In Caucasians, maternal allo-immunization against human platelet antigen (HPA)-1a is the most common cause for fetal and neonatal alloimmune thrombocytopenia (FNAIT). Intracranial hemorrhage (ICH) is a serious complication. The lack of a consistent correlation between antibody concentration, severity of thrombocytopenia, and ICH makes it difficult to predict thrombocytopenia in subsequent pregnancies. HPA-1a is located on integrin β3, which forms heterodimers with different alpha chains. The αvβ3 heterodimer is present on endothelial cells (ECs), and a specific antibody interaction with fetal ECs may explain the sporadic nature of ICH. Analysis of human sera was consistent with the presence of anti-HPA-1a of the anti-αvβ3 type in ICH positive cases. We show evidence that these sera induce VEGFR2 fixation to the cell surface, lead to VEGFR2 phosphorylation, enhance ROS production, and interfere with endothelial sprouting. Analysis of blinded human FNAIT samples demonstrates that combined biochemical and functional testing correctly identifies 6/6 ICH positive and 4/4 ICH negative cases. In a murine model, female b3 knockout mice develop anti-platelet antibodies after immunization with wild-type platelets. After mating with b3 wildtype males, only pups born to mice with anti-αvβ3 develop ICH. Matching our findings obtained with human material, only murine sera containing anti-αvβ3 induce VEGFR2 fixation on the cell surface, lead to VEGFR2 phosphorylation, induce ROS production, and hamper endothelial loop formation. In conclusion, we find evidence that ICH in FNAIT is based on vascular impairment and substantiate previous reports that impaired angiogenesis could represent a mechanism of ICH in FNAIT.

Factor VIII Aurora (FVIII-R571S) is the first described naturally occurring enhanced-potency FVIII variant identified in a patient with recurrent thrombosis and early mortality. The patient's plasma exhibited increased procoagulant activity and reduced responsiveness to activated protein C (APC). To define the mechanism, we generated recombinant FVIII-R571S and performed in vitro and in vivo studies. Consistent with the clinical phenotype, FVIII-R571S demonstrated a 6-fold increase in one-stage assay activity, while chromogenic substrate assay activity was comparable to wild-type FVIII (FVIII-WT). This discrepancy was explained by biochemical studies showing that activated FVIII-R571S (FVIIIa-R571S) has 10-20-fold higher affinity for FIXa; notably, the chromogenic assay is insensitive to differences in FVIIIa-FIXa affinity. Additional analyses demonstrated that FVIII-R571S is inactivated by APC and protein S analogous to FVIII-WT, indicating that the variant is not intrinsically APC-resistant. However, the increased affinity of FVIIIa-R571S for FIXa confers FIXa-dependent reduced A2-domain dissociation and APC-mediated inactivation in purified and plasma-based studies. These enhanced biochemical properties translated in vivo to a more potent procoagulant phenotype in hemophilia A mice. In the tail clip assay, FVIII-R571S exhibited a 4-5-fold increase in potency compared to FVIII-WT. In a thrombosis model, FVIII-R571S promoted significantly increased platelet and fibrin accumulation relative to FVIII-WT at equivalent antigen levels. Collectively, these data demonstrate that the prothrombotic phenotype of FVIII-R571S is driven by increased FIXa affinity, which enhances FVIIIa-FIXa complex assembly and function. This same mechanism confers reduced A2 dissociation and functional APC resistance, providing a unifying explanation for the observed gain-of-function phenotype.

Anemia, the most prevalent hematologic disorder in older adults, imposes a significant burden of cardiovascular events, cognitive decline, and mortality. However, the mechanisms underlying aging-related anemia, especially epigenetic dysregulation in hematopoietic stem and progenitor cells (HSPCs), remain incompletely understood. Although the gut microbiota is critical for hematopoiesis, its specific contribution to aging-related erythropoiesis impairment remains unclear. Here, we reveal that aging markedly activates phenylalanine metabolism and elevates plasma phenylacetic acid (PAA) levels in both humans and mice. We identify Odoribacter splanchnicus (O.splanchnicus) as a key gut symbiont whose abundance is significantly increased in aged mice and which directly drives PAA production from phenylalanine via the oxoacid:ferredoxin oxidoreductase (OFOR) superfamily encoded by porA,nifJ, and iorA/iorB. Rifaximin treatment selectively reduces O.splanchnicus and plasma PAA, thereby alleviating aging-related anemia. Mechanistically, PAA promotes a novel post-translational modification (PTMs) termed histone lysine phenylacetylation (Kpa) through the acetyltransferases HBO1. Elevated histone Kpa increases chromatin accessibility at the GATA2 promoter, disrupts the GATA switch, and blocks erythroid differentiation of HSPCs. In vivo, supplementation with sodium phenylacetate (NaPA) exacerbates anemia in microbiota-depleted mice, whereas the HBO1 inhibitor WM-3835 restores erythropoiesis by reversing histone Kpa and normalizing the GATA switch. Furthermore, dietary phenylalanine restriction lowers circulating PAA and effectively ameliorates aging-related anemia in both naturally aged mice and O.splanchnicus-colonized mice. These findings provide the first evidence that gut microbiota-derived PAA plays a critical role in the development of aging-related erythropoiesis impairment and offer multiple translatable strategies for treating this condition.

Neutrophils are acute responders to bacterial and fungal infection; however, their roles in disease extend far beyond their critical roles in countering pathogens. In this review, we seek to provide some broader context based on recent literature that highlights the contributions of the neutrophil lineage to cancer and chronic inflammation. Their importance for immunomodulation is underscored by their capacity to generate cytokines and chemokines, reactive oxygen species, granule secretory proteins, and use regulated inflammatory cell death pathways for the release of neutrophil extracellular traps. The potential to alter the balance between circumscribed inflammation and collateral tissue damage is most notable when considering the diverse contents and functions of secretory vesicles, and tertiary, specific, and azurophilic granules. We still have much to learn about the generation of granules in neutrophil progenitors, the factors controlling their release from mature neutrophils, and the cellular consequences for misfolding, misplacement, and premature release of granule contents from neutrophils. This review discusses recent advances in our understanding of neutrophil biology, roles in disease, and opportunities for depletion, inhibition, or supplementation and restoration of function.

Sickle cell disease attenuates nitric oxide signaling, limiting soluble guanylyl cyclase (sGC) stimulation needed for hemo-vascular function. Cytochrome b5 reductase 3 (CYB5R3) regulates sGC activity and expression. We show CYB5R3 T117S associates with reduced efficacy of the sGC stimulator riociguat. (NCT02633397).

The impact of chimeric antigen receptor (CAR)-T cell expansion and persistence on clinical outcomes and treatment-related morbidity in patients with relapsed/refractory multiple myeloma (RRMM) remains incompletely defined, in part due to limited availability of standardized CAR-T cell quantification assays. We evaluated CAR-T cell kinetics and their association with efficacy and toxicity in RRMM patients treated with idecabtagene vicleucel (ide-cel) or ciltacabtagene autoleucel (cilta-cel). Using a uniform flow cytometry-based platform (N=90; cilta-cel, n=54; ide-cel, n=36), we observed significantly greater CAR-T cell expansion with cilta-cel than with ide-cel (median 106 vs 49 cells/uL). Peak CAR-T cell expansion was associated with clinical response in the ide-cel cohort but not with cilta-cel, where rapid and excessive expansion was instead associated with an increased risk of delayed neurotoxicities (DNTs), a complication with potential long-term functional consequences (median peak 1,009 vs 96 cells/uL). To identify clinically accessible biomarkers of CAR-T cell expansion, we analyzed absolute lymphocyte count (ALC) as a surrogate biomarker in a larger multicenter cohort (N=532; cilta-cel, n=256; ide-cel, n=276). Higher peak ALC was significantly associated with the development of DNTs, particularly Parkinsonism after cilta-cel. A peak ALC [3]3000/uL - or [3]2500/uL following a daily twofold increase - predicted elevated DNT risk (sensitivity 81%, specificity 59%). Together, these findings delineate distinct expansion-toxicity relationships in cilta-cel and ide-cel therapy, establish ALC as a practical, uniformly available surrogate for CAR-T cell expansion, and define quantitative thresholds that may enable early recognition of patients at risk for DNT, informing preemptive strategies to mitigate morbidity following cilta-cel.