Platelet integrin αIIbβ3 is essential for hemostasis, thrombosis, and inflammation. We found that ligation of αIIbβ3 by von Willebrand factor or fibrin under flow triggered its accumulation in plasma membrane extensions or "platelet-derived integrin- and tetraspanin-enriched tethers" (PITTs). PITTs remained anchored to leukocytes or endothelial cells, whereas the partially αIIbβ3-deficient platelet body detached. Although still responsive to stimuli, αIIbβ3-deficient platelets did not support thrombus formation. PITTs promoted leukocyte activation and vascular inflammation in mouse models of infection and endotoxemia, and αIIbβ3 blockade reduced immune-mediated tissue damage. In patients with sepsis, COVID-19, or severe infections, PITT formation and platelet αIIbβ3 loss correlated with disease severity and adverse outcomes. We propose that PITTs are proinflammatory structures that amplify immune responses while contributing to platelet dysfunction in thrombo-inflammatory disease.

Summary Neutrophil recruitment into inflamed tissues requires coordinated transmigration across the multilayered venular wall, yet how this process is regulated beyond the endothelium remains poorly understood. Here, we identify a previously unrecognized platelet-neutrophil circuit that controls this post-endothelial phase. Intravital microscopy of inflamed cremasteric venules showed that pioneer neutrophil transmigration enabled platelet entry into the sub-endothelial compartment of venular walls. There, platelets became activated and released CXCL7 in a GPVI- and GPIbα-dependent manner, generating spatially confined chemokine microdomains. These platelet-derived cues directed follower neutrophil migration and promoted their exit across the pericyte layer into the interstitial tissue. Collectively, these findings identify extraluminal platelets as spatial organizers of neutrophil trafficking and uncover a localized intramural feedback mechanism in which neutrophils amplify their own recruitment within venular walls. Graphical summary

Abstract Glycoprotein (GP) VI is a platelet-specific activating receptor for collagen and fibrin(ogen), and a promising target for antithrombotic therapy. Inhibitory Fab fragments against GPVI provide robust protection in mouse models of arterial thrombosis and ischemic stroke without impairing hemostasis in mice or humans. However, their short in vivo half-life limits their suitability for long-term therapy. In contrast, GPVI-targeting IgGs (e.g., JAQ1-3) induce complete receptor depletion and a GPVI-knockout-like (GPVIKO-like) phenotype in mice, a mechanism also observed in humans with anti-GPVI autoantibodies. While GPVI deficiency causes only moderate hemostatic defects, bleeding risk increases when combined with high-dose aspirin (100 mg/kg). Using a humanized GPVI (hGP6 tg/tg ) mouse model, we show that high-affinity anti-human GPVI IgGs (Emf1 KD 0.490 nM, Emf2) induce a GPVIKO-like phenotype with antithrombotic efficacy. In contrast, JAQ1 IgG, which binds hGPVI with low affinity (KD: 9.6 nM), induces only partial (~50%) GPVI downregulation, generating a stable low-density GPVI (GPVILO) phenotype lasting up to 10 days. GPVILO platelets showed impaired aggregation, abolished procoagulant activity, and—similar to Fab-mediated inhibition—conferred protection from arterial thrombosis and LPS-induced thrombo-inflammation without increasing bleeding, even when combined with high-dose aspirin. The low-affinity anti-mouse GPVI antibody JAQ4 (KD = 21 nM) induced a comparable GPVILO phenotype in wild-type mice. Transfusion of human platelets in NOD/SCID mice showed that complete and partial GPVI depletion, respectively, also occurs in human platelets in vivo. These findings establish that partial, affinity-dependent GPVI downregulation can provide sustained antithrombotic protection while preserving hemostasis, offering a promising strategy for long-term platelet inhibition.

The vasculature within the bone marrow plays a pivotal role in shaping the hematopoietic environment, significantly influencing the development and function of blood cells. However, the intricate shifts in vascular architecture, especially as the body ages or under physiological stress, remain largely uncharted territory. In this study, we explored these vascular dynamics using light sheet fluorescence microscopy paired with advanced image analysis, focusing on both large, CD31hi, and smaller, CD105+, vessels within murine femora. We compared young adult (aged 8 weeks) mice with moderately aged (52 weeks) mice, and assessed the impact of hematological stress induced by previous blood sampling. Our findings revealed an intriguing balance of resilience and adaptability: larger vessels show a remarkable structural stability across ages, whereas smaller vessels undergo pronounced age-associated remodeling. In aged mice, vascular branching complexity surges, with higher branch density and narrower vessel diameters in specific femur regions, clear hallmarks of vascular aging. Interestingly, even after previous blood sampling, the bone marrow vasculature maintains its integrity, hinting at a robust resilience to hematological stress. This resilience underscores the potential to streamline research models by reusing animals for both cell isolation and histology, supporting the 3R principles (replacement, reduction, and refinement) to minimize animal use. These insights into the stability, adaptability, and regional-specific remodeling of bone marrow vasculature, highlighting its remarkable resilience, provide a deeper understanding of age-related vascular remodeling, and offer valuable implications for experimental design in hematopoiesis research.

Our research presents a new animal model of transient ischemic attack (TIA) that mimics brief episodes without cell loss, but results in neuronal and behavioral deficits. We identified excessive microglial reactivity, driven by acute ATP release, as a key factor in post-TIA neurological deficits, which were ameliorated by inhibiting the P2Y12 receptor, a microglia-specific purinergic receptor in the brain parenchyma responsible for activity-dependent microglial cell-cell interactions. This finding suggests that modulation of microglial reactivity offers a promising strategy to prevent cognitive impairment in TIA patients, opening avenues for future research in this underexplored area.

Macrophages have a dual role in tissue healing after injury as they perform tissue repair functions but can also precipitate tissue damage or promote fibrosis. Platelets, beyond their role in thrombosis and hemostasis, are crucial mediators of inflammation and interact with macrophages. Platelet-macrophage interactions have been proposed to modulate macrophage phenotype, including their profibrotic functions, but the full extent of the platelet impact on the macrophage transcriptome is unknown. Here, we aimed to investigate how platelets affect macrophage activation in vitro. Using experimental myocardial infarction (MI) in mice as a model of sterile tissue injury, we readily visualized the direct interaction of platelets with macrophages in the ischemic heart using fluorescence microscopy. Bulk RNA-sequencing of mouse bone marrow-derived macrophages co-cultured in vitro with thrombin-activated platelets showed a widespread proinflammatory activation, with upregulation of genes associated with inflammation (Il1b, Trem1, Tlr2, Cd14), angiogenesis (Vegfa) and response to hypoxia (Hif1a). Resting platelets also led to activation of inflammatory gene expression by macrophages, albeit to a much lesser extent. Activated or resting platelets, or the platelet-derived chemokine CXCL4, had a limited impact on macrophage expression of profibrotic genes (Spp1, Fn1). Using a transwell assay, we further demonstrate that the proinflammatory effects of platelets on the macrophage transcriptome were largely contact dependent. Altogether, our work shows that platelets interact with macrophages in the ischemic heart and polarize macrophages towards a proinflammatory phenotype in vitro, with potential implications for cardiac macrophage inflammatory activation after acute experimental MI.

BACKGROUND:Ischemic stroke (IS) is a prevalent cause of death and disability worldwide. Cerebral ischemia induces profound changes at the blood-brain barrier, which lead to a remarkable increase in paracellular permeability, worsening outcomes. Platelets are well known for safeguarding vascular integrity and the prevention of bleeding complications. On the other hand, platelet activation contributes to infarct progression in the context of IS. The manifold, context-dependent roles of platelets, however, have not yet been resolved. METHODS:IS was mimicked by transient middle cerebral artery occlusion in wild-type, transgenic, or treated mice, and vascular leakage was assessed by intravital 2-photon microscopy, as well as Western blotting and immunohistochemistry. Barrier property of primary murine brain microvascular endothelial cells was measured as transendothelial electrical resistance of cellular monolayers in response to platelet releasate or recombinant proteins. RESULTS:IS induces blood-brain barrier breakdown characterized by time-dependent leakage of albumin in the brain parenchyma. We could show that local platelet activation triggers the release of PDGF (platelet-derived growth factor)-A from α-granules, which induces the loss of brain endothelial cell layer integrity. This translates to a comprised vascular integrity in vivo. In the absence of α-granule content (Nbeal2-/-) or pharmacological blockade of PDGF, no disruption of the endothelial layer or vascular leakage was observed. CONCLUSIONS:PDGF-A released from platelets impairs blood-brain barrier integrity, resulting in detrimental vascular leakage and infarct progression. These findings provide important insights on the pivotal role of platelets in IS further elucidating the mechanisms of thrombo-inflammation in the brain.

Platelets, small anucleate blood cells, are essential not only for maintaining vascular integrity but also for broader systemic homeostasis. Traditionally recognized for their role in preventing hemorrhage following injury, recent research has revealed their involvement in a range of metabolic processes. Notably, platelets contribute to the regulation of insulin secretion, glucose blood levels, adipose tissue metabolism, and liver function. Furthermore, they have been implicated in the development of metabolic disorders, such as type 2 diabetes, obesity, and liver diseases. This review highlights the latest insights into how platelets influence key organs involved in systemic metabolic regulation.

Background Beyond their role in hemostasis, platelets are recognized as key regulators of inflammatory responses in ischemic diseases, including cardiac ischemia/reperfusion (I/R) injury with key roles of platelet membrane glycoproteins (GP)VI and IIb/IIIa. However, whether platelet-driven thrombo-inflammatory pathways affect acute inflammation and cardiac repair processes in permanent, non-reperfused myocardial infarction (MI) is unknown. Methods We targeted GPVI and GPIIb/IIIa in experimental permanent MI in mice. Cardiac, bone marrow, and blood innate immune responses were evaluated by flow cytometry and single-cell RNA-sequencing. Survival and cardiac repair were assessed over the inflammatory and scar formation phase, until day 10 after permanent MI. Results Platelet GPVI immunodepletion by injection of the anti-GPVI antibody JAQ1 did not affect levels of neutrophil or monocyte subsets (Ly6Chi and Ly6Clow) in the bone marrow and blood, and did not alter accumulation of monocytes, macrophage subsets (defined by expression of MHCII and TIM4), or neutrophil subsets (SiglecFhi/low) in the infarcted heart on day 4. GPVI depletion only had a minimal effect on cardiac repair, slightly decreasing interstitial fibrosis in the infarct border zone on day 10. Four days after MI, GPIIb/IIIa inhibition by JON/A-F(ab′)2 had no effect on cardiac or systemic innate immune cell levels as measured by flow cytometry and did not affect composition and transcriptomic profile of the cardiac immune infiltrate as revealed by single-cell RNA-sequencing. GPIIb/IIIa inhibition did not improve cardiac remodeling, and was even associated with an increased mortality rate over 10 days post-MI. Conclusion Targeting the GPVI-GPIIb/IIIa axis only had minor effects on post-MI inflammatory responses and cardiac wound healing in permanent myocardial ischemia. Our findings demonstrate that the therapeutic benefits of inhibiting platelet-driven thrombo-inflammation are particularly relevant in the subacute reperfusion phase. ### Competing Interest Statement The authors have declared no competing interest. Deutsche Forschungsgemeinschaft, https://ror.org/018mejw64, 453989101

Platelets, small anucleate blood cells, are essential not only for maintaining vascular integrity but also for broader systemic homeostasis. Traditionally recognized for their role in preventing hemorrhage following injury, recent research has revealed their involvement in a range of metabolic processes. Notably, platelets contribute to the regulation of insulin secretion, glucose blood levels, adipose tissue metabolism, and liver function. Furthermore, they have been implicated in the development of metabolic disorders, such as type 2 diabetes, obesity, and liver diseases. This review highlights the latest insights into how platelets influence key organs involved in systemic metabolic regulation.

ABSTRACT:Throughout thrombopoiesis megakaryocytes (MKs) form proplatelets within the bone marrow (BM) and release platelets into BM sinusoids. Casein kinase 1α (CK1α) is a major player and thus, an important therapeutic target in several hematological malignancies. This study aimed to define the role of CK1α for the essential steps of thrombopoiesis and to dissect potential mechanisms of thrombocytopenia. MK-specific CK1α-deficiency resulted in a macrothrombocytopenia. Ck1αPf4Δ/Pf4Δ mice displayed a substantial BM hyperplasia with pivotal changes in MK nuclear lobulation and reduced contact to BM sinusoids. Ck1αPf4Δ/Pf4Δ MKs displayed a defective cytoskeleton organization reflected by a decreased amount of polymerized filamentous actin and disturbed microtubule dynamics due to p21/p53 accumulation and impaired Rho-associated protein kinase (ROCK)/LIM domain kinase (LIMK)/cofilin signaling. Further, pronounced defects in DMS (demarcation membrane system) polarization and proplatelet formation of Ck1αPf4Δ/Pf4Δ MKs, unraveled CK1α as a prerequisite for thrombopoiesis. Our findings could be translated into a human approach, because a CRISPR/Cas9-mediated genetic deletion of CSNK1A1 in MKs derived from human CD34+ progenitor cells resulted in a substantial defect in human MK maturation and platelet production. The present observations elucidated CK1α as an important signaling molecule in MK cytoskeletal dynamics and polarization, proplatelet formation, and polyploidization, thus highlighting the crucial role of CK1α in platelet biogenesis.

ABSTRACT:During thrombopoiesis, megakaryocytes (MKs) transform their cytoplasm into proplatelets through complex cytoskeletal rearrangements. The shear force of blood flow releases newly formed platelets from the proplatelets into the bloodstream. Defects at any phase of this process can impair platelet production. Although various noncoding RNAs have been identified as regulators of platelet production, the regulatory mechanisms of thrombopoiesis remain to be further investigated. Despite the high abundance of circular RNAs (circRNAs) in platelets, their role in platelet production is unclear. In this study, using RNA sequencing and bioinformatics analysis, we identified a circular RNA from the FUT8 gene (circFUT8) as a novel circRNA that increases as hematopoietic stem cells from human umbilical cord blood differentiate into mature MKs, showing high expression in these mature cells. Knockdown of circFUT8 led to diminished proplatelet formation (PPF) and abnormal demarcation membrane system formation in human cultured MKs. In addition, inhibition of circFut8 in vivo decreased murine platelet counts. circFut8 deficiency reduced the number of MKs in contact with sinusoids. Mechanistically, we revealed that circFUT8 interacts with insulin-like growth factor 2 messenger RNA (mRNA)-binding protein 2 to stabilize tensin-1 (TNS1) mRNA in an m6A-dependent manner. In human cultured MKs, TNS1 knockdown resulted in defective filamentous actin polymerization and assembly, impaired spreading on extracellular matrix proteins, and decreased PPF. Taken together, our research reveals the crucial functions of circRNAs in platelet production and has significant implications for the development of therapeutic strategies for thrombocytopenia and bleeding disorders.

Background and Aims 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. Methods Fab-fragments of the mouse anti-hGPVI IgG Emf6.1 were tested for functional GPVI inhibition in human platelets and in hGPVI expressing (hGP6(tg/tg)) mouse platelets. The in vivo effect of Emf6.1(Fab) 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 Emf6.1(Fab) (EMA601) was generated. Emf6.1(Fab)/EMA601 interaction with hGPVI was mapped in array format and kinetics and quantified by bio-layer interferometry. Results Emf6.1(Fab) (K-D: 0.427 nM) blocked GPVI function in human and hGP6(tg/tg) mouse platelets in multiple assays in vitro at concentrations >= 5 mu g/mL. Emf6.1(Fab) (4 mg/kg)-treated hGP6(tg/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. Emf6.1(Fab) binds to a so far undescribed membrane proximal epitope in GPVI. The humanized variant EMA601 displayed further increased affinity for hGPVI (K-D: 0.195 nM) and fully inhibited the receptor at 0.5 mu g/mL, corresponding to a >50-fold potency compared with ACT017. Conclusions 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.