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Slc35a1 deficiency and thrombocytopenia
an SLC35A1-related congenital disorder of glycosylation (SLC35A1-CDG). Clinical manifestations in these rare SLC35A1-CDG patients are complex, including neutrope- nia, opportunistic infections, delayed psychomotor devel- opment, epilepsy, ataxia, microcephaly and choreiform.20- 23 However, a common manifestation in these patients is macrothrombocytopenia. Accelerated platelet clearance, but not impaired proplatelet production, is considered to contribute to the thrombocytopenia based on in vitro analyses.23
CST is a critical transporter to exchange CMP-sialic acid for CMP across the Golgi membrane, which is essential for sialylation, using an antiport mechanism (Figure 1A).24 So far, no mouse model with deficiency of CMP-SA is available to study this key process. For this reason, we generated genetically-targeted mice with exons 3 and 4 of Slc35a1 being flanked by loxP sites (Slc35a1f/f). In this study, we established a mouse model lacking Slc35a1 specifically in megakaryocytes and platelets (Plt Slc35a1–/–) to determine whether desialylation affects platelet homeostasis. Plt Slc35a1–/– mice exhibited thrombocytope- nia with impaired megakaryocyte maturation and increased platelet clearance. This study provides new insights into the role of sialylation in platelet production and clearance.
Methods
Based on our published methods,7,25 we first generated condi- tional mice with the Slc35a1 gene flanked by loxP sites (Slc35a1f/f). Plt Slc35a1–/– mice were generated by crossing Slc35a1f/f mice with a transgenic mouse line expressing Cre recombinase under the control of the mouse Pf4 promoter (Pf4Cre mice, The Jackson Laboratory, #008535) (Figure 1B). Mice were bred and maintained in specific pathogen-free conditions in the Laboratory Animal Experimental Center at Soochow University and Oklahoma Medical Research Foundation. Mouse studies were approved by the Animal Use Committee of the First Affiliated Hospital of Soochow University and Oklahoma Medical Research Foundation.
Platelet preparation and analysis
Platelets were isolated and prepared based on our previous pub- lications.3,7,26 In brief, whole blood was obtained from the inferior vena cava and collected into test-tubes containing sodium citrate as an anticoagulant, diluted 1:1 with Tyrode buffer, and cen- trifuged at 100 g for 10 min at room temperature. The supernatant was collected and centrifuged at 800 g for 8 min to obtain the platelet pellet, followed by two subsequent washes with Tyrode buffer. Details are provided in the Online Supplementary Data.
Flow cytometry
Details of flow cytometry analysis of platelet sialylation profile, reticulated platelets, and glycoprotein expression on platelet sur- faces are provided in the Online Supplementary Data.
Analysis of bone marrow megakaryocytes
Purification of megakaryocytes from the bone marrow, megakaryocyte counts of bone marrow paraffin-embedded sec- tions, proplatelet formation assay, and assay of colony-forming unit-megakaryocyte are detailed in the Online Supplementary Data.
Western blot analysis
Freshly isolated megakaryocytes or platelets were washed
with phosphate-buffered saline, and the pellet was re-suspend- ed in cell lysis buffer (Cell Signaling Technology) containing protease inhibitor (1:100 dilution, Cell Signaling Technology). Further details are given in the Online Supplementary Data.
Microscopic analysis
Blood smears were fixed with methanol for 10 min at room temperature followed by Wright-Giemsa staining. For immuno- fluorescence microscopy, livers were obtained from mice after perfusion. OCT-embedded livers were cut into 8 mm sections and stained with anti-F4/80 (Abcam) and anti-CD41 antibodies for Küpffer cells and platelets, respectively. After mounting, immunostaining was detected using an inverted confocal fluo- rescence microscope (TCSSP8, Leica). For transmission electron microscopy, bone marrow was fixed with 2.5% glutaraldehyde and embedded in Epon as described previously.27 Further details are provided in the Online Supplementary Data.
Platelet glycan analysis
Glycan structure was analyzed based on our published meth- ods.7 In brief, platelets isolated from wild-type (WT) or Plt Slc35a1–/– mice were dissolved in Ambic buffer (50 mM ammo- nium bicarbonate). Samples were heated for 5 min at 100°C for denaturation and then digested with trypsin (37°C, overnight). After tryptic digestion, the N-glycans were released using PNGase F (New England BioLabs) and the N-linked glycan frac- tion was eluted through a C18 reversed phase cartridge. Subsequently, O-glycans were released by β-elimination. Both N-glycans and O-glycans were permethylated and analyzed by matrix-assisted laser-desorption time-of-flight (MALDI-TOF) tandem mass spectrometry (MS/MS) (Online Supplementary Figure S3).
Statistics
The unpaired Student t-test was used to determine P values as indicated in the figures.
Results
Generation of mice with megakaryocyte- and platelet-specific deletion of Slc35a1
To generate the Slc35a1f/f mice, exons 3 and 4 of the Slc35a1 gene were flanked with loxP sites (Figure 1B). Floxed Slc35a1f/+ embryonic stem cells were injected into blastocysts of C57BL/6J mice to obtain chimeric mice. The derived mice were mated with C57BL/6J mice to achieve germline transmission of the floxed allele. Slc35a1f/+ female mice were then mated with Slc35a1f/+ male mice to gener- ate Slc35a1f/f mice. Slc35a1f/f mice were crossed with Pf4Cre mice to generate Slc35a1f/f;Pf4Cre mice (Plt Slc35a1–/–). Mouse genotypes were confirmed by polymerase chain reaction (PCR) by using tail genomic DNA to amplify a 273-bp product and a 343-bp product from the WT allele and Slc35a1 floxed allele, respectively (Figure 1C). Slc35a1 expression in bone marrow megakaryocytes was abol- ished in Plt Slc35a1–/– mice as determined by quantitative reverse transcription PCR (Figure 1D).
To further confirm the Slc35a1 deletion in Slc35a1–/– platelets, we amplified the major coding exons 3 to 6 of Slc35a1 by reverse transcription PCR. We detected a PCR amplicon of the expected size in cDNA from WT but not from Slc35a1–/– platelets, indicating deletion of functional Slc35a1 transcripts in platelets from Plt Slc35a1–/– mice (Online Supplementary Figure S1).
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