI-binding Affimers as novel tools to probe GPVI function. Among the positive clones, M17, D22, and D18 bound GPVI with the highest affinities (dissociation constant (KD) in the nanomolar range). These Affimers inhibited GPVI-collagen-related peptide (CRP)-XL/collagen interactions, CRP-XL/collagen-induced platelet aggregation, and D22 also inhibited in vitro thrombus formation on a collagen surface under flow. D18 bound GPVI dimer but not monomer. GPVI binding was increased for D18 but not M17/D22 upon platelet activation by CRP-XL and adenosine 5'-diphosphate. D22 but not M17/D18 displaced nanobody 2 (Nb2) binding to GPVI, indicating similar epitopes for D22 with Nb2 but not for M17/D18. Mapping of binding sites revealed that D22 binds a site that overlaps with Nb2 on the D1 domain, whereas M17 targets a site on the D2 domain, overlapping in part with the glenzocimab binding site, a humanized GPVI antibody fragment antigen-binding fragment. D18 targets a new region on the D2 domain. We found that D18 is a stable noncovalent dimer and forms a stable complex with dimeric GPVI with 1:1 stoichiometry. Taken together, our data demonstrate that Affimers modulate GPVI-ligand interactions and bind different sites on GPVI D1/D2 domains. D18 is dimer-specific and could be used as a tool to detect GPVI dimerization or clustering in platelets. A dimeric epitope regulating ligand binding was identified on the GPVI D2 domain, which could be used for the development of novel bivalent antithrombotic agents selectively targeting GPVI dimer on platelets.
| S-EPMC11321386 | biostudies-literature
Project description:Platelet activators stimulate post-translational modification of signalling proteins to change their activity or their molecular interactions leading to signal propagation. One covalent modification is attachment of the small protein ubiquitin to lysine residues in target proteins. Modification by ubiquitin can either target proteins for degradation by the proteasome or act as a scaffold for other proteins. Pharmacological inhibition of deubiquitylases or the proteasome inhibition of platelet activation by collagen, demonstrating a role for ubiquitylation, but relatively few substrates for ubiquitin have been identified and the molecular basis of inhibition is not established. Here, we report the ubiquitome of human platelets and changes in ubiquitylated proteins following stimulation by collagen-related peptide (CRP-XL). Using platelets from six individuals over three independent experiments, we identified 1,634 ubiquitylated peptides derived from 691 proteins, revealing extensive ubiquitylation in resting platelets. Note that 925 of these peptides show an increase of more than twofold following stimulation with CRP-XL. Multiple sites of ubiquitylation were identified on several proteins including Syk, filamin and integrin heterodimer sub-units. This work reveals extensive protein ubiquitylation during activation of human platelets and opens the possibility of novel therapeutic interventions targeting the ubiquitin machinery.
| S-EPMC6327716 | biostudies-literature
Project description:ObjectivePlatelet transfusion is a life-saving therapy to prevent or treat bleeding in patients with thrombocytopenia or platelet dysfunction. However, for >6 decades, safe and effective strategies for platelet storage have been an impediment to widespread use of platelet transfusion. Refrigerated platelets are cleared rapidly from circulation, precluding cold storage of platelets for transfusion. Consequently, platelets are stored at room temperature with an upper limit of 5 days due to risks of bacterial contamination and loss of platelet function. This practice severely limits platelet availability for transfusion. This study is to identify the mechanism of platelet clearance after cold storage and develop a method for platelet cold storage. Approach and Results: We found that rapid clearance of cold-stored platelets was largely due to integrin activation and apoptosis. Deficiency of integrin β3 or caspase-3 prolonged cold-stored platelets in circulation. Pretreatment of platelets with EGTA, a cell impermeable calcium ion chelator, reversely inhibited cold storage-induced platelet activation and consequently prolonged circulation of cold-stored platelets. Moreover, transfusion of EGTA-treated, cold-stored platelets, but not room temperature-stored platelets, into the mice deficient in glycoprotein Ibα significantly shortened tail-bleeding times and diminished blood loss.ConclusionsIntegrin activation and apoptosis is the underlying mechanism of rapid clearance of platelets after cold storage. Addition of a cell impermeable calcium ion chelator to platelet products is potentially a simple and effective method to enable cold storage of platelets for transfusion.
| S-EPMC8158249 | biostudies-literature
Project description:low temperature storage Raw sequence reads
| PRJNA861369 | ENA
Project description:CRP cytosol donor 1
2011-12-05 | PRD000535 | Pride
Project description:Introduction: Luteolin inhibits platelet activation and thrombus formation, but the mechanisms are unclear. This study investigated the effects of luteolin on GPVI-mediated platelet activation in vitro and explored the effect of luteolin on thrombosis, coagulation, and platelet production in vivo. Methods: Washed human platelets were used for aggregation, membrane protein expression, ATP, Ca2+, and LDH release, platelet adhesion/spreading, and clot retraction experiments. Washed human platelets were used to detect collagen and convulxin-induced reactive oxygen species production and endogenous antioxidant effects. C57BL/6 male mice were used for ferric chloride-induced mesenteric thrombosis, collagen-epinephrine induced acute pulmonary embolism, tail bleeding, coagulation function, and luteolin toxicity experiments. The interaction between luteolin and GPVI was analyzed using solid phase binding assay and surface plasmon resonance (SPR). Results: Luteolin inhibited collagen- and convulxin-mediated platelet aggregation, adhesion, and release. Luteolin inhibited collagen- and convulxin-induced platelet ROS production and increased platelet endogenous antioxidant capacity. Luteolin reduced convulxin-induced activation of ITAM and MAPK signaling molecules. Molecular docking simulation showed that luteolin forms hydrogen bonds with GPVI. The solid phase binding assay showed that luteolin inhibited the interaction between collagen and GPVI. Surface plasmon resonance showed that luteolin bonded GPVI. Luteolin inhibited integrin αIIbβ3-mediated platelet activation. Luteolin inhibited mesenteric artery thrombosis and collagen- adrenergic-induced pulmonary thrombosis in mice. Luteolin decreased oxidative stress in vivo. Luteolin did not affect coagulation, hemostasis, or platelet production in mice. Discussion: Luteolin may be an effective and safe antiplatelet agent target for GPVI. A new mechanism (decreased oxidative stress) for the anti-platelet activity of luteolin has been identified.
| S-EPMC10644720 | biostudies-literature
Project description:Recombination, the precise physical breakage and rejoining of DNA between homologous chromosomes, plays a central role in mediating the orderly segregation of meiotic chromosomes in most eukaryotes. Despite its importance, the factors that control the number and placement of recombination events within a cell remain poorly defined. The rate of recombination exhibits remarkable species specificity, and, within a species, recombination is affected by the physical size of the chromosome, chromosomal location, proximity to other recombination events (i.e., chiasma interference), and, intriguingly, the sex of the transmitting parent. To distinguish between simple genetic and nongenetic explanations of sex-specific recombination differences in mammals, we compared recombination in meiocytes from XY sex-reversed and XO females with that in meiocytes from XX female and XY male mice. The rate and pattern of recombination in XY and XO oocytes were virtually identical to those in normal XX females, indicating that sex, not genotype, is the primary determinant of meiotic recombination patterns in mammals.
| S-EPMC1275616 | biostudies-literature