Cloning and characterization of a cDNA coding for the lipoprotein-associated coagulation inhibitor shows that it consists of three tandem Kunitz-type inhibitory domains

T. C. Wun, K. K. Kretzmer, T. J. Girard, J. P. Miletich, G. J. Broze

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Abstract

Human plasma contains a lipoprotein-associated coagulation inhibitor (LACI) which inactivates factor X(a) directly, and in a X(a)-dependent fashion also inhibits the VII(a)-tissue factor complex of the extrinsic coagulation pathway. Rabbit polyclonal anti-LACI anti-serum was used to screen human placental and fetal liver λgt11 cDNA libraries for the expression of LACI antigens. Immunologically positive clones were further tested for their ability to bind 125I-factor X(a). Seven clones were obtained which are immunologically and functionally active. The longest cDNA insert (λP9) of these isolates is 1.4 kilobases (kb) while other clones are 1.0 kb in length. Nucleotide sequence analysis shows that λP9 consists of 1431 bases that include a 5'-noncoding sequence of 132 nucleotides, an open reading frame of 912 nucleotides, and a 3'-noncoding region of 387 nucleotides. The open reading frame encodes a signal peptide of 28 residues followed by a 32-kilodalton protein of 276 residues. The predicted sequence of mature LACI contains 18 cysteines and three potential N-linked glycosylation sites. The amino acid sequence analysis of purified LACI's NH2 terminus and two of its proteolytic fragments match exactly those deduced from the cDNA sequence, indicating that the cDNA codes for LACI. The translated amino acid sequence of LACI shows several discernible domains, including a highly negatively charged NH2 terminus, three tandem Kunitz-type inhibitory domains, and a highly positively charged carboxyl terminus. Northern blot analysis shows that the following liver-derived cell lines, Chang liver, HepG2 hepatoma, and SK hepatoma all, contain two major species of mRNA (1.4 and 4.4 kb) which hybridize with LACI cDNA.

Original languageEnglish
Pages (from-to)6001-6004
Number of pages4
JournalJournal of Biological Chemistry
Volume263
Issue number13
StatePublished - 1988

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