No abstract

Protein Z is a vitamin K-dependent plasma glycoprotein that is involved in the regulation of blood coagulation. Plasma concentrations of protein Z vary widely between subjects and are greatly reduced during warfarin therapy. We developed a sensitive and quantitative assay for protein secretion using a secretory luciferase to explore the mode of secretion of protein Z compared with that of factor X. Protein Z secretion was much less efficient than factor X and was totally dependent upon added vitamin K, while factor X secretion was not. Protein Z secretion was highly sensitive to warfarin treatment of the synthesizing cells. In contrast, although factor X secretion was not precluded by warfarin, its ␥-carboxylation was completely blocked. An exchange of the propeptide and/or ␥-carboxyglutamic acid domain between protein Z and factor X reproduced the inefficient and warfarinsensitive secretion pattern of protein Z, and vice versa. Joining of the propeptide and ␥-carboxyglutamic acid domain to luciferase also demonstrated that the ␥-carboxyglutamic acid domain of protein Z was responsible for its warfarinsensitive secretion. Thus, it was concluded that the difference observed in secretion patterns of protein Z and factor X was mainly based on the structure of their ␥-carboxyglutamic acid domains. IntroductionProtein Z (PZ) is a vitamin K (Vit.K)-dependent plasma glycoprotein homologous to coagulation factors VII, IX, and X (FVII, FIX, and FX), and protein C. Mature human PZ consists of 360 amino acids containing 13 ␥-carboxyglutamic acid (Gla) residues, and 1 Gla, 2 epidermal growth factors, and 1 serine protease domains. 1,2 PZ is not the zymogen of a serine protease, because it lacks the His and Ser residues of the catalytic triad. The human PZ gene is localized to chromosome 13q34, where the genes for FVII and FX exist side by side, and it spans approximately 14 kilobases consisting of 9 exons including 1 alternative exon. 3 PZ forms a calcium ion-dependent complex with activated FX (FXa) on the phospholipid surface and thereby serves as a cofactor for the inhibition of FXa by a PZ-dependent protease inhibitor. 4 Although PZ-null mice have an apparently normal phenotype, PZ deficiency dramatically enhances the thrombotic phenotype in mice carrying the factor V Leiden genotype. 5 These findings indicate that PZ plays an important role in the regulation of blood coagulation.Vit.K is required for the posttranslational formation of Gla from glutamic acid residue, which is present in several plasma proteins that are involved in hemostasis: prothrombin, FVII, FIX, and FX, and proteins C, S, and PZ. [6][7][8] Gla-mediated Ca 2ϩ binding in these proteins is necessary for their association with phospholipid surfaces and is critical for their hemostatic function. The conversion of glutamate to Gla is catalyzed by a microsomal membrane protein, Vit.K-dependent ␥-glutamylcarboxylase (GCX). 9 The formation of the GCX-substrate complex is mediated by the interaction between a propeptide (PP) in the substrate protein and the ...