Abstract Number: PB0188
Meeting: ISTH 2020 Congress
Background: Tissue Factor Pathway Inhibitor (tfpi) is an anticoagulant that inhibits both VIIa and Xa in the blood coagulation pathways. There are three Kunitz domains, K1, K2, and K3 in tfpi. K1 and K2 mainly inhibit factor VIIa and Xa, respectively. The role of K3 in vivo is not studied.
Aims: To establish the role of K3 using the zebrafish model.
Methods: Cas9 DNA was made by a plasmid DNA preparation method. gDNA template was amplified by PCR with primers containing a T7 promoter. kPT, kPTT assays, qRT-PCRs, in vitro transcriptions, laser-induced thrombosis, and piggyback knockdowns were performed according to published methods. Student’s t-test was used in statistical analysis.
Results: To address the role of K3, we transcribed both Cas9 DNA and gDNA template corresponding to K3, and this mixture was injected into single cell-stage zebrafish embryos. Genomic DNA from the tail clips of adult fish resulting from the injections was subjected to PCR across the K3 domain coding region. From these, a founder line with a deletion in the K3 region was identified. We then reared homozygous mutants and tested for kPT, kPTT, and laser-induced thrombosis using control siblings, and found that there were increased kPT and kPTT activities and decreased venous TTO but not arterial TTO. We performed piggyback knockdowns of tfpi, confirmed the knockdowns by qRT-PCRs, and found similar coagulation activities. In both tfpi and factor VIII knockdowns, factor VIII deficiency was rescued. tfpi RNA level of knockout fish was lower compared to the control. Since the results between knockout and knockdown fish are similar, we believe that K3 is not playing a role in vivo.
Conclusions: We generated a knockout tfpi zebrafish where the K3 domain is missing, a tfpi knockdown zebrafish, and assayed for clotting. The results suggest that the K3 domain does not have any in vivo function.
To cite this abstract in AMA style:Raman R, Ryon M, Al Qaryoute A, Jagadeeswaran P. Role of Kunitz Domain 3 of Tissue Factor Pathway Inhibitor in Zebrafish [abstract]. Res Pract Thromb Haemost. 2020; 4 (Suppl 1). https://abstracts.isth.org/abstract/role-of-kunitz-domain-3-of-tissue-factor-pathway-inhibitor-in-zebrafish/. Accessed March 4, 2024.
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