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Slc35a1 deficiency and thrombocytopenia
observed on either WT or Slc35a1–/– platelets (data not shown). RCA 1 (specific for terminal β-galactose) binding was increased in Slc35a1–/– platelets compared to that in WT platelets (Figure 2A). In contrast, MAL II (specific for terminal a2,3-sialic acid) binding was statistically decreased in Slc35a1–/– platelets when compared to its bid- ing in WT platelets (Figure 2A). To determine whether platelet size affected the lectin measurement, we co- stained lectins with anti-CD41 antibody and calculated ratios of their mean fluorescent intensity (MFI). The MFI of RCA 1/CD41 increased significantly while the MFI of MAL II/CD41 decreased significantly in Slc35a1–/– platelets compared to the intensities in WT platelets (Figure 2A). All Slc35a1–/– megakaryocytes were positive for RCA 1 stain- ing by both flow cytometry and confocal microscopy analysis (Figure 2B and C). However, for unknown rea- sons, we were unsuccessful in acquiring reproducible MAL II staining results either by flow cytometric analysis or immunostaining of megakaryocytes. These results demonstrated that disruption of Slc35a1 results in a signif- icant reduction of sialylation in Slc35a1–/– megakaryocytes and platelets.
To complement these lectin-based analyses, we released, purified, and permethylated N- and O-linked glycans from each platelet sample, then performed MALDI-TOF mass spectrometry to determine the platelet sialylation pattern, followed by nanospray ionization- MS/MS to confirm several structures by collision-induced dissociation (Figure 2D and E; Online Supplementary Figures S3 and S4). For N-glycosylation, there was a significant increase of un-sialylation (from 28.6% to 50.1%) in Slc35a1–/– platelets, whereas N-glycolylneuraminic acid (NeuGc), a major isoform of sialic acids in non-human mammals, exhibited a decreased trend (from 71.4% to 49.9%). Moreover, NeuGc isoform analysis showed that one of the NeuGc isoforms was undetectable in Slc35a1–/– platelets (Online Supplementary Figure S4A). Glycan struc- ture analysis indicated that O-glycans such as sialylated (NeuGc) core 1 structures at m/z 669 decreased signific- antly in Slc35a–/– platelets (Online Supplementary Figure S4B, Online Supplementary Table S1).
We noticed that residual sialylation was detected by the high resolution MALDI-TOF-MS/MS analysis in Slc35a1–/– platelets. As Plt Slc35a1–/– mice have normal sialylation in cells including hematopoietic cells other than mega- kyocytes/platelets, we hypothesized that residual sialyla- tion in Slc35a1–/– platelets is caused by exogenous plasma sialylated molecules, such as fibrinogen and IgG which are abundant in the plasma and known to be sialylated, inter- nalized by circulating platelets.28-31 In addition, exogenous IgG is also commonly found on the surface of platelets (PAIgG).30,31 This can be reflected in the fact that the rela- tive ratios of singly to doubly-sialylated glycoforms remained consistent to each other in the WT and Slc35a1– /– platelets, rather than the number of doubly-sialylated glycoforms decreasing in addition to the singly-sialylated and non-sialylated species increasing in incomplete Slc35a1–/– platelets. To test the hypothesis that exogenous sialylated plasma proteins contributed to the residual sialic acids in the Slc35a1–/– platelets, we immunoprecipitated IgG from platelet lysates using protein A/G beads, and then blotted the immunoprecipitated IgG with MALII lectin that binds to a2,3-sialic acids. Our results indicated that both WT and Slc35a1–/– platelets contained MALII- positive IgG (Online Supplementary Figure S5). As
megakaryocytes and platelets do not synthesize IgG, this result indicates that Slc35a1–/– platelets contain exogenous sialylated proteins, which supports our hypothesis. On the basis of all these findings, we concluded that Slc35a1– /– megakaryocytes and platelets have significantly reduced sialylation (Figure 2; Online Supplementary Figures S4 and S5; Online Supplementary Table S1), thus being an appropri- ate model to study the effect of sialylation on endogenous sialylated molecules in megakaryogenesis and platelet homeostasis.
To determine whether abnormal sialic acid accumula- tion existed or not, we checked free sialic acid levels in serum and found that the sialic acid levels were identical in serum from WT and Slc35a1–/– mice (Online Supplementary Figure S6).
Plt Slc35a1–/– mice exhibit macrothrombocytopenia The peripheral platelet count of Plt Slc35a1–/– mice was significantly lower than that of WT mice, and the mean platelet volume of Slc35a1–/– platelets was significantly larger than that of WT platelets (Figure 3A and Online Supplementary Figure S7A). Giemsa staining of blood smears also confirmed fewer platelets in Plt Slc35a1–/– mice (Figure 3B). Tail bleeding time analysis showed no signifi- cant difference between WT and Plt Slc35a1–/– mice (Online Supplementary Figure S7B). Since thrombopoietin regulates megakaryopoiesis and platelet formation, we measured plasma thrombopoietin level and found that it was signif- icantly increased in Plt Slc35a1–/– mice (Figure 3C). However, although the percentage of reticulated platelets was higher, the absolute count of reticulated platelets in Plt Slc35a1–/– mice was lower than that in WT mice (Figure 3D and E). These results suggest that platelet production
was impaired in Plt Slc35a1–/– mice.
There was also no difference in leukocyte count and
hemoglobin content in peripheral blood between WT and Plt Slc35a1–/– mice (Online Supplementary Figure S7C and D). Histology showed no abnormality of major organs (liver, spleen, heart and kidney) in Plt Slc35a1–/– mice (Online Supplementary Figure S8).
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Slc35a1 platelets express lower levels of GPIba
Platelet GPIba (CD42b) and integrin aIIbβ3 (CD41, CD61) are heavily glycosylated. To examine whether Slc35a1 deficiency impaired the surface levels of platelet glycoproteins, we measured GPIba and integrin aIIbβ3 on WT and Slc35a1–/– platelets by flow cytometry. Our results showed that the level of GPIba was decreased on Slc35a1–/– platelets (Figure 4A). However, no difference in integrin aIIbβ3 level was observed between WT and Slc35a1–/– platelets (Figure 4A). Likewise, there was no significant differences in GPIba and integrin aIIbβ3 lev- els between WT and Slc35a1–/– megakaryocytes (Figure 4B). Western blot analysis indicated reduced total GPIba content in Slc35a1–/– platelets (Online Supplementary Figure S9), suggesting that GPIba shedding, but not internalization, is likely the cause of the reduced level of GPIba on Slc35a1–/– platelets.33,34
Deletion of Slc35a1 results in impaired megakaryocyte differentiation and maturation
Platelets are primarily produced by megakaryocytes in the bone marrow. Plt Slc35a1–/– mice exhibited severe thrombocytopenia, raising the question of whether platelet production was affected by the absence of Slc35a1.
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