Hypoxia is associated with thrombosis, yet its effects on platelet function remain controversial due to variable experimental conditions. In this study, we systematically examined the effects of graded normobaric hypoxia on platelet function and arterial thrombosis in mice. Exposure to 8% O2 for 6 days increased red blood cell counts, hemoglobin, and hematocrit, recapitulating human hypoxic adaptation. Platelets from hypoxic mice show reduced activation and aggregation in response to multiple agonists, including thrombin and collagen-related peptide, with defects accentuated by more severe (8% vs. 12% O2) or prolonged (6-day vs. 4-day) hypoxia and most pronounced after glycoprotein VI (GPVI) stimulation. Similar platelet dysfunction occurs in humans after 4-day hypoxia (∼12% O2) followed by 4-day normoxia. Quantitative mass spectrometry identifies downregulation of lysozyme, Hsp90 signaling molecules, and GPVI. Pathway and systems-level network analyses reveal significant downregulation of Hsp90-involved protein folding and GPVI-mediated signaling pathways. Deletion of platelet lysozyme or Hsp90β, however, minimally affects platelet function, indicating regulation by multiple signaling pathways under hypoxia. We confirmed that GPVI levels are significantly reduced in platelets, while ADAM10 levels remain unchanged, suggesting hypoxia-induced GPVI downregulation. Hypoxic blood forms smaller thrombi on collagen under arterial shear. Paradoxically, hypoxic mice exhibit enhanced arterial thrombosis and shortened tail bleeding times. Biochemical and mass spectrometry analyses of mouse plasma demonstrate elevated circulating von Willebrand factor under hypoxia. These results uncover a paradox under hypoxia: despite GPVI downregulation and impaired agonist-induced platelet activation, elevated plasma von Willebrand factor drives enhanced arterial thrombosis. This multilayered regulation provides mechanistic insight into hypoxia-associated thrombotic risk and may inform therapeutic strategies.

JPT2/HN1L functions as an NAADP-binding protein in a cell-type-specific manner.

Regulation of platelet contractility by agonists present across a thrombus

Platelets play a major role in thrombo-inflammatory cardiovascular diseases such as myocardial infarction. Although platelet function is crucially determined by kinases, the impact of Casein Kinase 2α (CK2α) on platelet activation during arterial thrombosis and myocardial remodeling following ischemia and reperfusion (I/R) injury is not known. Using platelet-specific deletion of Csnk2a1 in mice, the evaluation of the CK2α-dependent platelet phosphoproteome revealed a diminished phosphorylation of the IP3 receptor type-1 in Csnk2a1-deficient mice. This finding was accompanied by attenuated IP3-induced Ca2+ mobilization, impaired integrin αIIbβ3 activation, abrogated platelet aggregation and secretion, as well as defective spreading on fibrinogen in response to collagen-related peptide. Accordingly, without affecting primary hemostasis, thrombotic vascular occlusion in vivo was diminished in Csnk2a1-deficient mice. When subjected to a myocardial I/R injury model, these mice displayed improved cardiac outcome when compared with wildtype mice. Raman spectromics, spatial metabolomics and molecular approaches revealed locally a CK2α-dependent release of chondroitin sulfate and transforming growth factor-β from platelets, which was associated with significantly reduced ventricular fibrosis and improved heart function in Csnk2a1-deficient mice. Altogether, our results disclose CK2α as pivotal player in platelet activation and pathogenesis of post-ischemic myocardial remodeling, including myocardial fibrosis and left ventricular impairment following myocardial ischemia.

ABSTRACT Xanthone, a naturally occurring oxygenated heterocyclic compound from the Garcinia family with known anti-cancer, antimicrobial, antioxidant, anti-inflammatory, and antiviral properties, has an unclear role in platelet function. This study investigated its effects by incubating human platelets with xanthone at doses of 0, 5, 10, and 20 μM for 1 hour to analyze platelet aggregation, granule release, activation, receptor expression, spreading, and clot retraction, while also administering xanthone (10 mg/kg) to mice to evaluate its impact on hemostasis, arterial, and venous thrombosis. Our findings demonstrated that xanthone dose-dependently reduced platelet aggregation and granule release induced by collagen-related peptide (CRP) or thrombin without altering the surface expression of receptors αIIbβ3, GPIbα, and GPVI; it also significantly inhibited platelet spreading on collagen or fibrinogen, thrombin-mediated clot retraction, and decreased phosphorylation of c-Src and PLCγ2 in treated platelets. In vivo, xanthone-administered mice exhibited prolonged tail bleeding time and impaired arterial and venous thrombosis. Mechanistically, xanthone inhibited NF-κB activation, phosphorylation of ERK1/2 and p38, calcium mobilization, and platelet procoagulant activity. These findings indicate that xanthone impairs platelet activation and both arterial and venous thrombus formation, suggesting its potential as a novel agent for treating thrombotic or cardiovascular diseases.

rFVIIIa-platelet binding enhances platelet procoagulant activity independently of thrombin generation.

Platelet dysfunction in Ehlers–Danlos syndrome is associated with low GPVI and PAR1 expression on platelets

Enhanced clot formation and altered platelet subpopulations in mesothelioma characterized by a high-dimensional analytical platform

Novel small molecule inhibitors of mitochondrial permeability transition pore (mPTP) block platelet procoagulant activity

BackgroundPlatelets interact with leukocytes to fine-tune their functions, thus providing essential regulation of (patho-) physiologic immune responses in various diseases. Circulating platelet-leukocyte aggregates (PLAs) represent a sensitive biomarker to estimate disease severity both in patients and murine models. However, a limited understanding of the sensitivity of PLA measurements to methodological variables may undermine their accuracy and comparability.ObjectivesTo elucidate how blood draw techniques, anticoagulation, processing delay and assay temperature affect murine platelet-leukocyte interactions.MethodsMurine blood was obtained via retro-orbital, vena cava or cardiac puncture, anticoagulated with heparin, citrate or acid-citrate-dextrose (ACD) +/- recalcification and stored for 30-120 min before stimulation at room temperature or 37°C with adenosine diphosphate (ADP), cross-linked collagen-related peptide (CRP-XL) and protease-activated receptor 4-activating peptide (PAR4-AP). PLA formation and leukocyte activation were analyzed by flow cytometry.ResultsBasal PLAs were minimally affected by blood sampling and anticoagulant, though delayed processing significantly raised basal PLAs. Agonist-induced PLA formation was independent of anticoagulation, and sampling technique did not affect ADP- or PAR4-AP-induced PLA levels. However, CRP-XL sensitivity was elevated in blood obtained by cardiac puncture. Contrarily, both delayed processing and stimulation at 37°C impaired CRP-XL sensitivity, but augmented ADP and PAR4-AP responses. Regulation of leukocyte activation did not follow PLA patterns, with monocytes and neutrophils displaying distinct susceptibility to anticoagulation, storage and temperature.ConclusionsVariations in preparing murine blood samples exert distinct influences on platelet-leukocyte interactions in vitro, underlining the critical need for fastidious assay optimization to support data reproducibility and comparability.

Phosphodiesterase enzymes (PDEs) play a pivotal role in regulating platelet activity by modulating intracellular levels of cAMP and cGMP. Modulation of PDE-2, -3 and -5 activity by suitable inhibitors has been found to reduce platelet activity, and thus thrombus formation. Our aim was to study Ibudilast effects on platelet activation, degranulation and aggregation. Therefore, we used the nonspecific PDE inhibitors IBMX as well as the PDE-5 inhibitor Sildenafil as controls. Platelet agonists collagen-related peptide (CRP-A), adenosine diphosphate (ADP) and thrombin receptor activator peptide (TRAP6) were used to induce distinct activation pathways. PDE inhibition was quantified by western blot analysis. Platelet activity was assessed using flow cytometry, light transmission aggregometry and in vitro thrombus formation. Inhibition of all platelet PDEs by IBMX substantially reduced platelet activation and aggregation in response to all tested platelet agonists. Ibudilast preferentially inhibits PDE-3 in platelets. Ibudilast decreased platelet activation and aggregation induced by ADP and TRAP, but not CRP-A. Sildenafil alone induced no reduction in PDE activity, platelet activation or aggregation. However, the combination of Sildenafil and Ibudilast had an additive effect on platelet activation. Interestingly, all tested PDE inhibitors demonstrated a significant effect on platelet-dependent thrombus formation. In conclusion, the effect of PDE inhibitors on platelet function is influenced by two primary factors: the pharmacological target of the inhibitor and the cAMP/cGMP interaction with the activation pathways induced. Platelet activation by ADP via P2Y12 and TRAP via PAR1 showed a greater response to PDE inhibitors than platelet activation by CRP via GPVI.

Phosphorylation at Y317 on Syk negatively regulates both ITAM- and hemITAM-mediated signaling and function in platelets

BackgroundCoronary artery disease is characterized by chronic immune‐inflammation, excessive endoplasmic reticulum (ER) stress, and platelet hyperactivity; however, whether there is a signaling hub linking these events remains unclear. Here, we identified that TREM2 (triggering receptor expressed on myeloid cells 2), an important pattern recognition receptor of the innate immune system, may serve as one such hub.MethodsTREM2 expression and ER stress were assessed in platelets. Transcriptional repression of TREM2 by excessive ER stress was evaluated using luciferase assay, chromatin immunoprecipitation, and electrophoretic mobility shift assay. The effects of TREM2 deficiency on platelet function, mouse FeCl3‐induced mesenteric arterial thrombosis, and myocardial infarction were explored. A TREM2‐activating antibody was also evaluated for its antiplatelet, antithrombotic, and cardioprotective potential against myocardial infarction.ResultsWe found that platelets express TREM2, and its expression is reduced in platelets from patients with coronary artery disease. Excessive ER stress downregulated TREM2 through the CHOP (C/EBP‐homologous protein)‐C/EBPα axis. TREM2 deficiency enhanced platelet activation in response to adenosine diphosphate, collagen, and CRP (collagen‐related peptide). TREM2 deficiency exacerbated mouse mesenteric arterial thrombosis and aggravated experimental myocardial infarction. Furthermore, a TREM2‐activating antibody inhibited platelet activation, reduced thrombosis, and alleviated experimental myocardial infarction. Mechanistically, the TREM2/DAP12 (DNAX activating protein of 12 kDa)/SHIP1 (Src homology 2 domain‐containing inositol 5‐phosphatase) axis negatively regulated platelet activation through reducing phosphatidylinositol (3,4,5)‐trisphosphate levels and inhibiting Akt phosphorylation. Sphingosine‐1‐phosphate was identified as a physiological TREM2 agonist.ConclusionsTREM2 integrates ER stress, immune inflammation, and platelet function. ER stress‐induced TREM2 downregulation contributes to platelet hyperactivation in coronary artery disease, suggesting TREM2 activation as a novel therapeutic target.

BACKGROUND:Recent clinical trials demonstrated a reduction in biomarkers of thrombosis and inflammation in patients with very high cardiovascular risk treated with the anti–IL-6 (interleukin 6) monoclonal antibody ziltivekimab. However, if and how direct IL-6 inhibition exerts antithrombotic effects remains unknown. This translational project aimed to investigate the effect of direct IL-6 inhibition on experimental arterial thrombus formation and its underlying cellular mechanisms.METHODS:Three-month-old C57BL/6J male and female mice received very low dose lipopolysaccharide for 4 weeks; in addition to lipopolysaccharide, during the fourth week, mice were randomized to receive either anti-mouse IL-6 monoclonal antibody 200 μg or IgG1 isotype control. Thrombosis of the right common carotid artery was induced by endothelial-targeted laser injury. Coagulation factors and platelet reactivity were assessed in treated mice and controls. Platelets were isolated from whole blood and their reactivity to different chemical stimuli was measured by fluorescence-activated cell sorting. Additionally, whole blood samples from patients with a history of percutaneous coronary intervention were incubated ex vivo with either ziltivekimab biosimilar or IgG1 isotype control. Platelet reactivity at rest and in response to diverse chemical stimuli was quantified by fluorescence-activated cell sorting.RESULTS:Mice with low-grade chronic inflammation treated with anti–IL-6 monoclonal antibody displayed significantly blunted thrombus formation, without any significant difference in coagulation factors. Ex vivo stimulation with Collagen-rP (collagen-related peptide) significantly activated platelets isolated from control mice but not those obtained from mice treated with anti–IL-6 monoclonal antibody. Similarly, platelet reactivity from patients with previous percutaneous coronary intervention fell significantly after ex vivo treatment with ziltivekimab biosimilar.CONCLUSIONS:Direct IL-6 inhibition blunts thrombus formation by reducing collagen-induced platelet activation. These findings offer a potential mechanistic explanation for the results observed in the RESCUE trial and support the rationale of the ongoing ZEUS trial (Ziltivekimab Cardiovascular Outcome Study).

Platelets are highly sensitive to subtle changes in their microenvironment, making functional analyses challenging and prone to variation. Advances in understanding how experimental procedures influence human platelet activation have improved the accuracy and comparability of diagnostic and research data. However, despite the pivotal role of murine models, the effects of methodological variations on murine platelets remain incompletely understood. To elucidate how blood draw techniques, anticoagulation, processing delay, and assay temperature affect murine platelets. Blood was obtained by retro-orbital, vena cava, or cardiac puncture and anticoagulated with heparin, citrate, or acid-citrate-dextrose ± recalcification. After 30 to 120 minutes, blood was stimulated at room temperature or 37 °C with adenosine diphosphate (ADP), protease-activated receptor 4-activating peptide (PAR4-AP), or cross-linked collagen-related peptide (CRP-XL), and platelets were analyzed by flow cytometry for CD62P, CD63, CD40L, and activated glycoprotein IIb/IIIa. Blood sampling had minimal impact on ADP-induced platelet activation. However, platelets isolated via vena cava or cardiac puncture exhibited heightened responsiveness to PAR4-AP and CRP-XL, respectively, compared with retro-orbital sampling. Citrate and acid-citrate-dextrose significantly impaired PAR4-AP responses compared with heparin, whereas CRP-XL sensitivity was anticoagulant-independent. Processing delays as brief as 60 minutes significantly altered platelet reactivity to CRP-XL and PAR4-AP, with further delays producing minimal additional impact. Finally, ADP- and CRP-XL-induced platelet activation was significantly reduced at 37 °C compared with room temperature. Common variations in murine platelet handling influence in vitro responsiveness of platelets in an agonist-specific manner, highlighting the critical need for meticulous assay optimization to ensure experimental consistency and comparability.

Various noncovalent interactions have been introduced to explore their impacts in folding a collagen triple helix. Among these interactions, the cation-π interaction represents one of the compelling forces stabilizing the triple helix. Still, the effects depend on the pairwise components and the orientation between the cationic and aromatic moieties. To gain more insights into this interaction within a collagen trimer, we prepared a series of collagen-mimetic peptides (CMPs) with cationic residues and aromatic residues incorporated to examine the contributions of two types of axial cation-π pairs (N → C and C → N cationic-to-aromatic pairwise) and the lateral cation-π pair. Circular dichroism (CD) measurements indicate that the N → C axial pairs have a significant stabilization effect. In contrast, the lateral and the C → N axial pairs destabilize the fold, and the lateral pairs cause the most destabilization consequences. We further designed and prepared the CMPs containing various lateral and axial cation-π pairs to investigate the coupling consequences in homotrimers and heterotrimers. From CD data, we found that the predicted differences in melting temperatures using individual cation-π pairwise contributions were comparable to the observed values for the designed homotrimers. CD and NMR measurements showed favorable cation-π interactions could effectively induce the folding of heterotrimers, in which the CMPs with more N → C axial pairs formed a more stable trimer than those containing a smaller number of N → C axial pairs. In this study, we have disclosed more valuable information about the properties of cation-π pairwise effects within a collagen triple helix, which can be considered in designing collagen-related peptides and materials.

Isorhynchophylline is a Chinese herbal medicine and has multiple effects such as anti-inflammatory and neuroprotective effects. Whether isorhynchophylline has antithrombotic property is unknown. This study aims to evaluate its role in platelet function. Human platelets were incubated with isorhynchophylline (0, 10, 20, and 40 μM) at 37°C for 1 hour to detect platelet aggregation and activation, receptors level, spreading, and calcium mobilization. In addition, isorhynchophylline (5 mg/kg) was injected into mice to measure in vivo hemostasis and thrombosis. Isorhynchophylline dose-dependently reduced platelet aggregation, adenosine triphosphate secretion, P-selectin expression, and α IIb β 3 activation induced by collagen-related peptide or thrombin without affecting surface level of receptors α IIb β 3 , GPIbα, and glycoprotein VI. Meanwhile, isorhynchophylline-treated platelets showed reduced spreading. Moreover, isorhynchophylline reduced platelet calcium mobilization, phosphatidylserine exposure, and the phosphorylation of PLCγ2 and PKCα. Furthermore, administration of isorhynchophylline into mice impaired platelet hemostatic function and arterial/venous thrombosis without affecting coagulation. In conclusion, isorhynchophylline impairs platelet function and arterial/venous thrombosis, implying its potential to be a novel agent for treating thrombotic or cardiovascular diseases.

BackgroundBeyond their conventional roles in hemostasis and wound healing, platelets have been shown to facilitate hematogenous metastasis by interacting with cancer cells. Depending on the activation route, platelets also generate different platelet-derived extracellular vesicles (PEVs) that may educate cancer cells in the circulation or within the tumor microenvironment. We engaged different platelet-activating receptors, including glycoprotein VI and C-type lectin-like receptor 2, to generate a spectrum of PEV types. This allowed us to investigate the differential capacity of PEVs to alter cancer hallmark functions such as proliferation, invasion, and pro-angiogenic potential using melanoma as a model. Additionally, we analyzed changes in the cell transcriptomes and cancer EV profiles.MethodsTwo human melanoma cell lines (MV3 and A2058) with differential metastatic potential were studied in the 3D spheroid cultures. Human platelets were activated with collagen related peptide (CRP), fucoidan from Fucus vesiculosus (FFV), thrombin & collagen co-stimulus and Ca2+ ionophore, and PEVs were isolated by size-exclusion chromatography followed by ultrafiltration. Spheroids or cells were treated with PEVs and used in functional assays of proliferation, invasion, and endothelial tube formation as well as for the analysis of cancer EV production and their tetraspanin profiles. Differentially expressed genes and enriched signaling pathways in the PEV-treated spheroids were analyzed at 6 h and 24 h by RNA sequencing.ResultsAmong the studied PEVs, those generated by CRP and FFV exhibited the most pronounced effects on altering cancer hallmark functions. Specifically, CRP and FFV PEVs increased proliferation in both MV3 and A2058 spheroids. Distinct tetraspanin signatures of melanoma EVs were induced by all PEV types. While the PI3K-Akt and MAPK signaling pathways were activated by both CRP and FFV PEVs, they differently upregulated the immunomodulatory TGF-β and type-I interferon signaling pathways, respectively.ConclusionsOur study revealed both shared and distinct, cancer-promoting functions of PEVs, which contributed to the transcriptome and metastatic capabilities of the melanoma spheroids. Inhibiting the platelet receptors that modulate the PEVs’ cancer-promoting properties may open up new strategies for identifying promising treatment targets for cancer therapy.

Impaired Platelet Function in Ehlers-Danlos Syndrome: Insights from Human Subjects and a Murine Model

Platelets of Patients Who Lack Bruton Tyrosine Kinase (BTK) Do Not Aggregate in Response to Fcγriia Activation: Implications for Developing Safe Treatments for Patients with Thrombotic Disorders Related to Fcγriia Activation