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FNAIT is a pregnancy-associated condition caused by maternal alloantibodies against paternally-inherited platelet antigens, most frequently HPA-1a on integrin ß3. The clinical effects range from no symptoms to fatal intracranial hemorrhage, but underlying pathophysiological determinants are poorly understood. Accumulating evidence suggests that differential antibody-Fc-glycosylation, activation of complement/effector cells, and integrin function-blocking effects contribute to clinical outcome. Furthermore, some antibodies preferentially bind platelet integrin ¿IIbß3, but others bind ¿vß3 on endothelial cells and trophoblasts. Defects in endothelial cells and angiogenesis may therefore contribute to severe anti-HPA-1a associated FNAIT. Moreover, anti-HPA-1a antibodies may cause placental damage, leading to intrauterine growth restriction. We discuss current insights into diversity and actions of HPA-1a antibodies, gathered from clinical studies, in vitro studies, and mouse models. Assessment of all factors determining severity and progression of anti-HPA-1a-associated FNAIT may importantly improve risk stratification and potentially reveal novel treatment strategies, both for FNAIT and other immunohematological disorders.

Background: Patients with gray platelet syndrome (GPS) and Neurobeachin-like 2 (NBEAL2) deficiency produce platelets lacking alpha-granules (AGs) and present with lifelong bleeding symptoms. AGs are lysosome-related organelles and store the hemostatic protein von Willebrand factor (VWF) and the transmembrane protein P-selectin. Weibel-Palade bodies (WPBs) are lysosome-related organelles of endothelial cells and also store VWF and P-selectin. In megakaryocytes, NBEAL2 links P-selectin on AGs to the SNARE protein SEC22B on the endoplasmic reticulum, thereby preventing premature release of cargo from AG precursors. In endothelial cells, SEC22B drives VWF trafficking from the endoplasmic reticulum to Golgi and promotes the formation of elongated WPBs, but it is unclear whether this requires NBEAL2. Objectives: To investigate a potential role for NBEAL2 in WPB biogenesis and VWF secretion using NBEAL2-deficient endothelial cells. Methods: The interaction of SEC22B with NBEAL2 in endothelial cells was investigated by interatomic mass spectrometry and pull-down analysis. Endothelial colony forming cells were isolated from healthy controls and 3 unrelated patients with GPS and mutations in NBEAL2. Results: We showed that SEC22B binds to NBEAL2 in ECs. Endothelial colony forming cells derived from a patient with GPS are deficient in NBEAL2 but reveal normal formation and maturation of WPBs and normal WPB cargo recruitment. Neither basal nor histamine-induced VWF secretion is altered in the absence of NBEAL2. Conclusions: Although NBEAL2 deficiency causes the absence of AGs in patients with GPS, it does not impact WPB functionality in ECs. Our data highlight the differences in the regulatory mechanisms between these 2 hemostatic storage compartments.

Von Willebrand factor (VWF) is a glycoprotein that is secreted into the circulation and controls bleeding by promoting adhesion and aggregation of blood platelets at sites of vascular injury. Substantial inter-individual variation in VWF plasma levels exists among the healthy population. Prior to secretion, VWF polymers are assembled and condensed into helical tubules, which are packaged into Weibel-Palade bodies (WPBs), a highly specialized post-Golgi storage compartment in vascular endothelial cells. In the inherited bleeding disorder Von Willebrand disease (VWD), mutations in the VWF gene can cause qualitative or quantitative defects, limiting protein function, secretion, or plasma survival. However, pathogenic VWF mutations cannot be found in all VWD cases. Although an increasing number of genetic modifiers have been identified, even more rare genetic variants that impact VWF plasma levels likely remain to be discovered. Here, we summarize recent evidence that modulation of the early secretory pathway has great impact on the biogenesis and release of WPBs. Based on these findings, we propose that rare, as yet unidentified quantitative trait loci influencing intracellular VWF transport contribute to highly variable VWF levels in the population. These may underlie the thrombotic complications linked to high VWF levels, as well as the bleeding tendency in individuals with low VWF levels.

Von Willebrand factor (VWF) is a multimeric hemostatic protein primarily synthesized in endothelial cells. VWF is stored in endothelial storage organelles, the Weibel-Palade bodies (WPB), whose biogenesis strongly depends on VWF anterograde trafficking and Golgi architecture. Elongated WPB morphology is correlated to longer VWF strings with better adhesive properties. We previously identified the SNARE SEC22B, which is involved in anterograde endoplasmic reticulum-to-Golgi transport, as a novel regulator of WPB elongation. To elucidate novel determinants of WPB morphology we explored endothelial SEC22B interaction partners in a mass spectrometry-based approach, identifying the Golgi SNARE Syntaxin 5 (STX5). We established STX5 knockdown in endothelial cells using shRNA-dependent silencing and analyzed WPB and Golgi morphology, using confocal and electron microscopy. STX5-depleted endothelial cells exhibited extensive Golgi fragmentation and decreased WPB length, which was associated with reduced intracellular VWF levels, and impaired stimulated VWF secretion. However, the secretion-incompetent organelles in shSTX5 cells maintained WPB markers such as Angiopoietin 2, P-selectin, Rab27A, and CD63. In brief, we identified SNARE protein STX5 as a novel regulator of WPB biogenesis.

Integrins are transmembrane receptors that transduce biochemical and mechanical signals across the plasma membrane and promote cell adhesion and migration. In addition, integrin adhesion complexes are functionally and structurally linked to components of the intracellular trafficking machinery and accumulating data now reveal that they are key regulators of endocytosis and exocytosis in a variety of cell types. Here, we highlight recent insights into integrin control of intracellular trafficking in processes such as degranulation, mechanotransduction, cell-cell communication, antibody production, virus entry, Toll-like receptor signaling, autophagy, and phagocytosis, as well as the release and uptake of extracellular vesicles. We discuss the underlying molecular mechanisms and the implications for a range of pathophysiological contexts, including hemostasis, immunity, tissue repair, cancer, and viral infection.