We describe here the mechanism of platelet adhesion to immobilized von Willebrand factor (VWF) and subsequent formation of platelet-derived microparticles mediated by glycoprotein Ib␣ (GPIb␣) under high shear stress. As visualized in whole blood perfused in a flow chamber, platelet attachment to VWF involved one or few membrane areas of 0.05 to 0.1 m 2 that formed discrete adhesion points (DAPs) capable of resisting force in excess of 160 pN. Under the influence of hydrodynamic drag, membrane tethers developed between the moving platelet body and DAPs firmly adherent to immobilized VWF. Continued stretching eventually caused the separation of many such tethers, leaving on the surface tubeshaped or spherical microparticles with a diameter as low as 50 to 100 nm. Adhesion receptors (GPIb␣, ␣IIb␤3) and phosphatidylserine were expressed on the surface of these microparticles, which were procoagulant. Shearing platelet-rich plasma at the rate of 10 000 s ؊1 in a cone-and-plate viscosimeter increased microparticle counts up to 55-fold above baseline. Blocking the GPIb-VWF interaction abolished microparticle generation in both experimental conditions. Thus, a biomechanical process mediated by GPIb␣-VWF bonds in rapidly flowing blood may not only initiate platelet arrest onto reactive vascular surfaces but also generate procoagulant microparticles that further enhance thrombus formation. IntroductionThe integrity of the vessel wall is key for the normal circulation of blood and is constantly surveyed by platelets. 1 In arterial flow, platelets are positioned at high density near the endothelial-cell layer, while erythrocytes are lifted away from it through a hemodynamic process called axial migration. 2,3 When damage to the vascular surface occurs, von Willebrand factor (VWF) binds rapidly to exposed subendothelial structures 4,5 and enables platelet arrest from fast-flowing blood through the interaction of its A1 domain (VWFA1) with the platelet glycoprotein Ib␣ (GPIb␣) receptor. 6 The VWFA1-GPIb␣ bond has a short half-life and by itself cannot provide irreversible adhesion. Consequently, the torque imposed by flowing blood causes platelets to translocate over immobilized VWF until receptors such as glycoprotein VI or integrin ␣IIb␤3 engage their respective ligands and mediate permanent adhesion, spreading, and aggregation. 7 Under the effect of shear stress, platelet-derived microparticles (PMPs) can be generated in blood through a process that was reported to be dependent on the VWF-GPIb␣ interaction by some investigators 8 but not others. 9 Owing to their ability to bind coagulation factors, and the exposure on their surface of phosphatidylserine 9 as well as adhesion receptors 10 and possibly tissue factor, 11,12 PMPs have been suggested to play a role in blood clotting and thrombus formation. 13 Lacking so far, however, is a direct visualization and explanation of how shear stress can induce the generation of microparticles from platelets and how this may be linked to the subsequent development of thrombi. Becaus...

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ObjectivesNeuromyelitis optica spectrum disorder (NMOSD) is a severe inflammatory disorder of the central nervous system (CNS) targeted against aquaporin‐4 (AQP4). The origin and trafficking of AQP4‐specific B cells in NMOSD remains unknown.MethodsPeripheral (n = 7) and splenic B cells (n = 1) recovered from seven NMOSD patients were sorted into plasmablasts, naïve, memory, and CD27‐IgD‐ double negative (DN) B cells, and variable heavy chain (VH) transcriptome sequences were generated by deep sequencing. Peripheral blood (PB) VH repertoires were compared to the same patient's single‐cell cerebrospinal fluid (CSF) plasmablast (PB) VH transcriptome, CSF immunoglobulin (Ig) proteome, and serum Ig proteome. Recombinant antibodies were generated from paired CSF heavy‐ and light chains and tested for AQP4 reactivity.ResultsApproximately 9% of the CSF VH sequences aligned with PB memory B cells, DN B cells, and plasmablast VH sequences. AQP4‐specific VH sequences were observed in each peripheral B‐cell compartment. Lineage analysis of clonally related VH sequences indicates that CSF AQP4‐specific B cells are closely related to an expanded population of DN B cells that may undergo antigen‐specific B‐cell maturation within the CNS. CSF and serum Ig proteomes overlapped with the VH sequences from each B‐cell compartment; the majority of matches occurring between the PB VH sequences and serum Ig proteome.InterpretationDuring an acute NMOSD relapse, a dynamic exchange of B cells occurs between the periphery and CNS with AQP4‐specific CSF B cells emerging from postgerminal center memory B cells and plasmablasts. Expansion of the PB DN B‐cell compartment may be a potential biomarker of NMOSD activity.

Background: Patient-derived aquaporin 4 (AQP4)-specific recombinant monoclonal antibodies (rAbs) cause neuromyelitis optica (NMO)-specific nervous system injury in animal models.Results: AQP4-specific rAbs bind human AQP4 based on differential sensitivity to loop A and C mutations.Conclusion: AQP4-specific rAbs derived from NMO patients recognize multiple conformational epitopes within the extracellular domains of human AQP4.Significance: High resolution mapping of AQP4 autoantibody epitopes identifies target regions for potential blocking therapies.

The study attempted to determine the incidence of HIV among pregnant women in Kabarole District, Uganda, and to identify socio-demographic and behavioral risk factors for seroconversion during pregnancy.