Mouse Platelets Research Articles

Critical role for platelet Ral GTPases in regulating venous thrombosis in mice

Deep vein thrombosis (DVT) is a major cause of morbidity and mortality globally. While its pathophysiology is complex, increasing evidence suggests a more prominent role for platelets than previously suspected. Genetic deletion of Ral GTPases, RalA and RalB, conditionally in mouse platelets (RalAB DKO), results in a near complete defect in P-selectin externalisation upon activation, while other platelet activation responses and arterial thrombosis are preserved. Given the critical role of P-selectin in mediating platelet-neutrophil interaction and thromboinflammation, we sought to investigate whether platelet Rals would also play critical roles in venous thrombosis, a thromboinflammatory disease, using RalAB DKO mice. DVT was induced by surgical partial ligation of the caudal (inferior) vena cava for 24-h or 48-h before venous thrombi were assessed by histology and immunofluorescence microscopy. RalAB DKO mice show a reduction in venous thrombus formation after 24-h, and near complete ablation of venous thrombosis by 48-h post IVC ligation. Immunofluorescence microscopy revealed that cross sections of thrombi from wildtype mice consist of an organised scaffolded structure of platelets surrounding leukocytes/neutrophils producing NETs to stabilize and propagate the thrombus. This organised structure was absent in platelet-specific conditional RalAB DKO thrombi. In vitro analysis of platelet-mediated NET formation was also significantly reduced when platelets lacked RalAB or when platelets were treated with the Ral inhibitor RBC8. We identify platelet Rals as novel, potentially critical regulators of venous thrombus stability, through their ability to regulate neutrophil NET formation via platelet P-selectin.

Suppressed ORAI1-STIM1-dependent Ca2+ entry by protein kinase C isoforms regulating platelet procoagulant activity

Agonist-induced rises in cytosolic Ca2+ control most platelet responses in thrombosis and hemostasis. In human platelets we earlier demonstrated that the ORAI1-STIM1 pathway is a major component of extracellular Ca2+ entry, in particular when induced via the ITAM-linked collagen receptor, glycoprotein VI (GPVI). In the present paper, using functionally defective platelets from patients with a loss-of-function mutation in ORAI1 or STIM1, we show that Ca2+ entry induced by the endoplasmic reticulum ATPase inhibitor, thapsigargin, fully relies on this pathway. We demonstrate that both the GPVI-induced and thapsigargin-induced Ca2+ entry is strongly suppressed by protein kinase C (PKC) activation, while leaving intracellular Ca2+ mobilization unchanged. Comparing effects of a PKC inhibitory panel pointed to redundant roles of beta and theta PKC isoforms in Ca2+-entry suppression. In contrast, tyrosine kinases positively regulated GPVI-induced Ca2+ entry and mobilization. Label-free and stable isotope phosphoproteome analysis of GPVI-stimulated platelets suggested a regulatory role of bridging integrator-2 (BIN2), known as important mediator of the ORAI1-STIM1 pathway in mouse platelets. Identified were 25-45 regulated phospho- sites in BIN2 and 16-18 in STIM1. Five of these were characterized as direct substrates of the expressed PKC isoforms alpha, beta delta and theta. Functional platelet testing indicated that the downregulation of Ca2+ entry by PKC resulted in suppressed phosphatidylserine exposure and plasmatic thrombin generation. Conclusively, our results indicate that in platelets multiple PKC isoforms constrain the store-regulated Ca2+ entry via ORAI1-BIN2-STIM1, and hence downregulate platelet-dependent coagulation.

The humanized platelet glycoprotein VI Fab inhibitor EMA601 protects from arterial thrombosis and ischaemic stroke in mice.

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

Platelet Ido1 expression is induced during Plasmodium yoelii infection, altering plasma tryptophan metabolites

AbstractPlatelets are immune responsive in many diseases as noted by changes in platelet messenger RNA in conditions such as sepsis, atherosclerosis, COVID-19, and many other inflammatory and infectious etiologies. The malaria causing Plasmodium parasite is a persistent public health threat and significant evidence shows that platelets participate in host responses to infection. Using a mouse model of nonlethal/uncomplicated malaria, Plasmodium yoelii XNL (PyNL)-infected but not control mouse platelets expressed Ido1, a rate limiting enzyme in tryptophan metabolism that increases kynurenine at the expense of serotonin. Interferon-γ (IFN-γ) is a potent inducer of Ido1 and mice treated with recombinant IFN-γ had increased platelet Ido1 and IDO1 activity. PyNL-infected mice treated with anti-IFN-γ antibody had similar platelet Ido1 and metabolic profiles to that of uninfected controls. PyNL-infected mice become thrombocytopenic by day 7 after infection and transfusion of platelets from IFN-γ–treated wild-type mice but not Ido1−/− mice increased the plasma kynurenine-to-tryptophan ratio, indicating that platelets are a source of postinfection IDO1 activity. We generated platelet-specific Ido1 knockout mice to assess the contribution of platelet Ido1 during PyNL infection. Platelet-specific Ido1−/− mice had increased death and evidence of lung thrombi, which were not present in infected wild-type mice. Platelet Ido1 may be a significant contributor to plasma kynurenine in IFN-γ-driven immune processes and the loss of platelets may limit total Ido1, leading to immune and vascular dysfunction.

Biomedical application of TiO2NPs can cause arterial thrombotic risks through triggering procoagulant activity, activation and aggregation of platelets.

Titanium dioxide nanoparticles (TiO2NPs) are widely used in medical application. However, the relevant health risk has not been completely assessed, the potential of inducing arterial thrombosis (AT) in particular. Alterations in platelet function and susceptibility to arterial thrombosis induced by TiO2NPs were examined using peripheral blood samples from healthy adult males and an in vivo mouse model, respectively. Here, using human platelets (hPLTs) freshly isolated from health volunteers, we demonstrated TiO2NP treatment triggered the procoagulant activity of hPLTs through phosphatidylserine exposure and microvesicles generation. In addition, TiO2NP treatment increased the levels of glycoprotein IIb/IIIa and P-selectin leading to aggregation and activation of hPLTs, which were exacerbated by providing physiology-mimicking conditions, including introduction of thrombin, collagen, and high shear stress. Interestingly, intracellular calcium levels in hPLTs were increased upon TiO2NP treatment, which were crucial in TiO2NP-induced hPLT procoagulant activity, activation and aggregation. Moreover, using mice in vivo models, we further confirmed that TiO2NP treatment a reduction in mouse platelet (mPLT) counts, disrupted blood flow, and exacerbated carotid arterial thrombosis with enhanced deposition of mPLT. Together, our study provides evidence for an ignored health risk caused by TiO2NPs, specifically TiO2NP treatment augments procoagulant activity, activation and aggregation of PLTs via calcium-dependent mechanism and thus increases the risk of AT.

LAIR-1 and PECAM-1 function via the same signaling pathway to inhibit GPVI-mediated platelet activation

BackgroundInhibition of platelet responsiveness is important for controlling thrombosis. It is well established that platelet endothelial cell adhesion molecule-1 (PECAM-1) serves as a physiological negative regulator of platelet-collagen interactions. We recently demonstrated that leukocyte-associated immunoglobulin-like receptor-1 (LAIR-1) is a negative regulator of platelet production and reactivity. It is however not known if LAIR-1 and PECAM-1 function in the same or different inhibitory pathways. ObjectivesIn this study, we investigated the role of LAIR-1 alongside PECAM-1 in megakaryocyte development and platelet production and determined the functional redundancy through characterization of a LAIR-1/PECAM-1 double knockout (DKO) mouse model. MethodsLAIR-1 and PECAM-1 expression in megakaryocytes were evaluated by western blotting. Megakaryocyte ploidy and proplatelet formation were evaluated by flow cytometry and fluorescent microscopy. Platelet function and signalling were compared in wild-type, LAIR-1−/−, PECAM-1−/− and DKO mice using aggregometry, flow cytometry and western blotting. Thrombosis was evaluated using the FeCl3 carotid artery model. ResultsWe show that LAIR-1/PECAM-1 DKO mice exhibit a 17% increase in platelet count. Bone marrow-derived megakaryocytes from all 3 mouse models had normal ploidy in vitro, suggesting that neither LAIR-1 nor PECAM-1 regulates megakaryocyte development. Furthermore, relative to wild-type platelets, platelets derived from LAIR-1, PECAM-1, and DKO mice were equally hyperresponsive to collagen in vitro, indicating that LAIR-1 and PECAM-1 participate in the same inhibitory pathway. Interestingly, DKO mice exhibited normal thrombus formation in vivo due to DKO mouse platelets lacking the enhanced Src family kinase activation previously shown in platelets from LAIR-1-deficient mice. ConclusionFindings from this study reveal that LAIR-1 and PECAM-1 act to inhibit GPVI-mediated platelet activation via the same signaling pathway. Mice lacking LAIR-1 and PECAM-1 do not however exhibit an increase in thrombus formation despite minor increase in platelet count and reactivity to collagen. This study adds to the growing evidence that immunoreceptor tyrosine-based inhibition motif–containing receptors are important regulators of platelet count and function.

Thrombocytopenia in SFTS due to platelets with altered function undergoing cell death pathways.

Thrombocytopenia is the major clinical feature associated with the severity of SFTS, but the mechanism by which it occurs remains unclear. RNA transcriptome analyses were performed on platelets purified from SFTS patients and SFTSV-infected mice. The functions of differentially expressed genes (DEGs) in the platelets were characterized. ELISA, flow cytometry, and qRT-PCR were used to measure the levels of platelet activation, SFTSV infection in platelets, formation of neutrophil extracellular traps (NETs), transcription of DEGs and percent of platelets undergoing cell death. Enhanced neutrophil activation and interferon (IFN) signaling involved in the viral life cycle were common platelet responses in SFTS, which may consume increasing numbers of platelets. Other functional changes may be associated with different outcomes of SFTS. SFTSV infection led to platelet destruction by pyroptosis, apoptosis, necroptosis, and autophagy. In contrast to SFTS patients, platelets in SFTSV-infected mice mainly play a role in adaptive immunity, and platelet death was not as severe as in humans. The altered functions of platelets, such as mediating leukocyte activation and undergoing cell death, contribute to thrombocytopenia in SFTS patients. The different mechanisms of thrombocytopenia in mice, suggest that platelet functions should be considered in experimental animal models.

Antidiabetic potential of Selenium nanoparticles and plasma-rich platelets in diabetic mice

Diabetes mellitus is a widespread endocrine disorder, which is categorized as the fourth leading cause of global mortality. Allopathic medicine has yet to provide a satisfactory cure for this condition. Consequently, there is an urgent demand for innovative antidiabetic treatment approaches with enhanced management and minimum side effects and costs. The study investigated the synergistic antidiabetic potential of combining selenium nanoparticles (Se NPs) and plasma-rich platelets (PRP) in diabetic mice. Antidiabetic activity of the proposed combination (Se NPs and PRP) was evaluated from histopathological and biochemical perspectives. The experiment involved alloxan monohydrate induced diabetic mouse model. In the in vivo study, several biochemical parameters for assessing the antidiabetic effect of the novel combination of (Se NPs and PRP) were performed such as blood glucose levels, body weight, lipid profiles, and liver damage markers (AST and ALT). Scavenging antioxidant activity was assessed by evaluation levels of hepatic and renal GSH, MDA, SOD, and CAT activities. Complete histopathological examinations of vital internal organs were carried out. Results revealed that combining Se NPs and PRP presents a novel approach for better diabetes management and reduced complications associated with the disease. These findings have therapeutic implications for managing diabetes mellitus.

Associations of Cadmium Exposure with Peripheral Blood Cell Subtype Counts and Indices in Cadmium-Poisoned Mice.

This research was designed to investigate the effects of cadmium on blood cell injury in cadmium-poisoned mice. Twenty mice were randomly divided into two groups: control group and model group. The control group was intraperitoneally injected with normal saline every day and the model group was intraperitoneally injected with 1.4mg/kg cadmium solution every day. The experimental period was 28days. The blood of the mice was collected for detection and hematological analysis. The results demonstrated that cadmium increased the number of white blood cells and the number of neutrophils in mice. Cadmium reduced the number of eosinophils, the number of basophils, the number of monocytes, the amount of lymphocytes, the number of red blood cells, the hemoglobin concentration, mean corpusular volume, mean corpusular hemoglobin, mean corpusular hemoglobin concentration, and the number of platelets in mice. In summary, cadmium caused some damage to white blood cells, red blood cells, and platelets in mice, but the direction of damage to different cells was inconsistent. The possible reason for this result is that cadmium damages the generation of blood cells, and the body takes corresponding defense measures.

Hydrogen Sulfide Promotes Platelet Autophagy via PDGFR-α/PI3K/Akt Signaling in Cirrhotic Thrombocytopenia.

The role of platelet autophagy in cirrhotic thrombocytopenia (CTP) remains unclear. This study aimed to investigate the impact of platelet autophagy in CTP and elucidate the regulatory mechanism of hydrogen sulfide (H2S) on platelet autophagy. Platelets from 56 cirrhotic patients and 56 healthy individuals were isolated for in vitro analyses. Autophagy markers (ATG7, BECN1, LC3, and SQSTM1) were quantified using enzyme-linked immunosorbent assay, while autophagosomes were visualized through electron microscopy. Western blotting was used to assess the autophagy-related proteins and the PDGFR/PI3K/Akt/mTOR pathway following treatment with NaHS (an H2S donor), hydroxocobalamin (an H2S scavenger), or AG 1295 (a selective PDGFR-α inhibitor). A carbon tetrachloride-induced cirrhotic BALB/c mouse model was established. Cirrhotic mice with thrombocytopenia were randomly treated with normal saline, NaHS, or hydroxocobalamin for 15 days. Changes in platelet count and aggregation rate were observed every three days. Cirrhotic patients with thrombocytopenia exhibited significantly decreased platelet autophagy markers and endogenous H2S levels, alongside increased platelet aggregation, compared to healthy controls. In vitro, NaHS treatment of platelets from severe CTP patients elevated LC3-II levels, reduced SQSTM1 levels, and decreased platelet aggregation in a dose-dependent manner. H2S treatment inhibited PDGFR, PI3K, Akt, and mTOR phosphorylation. In vivo, NaHS significantly increased LC3-II and decreased SQSTM1 expressions in platelets of cirrhotic mice, reducing platelet aggregation without affecting the platelet count. Diminished platelet autophagy potentially contributes to thrombocytopenia in cirrhotic patients. H2S modulates platelet autophagy and functions possibly via the PDGFR-α/PI3K/Akt/mTOR signaling pathway.

Isaridin E Protects against Sepsis by Inhibiting Von Willebrand Factor-Induced Endothelial Hyperpermeability and Platelet-Endothelium Interaction.

Endothelial hyperpermeability is pivotal in sepsis-associated multi-organ dysfunction. Increased von Willebrand factor (vWF) plasma levels, stemming from activated platelets and endothelium injury during sepsis, can bind to integrin αvβ3, exacerbating endothelial permeability. Hence, targeting this pathway presents a potential therapeutic avenue for sepsis. Recently, we identified isaridin E (ISE), a marine-derived fungal cyclohexadepsipeptide, as a promising antiplatelet and antithrombotic agent with a low bleeding risk. ISE's influence on septic mortality and sepsis-induced lung injury in a mouse model of sepsis, induced by caecal ligation and puncture, is investigated in this study. ISE dose-dependently improved survival rates, mitigating lung injury, thrombocytopenia, pulmonary endothelial permeability, and vascular inflammation in the mouse model. ISE markedly curtailed vWF release from activated platelets in septic mice by suppressing vesicle-associated membrane protein 8 and soluble N-ethylmaleide-sensitive factor attachment protein 23 overexpression. Moreover, ISE inhibited healthy human platelet adhesion to cultured lipopolysaccharide (LPS)-stimulated human umbilical vein endothelial cells (HUVECs), thereby significantly decreasing vWF secretion and endothelial hyperpermeability. Using cilengitide, a selective integrin αvβ3 inhibitor, it was found that ISE can improve endothelial hyperpermeability by inhibiting vWF binding to αvβ3. Activation of the integrin αvβ3-FAK/Src pathway likely underlies vWF-induced endothelial dysfunction in sepsis. In conclusion, ISE protects against sepsis by inhibiting endothelial hyperpermeability and platelet-endothelium interactions.

Thioredoxin-related transmembrane protein 1 negatively regulates coagulation and phosphatidylserine exposure

BackgroundFive secreted platelet protein disulfide isomerases (PDIs) and 1 transmembrane PDI regulate platelet function and thrombosis. Thioredoxin-related transmembrane protein 1 (TMX1) was the first member of the PDI family found to negatively regulate platelet aggregation and platelet accumulation in vivo. The effect of TMX1 on coagulation is unknown. ObjectivesTo determine the effect of TMX1 on coagulation. MethodsTMX1-/- mice were used to study platelet accumulation and fibrin deposition in vivo in the laser-induced thrombosis injury model. Annexin V deposition at the site of vascular injury was studied using conditional TMX1 knockout mice. Annexin V binding to platelets was studied using human platelets, anti-TMX1 antibodies, and TMX1-deficient platelets. ResultsTMX1-/- mice had increased fibrin deposition that was reversed with infusion of recombinant TMX1. Infusion of recombinant TMX1 inhibited platelet accumulation and fibrin deposition in wild-type mice and inhibited fibrin deposition in β3-null mice. Platelet accumulation is absent in β3-null mice, suggesting that TMX1 inhibits coagulation independently of platelets. Annexin V binding was increased in activated human platelets incubated with an anti-TMX1 antibody and mouse platelets lacking TMX1. Addition of recombinant TMX1 decreased annexin V binding to platelets. Annexin V binding was increased at the site of vascular injury in Tie2-Cre/TMX1fl/fl mice deficient in endothelial cell TMX1. ConclusionTMX1 decreases coagulation at the site of vascular injury and negatively regulates phosphatidylserine exposure on endothelial cells and platelets.

Biotin labeling allows for post-transfusion functional assessment of stored human platelets in mice.

Platelet radiolabeling with radioisotopes is currently used for human platelet recovery and survival studies. Biotinylation enables ex vivo post-transfusion platelet function testing. Whether platelet biotinylation itself affects platelet function is controversial. Platelet concentrates from healthy humans were stored for 6 days. Samples were obtained at 1 or 2 and 6 days, and platelets were labeled following a radiolabeling protocol using saline instead of radioactive indium-111 (sham radiolabeling [sham-RL]). Alternatively, a newly developed biotinylation protocol, a washing protocol, or an unmanipulated control sample were used. Platelet function was assessed by flow cytometry after stimulation with platelet agonists and labeling of platelets with platelet activation markers. To test whether platelets can be activated after transfusion, labeled platelets were transfused into nonobese diabetic/severe combined immunodeficiency mice, and samples were obtained 1 h after transfusion. The activation profile of biotinylated platelets was comparable to sham-RL platelets before transfusion except for significantly less α-degranulation and more phosphatidyl serine exposure on storage day 1/2. There was no significant difference between sham-RL and biotinylated platelets on storage day 6. Sham-RL and biotinylated platelets were significantly less activatable than washed and unmanipulated control platelets. After transfusion, the activation profile of biotinylated platelets was largely indistinguishable from unmanipulated ones. The decrease in activation level in biotinylated platelets we and others observed appears mainly due to the physical manipulation during the labeling process. In conclusion, biotinylated platelets allow for post-transfusion function assessment, a major advantage over radiolabeling.

The effect of near-infrared low-level light on the in vitro quality of platelets during storage.

Near-infrared (NIR) light has been successfully applied to improve the quality of mouse platelets during storage. Because it is suspected that the mitochondria contain the primary photon acceptor, we hypothesized that human platelets for transfusion may be affected similarly and could benefit from NIR light treatment. The optimal light dose was determined using portions of platelet concentrates (PCs) in PAS-E. A pool-and-split design was used to prepare PCs in PAS-E or plasma (n = 6). On day 1, one unit of both pairs was illuminated with 830 nm light (light-emitting diodes, 15 J/cm2). PCs were stored at 22°C and sampled regularly for analysis. Data were compared with their corresponding controls with a paired two-sided t-test. Illuminated platelets in PAS-E were less activated with significantly lower CD62P expression (day 8: 10.8 ± 1.8 vs. 12.2 ± 2.6, p < 0.05) and lower Annexin A5 binding (day 8: 11.8 ± 1.9 vs. 13.1 ± 2.4, ns). They produced significantly less lactate resulting in a higher pH (days 6-10). ATP content and mitochondrial membrane potential were not affected. Although these trends were also observed for PCs in plasma, the differences did not reach statistical significance as compared with the control group. Our study demonstrates that the glycolysis rate of human platelets can be modulated through the use of NIR, possibly through mitochondrial aerobic metabolism, but this requires confirmation. If NIR illumination can be further optimized, it may potentially become a useful tool in situations in which glycolysis and platelet activation are exacerbated.

Essential role of glycoprotein Ibα in platelet activation

AbstractGlycoprotein Ibα (GPIbα), the ligand-binding subunit of platelet GPIb-IX complex, interacts with von Willebrand factor (VWF) exposed at the injured vessel wall, initiating platelet adhesion, activation, hemostasis, and thrombus formation. The cytoplasmic tail of GPIbα interacts with 14-3-3ζ, regulating the VWF-GPIbα–elicited signal transduction and VWF binding function of GPIbα. However, we unexpectedly found that the GPIbα–14-3-3ζ association, beyond VWF-dependent function, is essential for general platelet activation. We found that the myristoylated peptide of GPIbα C-terminus MPαC, a potential GPIbα inhibitor, by itself induced platelet aggregation, integrin αIIbβ3 activation, granule secretion, and phosphatidylserine (PS) exposure. Conversely, the deletion of the cytoplasmic tail of GPIbα in mouse platelets (10aa−/−) decreased platelet aggregation, integrin αIIbβ3 activation, granule secretion, and PS exposure induced by various physiological agonists. Phosphoproteome-based kinase activity profiling revealed significantly upregulated protein kinase C (PKC) activity in MPαC-treated platelets. MPαC-induced platelet activation was abolished by the pan-PKC inhibitor and PKCα deletion. Decreased PKC activity was observed in both resting and agonist-stimulated 10aa−/− platelets. GPIbα regulates PKCα activity by sequestering 14-3-3ζ from PKCα. In vivo, the deletion of the GPIbα cytoplasmic tail impaired mouse hemostasis and thrombus formation and protected against platelet-dependent pulmonary thromboembolism. Therefore, our findings demonstrate an essential role for the GPIbα cytoplasmic tail in regulating platelet general activation and thrombus formation beyond the VWF–GPIbα axis.

Abstract 138: Platelet RIPK1 Promotes Platelet Activation And Aggregation In Arteriolar Thrombosis And Ischemic Stroke

Receptor-interacting protein Ser/Thr kinase 1 (RIPK1) is a molecular switch for inflammation and cell death in nucleated cells, and its function is controlled by phosphorylation at the Ser166 residue. However, it is unknown whether RIPK1 regulates platelet function in arterial thrombosis. Intravital microscopy using megakaryocyte-specific Ripk1 conditional knockout (CKO) mice ( Ripk1 fl/fl;Pf4-cre ) revealed that platelet Ripk1 promoted platelet thrombus formation at sites of laser-induced cremaster arteriolar thrombosis. Deletion of platelet Ripk1 prolonged the time to occlusion in FeCl 3 -induced carotid arterial thrombosis without affecting tail bleeding times. Further, compared to wild-type (WT) mice, Ripk1 CKO mice exhibited improved neurological deficits and reduced infarct volume in ischemic stroke induced by transient middle cerebral artery occlusion. Flow cytometry and aggregation assays showed that deletion of platelet Ripk1 reduced P-selectin exposure, αIIbβ3 integrin activation, and platelet aggregation induced by thrombin, collagen-related peptides (CRP), and U46619, but not ADP. Pretreatment of Ripk1-null platelets with ADP rescued the aggregation defect. Treatment of platelets with Nec-1s, a RIPK1 inhibitor that blocks RIPK1-Ser166 phosphorylation, did not affect platelet activation and aggregation. Instead, RIPK1-Ser320/321 was rapidly phosphorylated in thrombin- or CRP-stimulated human and mouse platelets. Treatment of platelets with an inhibitor of phospholipase C, p38, or MAPK-activated protein kinase 2 (MK2), but not a protein kinase C inhibitor, decreased RIPK1-Ser320/321 phosphorylation after thrombin or CRP stimulation, implying that the p38-MK2 axis phosphorylates RIPK1-Ser320/321. Using a genetic approach in human megakaryoblastic MEG-01 cells, we found that RIPK1 knockdown reduced granule secretion from stimulated MEG-01 cells, which was rescued by overexpressing WT RIPK1, but not mutant RIPK1-Ser320Ala, in the RIPK1 knockdown MEG-01 cells. These results suggest that platelet RIPK1 enhances granule secretion during platelet activation through p38-MK2-mediated phosphorylation of RIPK1-Ser320/321, thereby contributing to the pathology of thrombotic disease.

Differential Impact In Vivo of Pf4-ΔCre-Mediated and Gp1ba-ΔCre-Mediated Depletion of Cyclooxygenase-1 in Platelets in Mice.

Low-dose aspirin is widely used for the secondary prevention of cardiovascular disease. The beneficial effects of low-dose aspirin are attributable to its inhibition of platelet Cox (cyclooxygenase)-1-derived thromboxane A2. Until recently, the use of the Pf4 (platelet factor 4) Cre has been the only genetic approach to generating megakaryocyte/platelet ablation of Cox-1 in mice. However, Pf4-ΔCre displays ectopic expression outside the megakaryocyte/platelet lineage, especially during inflammation. The use of the Gp1ba (glycoprotein 1bα) Cre promises a more specific, targeted approach. To evaluate the role of Cox-1 in platelets, we crossed Pf4-ΔCre or Gp1ba-ΔCre mice with Cox-1flox/flox mice to generate platelet Cox-1-/- mice on normolipidemic and hyperlipidemic (Ldlr-/-; low-density lipoprotein receptor) backgrounds. Ex vivo platelet aggregation induced by arachidonic acid or adenosine diphosphate in platelet-rich plasma was inhibited to a similar extent in Pf4-ΔCre Cox-1-/-/Ldlr-/- and Gp1ba-ΔCre Cox-1-/-/Ldlr-/- mice. In a mouse model of tail injury, Pf4-ΔCre-mediated and Gp1ba-ΔCre-mediated deletions of Cox-1 were similarly efficient in suppressing platelet prostanoid biosynthesis. Experimental thrombogenesis and attendant blood loss were similar in both models. However, the impact on atherogenesis was divergent, being accelerated in the Pf4-ΔCre mice while restrained in the Gp1ba-ΔCres. In the former, accelerated atherogenesis was associated with greater suppression of PGI2 biosynthesis, a reduction in the lipopolysaccharide-evoked capacity to produce PGE2 (prostaglandin E) and PGD2 (prostanglandin D), activation of the inflammasome, elevated plasma levels of IL-1β (interleukin), reduced plasma levels of HDL-C (high-density lipoprotein receptor-cholesterol), and a reduction in the capacity for reverse cholesterol transport. By contrast, in the latter, plasma HDL-C and α-tocopherol were elevated, and MIP-1α (macrophage inflammatory protein-1α) and MCP-1 (monocyte chemoattractant protein 1) were reduced. Both approaches to Cox-1 deletion similarly restrain thrombogenesis, but a differential impact on Cox-1-dependent prostanoid formation by the vasculature may contribute to an inflammatory phenotype and accelerated atherogenesis in Pf4-ΔCre mice.

Characterization of a novel mouse platelet transfusion model.

Platelet transfusions are increasing with medical advances. Based on FDA criteria, platelet units are assessed by in vitro measures; however, it is not known how platelet processing and storage duration affect function in vivo. Our study's aim was to develop a novel platelet transfusion model stored in mouse plasma that meets FDA criteria adapted to mice, and transfused fresh and stored platelets are detectable in clots in vivo. Platelet units stored in mouse plasma were prepared using a modified platelet-rich plasma (PRP) collection protocol. Characteristics of fresh and stored units, including pH, cell count, in vitro measures of activity, including activation and aggregation, and post-transfusion recovery (PTR), were determined. Lastly, a tail transection assay was conducted using mice transfused with fresh or stored units, and transfused platelets were identified by confocal imaging. Platelet units had acceptable platelet and white cell counts and were negative for bacterial contamination. Fresh and 1-day stored units had acceptable pH; the platelets were activatable by thrombin and adenosine diphosphate, agreeable with thrombin, had acceptable PTR, and were present in vivo in clots of recipients after tail transection. In contrast, 2-day stored units had clinically unacceptable quality. We developed mouse platelets for transfusion analogous to human platelet units using a modified PRP collection protocol with maximum storage of 1 day for an 'old' unit. This provides a powerful tool to test how process modifications and storage conditions affect transfused platelet function in vivo.

A novel GARP humanized mouse model for efficacy assessment of GARP-targeting therapies

Although breakthroughs have been achieved with immune checkpoint inhibitors (ICI) therapy, some tumors do not respond to those therapies due to primary or acquired resistance. GARP, a type I transmembrane cell surface docking receptor mediating latent transforming growth factor-β (TGF-β) and abundantly expressed on regulatory T lymphocytes and platelets, is a potential target to render these tumors responsive to ICI therapy, and enhancing anti-tumor response especially combined with ICI. To facilitate these research efforts, we developed humanized mouse models expressing humanized GARP (hGARP) instead of their mouse counterparts, enabling in vivo assessment of GARP-targeting agents. We created GARP-humanized mice by replacing the mouse Garp gene with its human homolog. Then, comprehensive experiments, including expression analysis, immunophenotyping, functional assessments, and pharmacologic assays, were performed to characterize the mouse model accurately. The Tregs and platelets in the B-hGARP mice (The letter B is the first letter of the company's English name, Biocytogen.) expressed human GARP, without expression of mouse GARP. Similar T, B, NK, DCs, monocytes and macrophages frequencies were identified in the spleen and blood of B-hGARP and WT mice, indicating that the humanization of GARP did not change the distribution of immune cell in these compartments. When combined with anti-PD-1, monoclonal antibodies (mAbs) against GARP/TGF-β1 complexes demonstrated enhanced in vivo anti-tumor activity compared to monotherapy with either agent. The novel hGARP model serves as a valuable tool for evaluating human GARP-targeting antibodies in immuno-oncology, which may enable preclinical studies to assess and validate new therapeutics targeting GARP. Furthermore, intercrosses of this model with ICI humanized models could facilitate the evaluation of combination therapies.

Preservation of recipient plasma sphingosine-1-phosphate levels reduces transfusion-related acute lung injury.

Cold-stored (CS) platelets are once again being reintroduced for clinical use. Transfused CS platelets offer benefits over room temperature-stored (RTS) platelets such as increased hemostatic effects and prolongation of shelf-life. Despite these advantages little is known about their association with transfusion-related acute lung injury (TRALI). TRALI is associated with prolonged storage of RTS platelets and has a mortality of >15%. Determining the safety of CS platelets is important considering their proposed use in TRALI-vulnerable populations with inflammation such as surgical patients or patients with trauma. Donor platelet-derived ceramide causes TRALI, whereas donor platelet sphingosine-1-phosphate (S1P) is barrier protective. Females have higher plasma levels of S1P than males. Cold temperatures increase S1P levels in cells. Therefore, we hypothesized that female (donors or recipients) and/or CS platelets would decrease TRALI. To test this, we compared how male and female donor and recipient allogeneic platelet transfusions of CS (4°C) versus RTS (23°C) platelets stored for 5 days influence murine TRALI. Transfusion of CS platelets significantly reduced recipient lung tissue wet-to-dry ratios, bronchoalveolar lavage total protein, lung tissue myeloperoxidase enzyme activity, histological lung injury scores, and increased plasma sphingosine-1-phosphate (S1P) levels compared with RTS platelet transfusions. Female as opposed to male recipients had less TRALI and higher plasma S1P levels. Female donor mouse platelets had higher S1P levels than males. Mouse and human CS platelets had increased S1P levels compared with RTS platelets. Higher recipient plasma S1P levels appear protective considering females, and males receiving platelets from females or male CS platelets had less TRALI.NEW & NOTEWORTHY Transfusion-related acute lung injury (TRALI) though relatively rare represents a severe lung injury. The sphingolipid sphingosine-1-phosphate (S1P) regulates the severity of platelet-mediated TRALI. Female platelet transfusion recipient plasmas or stored platelets from female donors have higher S1P levels than males, which reduces TRALI. Cold storage of murine platelets preserves platelet-S1P, which reduces TRALI in platelet-transfused recipients.