Figure 2.
St3gal1 deletion in MKs leads to TF antigen expression and thrombocytopenia that is not due to accelerated clearance. (A) Binding of PNA specific for TF antigen in various BM cell types: MKs, RBCs, leukocytes (white blood cells [WBCs]), and myeloid cells. Dashed line at 1 indicates fold, WPQD3. change over controls (n = 5-7). (B) Binding of various lectins (PNA, Sambucus nigra lectin [SNA], Ricinus communis agglutinin [RCA], and Erythrina crista-galli lectin [ECL]) to platelets as evaluated by flow cytometry. Dashed line indicates fold change over controls (n = 3-8). (C) Platelet count in control mice (blue) and St3gal1MK−/− mice (red) (n = 13-14). (D) Endogenous platelet clearance was measured by daily monitoring (via flow cytometry) of the percent circulating biotinylated platelets and fluorescein isothiocyanate–streptavidin (n = 3). (E) Platelet glycoprotein expression was measured by flow cytometry using specific antibodies. Size-normalized mean fluorescence intensity (MFI) from analysis of ST3Gal1MK−/− platelets is shown. Dashed line indicates fold change over controls. (F) MFI of platelet surface IgG measured by using flow cytometry. (G) IgG levels in plasma and BM supernatant. (H) Platelet counts of mice weeks postspenectomy, measured at the indicated time points (n = 3 for both phenotypes). (I) Platelet clearance of fluorescently labeled St3gal1MK−/− platelets transfused into wild-type (WT) or AMR-null mice (n = 3). (J) TPO messenger RNA expression in control and St3gal1MK−/− mouse livers (n = 4-5). (K) Control, St3gal1MK−/−, and St3gal4−/− platelet lysate were subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis and proved with anti-GPIbα and anti–β-actin antibodies (n = 3-4). Full blots (uncropped) are shown in supplemental Figure 2. For comparison of the 2 groups (A-B,E-G,J), a 2-tailed unpaired Student t test was performed. The Student t test was performed comparing MFI of WT vs St3gal1MK−/− of the measured parameters (A-B,E). For comparison of 2 groups over a treatment period (D,H-I), a multiple Student t test with Holm-Šídák method correction was performed. *P < .05, **P < .01, ****P < .0001. Only significant changes are annotated.

St3gal1 deletion in MKs leads to TF antigen expression and thrombocytopenia that is not due to accelerated clearance. (A) Binding of PNA specific for TF antigen in various BM cell types: MKs, RBCs, leukocytes (white blood cells [WBCs]), and myeloid cells. Dashed line at 1 indicates fold, WPQD3. change over controls (n = 5-7). (B) Binding of various lectins (PNA, Sambucus nigra lectin [SNA], Ricinus communis agglutinin [RCA], and Erythrina crista-galli lectin [ECL]) to platelets as evaluated by flow cytometry. Dashed line indicates fold change over controls (n = 3-8). (C) Platelet count in control mice (blue) and St3gal1MK−/− mice (red) (n = 13-14). (D) Endogenous platelet clearance was measured by daily monitoring (via flow cytometry) of the percent circulating biotinylated platelets and fluorescein isothiocyanate–streptavidin (n = 3). (E) Platelet glycoprotein expression was measured by flow cytometry using specific antibodies. Size-normalized mean fluorescence intensity (MFI) from analysis of ST3Gal1MK−/− platelets is shown. Dashed line indicates fold change over controls. (F) MFI of platelet surface IgG measured by using flow cytometry. (G) IgG levels in plasma and BM supernatant. (H) Platelet counts of mice weeks postspenectomy, measured at the indicated time points (n = 3 for both phenotypes). (I) Platelet clearance of fluorescently labeled St3gal1MK−/− platelets transfused into wild-type (WT) or AMR-null mice (n = 3). (J) TPO messenger RNA expression in control and St3gal1MK−/− mouse livers (n = 4-5). (K) Control, St3gal1MK−/−, and St3gal4−/− platelet lysate were subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis and proved with anti-GPIbα and anti–β-actin antibodies (n = 3-4). Full blots (uncropped) are shown in supplemental Figure 2. For comparison of the 2 groups (A-B,E-G,J), a 2-tailed unpaired Student t test was performed. The Student t test was performed comparing MFI of WT vs St3gal1MK−/− of the measured parameters (A-B,E). For comparison of 2 groups over a treatment period (D,H-I), a multiple Student t test with Holm-Šídák method correction was performed. *P < .05, **P < .01, ****P < .0001. Only significant changes are annotated.

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