Biology subjects

Margadant, C.

Publications and source records attributed to Margadant, C..

3 recordsLinked to original sources

bioRxiv2026

High-resolution cryo-EM structure of integrin αIIbβ3 bound to disease-causing maternal HPA-1a antibody that blocks integrin activation

Integrins promote immunity, embryonic development, wound healing, and hemostasis, and are activated by bent/closed to extended/open conformational changes. Integrin IIb{beta}3, being crucial for platelet activation and aggregation, is a therapeutic target for bleeding disorders and thrombosis. Human Platelet Antigen-1a (HPA-1a) on {beta}3 is recognized by pregnancy-associated maternal alloantibodies, potentially causing fetal/neonatal alloimmune thrombocytopenia (FNAIT) and even intracranial hemorrhage or perinatal death. However, severe disease determinants are largely unknown. We report the first structure of an anti-HPA-1a antibody fragment (Fab 26.4) in complex with integrin IIb{beta}3 at high resolution by cryo-electron microscopy. Fab 26.4 binding traps IIb{beta}3 in the inactive, bent/closed conformation, is incompatible with integrin extension, and inhibits IIb{beta}3-dependent fibrinogen binding and platelet aggregation. Thus, anti-HPA-1a antibodies directly impair integrin activation by preventing required conformational changes. These insights will improve FNAIT diagnostics and treatment, and spark the development of novel allosteric inhibitors against {beta}3 integrins for future therapeutic applications.

biophysics↗

bioRxiv2022

Mutations in the SEC22B interactor NBEAL2 do not impact Weibel-Palade body biogenesis and Von Willebrand factor secretion in Gray Platelet Syndrome Endothelial Colony Forming Cells

BackgroundGray Platelet Syndrome (GPS) patients with Neurobeachin-like 2 (NBEAL2) deficiency produce platelets lacking alpha-granules (AGs) and present with lifelong bleeding symptoms. AGs are lysosome-related organelles (LROs) and store the hemostatic protein Von Willebrand factor (VWF) and the transmembrane protein P-selectin. Weibel-Palade bodies (WPBs) are LROs 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 (ER), thereby preventing premature release of cargo from AG precursors. In endothelial cells, SEC22B drives VWF trafficking from ER to Golgi and promotes the formation of elongated WPBs, but it is unclear if this requires NBEAL2. ObjectivesTo investigate a potential role for NBEAL2 in WPB biogenesis and VWF secretion using NBEAL2 deficient endothelial cells. MethodsInteraction of SEC22B with NBEAL2 in endothelial cells was investigated by interactomic mass spectrometry and pull down analysis. Endothelial Colony Forming Cells (ECFCs) were isolated from healthy controls and 3 unrelated GPS patients with mutations in NBEAL2. ResultsWe show that SEC22B binds to NBEAL2 in ECs. GPS patient-derived ECFCs are deficient of NBEAL2, but reveal normal formation and maturation of WPBs and normal WPB cargo recruitment. Neither basal nor histamine-induced VWF secretion are altered in the absence of NBEAL2. ConclusionsWhile NBEAL2 deficiency causes absence of AGs in GPS patients, it has no impact on WPB functionality in ECs. Our data highlight the difference in regulatory mechanisms between these two hemostatic storage compartments. EssentialsO_LIWe characterized Gray Platelet Syndrome patient-derived endothelial cells with biallelic NBEAL2 mutations ex vivo. C_LIO_LINBEAL2 is not essential for Weibel-Palade body biogenesis, maturation, and Von Willebrand factor secretion from endothelial cells. C_LI

cell biology↗

bioRxiv2021

Syntaxin 5 determines Weibel-Palade body size and Von Willebrand factor secretion by controlling Golgi architecture

Von Willebrand factor (VWF) is a multimeric hemostatic protein primarily synthesized in endothelial cells (ECs). VWF is stored in endothelial storage organelles, the Weibel-Palade bodies (WPBs), 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 ER-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 spectrometrybased approach, identifying the Golgi SNARE Syntaxin 5 (STX5). We established STX5 knockdown in ECs using shRNA-dependent silencing and analyzed WPB and Golgi morphology, using confocal and electron microscopy. STX5-depleted ECs 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. Taken together, our study has identified SNARE protein STX5 as a novel regulator of WPB biogenesis.

cell biology↗