Thrombomodulin with the Asp468Tyr mutation is expressed on the cell surface with normal cofactor activity for protein C activation

Thrombomodulin (TM) is an endothelial cell glycoprotein that acts as an ant icoagulant. Mutation in the TM gene is a potential risk factor for thrombos is. The first TM mutation identified tvas a heterozygous substitution of T for G at nucleotide position 1456, which predicted Asp468 with Tyr in a Ser /Thr-rich domain. To evaluate the reported TM gene mutation as a possible c ause of thrombosis, rye transiently tranfected a vector for TM gene carryin g the mutation to mammalian COS7 cells. TM antigen levels in lysates of cells transfected with variant TM were comp arable to those in preparations of normal TM. The TM cofactor activity for protein C (PC) activation on the variant TM-expressing cells was similar to that of the control. The Michaelis constant K-m and V-max of variant TM fo r PC activation were shown to be similar compared to those of normal TM. Th e affinity of each TM for thrombin in PC activation was also similar. We ob tained several stable cell lines expressing normal and variant TM. Lysate o f the cell lines with normal and variant TM genes had a similar expression level of TM antigen, Pulse-chase analysis showed that normal and variant TM were glycosylated and resistant to endoglycosidase Ii, indicating that the variant TM was expressed on the cell surface In a mature form. Variant TM protein is apparently expressed on the cell surface with normal cofactor activity for PC activation. It is unlikely that the TM variant dir ectly causes thrombosis by mechanism of reduced expression or impaired cofa ctor activity for PC activation, which comprises a major anticoagulant acti vity of TM.