Abstract
Thrombosis of arteries and veins is fundamentally important in human diseases such as myocardial infarction, stroke, and venous thrombotic events. Thrombus formation in response to tissue factor (TF) is highly regulated and results from the complex interactions of pro-, anti-coagulant and fibrinolytic effectors. We have taken a mouse genetic approach to identify novel molecules and pathways that regulate the response to a thrombotic trigger. Previously, we observed that the wild-derived inbred mouse strain Cast/EiJ (Cast) was completely resistant to an otherwise lethal dose of TF in an in vivo model of pulmonary embolism (PE). TF-resistance was 100% in the F1 offspring of an intercross to a susceptible strain, C57Bl6/J (B6). To identify the gene(s) mediating TF-resistance, we performed genome-wide linkage analysis on 200 N2 generation animals from a Cast-B6 backcross and found several TF-resistance loci. One such locus was on the proximal end of chromosome 11 (chr11, rs3088940). At the chr11 marker, animals heterozygous for B6 and Cast alleles were significantly protected in the PE model (median survival times of 770 vs. 210 seconds, p<0.05). To confirm linkage, we measured TF-resistance in a congenic strain carrying the Cast chr11 on a B6 background (11C). We found that mice homozygous for Cast chr11 showed significant protection in the PE model; B6, 11C, and Cast strains demonstrated survival fractions of 0, 0.5, and 1 and median survival times of 225, 772 and >1200 seconds, respectively (p<0.0001). To explore the mechanism of TF-resistance and to identify potential candidate genes within the chr11 locus, we performed in vitro analyses of coagulation. Whole blood clotting times triggered with dilute TF were significantly prolonged in 11C mice as compared to B6 (53.7±0.4 vs. 48.9±0.4 sec, p<0.0001). To determine the contribution of platelets, we repeated dilute TF-triggered clotting in platelet poor plasma and found that clotting times remained prolonged in plasma from 11C animals as compared to B6 (52.2±0.5 vs. 46.7±0.9 sec, p<0.05). Next, we measured the aPTT and PT and found that while the PT was similar, the aPTT was prolonged in 11C versus B6 plasma (mean time 50.8±5.3 vs.44.8±6.4, p<0.05). Thus, 11C mice exhibited prolonged clotting times in whole blood and in plasma in response to dilute TF, and had prolonged aPTT. These findings suggested that the principal mechanism of protection was within the intrinsic pathway or in the thrombin feedback pathway. To further investigate, we measured the activity of factors II, V, VII, VIII, IX, X and XI using human factor deficient plasma. There were no significant differences in factor activity between 11C and B6 mice, with the exception of factor XI. Factor XI activity in 11C animals was reduced to 59.1% of B6 activity. These results were confirmed using a chromogenic substrate for factor XI. Activity was decreased in 11C mice as compared to B6 (EC50 values of 74.4±0.3 vs. 70.9±0.6, p<0.0001). The 40% absolute reduction in plasma factor XI activity suggests a potential mechanism for the observed resistance to thrombosis, and supports our theory that the defect lies in the intrinsic or feedback pathway. Since there are no obvious coagulation related genes on mouse chr11, we speculate that the Cast chr11 locus contains a gene(s) that increases the expression and/or activity of a novel inhibitor of factor XI activity, thereby mediating resistance to thrombus formation in vivo.
A role for factor XIIa–mediated factor XI activation in thrombus formation in vivo