Figure 3.

Comparison of activation of coagulation by 5.8S rRNA, 5'-119 SmaI RNA, 5.8S rRNA-S, 3'-35 RNA, and 5'-119 RNA. (A) Left: zebrafish gel-purified 5.8S rRNA was used as a template to generate 5.8S DNA by using RT-PCR. The bands corresponding to 5.8S RNA and 5.8S DNA are shown on 5% polyacrylamide gel. Middle: SmaI digestion of 5.8S DNA to generate 5'-119 SmaI RNA. Right: 5.8S DNA sequence containing 2 SmaI restriction sites (indicated by arrows). (B-C) Left graphs show increasing fibrin formation with increasing time in the kinetic coagulation assay. (B) Left graph: curves were obtained from zebrafish plasma that contained 5.8S rRNA or 5'-119 SmaI RNA or had no RNA. Bar graph shows a significant shortening of time to half-maximal fibrin formation (using data obtained from the left graph) of the zebrafish plasma with 5.8S rRNA compared with zebrafish plasma with 5'-119 SmaI RNA (n = 4). The data for time to half-maximal fibrin formation (in minutes) were analyzed using Student t test and are shown as mean ± SEM. (C) Left graph: curves were obtained from zebrafish plasma that contained 5.8S rRNA-S, 3'-35 RNA, or 5'-119 RNA, or with no RNA. Bar graph shows a significant shortening of the time to half-maximal fibrin formation (obtained from the left graph) of zebrafish plasma with 5.8S rRNA-S or 3'-35 RNA compared with 5'-119 RNA (n = 4). The data for time to half-maximal fibrin formation (in minutes) were analyzed using one-way analysis of variance (ANOVA) and are show as mean ± SEM. For panel B and C curves, the time (in minutes) was plotted against the absorbance at 405 nm at 25°C. For more detail, see supplemental Table 2. ****P < .0001. NS, not significant.