title: Stable expression of a secretable deletion mutant of recombinant human thrombomodulin in mammalian cells.
abstract: Thrombomodulin is an endothelial cell membrane protein which plays a central regulatory role in the protein C anticoagulant pathway. The human thrombomodulin intronless gene was isolated from a genomic DNA library and used to isolate the coding region. A mammalian expression vector, phd-TMD1, encoding all the extracellular domains of human thrombomodulin but lacking the transmembrane and cytoplasmic domains was constructed. Stable phd-TMD 1 transformants, in both hamster AV12-644 and human 293 cells, expressed functionally active recombinant thrombomodulin as a secreted, soluble product. Soluble thrombomodulin was secreted as two major proteins of 105 kDa and 75 kDa, both of which were purified to homogeneity. The kinetic properties for protein C activation of the two proteins were very different: the Kd for thrombin, Km for protein C, and Ca2+ optima were 3.0 nM, 1.5 microM, and 1-3 mM for the 105-kDa protein and 16 nM, 2.3 microM, and 0.2-0.5 mM for the 75-kDa protein. In clotting and platelet activation assays, the 105-kDa protein was a much more potent anticoagulant than the 75-kDa protein. Both forms of the protein had the amino-terminal sequence Ala19-Pro-Ala-Glu-Pro-Gln. Amino acid composition analysis indicated that both forms of the protein had the same amino acid content which was consistent with the predicted protein comprising residues Ala19 to Ser515. The difference in size appeared to be due to glycosylation as both forms were of similar size following chemical deglycosylation. These studies suggest that (1) secretable thrombomodulin derivatives can be used to study structure-function relationships of the extracellular domains of this important regulatory protein, (2) the extent of glycosylation has profound effects on the kinetic and anticoagulant properties of human thrombomodulin, and (3) soluble recombinant human thrombomodulins may be developed as clinically significant therapeutic anticoagulants.
authors.affiliation: Lilly Research Laboratories, Indianapolis, Indiana 46202.
authors.forename: J F
authors.initials: JF
authors.lastname: Parkinson
authors.forename: B W
authors.initials: BW
authors.lastname: Grinnell
authors.forename: R E
authors.initials: RE
authors.lastname: Moore
authors.forename: J
authors.initials: J
authors.lastname: Hoskins
authors.forename: C J
authors.initials: CJ
authors.lastname: Vlahos
authors.forename: N U
authors.initials: NU
authors.lastname: Bang
mesh_terms: D000596:Amino Acids / Q000032:analysis; D000818:Animals; D000925:Anticoagulants; D001483:Base Sequence; D002118:Calcium / Q000378:metabolism; D002478:Cells, Cultured; D002808:Chondroitin Lyases / Q000494:pharmacology; D006224:Cricetinae; D006023:Glycoproteins / Q000032:analysis; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009154:Mutation; D015345:Oligonucleotide Probes; D011486:Protein C / Q000378:metabolism; D011956:Receptors, Cell Surface / Q000235:genetics* / Q000378:metabolism; D018179:Receptors, Thrombin; D011994:Recombinant Proteins; D013329:Structure-Activity Relationship
chemical_list: D000596:Amino Acids; D000925:Anticoagulants; D006023:Glycoproteins; D015345:Oligonucleotide Probes; D011486:Protein C; D011956:Receptors, Cell Surface; D018179:Receptors, Thrombin; D011994:Recombinant Proteins; D002808:Chondroitin Lyases; D002118:Calcium