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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 approximately 21 amino acids, an amino-terminal ligand-binding domain of approximately 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.(ABSTRACT TRUNCATED AT 250 WORDS)

Protein C activation by thrombin is significantly accelerated by the endothelial cell cofactor, thrombomodulin. In this study, we have developed a radioimmunoassay for thrombomodulin and have measured the cofactor content in several human tissues. The assay method detects as little as 2 ng of thrombomodulin. The highest thrombomodulin content was found in lung and placenta, but the antigen was also detected in spleen, pancreas, liver, kidney, skin, heart, and aorta. Unexpectedly, thrombomodulin was absent from brain. Extracts from cerebral cortex, cerebellum, centrum semiovale, midbrain, basal ganglia, pons, and medulla were devoid of thrombomodulin. In contrast, thrombomodulin antigen is present in extracerebral intracranial vessels, including basilar and internal carotid arteries and choroid plexus, as well as in endothelium of the pia-arachnoid.

In previous reports it has been shown that all of the reactions of fatty acid synthesis in Escherichia coli occur with the substrates bound in thioester linkage to the acyl carrier protein (ACP). -10 The nature of the substrate binding site of ACP is of great interest as several types of reactions occur with the substrates bound to this protein. Thus, thioesters of ACP are substrates in the condensation-decarboxylation reaction,6' 7, 10 the 2 reductions,7 8 and the enoyl hydrase reaction of fatty acid synthesis.9 In each of these reactions acyl-S-ACP derivatives are either much more reactive than the corresponding acyl-S-CoA compounds, or the latter do not react at all. Initially it was reported6 that the sulfhydryl group at the binding site of ACP is a cysteine residue. Wakil1' reported that the sulfhydryl residue is accounted for by thioethanolamine and that ACP contains one mole of f3-alanine per mole of protein. These findings have been confirmed in this laboratory. Further investigation has shown that the sulfhydryl residue is part of a covalently bound prosthetic group. This report presents experiments which establish the binding site of ACP as 4'-phosphopantetheine which is probably bound through a phosphodiester linkage to a serine residue of ACP.Materials and Methods.-2-CI4-Malonyl CoA was synthesized as described previously.'2 ACP was purified as described previously from either E. coli B or from E. coli K12.A Dowex resins were purchased from Calbiochem (Biorad). P32-orthophosphate was obtained from Oak Ridge National Laboratory. 2-C14-Malonic acid was purchased from the New England Nuclear Co. Pronase was purchased from Calbiochem. Pepsin 2 X crystallized and E. coli alkaline phosphatase