Identification and characterization of a thrombomodulin gene mutation coding for an elongated protein with reduced expression in a kindred with myocardial infarction

Thrombomodulin is an endothelial cell receptor for thrombin. It functions a s a natural anticoagulant by greatly accelerating activation of protein C b y thrombin, Using a direct gene screening strategy we identified a frameshi ft insertion mutation, insT 1689, in the thrombomodulin gene of a patient w ith myocardial infarction, The mutation predicts an elongated gene product because of substitution of the 12 C-terminal amino acids by 61 abnormal res idues. Pedigree analysis showed that the mutation was also likely to have b een present in a sibling who had had fatal myocardial infarction, Carriers of the mutant allele express significantly lower amounts of thrombomodulin on the surface of their monocytes detected by flow cytometry and have lower levels of soluble thrombomodulin in plasma. Wild type and the mutant throm bomodulin were expressed in COS-7 cells. Cellular distribution of the expre ssed proteins was evaluated by immunofluorescence microscopy, which showed reduced cell surface expression and intense juxtanuclear localization of th e abnormal protein. This suggests impaired, translocation through the endop lasmic reticulum/Golgi apparatus. Cells expressing abnormal thrombomodulin had reduced ability (similar to 2.5-fold) to accelerate the thrombin mediat ed activation of protein C. This is the first demonstration of reduced expr ession arising from a natural thrombomodulin gene mutation. The results pro vide support for the suggestion that gene mutation of thrombomodulin may be important in the pathogenesis of some cases of occlusive thrombotic diseas e.