Human thrombomodulin: complete cDNA sequence and chromosome localization of the gene

Abstract

A human umbilical vein endothelial cell cDNA library in .lambda.gt11 was screened for expression of thrombomodulin antigens with affinity-purified rabbit polyclonal anti-thrombomodulin immunoglobulin G (IgG) and mouse monoclonal anti-human thrombomodulin IgG. Among 7 million recombinant clones screened, 12 were recognized by both antibodies. Two of these, .lambda.HTm10 and .lambda.HTm12, were shown to encode thrombomodulin by comparison of the amino acid sequence deduced from the nucleotide sequence to the amino acid sequence determined directly from tryptic peptides of thrombomodulin. Thrombomodulin mRNA was estimated to be 3.7 kilobases in length by Northern blot analysis of endothelial cell and placental poly(A)+ RNA. Thrombomodulin mRNA was not detected in human brain, HepG2 hepatoma cells, or the monocytic U937 cell line. Additional cDNA clones were selected by hybridization with the 1.2-kilobase insert of .lambda.HTm10. One isolate, .lambda.HTm15, contained a 3693 base pair cDNA insert with an apparent 5''-noncoding region of 146 base pairs, an open reading frame of 1725 base pairs, a stop codon, a 3''-noncoding region of 1779 base pairs, and a poly(A) tail of 40 base pairs. The cDNA sequence encodes a 60.3-kDa protein of 575 amino acids. The predicted protein sequence includes a signal peptide of .apprx. 21 amino acids, an amino-terminal ligand-binding domain of .apprx. 223 amino acids, an epidermal growth factor (EGF) homology region of 236 amino acids, a serine/threonine-rich segment of 34 amino acids, a membrane-spanning domain of 23 amino acids, and a cytoplasmic tail of 38 amino acids. The EGF-homology region consists of six tandemly repeated EGF-like domains. The organization of thrombomodulin is similar to that of the low-density lipoprotein receptor, and the protein is homologous to a large number of other proteins that also contain EGF-like domains, including factor VII, factor IX, factor X, factor XII, protein C, tissue plasminogen activator, and urokinase. The gene for thrombomodulin has been localized to chromosome 20 by hybridization of cDNA probes to purified human chromosomes.