Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues
Reexamination Certificate
1998-01-27
2001-07-17
Kemmerer, Elizabeth (Department: 1646)
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
C530S300000, C530S328000, C530S329000, C530S345000, C530S387100, C530S402000
Reexamination Certificate
active
06262233
ABSTRACT:
The present invention relates to a novel human gene encoding a polypeptide which is a member of the Kunitz-type protease inhibitor family. More specifically, isolated nucleic acid molecules are provided encoding a human polypeptide named Tissue Factor Pathway Inhibitor-3 hereinafter referred to TFPI-3. TFPI-3 polypeptides are also provided, as are vectors, host cells and recombinant methods for producing the same. Also provided are diagnostic methods for detecting disorders related to vascular hemostatsis and therapeutic methods for treating such disorders. The invention further relates to screening methods for identifying agonists and antagonists of TFPI-3.
BACKGROUND OF THE INVENTION
Proteases are responsible, either directly or indirectly, for all bodily functions, including cell growth, differentiation and death (apoptosis), cell nutrition, intra- and extracellular protein turnover (housekeeping and repair), cell migration and invasion, and fertilization and implantation. These functions extend from the cellular level to the organ and organism level to produce cascade systems such as hemostatis and inflamation, and complex processes at all levels of physiology and pathophysiology.
Maintenance of vascular integrity is an important host response to injury. Complex hemostatis mechanisms of coagulation, platelet function, and fibrinolysis exist to minimize adverse consequences of vascular injury and to accelerate vascular repair. Many of these hemostatic mechanisms are initiated and/or regulated by cells of the wall of the blood vessel.
Tissue-factor-pathway inhibitor (TFPI) is a cell-surface associated glycoprotein which plays a key role in the regulation of tissue factor-initiated blood coagulation. Human TFPI is a trace 42-kDa plasma glycoprotein that is synthesized primarily by endothelial cells and consists of a negatively charged amino terminal region, three tandem Kunitz-type inhibitor domains, and a highly basic carboxyl-terminal tail (Wun, T. C., et al., J. Biol. Chem. 263:6001 (1988)). After a 22-residue signal peptide, the mature protein contains 213 amino acids with 18 cysteines. TFPI forms a complex with factor Xa and inhibits its amidolytic and proteolytic activity. The factor Xa-TFPI complex rapidly inhibits activity of the factor VIIa-tissue factor complex.
The cloning and characterization of a gene coding for a second tissue factor pathway inhibitor (TFPI-2), has been reported (Sprecher, C. A. et al., Proc. Natl. Acad, Sci. USA, 91, 3353-3357 (1994)). This gene was initially identified on the basis of primary sequence homology and structural similarity. Subsequent characterization has confirmed its predicted activity as a protease inhibitor. Alterations of the hemostatic system can result from such causes as neoplasia and trauma. Such alterations result in an increased incidence of thrombotic disorders such as venous thrombosis, pulmonary embolism, atrial fibrillation, cerebral thrombosis, and hemophilia. Thus, there is a need for identification and characterization of polypeptides that function as inhibitors of the coagulation pathway which can play a role in detecting, preventing, ameliorating or correcting such disorders.
SUMMARY OF THE INVENTION
The present invention provides isolated nucleic acid molecules comprising a polynucleotide encoding at least a portion of the TFPI-3 polypeptide having the complete amino acid sequence shown in SEQ ID NO:2 or the complete amino acid sequence encoded by the cDNA clone deposited as plasmid DNA as ATCC Deposit Number 97797 on Nov. 20, 1996. The nucleotide sequence determined by sequencing the deposited TFPI-3 clone, which is shown in SEQ ID NO:1, contains an open reading frame encoding a complete polypeptide of 252 amino acid residues, including an initiation codon encoding an N-terminal methionine at nucleotide positions 361 to 363 and a predicted molecular weight of about 28.2 kDa.
The TFPI-3 protein of the present invention shares sequence homology with the translation product of the human MRNA for Tissue Factor Pathway Inhibitor (TFPI), TFPI-2 and aprotinin, including the following conserved domains: (a) a first predicted Kunitz-type domain of about 51 amino acids; and (b) a second predicted Kunitz-type domain also of about 51 amino acids, both of which are thought to be important in regulating blood coagulation, and are underscored with stars in
FIGS. 1A and 1B
. The homology between human TFPI, TFPI-2, aprotinin and TFPI-3, shown in
FIG. 2
, indicates that TFPI-3 is also a protease and may be involved in regulating blood coagulation. Protease inhibition assays described herein confirm the ability of TFPI-3 polypeptides to inhibit protease activity.
The encoded polypeptide has a predicted leader sequence of 27 amino acids underlined in
FIGS. 1A and 1B
; and the amino acid sequence of the predicted mature TFPI-3 protein is also shown in
FIG. 1
, and as amino acid residues 1-225 (SEQ ID NO:2).
Thus, one aspect of the invention provides an isolated nucleic acid molecule comprising a polynucleotide having a nucleotide sequence selected from the group consisting of: (a) a nucleotide sequence encoding a polypeptide comprising the predicted second Kunitz-type domain of the TFPI-3 polypeptide having the amino acid sequence at positions 106 to 156 in SEQ ID NO:2 or as encoded by the cDNA clone contained in ATCC Deposit No. 97797; (b) a nucleotide sequence encoding a polypeptide comprising the consensus Kunitz-type domain having the amino acid sequence shown as SEQ ID NO:28; and (c) a nucleotide sequence complementary to any of the nucleotide sequences in (a) or (b) above; wherein said nucleic acid sequence in (a) or (b) does not encode a polypeptide comprising a sequence shown as SEQ ID NO:29, SEQ ID NO:30, or SEQ ID NO:31.
Further embodiments of the invention include isolated nucleic acid molecules that comprise a polynucleotide having a nucleotide sequence at least 90% identical, and more preferably at least 95%, 96%, 97%, 98% or 99% identical, to any of the nucleotide sequences in (a), (b) or (c), above, or a polynucleotide which hybridizes under stringent hybridization conditions to a polynucleotide in (a), (b) or (c), above. This polynucleotide which hybridizes does not hybridize under stringent hybridization conditions to a polynucleotide having a nucleotide sequence consisting of only A residues or of only T residues. An additional nucleic acid embodiment of the invention relates to an isolated nucleic acid molecule comprising a polynucleotide which encodes the amino acid sequence of an epitope-bearing portion of a TFPI-3 polypeptide having an amino acid sequence in (a) or (b), above.
The present invention also relates to recombinant vectors, which include the isolated nucleic acid molecules of the present invention, and to host cells containing the recombinant vectors, as well as to methods of making such vectors and host cells and for using them for production of TFPI-3 polypeptides or peptides by recombinant techniques.
The invention further provides an isolated TFPI-3 polypeptide comprising an amino acid sequence selected from the group consisting of: (a) the amino acid sequence of the polypeptide comprising the predicted second Kunitz-type domain of the TFPI-3 polypeptide having the amino acid sequence at positions 106 to 156 in SEQ ID NO:2 or as encoded by the cDNA clone contained in ATCC Deposit No. 97797; or (b) the amino acid sequence of the polypeptide comprising the consensus Kunitz-type domain having the amino acid sequence of SEQ ID NO:28; wherein said nucleic acid sequence in (a) or (b) does not encode a polypeptide comprising a sequence shown as SEQ ID NO:29, SEQ ID NO:30, or SEQ ID NO:31.
The polypeptides of the present invention also include polypeptides having an amino acid sequence at least 80% identical, more preferably at least 90% identical, and still more preferably 95%, 96%, 97%, 98% or 99% identical to those described in (a) or (b) above, as well as polypeptides having an amino acid sequence with at least 90% similarity, and more preferably at least 95% similarity, to those above.
An a
Gentz Reiner L.
Hsu Tsu-An
Ni Jian
Rosen Craig A.
Human Genome Sciences Inc.
Kemmerer Elizabeth
Sterne Kessler Goldstein & Fox P.L.L.C.
LandOfFree
Tissue factor pathway inhibitor-3 does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Tissue factor pathway inhibitor-3, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Tissue factor pathway inhibitor-3 will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-2564132