EP0471759A1 - Hohe expression von funktionellem, menschlichem plasminogenaktivator-inhibitor (pai-1) in e-coli - Google Patents
Hohe expression von funktionellem, menschlichem plasminogenaktivator-inhibitor (pai-1) in e-coliInfo
- Publication number
- EP0471759A1 EP0471759A1 EP90907745A EP90907745A EP0471759A1 EP 0471759 A1 EP0471759 A1 EP 0471759A1 EP 90907745 A EP90907745 A EP 90907745A EP 90907745 A EP90907745 A EP 90907745A EP 0471759 A1 EP0471759 A1 EP 0471759A1
- Authority
- EP
- European Patent Office
- Prior art keywords
- pai
- rpai
- coli
- val
- protein
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Withdrawn
Links
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/81—Protease inhibitors
- C07K14/8107—Endopeptidase (E.C. 3.4.21-99) inhibitors
- C07K14/811—Serine protease (E.C. 3.4.21) inhibitors
- C07K14/8121—Serpins
- C07K14/8132—Plasminogen activator inhibitors
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P7/00—Drugs for disorders of the blood or the extracellular fluid
- A61P7/04—Antihaemorrhagics; Procoagulants; Haemostatic agents; Antifibrinolytic agents
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/86—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving blood coagulating time or factors, or their receptors
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/81—Protease inhibitors
- G01N2333/8107—Endopeptidase (E.C. 3.4.21-99) inhibitors
- G01N2333/811—Serine protease (E.C. 3.4.21) inhibitors
- G01N2333/8121—Serpins
- G01N2333/8132—Plasminogen activator inhibitors
Definitions
- t-PA Human tissue-type plasminogen activator
- PAI-1 The principal physiological regulator of t-PA appears to be a specific, fast-acting, plasminogen activator inhibitor (PAI-1).
- PAI-1 is a protein of Mr 50,000 which binds to t-PA in a 1:1 complex, and inactivates the serine protease (Van Mourik et al. (1984) J. Biol. Chem. 259:14914-14921; Colucci et a l. (1985) J. Clin. Invest. 75:818-824; Aimer and Ohlin (1987) Thromb. Research 47:335-339).
- Ny et al. disclosed the expression of functional rPAI in E. coli. using a phage lambda gtll-derived vector.
- rPAI was expressed as a beta-gal actosidase-PAI fusion protein of about 180 kDa, with the PAI coding sequence fused to the E. coli beta- galactosidase coding sequence.
- a PAI-derived fragment of about 40 kDa was also detected by immunoblot and by reverse fibrin autography assay. This 40 kDa form of rPAI was not characterized by Ny et al., but likely arises by translation initiation at Met/aa #3, since an E.
- coli translation initiatieon start signal AGGA fortuitously occurs 5' to this Met (see Table 1).
- Wun and Kretzmer similarly used a lambda gt11 expression vector to express functional rPAI in E. coli.
- Pannekoek et al. reported the expression of a functional 43 kDa form of rPAI in E. coli using a pUC8-derived vector, as assayed by reverse fibrin autography.
- PAI-1 isolated from many mammalian cell types is obtained in a partially active latent form; the latent form can be converted to a fully active form, with about 6-fold increased specific activity relative to the latent form, by treatment with denaturants such as sodium dodecylsulfate (SDS) (Hekman and Loskutoff (1985) J. Biol. Chem. 260:11581).
- SDS sodium dodecylsulfate
- rPAI-1 is expressed in E. coli almost exclusively in an inactive, latent form (Lambers et al . Fibrinolysis (1988) 2, Supp. 1:33).
- the invention provides substantially pure, biologically functional, nonfused mature form E. coli-expressed human PAI-1 protein having a specific activity of about 0.3 unit/ng, where a unit is defined as the amount of protein required to neutralize 1 international unit of t-PA in an S2288 chromogenic assay, where the amidolytic activity of t-PA is measured.
- the invention also relates to substantially pure
- the invention relates further to a recombinant plasmid expression vector capable of expression in E. coli of soluble, biologically functional human PAI-1, wherein the DNA coding sequence for mature PAI-1 is operatively linked to a phage lambda P L promoter or to a tac promoter, preferably to a phage lambda, P L , promoter, which gives particularly high level expression.
- a plasmid which is or which has the identifying characteristics of pCE1200.
- the invention also relates to an E. coli cell line transformed with such a plasmid, preferably an E. coli cell line that is or that has the identifying
- the invention relates further to biologically active recombinant mature human PAI-1 expressed by such a cell line.
- the invention also provides a method for identifying
- inhibitors of the binding of t-PA and PAI-1 comprising the steps of:
- PAI-1 protein the PAI-1 protein being attached to the surface by a monoclonal antibody specific for PAI-1, said monoclonal antibody characterized in that it binds to PAI- 1 at a domain not involved in the interaction of t-PA and PAI-1;
- the invention relates further to a pharmaceutical composition
- a pharmaceutical composition comprising, in a pharmaceutically acceptable vehicle, an amount of rPAI-1 of the invention, that is therapeutically effective to counteract excessive or inappropriate fibrinolysis.
- a method of treating inappropriate or excessive fibrinolysis in a mammal suffering from such a condition comprising administering to the mammal an effective dose of E. coli-expressed rPAI of the invention.
- the invention relates further to a recombinant human PAI-1 peptide having an ami no acid sequence corresponding to that of Table 1, with an N-terminal sequence of Met-Ser-Ile-Val-His or Ser-Ile-Val-His, where Val is amino acid #24 of the PAI-1 precursor protein, and to a recombinant human PAI-1 peptide having an amino acid sequence corresponding to that of Table 1, and having an N-terminal sequence of Met-Val-His or Val -His, where Val is amino acid #24 of the PAI-1 precursor protein.
- FIG. 1 Scatchard analysis of binding data for the binding of 125 I-t-PA to PAI-1 in the assay of the invention, characterized in that PAI-1 is bound to a solid support using a PAI-1-specific monoclonal antibody.
- PAI-1-containing wells, prepared as described in Example 5 were incubated with 100 fmoles of 125 I-t-PA and increasing concentration of unlabeled t-PA (ranging from 10 fmoles to 10 nmoles). The total t-PA bound in each well was determined, and was plotted versus the bound/free ratio. Each point represents the average value of triplicate samples.
- the "tac" promoter-containing expression vector only produced rPAI at levels of about 1% of total cell protein.
- E. coli- expressed nonfused mature form rPAI purified to homogeneity exhibits full biological activity.
- the purified E. coli-expressed rPAI exhibited specific functional activity exceeding that of purified human fibrosarcoma cell PAI by 5 to 10-fold, depending on the assay employed.
- E. coli-expressed purified rPAI did not require an activation step to convert the protein from a less active to a more active form.
- rPAI obtained using our process exhibits functional activity exceeding that of human fibrosarcoma PAI by 5 to 10-fold.
- Other recent reports (Lambers et al . (1988) Fibrinolysis 2, Supp.1:33) state that rPAI is obtained from E. coli almost exclusively in an inactive latent form.
- rPAI obtained by the methods of the invention exhibits full functional activity that is not enhanced by and does not require any activation procedures.
- a further aspect of the invention involves a process for detecting inhibitors of the interaction of PAI and t-PA.
- This process employs rPAI which is immobilized to a solid support using a PAI-specific monoclonal antibody which is first attached to the solid support. The binding of radio! abeled t-PA to the immobilized PAI is quantitated by separating the bound and free t-PA.
- Previous assays for the quantisation of PAI and t-PA binding which have used immobilized PAI did not utilize a PAI-specific monoclonal antibody to capture the PAI on the solid support and present it for binding to t-PA. We have found that the use of such a capture antibody markedly improves the sensitivity of the binding assay.
- cDNA lambda gtll library was constructed using published methods (Gubler and Hoffman (1983) Gene 25:263). Oligonucleotide probes to screen the library were designed based on published PAI-1 sequences (Ny et al (1986)
- the 2.1 kbp PAI-1-containing EcoRI to EcoRI fragment was inserted at the EcoRI site and 3' to the trp promoter in a plasmid derivative of pKGP36-trp (Ivanoff et al. (1986) Proc. Natl. Acad. Sci. USA 83:5392-5396; ATCC No. 39413) which contains a unique EcoRI site 3' to the trp promoter.
- the resultant plasmid which contains the PAI coding sequence inserted in the correct
- E. coli containing pPAI2.1 expressed an approximately 50 kDa form of rPAI-1 which was detected by reverse fibrin autography assay (Loskutoff et al., supra). However, rPAI-1 protein levels produced in E. coli containing pPAI2.1 were not detectable (estimated to be less than 0.1% of total E. coli protein) by Coomassie blue staining of SDS-PAGE gels.
- the N-terminal leader peptide of the PAI-1 coding region was removed by a partial ApaLI and Nsil digest of Haelll methyl ase treated pECE3-l DNA.
- the approximately 1800 bp fragment containing the PAI coding sequence was modified by adding a synthetic DNA adaptor to the 5' end of the fragment as shown below:
- E. coli strains HB101 or JM109 (mcrB-) containing ptac- PAI produced functional mature PAI-1 (i.e., PAI-1 lacking N- terminal leader peptide that is normally removed during
- translocation in the eukaryotic cell as determined by SDS-PAGE, reverse fibrin autography, amidolytic assay, t-PA binding activity, and immunoblot analysis (see below for details of these assays).
- cells containing this plasmid produced recombinant mature form PAI-1 at a level of approximately 1% of total E. coli protein, as determined by Coomassie brilliant blue staining of SDS-PAGE gels.
- the apparent molecule weight on SDS- PAGE gels was 45 kDa.
- the PAI-derived product of ptac-PAI corresponds to one of the two forms of mature PAI-1 (the two- residue shorter form) found in mammalian cells.
- two forms of mature PAI-1 are found in approximately equal amounts: a form with an N-terminus of Ser-Ala-Val-His-His and a two-residue shorter form with N-terminus Val-His-His (Andreasen et al. (1986) FEBS Letters 209: 213-218) (see Table 1).
- the two forms of mature PAI-1 begin with aa #22 (Ser) and #24 (Val) (see Table 1).
- the leader sequence, amino acids #1-21 or #1-23, is removed in mammalian cells to yield the mature form PAI-1.
- Plasmid pCE1200 designed to express in E. coli the mature form of PAI with the N-terminus Ser-Ala-Val- His-His.
- the construction of plasmid pCE1200 is described below.
- the plasmid expression vector pCE31 (described below) was digested with Clal and the overhang was filled-in with DNA polymerase I Klenow fragment and the four deoxyribonucleotides.
- the vector was digested with BamHI and the resulting large fragment containing the lambda P L promoter was purified from an agarose gel.
- the 1206 bp PAI DNA fragment was then ligated into pCE31 at the filled-in Clal and BamHI sites, to yield plasmid pCE1200.
- nucleotide sequence of pCE1200 encoding the N- and C- terminal amino acid sequence of the PAI-1-derived protein was determined and is shown below.
- the predicted molecular weight of the protein expressed from pCE1200 is approximately 42 kDa based on an average of 110
- Da/ami no acid The product expressed by pCE1200 differs from the longer mature form of PAI by the conservative substitution of Ala residue #23 with He.
- the expression of rPAI-1 encoded by pCE1200 is under the control of the phage lambda P L promoter.
- the plasmid pCE1200 in E. coli host TAP106 contains a defective lambda prophage including a temperature sensitive, mutant repressor gene (cI857).
- the mutant repressor At low temperatures (32°C), the mutant repressor is active and transcription from P L is shut off; however, when the temperature is raised to 42°C the repressor is inactivated and transcription initiating at the P L promoter can proceed (Reznikoff and Gold, Maximizing Gene Expression,
- the genotype of the TAP106 host is leu-, bio-, gal K (am), bl-, ⁇ lacU.169, rpsL, Sup E44, ⁇ def [ ⁇ BamHI, N-, Kan r , CI857, ⁇ bio].
- Production of PAI-1 involves growing the culture under conditions at which the repressor is active, preferably at temperature of 32°C.
- the culture may then be induced to produce PAI-1 by changing to conditions which inactivate the repressor and allow expression from the P L promoter, e.g., raising the temperature to 42°C.
- Other conditions can be used to inactivate the repressor, e.g., adding nalidixie acid or mitomycin C.
- Typical large scale induction conditions require growth at 32°C in LB media supplemented with ampicillin (100 ⁇ g/mL) and glucose (3%) until mid-log growth phase and then the culture is induced by raising the temperature of the fermenter to 42°C (Sisk et al. (1986) Gene 48:183-193). The culture is grown at the elevated temperature for 4 to 8 hours, after which time the cells are harvested by centrifugation and frozen at -70°C prior to PAI-1 purification.
- the yields of recombinant PAI-1 in E. coli using the CE1200 expression vector are about 10 to 15% of total E. coli protein, as estimated by Coomassie blue staining of SDS- PAGE gels.
- high level expression of recombinant PAI-1 means yields of at least 5%, and preferably 10% or greater.
- the E. coli-expressed PAI-1 protein of the invention was not only produced at high levels, but was also in a soluble, easily purified form.
- Plasmid pCE1200 in E. coli K12, TAP 106 (host) was deposited on March 15, 1989 in the Ameri can Type Culture Collection (ATCC), Rockville, MD, in accordance with the provisions of MPEP 608.01 (p) (C) (1) (2) and (3) and the Budapest Treaty.
- the ATCC accession number is 67911. Access to the culture will be available during pendency of the patent application to one determined by the Commissioner to be entitled thereto under 37 CFR 1.14 and 35 USC 122. Upon granting of a patent all
- Plasmid pCE31 was derived from plasmid pJL6, which is described by Lautenberoer et al. (Gene 23:75-84. 1983). Plasmid pJL6 was digested with restriction enzymes BstXI and Cl al and a synthetic DNA sequence was i nserted contai ni ng a consensus ribosome binding site (SD) and an ATG initiation codon. The sequence of the synthetic oligonucleotide used was as follows:
- Plasmid pCE31 provides the Clal site 3' to a translation start site allowing DNA coding sequences to be inserted and operatively linked to the phage lambda (P L ) promoter.
- P L phage lambda
- E. coli cells containing the PAI protein were suspended in ten volumes of 50 mM Na phosphate pH 6 and disrupted using a sonicator (Heat Systems Ultrasonics). The sonication was carried out for about 5 min using a medium tip probe. The sample was kept cold in an ice bath during the sonication. The sonicated sample was then centrifuged at 10,000 rpm for about 20 min and the supernatant saved. The pellet obtained was resuspended in 5 volumes of 50 mM Na phosphate buffer and resonicated using similar conditions as for the first sonication. The material obtained after the second sonication was centrifuged at 10,000 rpm as before. The supernatants obtained after the two
- centrifugation steps were pooled, filtered through a 5 micron filter and pumped onto a 4.4 x 30 cm Q Sepharose fast flow
- the column was washed at 10 mL/min with 50 mM Na phosphate buffer until the 280 nm absorbance was close to the baseline obtained with buffer alone (about 1 column volume).
- the protein was then eluted from the column at the same flow rate using a 0 to 1 M NaCl gradient in 50 mM Na phosphate pH 6.
- the fractions were assayed for PAI using sodium dodecyl sulfate polyacryl amide gel electrophoresis (SDS-PAGE) and analytical HPLC, using the methods outlined below.
- the PAI was found to be about 85 to 90% pure by SDS-PAGE and by analytical HPLC.
- the fractions containing PAI were pooled, diluted about 2.5 fold with water and loaded onto another 4.4 x 30 cm S Sepharose column equilibrated with 50 mM Na phosphate buffer pH 6. The column was washed with about 2 column volumes each of starting buffer and 50 mM Na phosphate buffer pH 8.0 successively. The PAI was eluted using 150 mM Na phosphate pH 8.6. The PAI obtained at this stage was found to be about 98% pure by SDS-PAGE and by analytical HPLC. PAI of greater than about 95% purity is considered
- substantially pure Other standard protein purification procedures may be used, as known to those skilled in the art. Generally speaking, the cells are disrupted, preferably by sonication. The soluble supernatant is then isolated, and the PAI-1 is purified from the soluble supernatant using column chromatography. In the method described above, Q Sepharose is an anion exchange column; S Sepharose is a cation exchange column. The described
- the fractions were assayed for PAI by SDS-PAGE and by analytical reverse phase HPLC.
- the SDS-PAGE was carried out using the Pharmacia Phast system (Pharmacia, Piscataway, NJ) utilizing the procedures outlined in the users manual. An 8 to 25% polyacrylamide gradient gel was used and the proteins were visualized by staining with Coomassie blue.
- Analytical HPLC was carried out using an HP 1090 M HPLC system. A 4.6 x 50 mm C4 Vydac column was used. The column was equilibrated in 0.1% TFA and the samples were applied to the column in the same solvent.
- the N-terminal sequence of purified E. coli-expressed rPAI-1 expressed from plasmid pCE1200 was determined to begin Ser-Ile- Val-His-His.
- the N-terminal Met residue of the nascent protein appears to be efficiently removed by amino peptidases in E. coli.
- short form mature PAI-1 (aa #24-402) or long form mature PAI-1 (aa#22-402) described above may be produced in the same or similar manner by introducing the desired modifications in the PAI-1 DNA coding sequence, as is known to those skilled in the art.
- Mature PAI-1 as defined herein includes both long form and short form proteins, from which the N-terminal leader sequence has been removed. Also included are mature PAI-1 proteins having substantially the same amino acid sequence and substantially the same activity.
- hPAI-1 The relative activities of purified recombinant E. coli- expressed human rPAI-1 products of both pCE1200 and ptac-PAI (98% pure, as described in Example 3), and purified nonrecombinant human PAI-1 expressed in a human fibrosarcoma cell line, designated hPAI-1, were compared using the assays described below.
- Purified hPAI-1 was obtained from American Diagnostica Inc. (New York, NY), with a purity of about 98%, as estimated by SDS-PAGE and Coomassie staining of SDS-PAGE gels.
- the S2251 assay measures the enzymatic ability of tissue plasminogen activator (t-PA) to generate plasmin from its plasminogen precursor by determining the level of amidolytic activity of generated plasmin on the chromogenic substrate
- rPAI-1 products of both pCE1200 and ptac-PAI were purified, as detailed in Example 3, and shown to have similar specific activities in the assays here described.
- a unit of PAI-1 as the amount of protein needed to neutralize 1 IU of t-PA, we determined the specific activity for the hPAI-1, in terms of units/ng, of 5/25 or 0.2 units/ng. A similar calculation with the rPAI-1 yields a specific activity of approximately 1 unit/ng.
- the rPAI-1 product of either pCE1200 or ptac-PAI was, therefore, approximately 5-fold more active than the hPAI-1 in this
- the substantially pure E. coli expressed rPAI- 1 of the invention will have a specific activity in the S2251 chromogenic assay of about lu/ng. Those skilled in the art will recognize that some variation in specific activity, e.g., in the range of ⁇ 10%, is to be expected.
- the PAI-1 isolated from many cell types is obtained in a partially inactive or latent form; the activity can be induced or increased by treatment with protein denaturants such as sodium dodecylsulfate (SDS) (Hekman and Loskutoff (1985) J. Biol. Chem. 260:11581).
- SDS sodium dodecylsulfate
- the activity of hPAI-1 from human fibrosarcoma cells was noted to be increased approximately 5.6-fold following an activation procedure which involved incubation of the protein for 1 h at 37° in the presence of 0.1% SDS, and dialysis of the material overnight against 150 mM Na 2 HPO 4 . This result indicates that the hPAI-1 contains a certain content of inactive or latent species.
- E. coli expressed ⁇ PAI- 1 the activity of which is not significantly increased by treatment with protein denaturants, e.g., less than about 10%, is considered to be within the invention.
- Our results establish an advantage of our rPAI-1 composition in comparison with the hPAI-1, based on its higher specific activity in all three functional assays and on the absence of any latent species or need for an activation step to obtain fully active PAI-1.
- the present results are surprising since they contradict those of Lambers et aL. (the Pannekoek lab) who reported that rPAI-1 expressed in E. coli was expressed almost exclusively in an inactive, latent form (Fibrinolysis (1988) 2, Supp.1, 33).
- the assay will prove useful in identifying specific domains on the t-PA and PAI-1 involved in their interaction, an important first step in the process of rationally designing inhibitors of binding of the t-PA and PAI-1.
- PAI t-PA Binding Assay: 50 ⁇ l of a murine monoclonal antibody specific for PAI-1 (#379, American Diagnostica Inc.), resuspended in 0.1 M Na 2 CO 3 buffer to a concentration of 2 ng/ ⁇ l, was added to wells of 96-well microtiter plates made using Removawell strip holders (Dynatech Labs). The plates were incubated at 4°C overnight, after which time the wells were washed with phosphate buffered saline (PBS), dried by patting on a paper towel, and incubated with 5% bovine serum albumin in PBS for 2 h at room temperature, or overnight at 4°C. Plates may be stored in this condition for weeks at 4°C. Following three washes with PBS (PBS), dried by patting on a paper towel, and incubated with 5% bovine serum albumin in PBS for 2 h at room temperature, or overnight at 4°C. Plates may be stored in this condition for weeks
- PAI-1-containing lysate from E. coli cells (containing
- the binding of t-PA to immobilized PAI was substantially lower when the capturing antibody was omitted.
- the antibody therefore captures PAI-1 in a conformation which permits t-PA to bind to it.
- the use of a monoclonal antibody to capture PAI-1 in a conformation which allows it to bind t-PA distinguishes the assay from other t-PA-PAI-1 binding assays which have been described.
- the PAI-specific capture antibody utilized in this Example is commercially available (#379 from American Diagnostica). The state of the art of hybridoma technology is such that production of other PAI-1-specific monoclonal antibodies to serve the same purpose is a
- Table 4 shows the % of the total t-PA input bound by immobilized PAI-1, where the PAI-1 was captured with different PAI-1 monoclonal antibodies.
- Six different monoclonal antibodies were tested, the first three of which were from the commercial vendor American Diagnostica (379, 380, and 3783) and the last three of which were generated in our laboratory using hybridoma technology known to those of skill in the art (FCD10, BBA4 and FAG9). All significantly increased the binding of t-PA to PAI-1 in comparison with a control test where no antibody was utilized. All the monoclonal antibodies in this experiment were utilized at a concentration of 1 ⁇ g per well.
- PAI-1-containing lysate up to 50 ⁇ l per well resulted in increased t-PA binding.
- ELISA studies revealed that approximately 100 ng of PAI-1 is the maximum amount that is immobilized by the 100 ng of capturing antibody.
- Table 8 compares the inhibitory effect of the purified heavy and light chains of t-PA (prepared by reduction of two-chain t-PA with dithiolthreitol) with that of intact t-PA on the binding of 1 25 I-t-PA to PAI-1.
- the purified light chain which contains the catalytic moiety of the t-PA, was a potent inhibitor of binding as would be predicted based on our knowledge that the catalytic site of a serine protease is involved in binding to its specific inhibitor.
- the purified heavy chain also displayed inhibi tory properties suggesti ng the presence of a domai n on thi s chain that al so interacts with PAI-1.
- the microtiter well environment of the assay will permit the screening of large numbers of test compounds for the desired activity, that is, inhibition of the binding of t-PA to PAI-1.
- PAI-1-coated wells may be prepared weeks in advance, thereby reducing the time, and increasing the efficiency of drug screening.
- our data indicates that in the assay of the invention the interaction between immobilized PAI-1 and 1 25 I-t-PA mimics the physiological reaction between these two proteins.
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US35026489A | 1989-05-11 | 1989-05-11 | |
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EP1485709A4 (de) * | 2002-02-19 | 2005-09-21 | Univ Vanderbilt | Therapieverfahren mit pai-1-inhibitoren und transgenes nichtmenschliches tier zum screening von pai-1-inhibitorkandidaten |
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