Cellular and functional characterization of three recombinant antithrombinmutants that caused pleiotropic effect-type deficiency

Inherited antithrombin deficiency is associated with a predisposition for f amilial venous thromboembolic disease. Pleiotropic effect-type mutants of a ntithrombin that have an amino acid replacement in a distal hinge region in cluding strands 1C, 4B, and 5B of the polypeptide chain are known to exhibi t impaired interactions with both thrombin and heparin, coupled with a secr etion defect. To examine the mechanism of pleiotropic effect-type antithrom bin deficiency, we expressed three mutants, Oslo (Ala404-->Thr), Kyoto (Arg 406-->Met), and Utah (Pro407-->Leu), in baby hamster kidney (BHK) cells, an d compared their secretion rates, affinities for heparin and abilities to f orm thrombin-antithrombin (TAT) complexes with those of wild-type (Wt) anti thrombin, Pulse-chase experiments showed that the Oslo- and Kyoto-mutants w ere secreted at rates similar to Wt antithrombin. In contrast, the Utah-mut ant underwent partial intracellular degradation. The intracellular degradat ion of the Utah-mutant was not inhibited by lysosomotropic inhibitors, but by proteasome inhibitors such as carbobenzoxy-L-leucyI-L-leucyl-L-leucinal (LLL) and lactacystin, indicating that a part of the Utah-mutant was degrad ed by proteasome through quality control in the endoplasmic reticulum (ER), Crossed immunoelectrophoresis in the presence of heparin showed that only the Oslo-mutant lacks heparin-binding ability. Incubation with thrombin sho wed that the Kyoto- and Utah-mutants, but not the Oslo-mutant, formed a wea k but detectable TAT complex. Furthermore, heparin enhanced the TAT complex formation by the Kyoto- and Utah-mutants, suggesting heparin cofactor acti vities of these mutants. These results show that each of the Oslo-, Kyoto-, and Utah-mutants exhibits different properties as to secretion, intracellu lar degradation and functional activity, although they are grouped as pleio tropic effect-type mutants.