David Stegner

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:

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.

Background: Classical myeloproliferative neoplasms (MPN)—essential thrombocythemia, polycythemia vera, and primary myelofibrosis—are characterized by clonal hematopoiesis, overproduction of mature blood cells, and a high burden of thromboembolic events. Although thrombosis is the leading cause of morbidity and mortality in MPN, the contribution of the vascular endothelium remains incompletely defined. We investigated patient-derived endothelial colony-forming cells (ECFCs) as a surrogate for vascular endothelium in individuals with JAK2 V617F–mutated MPN. Methods: ECFCs were cultured from peripheral blood of patients with MPN and healthy controls, phenotyped for thrombo-inflammatory and adhesive markers, tested for JAK2 V617F, and profiled by bulk RNA sequencing. Functional assays assessed endothelial-dependent factor Xa generation. Transcriptomes were benchmarked against public HUVEC reference datasets processed through an identical quantification pipeline. Results: ECFCs were obtained more frequently and in greater numbers from patients with MPN than from controls, indicating enhanced endothelial regenerative or activation potential. MPN ECFCs exhibited increased von Willebrand factor and P-selectin expression and release, along with elevated endothelial cell–dependent factor Xa generation, consistent with a thrombo-inflammatory, procoagulant phenotype. JAK2 V617F was not detected in any ECFC colonies, supporting a non-clonal origin of these endothelial abnormalities. Transcriptomic analysis identified 289 differentially expressed genes in MPN versus control ECFCs, with pathway enrichment revealing coordinated dysregulation of blood coagulation, platelet activation, plasminogen regulation, vascular permeability, extracellular matrix organization, and angiogenesis. Benchmarking against HUVEC datasets confirmed strong endothelial identity of ECFC-derived cells, with MPN-associated changes reflecting endothelial activation rather than loss of endothelialness. Conclusions: ECFCs from patients with JAK2-mutated MPN display functional and transcriptomic signatures of endothelial dysfunction in the absence of detectable driver mutations. These findings support a model in which a primed, thrombo-inflammatory endothelium cooperates with clonal hematopoiesis to promote the heightened thrombotic risk characteristic of MPN.

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.

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 (Ly6C ^hi and Ly6C ^low ) 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 (SiglecF ^hi/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.

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.