Targeted Replacement of the Murine Thrombomodulin Gene with Human Thrombomodulin Coding Sequence Results in Decreased Protein C Activation and Enhanced Thrombotic Response to Photochemical Injury.

Thrombomodulin is an endothelial transmembrane glycoprotein that regulates hemostasis and inflammation. Deletion of the thrombomodulin gene in mice results in embryonic lethality. To examine the function of human thrombomodulin in a murine model, we used gene targeting to replace the entire coding sequence of the intronless murine thrombomodulin gene with a human thrombomodulin cDNA. Mice derived from successfully targeted 129/SvEv embryonic stem cells were maintained on either a 129/SvEv background or a mixed (129/B6) background. Mice homozygous for the modified allele (hthm/hthm) were viable, and the total amount of thrombomodulin mRNA (measured by real time PCR) did not differ between hthm/hthm and wild-type (+/+) mice (p>0.05). In heterozygous (hthm/+) mice, the ratio of human to murine thrombomodulin mRNA was similar in lung, heart, and kidney, and expression of murine thrombomodulin transcripts was 50% of that in wild-type (+/+) mice. Expression of human thrombomodulin protein was detected in endothelium by immunohistochemistry and in lung lysates by enzyme linked immunoassay. Thrombomodulin cofactor activity, measured in a protein C activation assay with human protein C and thrombin, was decreased in the aorta of hthm/hthm mice (1.1±0.5 nmol/mm) compared with +/+ mice (3.6±0.3 nmol/mm; p<0.001). Thrombomodulin activity also was lower in lung lysates of hthm/hthm mice (0.7±0.1 nmol/mg protein) than in +/+ mice (2.5±0.5 nmol/mg protein; p<0.01). The relative difference in lung thrombomodulin activity between hthm/hthm and +/+ mice was even greater when measured with murine protein C and thrombin (0.7±0.6 vs. 5.2±0.9 nmol/mg protein; p<0.001). Hthm/+ mice had intermediate levels of cofactor activity with either human or murine reagents. Time to thrombotic occlusion of the carotid artery after photochemical injury (rose bengal and green laser) was shorter in hthm/hthm mice than in +/+ mice (10.4±3.0 vs. 34.6±10.6 minutes; p<0.04). We conclude that the humanized thrombomodulin gene is expressed normally in mice but produces a phenotype with markedly diminished anticoagulant activity. The increased susceptibility to experimental thrombosis in hthm/hthm mice suggests that this animal model may be useful for investigation of human thrombomodulin function in vivo.