Abstract
We have cloned a serum- and cycloheximide-inducible mRNA from AKR-2B murine fibroblasts which encodes a protein with significant sequence similarity to human tissue factor, a cellular initiator of the blood coagulation cascade. Information derived from this clone was used to establish the presence of a virtually identical sequence in mouse brain. Most importantly, cDNA-directed expression in a quail fibroblast cell line produced high levels of tissue factor procoagulant activity, confirming the identity of this protein as murine tissue factor. Additional studies demonstrate that transforming growth factor type beta 1 stimulates tissue factor gene transcription and is a potent inducer of tissue factor procoagulant activity in fibroblasts. Other tested mitogens such as platelet-derived growth factor, epidermal growth factor, and insulin were weak inducers. These results may reflect a role for transforming growth factor beta 1 in the maintenance of hemostasis or, alternatively, a role for tissue factor in cellular functions unrelated to blood coagulation.
Highlights
MATERIALS ANDMETHODS tissue factor procoagulant activity infibroblasts
+ free medium MCDB 402, stimulated with fresh medium to which growth factor, 20% fetal calf serum (FCS), or 20% FCS cycloheximidewas added as specified in each experiment
Tissue factor (TF)’ is a transmembraneglycoprotein which Construction of cDNALibrary-The construction and replica serves as acell surface receptor for plasma coagulation factor VII/VIIa, aserine protease
Summary
A search of the GenBank nucleic acid sequence data base revealed that the illustrated sequence was68.4% identical with aportion of human T F mRNA (nucleotides 43-1329) as recently reported not be retained in the mature protein. The amino-terminal 27 residues of c70 and the first 30 of Because of the unexpected similarity between the c70 se- human T F are largely hydrophobic and followedby a prequence and thatof human TF, itwas important to determine dicted (30) signal peptide cleavage site To achieve single nucleotide COOH terminus, both sequences exhibit a stretch of 23 conresolution, we employed the recently developed RAWTS tech- secutive hydrophobic amino acids followed bya short stretch c70Pr0 HUTF clOPro HuTF c7OPro HUTF
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