Abstract Number: OC 09.1
Meeting: ISTH 2021 Congress
Background: In humans, a quantitative defect in von Willebrand factor (VWF) causes Type 1 von Willebrand disease (VWD). ABO blood group and ST3GAL4, the glycosyltransferases, have been identified as regulators of VWF levels. However, in 30% of Type 1 VWD patients, the etiology of VWD remains unknown.
Aims: To identify novel glycosyltransferases that regulate plasma VWF levels.
Methods: We performed a piggyback knockdown screen of glycosyltransferase genes using adult zebrafish. Plasma Vwf levels were estimated by ELISA. Larval knockdowns and laser thrombosis assay were according to published protocols.
Results: Since VWF is heavily glycosylated, we hypothesize that other glycosyltransferases may also be involved in regulating VWF. To address this, we established the proof of principle by neuraminidase injections and st3gal4 knockdown experiments using functional assays. We also developed ELISA and confirmed the above results. We then performed a knockdown screen of 234 glycosyltransferase genes orthologous to 170 human glycosyltransferase genes using the simultaneous 3-gene knockdown approach and evaluated plasma Vwf levels by ELISA. We found that knockdown of st6galnac1.1, ugt5b5, extl3, pofut2, gys1, gys2, galnt18a, and galnt18b increased Vwf levels by 1.5-fold while knockdown of st3gal4, si:ch211-250e5.2, alg2, b3galt1a, b3gnt2a, and chsy1 decreased Vwf levels at least 2-fold compared to wild-type. Simultaneous knockdown of st3gal4, si:ch211-250e5.2, alg2, b3galt1a, and b3gnt2a decreased Vwf levels, and impaired ristocetin-mediated agglutination in wild-type and vwf +/- fish, and 4 vwf +/- fish also showed bleeding in their gut region.
Conclusions: Our data show the knockdown of 14 new glycosyltransferase genes independently, altered plasma Vwf levels in zebrafish. The sequencing of these genes in Type 1 VWD patients could help identify novel mutations to decipher the molecular basis for the unknown etiologies in Type 1 VWD. The approach developed here may be utilized for studying other factors regulating not only VWF levels but also other plasma proteins.
To cite this abstract in AMA style:Iyer N, Al Qaryoute A, Jagadeeswaran P. Piggyback Knockdown Screen of Glycosyltransferases in Zebrafish: Identification of Novel Regulators of von Willebrand Factor Levels [abstract]. Res Pract Thromb Haemost. 2021; 5 (Suppl 1). https://abstracts.isth.org/abstract/piggyback-knockdown-screen-of-glycosyltransferases-in-zebrafish-identification-of-novel-regulators-of-von-willebrand-factor-levels/. Accessed September 24, 2021.
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