Sialyltransferase ST3Gal-IV operates as a dominant modifier of hemostasis by concealing asialoglycoprotein receptor ligands

Abstract

A number of poorly characterized genetic modifiers contribute to the extensive variability of von Willebrand disease, the most prevalent bleeding disorder in humans. We find that a genetic lesion inactivating the murine ST3Gal-IV sialyltransferase causes a bleeding disorder associated with an autosomal dominant reduction in plasma von Willebrand factor (VWF) and an autosomal recessive thrombocytopenia. Although both ST3Gal-IV and ST6Gal-I sialyltransferases mask galactose linkages implicated as asialoglycoprotein receptor ligands, only ST3Gal-IV deficiency promotes asialoglycoprotein clearance mechanisms with a reduction in plasma levels of VWF and platelets. Exposed galactose on VWF was also found in a subpopulation of humans with abnormally low VWF levels. Oligosaccharide branch-specific sialylation by the ST3Gal-IV sialyltransferase is required to sustain the physiologic half-life of murine hemostatic components and may be an important modifier of plasma VWF level in humans.

Figure 1

ST3Gal-IV mutation in the germline of mice ablates the large sialyl motif and results in loss of RNA. (A) An ST3Gal-IV genomic isolate in germline configuration was used in conjunction with the pflox vector to construct a targeting vector in which exons 5–7 containing the large sialyl motif (16) were flanked by loxP sites for deletion (ST3Gal-IV^F[tkneo]). Restriction enzyme sites indicated are BamHI (B) AvrII (A), EcoRI (E), Hind III (H), KpnI (K), NotI (N), SalI (Sa), and SpeI (Sp). (B) Transient Cre expression in ST3Gal-IV gene-targeted R1 embryonic stem cells produces subclones heterozygous for the Δ (systemic deleted) or F (conditional) mutations illustrated. (C) Southern blot analysis of AvrII- and SpeI-digested embryonic stem cell DNA probed with a loxP probe confirmed the expected genomic structures. Three loxP sites are present in a targeted parental clone (–6), whereas one loxP site is present in each of two ST3Gal-IV^Δ/wt subclones (21-F1 and 21-D1), and two loxP sites are present in the ST3Gal-IV^F/wt subclones (21-A3 and 21-E1). (D) Tail DNA derived from offspring of parents heterozygous for the Δ allele was digested with HindIII and probed with the indicated genomic probe to reveal the 6.8-kb wild-type allele and the 5.3-kb deleted (Δ) allele. (E) ST3Gal-IV RNA expression in normal wild-type mouse tissues. (F) ST3Gal-IV RNA expression is deficient among ST3Gal-IV^Δ/Δ mice.

Figure 2

Increased bleeding time with decreased levels of VWF and Factor VIII occurs with ST3Gal-IV deficiency but not with ST6Gal-I deficiency. (A) Bleeding times were analyzed among 6- to 12-wk-old littermates of the indicated genotypes. Results are from over 30 mice of each ST3Gal-IV genotype and 11 of each ST6Gal-I genotype are expressed as means ± SEM. Significantly increased bleeding times for ST3Gal-IV^Δ heterozygotes and homozygotes were observed (P < 0.05 and P < 0.001, respectively). (B) VWF levels in plasma were measured from over 30 mice of each genotype and plotted as a percentage of the mean value obtained from ST3Gal-IV wild-type littermates. A significant reduction in plasma VWF was observed in both heterozygous and homozygous ST3Gal-IV-deficient mice to a mean of 57 and 50%, respectively (P < 0.001). No decrease in plasma VWF levels was observed in mice lacking ST6Gal-I. (C) Factor VIII (F VIII) levels were also reduced in mice analyzed in B (P < 0.001). ST6Gal-I deficiency had no effect on F VIII levels in circulation (not shown). (D) Levels of blood coagulation factors were analyzed from 40 mice of indicated genotypes. No alterations of hemostatic significance were observed.

Figure 3

VWF glycosylation deficiencies and multimer formation. Alterations in lectin binding are compared with results obtained by using purified sialidase treatment (*; see Methods). (A) RCA-I lectin binding to plasma-derived VWF from wild-type and ST3Gal-IV mutant mice indicates increased exposure of terminal β-linked Gal on VWF from both heterozygous and homozygous mutant samples. Reduced Ricin binding to VWF from ST6Gal-I-deficient mice was observed. (B) ECA-I lectin binding to Galβ1–4GlcNAc- termini on VWF is increased in mice heterozygous or homozygous for ST3Gal-IV gene mutation. Absence of ST6Gal-I also results as expected in increased ECA binding to VWF. (C) SNA lectin binding to VWF is eliminated in ST6Gal-I deficiency, as expected, but increased in ST3Gal-IV deficiency. (D) PNA lectin binding to Galβ1–3GalNAc- is unaltered on VWF glycans from ST3Gal-IV mutant mice. (E) Possible N-glycan branch termini structures of wild-type and mutant VWF. Wild-type VWF bears highly sialylated N-glycan branches with both α3- and α6-linked sialic acids. Absence of ST6Gal-I eliminates only α6-linked sialic acid, although a partial compensation by ST3Gal-IV may occur (lightly shaded sialic acid). Separately, ST3Gal-IV deficiency exposes ECA-reactive Galβ1–4 GlcNAc termini and may also expose the Galβ1–3GlcNAc- disaccharide. Partial compensation (shaded sialic acid linkage) by ST6Gal-I occurs, whereas no sialyltransferase exists that can add α6 linkages to Galβ1–3GlcNAc-. However, some degree of α3 sialylation of Galβ1–3GlcNAc- may occur (shaded sialic acid). No changes occur in the sialylation of the Core 1 O-glycan Galβ1–3GalNAc-. (F) Multimer analysis of plasma VWF from wild-type and ST3Gal-IV mutants indicates normal multimer formation in mutant samples. Data are representative of three separate experiments. Lectin binding in A–C was quantitated by ELISA and plotted as the ratio of lectin binding per unit of VWF antigen (see Methods). Data in A–C are means ± SEM as derived from analyses of 12 or more mice of each genotype in each assay. Saccharide symbols: GlcNAc, black square; GalNAc; open square; Gal, black circle; sialic acid, black diamond.

Figure 4

Decreased VWF half-life and platelet homeostasis corrected by blocking ASGPR. (A) Plasma fractions from mice of indicated genotypes were biotinylated and injected into wild-type mice in the presence or absence of 10 mg of pyrogen-free asialofetuin. Biotinylated VWF levels in blood were analyzed at the indicated times. Data are derived from three mice per treatment group and are representative of two separate experiments. In additional experiments that included native fetuin, only asialofetuin was able to block ASGP clearance and increase half-life (data not shown). (B) RCA lectin binding to platelets was increased among blood samples from mice homozygous for the ST3Gal-IV mutation as detected by flow cytometric analysis. A slight increase in RCA-I binding to platelets is seen in heterozygous samples. Data are representative of three separate experiments. (C) Mice homozygous for the ST3Gal-IV gene deletion were injected with asialofetuin or fetuin at 0, 4, and 8 h. Platelet levels were analyzed by flow cytometry of whole blood at the timepoints indicated. Data are the means ± SEM from 10 mice per group.

Figure 5

RCA/VWF ratios in ST3Gal-IV deficiency are similar to those in a subpopulation of humans bearing abnormally low plasma VWF levels. (A) Ratios of RCA-I binding to VWF antigen level in mouse plasma consist of low values in wild-type littermates. In ST3Gal-IV mutant mice, ratios are almost always increased to greater than 2 SD above the mean, as indicated by the dotted line. Data reflect more than 15 mice of each genotype. (B) A subpopulation of humans bearing abnormally low VWF levels shows increased RCA/VWF ratios more than 2 SD from the mean. No examples of similarly increased RCA/VWF ratios were found among samples bearing more than 50% the normal level of VWF.