Abstract Number: PB0249
Meeting: ISTH 2020 Congress
Background: Hemolytic disorders are prone to thrombosis. Previous work showed the RNA released from damaged cells activates clotting. The type of RNA released from hemolysis and its coagulation function is not known.
Aims: To test whether the hemolysis releases coagulation-inducing RNA and identify that RNA.
Methods: Zebrafish red cell lysate was gel-electrophoresed, and RNA-Seq was performed. RT-PCR, in vitro transcription, and restriction digestions were performed according to the manufacturer recommendations. Deoxy-oligonucleotides were synthesized. Coagulation assays and laser-induced thrombosis assays were according to published methods. Student’s t-test was used in statistical analysis.
Results: The lysate from zebrafish red cells showed a single band of RNA on gels and was identified as 5.8S RNA (5.8SR) by RNA-Seq analysis. 5.8SR activated coagulation in zebrafish plasma. In vitro synthesized 5.8SR, its shorter fragments and mutated versions were made using DNA templates produced by RT-PCR or synthetic deoxyoligonucleotides. We found 3′-end 26 nucleotides (3′-26) with the stem and loop were sufficient for inducing clotting activity. Similar results were obtained with human RBC lysates and plasma. We found prolonged clotting time and time to occlusion in laser-induced thrombosis assays in the presence of corn trypsin inhibitor and 5.8SR monoclonal antibody (5.8SRmAb). Similar inhibitions were found with human plasma. 5.8SRmAb injections into adult fish resulted in higher bleeding in mechanical injury assay. Since zebrafish has no factor XII and hepatocyte growth factor activator (HGFA) has similarities to factor XII, we knocked down the HGFA in adult zebrafish and found that the plasma from this fish does not respond to 3′-26.
Conclusions: We identified 5.8SR released in hemolysis to activate clotting in human and zebrafish plasma. Only 3′-26 is needed for the activity. 5.8SRmAb injections cause bleeding. Thus, it may have clinical utility in treating thrombosis. Furthermore, we showed that fish HGFA activates coagulation. Thus, factor XII may have evolved from HGFA.
To cite this abstract in AMA style:Alharbi A, Raman R, Iyer N, Al Qaryoute A, Burks D, Azad R, Jagadeeswaran P. Activation of Coagulation by 5.8S RNA [abstract]. Res Pract Thromb Haemost. 2020; 4 (Suppl 1). https://abstracts.isth.org/abstract/activation-of-coagulation-by-5-8s-rna/. Accessed May 6, 2021.
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