Abstract 840: Major gD-Type 3-O-Sulfotransfeases Can Produce Anticoagulant Heparan Sulfate
Introduction: Anticoagulant heparan sulfate (HSAT+) is a form of heparan sulfate with antithrombin (AT) binding sites. In vitro, HSAT+ catalyzes AT neutralization of coagulation proteases. HSAT+ is mainly derived from the enzyme 3-O-sulfotransferase-1 (3OST1; encoded by the Hs3st1 gene). Hs3st1−/− mice exhibit large reductions in HSAT+ but unexpectedly show normal hemostatic tone, which might reflect HSAT+ production by other 3OST isoforms. 3OST5 can generate HSAT+, but has extremely minor and limited expression. The remaining 3OST isoforms are classified as gD-type; they are not considered to produce significant HSAT+ and instead chiefly synthesizes a distinct motif, HSgD+.
Hypothesis: gD-Type 3OSTs can drive cellular synthesis of HSAT+.
Methods & Results: To identify isoforms that may produce HSAT+ in vivo, we studied Hs3st1+/+ and Hs3st1−/− liver and brain. These tissues show high HSAT+ levels in Hs3st1−/− mice. Expression of all seven 3OST isoforms was measured by real time PCR. In both genotypes, the predominant isoforms were major gD-type isoforms; 3OST3A/3OST3B in liver and 3OST2/3OST4 in brain. Liver lacked 3OST5; yet the probing of liver sections with fluorescently tagged AT and anti-CD31 revealed comparable levels of HSAT+ on Hs3st1+/+ and Hs3st1−/− sinusoidal endothelium. Combined these data suggests gD-type isoforms might generate vascular HSAT+ in vivo. Enzymatic analyses of extracts from transient transfectants revealed 3OST2, 3OST3A and 3OST4 can all generate HS AT+, but at 200- to 400-fold lower efficiency than 3OST1. To determine if high cellular expression can circumvent such low in vitro efficiencies, 3OST isoforms were expressed by retroviral transduction of a naïve cell line and cell surface HSAT+ was measured by flow cytometry. Transductants expressing the various gD-isoforms exhibited HSAT+ levels that were only 2- to 30-fold lower than for 3OST1 transductants. Moreover, HSAT+ levels of gD-type transductants were greater than that of primary endothelial cells.
Conclusions: Despite their low enzymatic efficiencies, gD-type 3OSTs can drive substantial cellular synthesis of HSAT+. Endothelial cell expression of gD-type isoforms might account for the lack of a procoagulant phenotype in Hs3st1−/− mice.