COMPLEMENTARY-DNA SEQUENCING OF CANINE TISSUE FACTOR PATHWAY INHIBITOR REVEALS A UNIQUE NANOMERIC REPETITIVE SEQUENCE BETWEEN THE 2ND AND 3RD KUNITZ DOMAINS

Tissue factor pathway inhibitor (TFPI) is a factor Xa-dependent inhibi tor of the factor VIIa-tissue factor complex of blood coagulation. The primary amino acid sequence of canine TFPI has been deduced from cDNA sequences obtained using the techniques of reverse transcription foll owed by amplification using PCR and conventional screening of a canine endothelial cell cDNA library. The open reading frame for canine TFPI encodes a signal peptide of 28 amino acids followed by a 40.7 kDa pro tein of 368 amino acids. Similar to human, rat and rabbit TFPI, canine TFPI contains a negatively-charged cluster of amino acids at its matu re amino-terminus, followed by three Kunitz-type proteinase inhibitory domains and a cluster of positively-charged amino acids near its carb oxy-terminus. In contrast to other TFPIs, following its second Kunitz- type proteinase inhibitory domain canine TFPI contains an additional a mino acid insert which includes a nanomeric peptide-sequence repeated six times. Recombinant canine TFPI was expressed in both bacterial- an d insect cell-expression systems for functional analysis and the gener ation of antibodies. The recombinant canine TFPI inhibits tissue facto r-induced coagulation in an in vitro canine system. Immunoprecipitatio n of TFPI from canine plasma, followed by Western-blot analysis, tenta tively identifies canine TFPI as an 80000 kDa protein. Anti-peptide an tibodies raised to the nanomeric peptide repeat immunoprecipitate an i dentical, cross-reactive, 80000 kDa protein.