Tail bleeding assay identifies three chromosomes with QTL for rebleeding in CSS
Using the tail bleeding/rebleeding assay as a surrogate marker for hemostasis and thrombosis, the CSS were screened. The bleeding times (Figure 4A) for the B6 and the A/J mice were not different, but 6six of the strains, B6-Chr5, 6, 8, 14, 15, and YA/J, had lower (P < 0.002) bleeding times (49–79 seconds) compared to the value for the B6 mice (121 sec). The rebleeding time (Figure 4B), determined as the time between bleeding cessation and initiation of the second bleeding, was markedly increased in the A/J mice compared to the B6 mice and two strains, B6-Chr11A/J (CSS-11) and B6-Chr17A/J (CSS-17), had significantly (P < 0.002) longer rebleeding times compared to the B6 mice that were similar to values from the A/J mice. Another strain, B6-Chr5A/J (CSS-5), approached significance at P < 0.008. The rebleeding time was stopped at 600 sec, and the percentage of mice with this value determined for the three strains: CSS-5–60%, CSS-11–60% and CSS-A17–46%. The percentage of B6 mice with a rebleeding time of 600 sec was 7% and for the A/J mice the percentage was 85%.
Figure 4  Bleeding and rebleeding time in the CSS. Panel A: Bleeding Time (seconds). Panel B: Rebleeding Time (seconds). Bars are the mean ± SEM of 7–28 mice. Statistical differences between B6 mice and CSS were determined with a t-test, and a Bonferroni correction was made to determine significance value (P < 0.002). Symbols indicate a significant difference between B6 values. *P < 0.002. Rebleeding time in the parent strains and CSS were compared to values from the progeny of mice that were crossed with B6 mice to produce heterosomic mice for the CSS-5, (B6xCSS-5)F1 and CSS-17 (B6xCSS-17)F1 strains (Figure 5). The rebleeding values for CSS-5F1 and CSS-17F1 mice were similar to the B6 rather than the A/J mice, suggesting the A/J trait is recessive. Bleeding and rebleeding in the progeny of the cross, B6-Chr5A/J × B6-Chr17A/J (CSS-5 × CSS-17) resulted in the recovery of the A/J phenotype in the progeny of these double heterosomic strains. Thus, despite the heterosomic chromosomes of 5 and 17 in the progeny of the CSS-5 × CSS-17, the phenotype was now similar to A/J. This suggested that traits on the A/J chromosomes were interacting to produce the phenotype.
Figure 5  Bleeding and rebleeding time in the CSS backcrosses. Panel A: Bleeding Time (seconds) and Panel B: Rebleeding Time (seconds) were tested in heterosomic mice, (B6xCSS-5)F1 (5F1) and (B6xCSS-17)F1 (17F1) and doubly heterosomic mice for chromosomes 5 and 17 (5 × 17). Bars are the mean ± SEM of 5–28 mice. Statistical differences between B6 mice and the CSS were determined with a t-test, and a Bonferroni correction was made to determine significance value (P < 0.01). Symbols indicate a significant difference between B6 values. *P < 0.01.