Control of human coagulation by recombinant serine proteases. Blood clotting is activated by recombinant factor XII deleted of five regulatory domains.
The availability of engineered serine proteases allows one to study the activation, substrate specificity and regulation of human coagulation and fibrinolytic activities. Human coagulation factor XII is composed of the protease catalytic region at the C-terminus, a hinge proline-rich region and regulatory domains at the N-terminus. From cDNA clones coding for factor XII, two DNA molecules were constructed, one being full length and the other being deleted of exons coding for the regulatory domains. Engineered factor-XII cDNA species were inserted by a homologous recombination technique into vaccinia viruses, which were used to infect the human hepatoma cell line HepG2. Two recombinant proteins were prepared from the culture media and identified by their antigenic properties and electrophoretic mobilities. The recombinant protein of larger size was identified as the full-length factor XII of 80 kDa and its specific activities and activation patterns, determined both by the coagulation and the amidolytic assays, are very similar to these of native human factor XII. The recombinant protein of smaller size was identified as a 319-amino-acid-deleted factor-XII protein of 32 kDa, containing only the entire protease region and part of the proline-rich hinge. This protein was expected to be the 'minimal' portion of factor XII able to sustain protease but unable to recognize substrates and surfaces necessary to activate the contact phase of coagulation. However, this 'minimal' factor-XII protein displays a marked protease activity and, although lacking five regulatory domains of factor XII, is bound and activated by negative charges and promotes coagulation with high efficiency.
Citarella
F
F
Dipartimento di Biopatologia Umana, Università di Roma La Sapienza, Italy.
Aiuti
A
A
La Porta
C
C
Russo
G
G
Pietropaolo
C
C
Rinaldi
M
M
Fantoni
A
A
D001483:Base Sequence; D001665:Binding Sites; D001777:Blood Coagulation / Q000502:physiology*; D006528:Carcinoma, Hepatocellular; D002460:Cell Line; D002872:Chromosome Deletion; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics; D005174:Factor XII / Q000235:genetics / Q000378:metabolism*; D015723:Gene Library; D006801:Humans; D007700:Kinetics; D008113:Liver Neoplasms; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides; D015202:Protein Engineering; D011994:Recombinant Proteins / Q000378:metabolism*; D015183:Restriction Mapping; D012697:Serine Endopeptidases / Q000235:genetics / Q000378:metabolism*
D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D005174:Factor XII; D004247:DNA; D012697:Serine Endopeptidases