[PMC free article] [PubMed] [Google Scholar]. functional characterization of megakaryocytes and platelets in mutant CoWas mice and patients with WAS with and without autoantibodies was performed. Platelet antigen expression was examined through a protein expression profile and cluster proteomic conversation network. Platelet immunogenicity was tested by using ELISAs and B-cell and platelet cocultures. Results: CoWas mice showed increased megakaryocyte numbers and normal thrombopoiesis female mice22 and Pf4-Cre male mice (Jackson Laboratory). All the mouse models (WT, WKO, WKO-experiments Platelet depletion Sermorelin Aceta was performed by means of intravenous injection of 0.5 g/g of rat purified anti-mouse GPIba (Emfret Analytics, Eibelstadt, Germany) in PBS. At different time points after injection, blood has been collected from the retro-orbital sinus to analyze platelet counts by using a hemogram and RT-PLTs by using Thiazole Orange staining. Platelet clearance was evaluated by mean of retro-orbital injection of 3 g of DyLight 488Clabeled or Alexa Fluor 647Clabeled antiCGPIb-V-IX (Emfret Analytics) antibody diluted in PBS.25 To analyze the percentage TWS119 of labeled platelets, 10 L of whole blood was incubated with 2 L of anti-CD61 for 15 minutes, followed by addition of 150 mL of PBS. To perform the adoptive transfer experiment, values of less than .05 were considered significant. Study approval Animal procedures TWS119 were performed according to IACUC 557 and 741 approved by the Italian Institutional Animal Care and OSR Committee. Human studies were performed according to the Helsinki Declaration and the TIGET PERIBLOOD or TIGET08B, TIGET02, and TIGET06 clinical protocols approved by the OSR Ethical Committee. The study was also approved by the National Institutes of Health Institutional Review Board (protocol 16-I-N139). RESULTS CoWas mice selectively lack WASp in platelets To understand the extent to which the lack of WASp expression affects platelet generation and survival, we generated mice lacking WASp only in the megakaryocytic lineage. We obtained compound heterozygous female (CoWas+/?) and CoWas mice, which were completely null for WASp expression in the megakaryocytic lineage (see Fig E1, represents a different mouse from 2 impartial experiments; in Fig 1, represents the analysis of single platelets within different TEM images. All the graphs report means SDs, and statistical analysis was performed with 1-way ANOVA and the Bonferroni postcorrection test. *< .05, **< .005, ***< .001, and ****< .0001. and identified as CD41+CD150+ cells gated around the Lin?c-Kit+Sca1? compartment in WT, WKO, and CoWas mice is usually shown. B, The number of MKcolony-forming units was counted with the microscope by using a 4 objective. C and D, MKs (CD61+CD41+ cells) are expressed as percentages on total BM cells (Fig 2, represents a different mouse from 1 (Fig 2, < .05, **< .005, ***<.001, and ****< .0001. normal proplatelet formation (see Fig E2, thrombopoiesis, we depleted platelets by means of platelet injection of anti-GPIb mAb (Fig 2, data might suggest normal thrombopoiesis in WKO and CoWas mice, at least in stressed situations, because mutant animals have higher numbers of MKs in the BM and do not reach an equilibrium of RT-PLTs in a shorter time period, we cannot exclude that thrombopoiesis per MK could be affected. Increased peripheral elimination of hyperactived injection of Dy-Light 488-anti-GPIb-V-IX antibody selectively labeling circulating platelets25 and observed a shorter lifespan in WKO and CoWas mice (Fig 3, and platelet half-life detection. B, Platelet half-life was analyzed as the percentage of GPIb-V-IX+ platelets 15 minutes after injection (time 0) and at different time points after injection. C, Differentially labeled WT and represents a different mouse from TWS119 1 (Fig 3, and TWS119 and and < .05, **< .005, ***< .001, and ****< .0001. ratios of 3.5 or greater and values of .05 or less were extracted as descriptors (see Table E2 in this articles Online Repository at www.jacionline.org). Hierarchical clustering obtained on processing descriptors showed that CoWas is an intermediate condition between WT and WKO (see Fig E3, protein-protein conversation network by using Cytoscape and STRING db (see Fig E4 in this articles Online Repository at www.jacionline.org). The network analysis (19 clusters) confirmed intermediate levels of expression of distinct biological processes/functional classes in CoWas platelets between WT and WKO platelets. This obtaining was more evident when focusing on a subnetwork of 8 clusters involving metabolic processes, cytoskeleton, ubiquitins, 14-3-3 proteins, and immunoglobulins (Fig 5, A). Specifically, we did not find any abnormalities in the total amount of actin-related proteins but relative TWS119 changes in some proteins related to this pathway. Metabolic profile perturbation with a reduction in lipid, glucose, and phosphate metabolism-related proteins was also found. On the other hand, ubiquitin-related proteins were upregulated in line with reduced lifespan and increased antigen processing, which is frequently observed in patients with autoimmune disorders. 33 The proteomic profile also revealed alterations in the immunoglobulin content,.