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WO1993025675A1 - Mutants des domaines du facteur de croissance epidermique de la thrombomoduline humaine - Google Patents

Mutants des domaines du facteur de croissance epidermique de la thrombomoduline humaine Download PDF

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Publication number
WO1993025675A1
WO1993025675A1 PCT/US1993/005585 US9305585W WO9325675A1 WO 1993025675 A1 WO1993025675 A1 WO 1993025675A1 US 9305585 W US9305585 W US 9305585W WO 9325675 A1 WO9325675 A1 WO 9325675A1
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analog
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PCT/US1993/005585
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Michael John Morser
Mariko Nagashima
John Francis Parkinson
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Schering Aktiengesellschaft
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Priority to AU45330/93A priority Critical patent/AU4533093A/en
Publication of WO1993025675A1 publication Critical patent/WO1993025675A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/745—Blood coagulation or fibrinolysis factors
    • C07K14/7455—Thrombomodulin

Definitions

  • TM is an endothelial cell surface glycoprotein that binds to thrombin with high affinity. Binding of TM to thrombin alters its conformation, leading to accelerated activation of protein C. Activated protein C then catalyzes the proteolytic inactivation of clotting factors Va and Villa in the presence of cofactor protein S, thereby inhibiting the coagulation cascade. Complex formation between thrombin and TM also results in direct inhibition of the procoagulant activities of thrombin, namely, fibrin formation and platelet activation. Thus, TM plays a critical role in maintenance of the anticoagulant surface.
  • Mature human TM is composed of a single polypeptide chain of 559 residues and consists of 5 domains: an amino- terminal "lectin-like" domain, an "6 EGF domain” comprising six epidermal growth factor (EGF) -like repeats, an 0- glycosylation domain, the transmembrane domain and cytoplasmic domain.
  • EGF epidermal growth factor
  • Fig. 1 is a comparison of the M388L analog, the wild type 6-EGF and p select controls in the cofactor activation assay described herein.
  • Figs. 2a-c compare the relative cofactor activity of the various alanine mutations described herein.
  • Fig. 3 provides estimates for the K d of 22 analogs.
  • TM analogs are defined by amino acid substitutions primarily in the last 3 EGF repeats. The substitutions are illustrated by alanine substitutions and in general provide sites of modification resulting in controllable and predictable changes in the cofactor activity of the analog. Although the work was done on analogs comprising only the 6-EGF domains , the cofactor activities are predictable for larger analogs comprising some or all of the other domains of native TM. Unless otherwise stated amino acids are referred to by either their full name, three letter designation or the standard single letter designation.
  • this invention provides for a thrombomodulin analog which has a modified cofactor activity upon binding to thrombin, as compared to TM E having natural sequence, said analog having an amino acid modification at a position corresponding to natural sequence at: a) 336 (asparagine) ; b) 337 (tyrosine) ; c) 340 (valine) ; d) 341 (aspartic acid) ; e) 365 (glutamic acid) ; f) 369 (leucine) ; g) 388 (methionine) ; h) 447 (isoleucine) ; or i) 454 (leucine) .
  • the modifications set forth above provide a level of cofactor activity which is less than or equal to about 25% of the wild type activity or and increased cofactor activity.
  • the analogs may comprise one or more of the above modifications. Preferred modifications are where said analog has modifications at both positions 365 and 369. These modifications result in about a 30% increase in cofactor activity.
  • the exemplified modifications are for alanine, however, aliphatic amino acids such as glycine, valine, leucine or isoleucine are also useful.
  • the above analogs are soluble and/or comprising at least one structural domain which is EGF4, EGF5, and EGF6. Also preferred are additional domains including: a) the lectin domain; b) EGF domains 1, 2, or 3; or c) the 0-linked glycosylation domain.
  • analogs may further comprise modifications at positions 456 or 457 which confer protease resistance to said analog.
  • Preferred substitutions are 456gly or 457gln.
  • a preferred analog has substitutions at positions 365, 369, 388, 456, and 457.
  • a further preferred analog provides for uniform termini during production of the recombinant protein.
  • Said analogs have terminal sequences of GPQP at the amino terminus, and LTPP at the carboxy terminus.
  • Nucleic acids encoding the above analogs are also described herein.
  • a second series of analogs is described herein having about 50% or less of the cofactor activity of the control TMgM388L. More particularly said thrombomodulin analog upon binding to thrombin, induces a modified cofactor activity as compared to binding with TM E M388L of less than or equal to 50%, said analog having an amino acid substitution at one or more positions corresponding to: aa) 349 (aspartic acid) ; ab) 355 (asparagine) ; ac) 357 (glutamic acid) ; ad) 358 (tyrosine) ; ae) 359 (glutamine) ; af) 363 (leucine) ; ag) 371 (valine) ; second letter represent the relative position of the modification with regard to other residues in the listing.
  • the analogs of this list may have modified modified K d for binding thrombin, a modified k cat /K m , or both.
  • the following analogs are a subset of the above list wherein the analogs have 25% or less of the cofactor activity of the control, TM E M388L.
  • Analogs having modifications in the latter two EGF repeats which resulted in reduced cofactor activity are listed below: be) 398 (aspartic acid) ; bd) 400 (aspartic acid) ; be) 402 (asparagine) ; bg) 408 (glutamic acid) ; bh) 413 (tyrosine) ; bi) 414 ( isoleucine) ; b j ) 415 (leucine) ; bk) 416 (aspartic acid) ; bl) 417 (aspartic acid); ca) 423 (aspartic acid) ; cb) 424 (isoleucine) ; cc) 425 (aspartic acid) ; cd) 426 (glutamic acid) ; cf) 429 (asparagine) ; ck) 439 (asparagine) ; en) 444 (phenylalanine) ; or cr) 461
  • EGF4 may also grouped by their respective domains as well as by their respective relative activity, i.e., EGF4, EGF5 or EFG6.
  • EGF4 having 50% of the control cofactor activity are: aa) 349 (aspartic acid) ; ab) 355 (asparagine) ; ac) 357 (glutamic acid) ; ad) 358 (tyrosine) ; ae) 359 (glutamine) ; af) 363 (leucine) ; ag) 371 (valine) ; ah) 374 (glutamic acid) ; ai) 376 (phenylalanine) ; aj) 384 (histidine); or ak) 385 (arginine) .
  • EGF4 having less than 25% of the cofactor activity of the control are: aa) 349 (aspartic acid) ; ac) 357 (glutamic acid) ; ad) 358 (tyrosine) ; ae) 359 (glutamine) ; ag) 371 (valine) ; or ai) 376 (phenylalanine) .
  • EGF5 EGF5
  • the following modifications resulted in analogs having at least a 50% reduction in cofactor activity: be) 398 (aspartic acid) ; bd) 400 (aspartic acid) ; be) 402 (asparagine) ; bf) 403 (threonine) ; bg) 408 (glutamic acid) ; bh) 413 (tyrosine) ; bi) 414 (isoleucine) ; bj) 415 (leucine); bk) 416 (aspartic acid) ; bl) 417 (aspartic acid) ; or bm) 420 (isoleucine) .
  • analogs are those where the analog has essentially an unmodified k cat /K m compared to TM E M388L.
  • the analogs can be further subgrouped according to those modifications resulted in analogs having at least a 75' reduction in cofactor activity: be) 398 (aspartic acid) ; bd) 400 (aspartic acid) ; be) 402 (asparagine) ; bg) 408 (glutamic acid) ; bh) 413 (tyrosine) ; bi) 414 (isoleucine) ; bj) 415 (leucine); bk) 416 (aspartic acid) ; or bl) 417 (aspartic acid) .
  • Nucleic acids encoding the above analogs are also claimed.
  • EGF6 the groups are provided below. Those having a cofactor activity of less than 50% of the control are: ca) 423 (aspartic acid) ; cb) 424 (isoleucine) ; cc) 425 (aspartic acid) ; cd) 426 (glutamic acid) ; ce) 428 (glutamic acid) ; cf) 429 (asparagine) ; eg) 432 (phenylalanine) ; eh) 434 (serine) ; ci) 436 (valine) ; cj) 438 (histidine); ck) 439 (asparagine) ; cl) 440 (leucine) ; cm) 443 (threonine) ; en) 444 (phenylalanine) ; co) 445 (glutamic acid) ; cp) 456 (arginine) ; cq) 458 (isoleucine) ;
  • Those having a cofactor activity of less than 25% of the control are: ca) 423 (aspartic acid) ; cb) 424 (isoleucine) ; cc) 425 (aspartic acid) ; cd) 426 (glutamic acid) ; cf) 429 (asparagine) ; ck) 439 (asparagine) ; cl) 440 (leucine) ; en) 444 (phenylalanine) ; or cr) 461 (aspartic acid) .
  • the preferred analogs are those set forth above with additional modifications for solubility, protease resistance, oxidation resistance as well as uniform terminal ends.
  • the nucleic acid encoding these analogs are also a part of the claimed invention. As with the other groups, these analogs include those wherein said analog has an essentially unmodified k cat /K m compared to TM ⁇ BBQL .
  • the analogs can be further subgrouped according to those possessing a modified amino acid at a position, wherein said analog has essentially equivalent K ⁇ for thrombin compared to an analog having at said position the native residue, wherein said position corresponds to: aa) 349 (aspartic acid) ; ab) 355 (asparagine) ; ac) 357 (glutamic acid) ; ad) 358 (tyrosine) ; or ae) 359 (glutamine) .
  • These analogs may have a modified k cat /K m of less than 30% of the control.
  • the following sites embrace describe analogs having a modified K ⁇ or k cat /K m compared to an analog having at said position the native residue, wherein said position corresponds to: af) 363 (leucine) ; ag) 371 (valine) ; ah) 374 (glutamic acid) ; ai) 376 (phenylalanine) ; aj) 384 (histidine); ak) 385 (arginine) ; be) 398 (aspartic acid) ; bd) 400 (aspartic acid) ; or be) 402 (asparagine) .
  • the following sites describe analogs having a lower cofactor activity and a K ⁇ or k cat /K m that is essentially equivalent when compared to an analog having at said position the native residue, wherein said position corresponds to: lutamic acid) ; yrosine) ; soleucine) ; eucine); spartic acid) ; spartic acid) ; soleucine) ; spartic acid) ; soleucine) ; spartic acid) lutamic acid) lutamic acid) sparagine) ; henylalanine) ; erine) ; aline) ; istidine) ; sparagine) ; eucine) ; threonine) ; henylalanine) ; lutamic acid) ; rginine) ; isoleucine) ; or aspartic acid) .
  • a method useful for screening for analogs of thrombomodulin which exhibit a modified K ⁇ for thrombin binding comprising the steps of: a) making an amino acid substitution at a position: bg) 408 (glutamic acid) ; bh) 413 (tyrosine) ; bi) 414 (isoleucine) ; bj) 415 (leucine); bk) 416 (aspartic acid) ; bl) 417 (aspartic acid) ; b ) 420 (isoleucine) ; ca) 423 (aspartic acid) ; cb) 424 (isoleucine) ; cc) 425 (aspartic acid) ; cd) 426 (glutamic acid) ; ce) 428 (glutamic acid) ; cf) 429 (asparagine) ; eg) 432 (phenylalanine) ; ch) 434 (s
  • Various embodiments of this invention include those wherein said K ⁇ is modified by at least 33%, or where said modification is an amino acid substitution, or wherein said control molecule is TMJJM388L.
  • a preferred grouping of modifications for use in the method are: bg) 408 (glutamic acid) ; bh) 413 (tyrosine) ; bi) 414 (isoleucine) ; bj) 415 (leucine); bk) 416 (aspartic acid) ; bl) 417 (aspartic acid) .
  • An another method is described herein wherein the method is useful for screening for analogs of thrombomodulin which induce a modified cofactor activity upon binding to thrombin, comprising the steps of: a) making an amino acid modification at a position: aa) 349 (aspartic acid) ; ab) 355 (asparagine) ; ac) 357 (glutamic acid) ; ad) 358 (tyrosine) ; ae) 359 (glutamine) ; and b) comparing the rate of cofactor activity upon binding to thrombin with the rate of a control molecule.
  • Cofactor activity refers to the relative ability of the TM analogs to complex with thrombin and potentiate the ability of thrombin to activate protein C.
  • the assay procedures used to measure cofactor activity are provided in Example 5.
  • Cofactor activity is also referred to as k cat when thrombin and protein C are at saturation concentrations.
  • K ⁇ refers to the relative binding affinity between the TM analog and thrombin. High K ⁇ values represent low binding affinity.
  • the precise assays and means for determining K ⁇ are provided in example 5.
  • KJJJ refers to the Michaelis constant and is derived in the standard way by measuring the rates of catalysis measured at different substrate concentrations. It is equal to the substrate concentration at which the reaction rate is half of its maximal value.
  • K is determined by keeping thrombin concentrations at a constant level e.g. (1 nM) and using saturation levels of TM (e.g. 100 nM or greater depending on the kd. Reactions are carried out using increasing concentration of protein C (e.g., 1-60 ⁇ M) . K ⁇ and k cat are then determined using Lineweaver-Burke plotting or nonlinear regression analysis.
  • ⁇ Modification or "modified amino acid” refers either a deletion or a substitution of a native amino acid residue for either a peptide bond or a substituent amino acid.
  • Natural sequence refers to the native sequence of thrombomodulin as provided in sequence ID. No. 1.
  • Thrombomodulin analog refers to a molecule having TM-like biological properties with regard to the activation of protein C and/or the binding to thrombin. Said analogs have substantial identity with the primary sequence of the domains of native TM that are responsible for the biological activity of TM. Analogs include deletion mutants, soluble analogs, protease resistant analogs and oxidation resistant analogs. n TM E " refers to an analog of TM consisting of the six EFG repeats.
  • a DNA sequence encoding the full-length native human thrombomodulin protein was isolated and described in (European Patent Application No. 88870079.6, which is incorporated herein by reference) .
  • the cDNA sequence encodes a 60.3 kDa protein of 575 amino acids, which includes a signal sequence of about 18 amino acids.
  • thrombomodulin exhibit a high degree of homology with one another.
  • domain refers to a discrete amino acid sequence that can be associated with a particular structure, function or characteristic.
  • the full length thrombomodulin gene encodes a precursor peptide containing the following domains:
  • the TM analogs of the present invention are modified in at least one of 22 amino acid residues in the 6-EGF region 227-462.
  • Modifications of the native sequence are indicated by the following formula: Z N Z' wherein Z and Z' are the single letter designation for the natural amino acids and where Z is the native residue and Z' is the substituted residue and wherein N is the number of the residue being modified.
  • M388L represents deletion of the native methionine at position 388 of TM and introduction of a leucine residue.
  • the modification is described as changing a parameter (e.g., K d , k cat or I ⁇ ) by at least %, the modification can be plus or minus. It being understood that a particular site of modification is determined as critical and substitutions being available therein to affect the desired change in %.
  • TM analogs of the present invention may be further modified to embrace the 6 epidermal growth factor [EGF] -like domain plus or minus the 0-linked glycosylation domain.
  • TM analogs preferably include any or all of the following characteristics: i) they are soluble in aqueous solution in the absence of detergents; ii) they retain activity after exposure to oxidants; iii) they are protease resistant through modification of residues 456 and 457; iv) they have uniform amino and carboxy termini; and, v) when bound to thrombin, they potentiate the thrombin-mediated activation of protein C and through the elimination of chondroitin sulfate have a reduced ability to inhibit the direct anti-coagulant activities of thrombin such as the conversion of fibrinogen to fibrin or the activation and aggregation of platelets.
  • Soluble TM analogs that retain activity after exposure to oxidants are termed "oxidation resistant". Such analogs are described in detail in co-pending co-assigned USSN 506,325 filed April 9, 1990, incorporated herein by reference.
  • a preferred TM analog are those rendered oxidation resistant by substitution of the methionine at position 388 ,in particular with leucine, M 388 L.
  • a "soluble TM analog” is a TM analog which is soluble in an aqueous solution and preferably can be secreted by a cell.
  • the soluble TM analog or an insoluble analog comprising the native cytoplasmic domain may optionally be combined with phospholipid vesicles, detergents or other similar compounds well known to those skilled in the art of pharmacological formulation.
  • the preferred TM analogs of the present invention are soluble in the blood stream, making the analogs useful in various anticoagulant and other therapies. These modifications do not significantly affect activities of native thrombomodulin such as affinity for thrombin or activity in protein C activation.
  • TM Analogs A. General Methods For Making TM Analogs. This invention relies upon molecular genetic manipulations that can be achieved in a variety of known ways.
  • the recombinant cells, plasmids, and DNA sequences of the present invention provide means to produce pharmaceutically useful compounds wherein the compound, secreted from recombinant cells, is preferably a soluble derivative of thrombomodulin.
  • Oligonucleotides that are not commercially available can be chemically synthesized according to the solid phase phosphoramidite triester method first described by S.L. Beaucage and M.H. Caruthers, (1981) Tetrahedron Letts . , 22(20) :1859-1862 using an automated synthesizer, as described in D.R. Needham-VanDevanter et al . , (1984) Nucleic Acids Res . , 12:6159-6168. Purification of oligonucleotides was by either native acrylamide gel electrophoresis or by anion-exchange
  • the sequence of the cloned genes and synthetic oligonucleotides can be verified using the chemical degradation method of A.M. Maxam et al . , (1980) Methods in Enzymology, 65:499-560. The sequence can be confirmed after the assembly of the oligonucleotide fragments into the double- stranded DNA sequence using the method of Maxam and Gilbert, supra, or the chain termination method for sequencing double- stranded templates of R.B. Wallace et al., (1981) Gene, 16: 21 - 26. Southern Blot hybridization techniques were carried out according to Southern et al . , (1975) J. Mol . Biol . , 58:503.
  • Embodiments of this invention involve the creation of novel peptides and genes by in vi tro mutagenesis.
  • Target genes are isolated in intermediate vectors and cloned for amplification in prokaryotes such as E. coli , Bacillus or Streptomyces. Most preferred is E. coli because that organism is easy to culture and more fully understood than other species of prokaryotes.
  • the Sambrook manual contains methodology sufficient to conduct all subsequently described clonings in E. coli .
  • Strain MH-1 is preferred unless otherwise stated. All E. coli strains are grown on Luria broth (LB) with glucose, or M9 medium supplemented with glucose and acid-hydrolyzed casein amino acids.
  • DNA sequence encoding human thrombomodulin and thrombin facilitates the preparation of genes and is used as a starting point to construct DNA sequences encoding TM peptides.
  • the peptides of the present invention are preferably soluble derivatives which lack the stop transfer sequence of TM in addition to having internal amino acid substitutions.
  • these analogs are secreted from eukaryotic cells which have been transfected or transformed with plasmids containing genes which encode these polypeptides. Methods for making modifications, such as amino acid substitutions, deletions, or the addition of signal sequences to cloned genes are known. Specific methods used herein are described below.
  • the full length gene for thrombomodulin can be prepared by several methods. Human genomic libraries are commercially available. Oligonucleotide probes, specific to these genes, can be synthesized using the published gene sequence. Methods for screening genomic libraries with oligonucleotide probes are known. The publication of the gene sequence for thrombomodulin demonstrates that there are no introns within the coding region. Thus a genomic clone provides the necessary starting material to construct an expression plasmid for thrombomodulin using known methods. A thrombomodulin encoding DNA fragment can be retrieved by taking advantage of restriction endonuclease sites which have been identified in regions which flank or are internal to the gene. (R.W. Jackman et al., (1987) Proc .
  • the full length genes can also be obtained from a cDNA bank.
  • messenger RNA prepared from endothelial cells provides suitable starting material for the preparation of cDNA.
  • a cDNA molecule containing the gene encoding thrombomodulin is identified as described above. Methods for making cDNA banks are well known (See Sambrook, supra) .
  • Genes encoding TM peptides may be made from wild- type TM genes first constructed using the gene encoding full length thrombomodulin.
  • a preferred method for producing wild- type TM peptide genes for subsequent mutation combines the use of synthetic oligonucleotide primers with polymerase extension on a mRNA or DNA template. This polymerase chain reaction (PCR) method amplifies the desired nucleotide sequence.
  • PCR polymerase chain reaction
  • Restriction endonuclease sites can be incorporated into the primers.
  • Genes amplified by the PCR reaction can be purified from agarose gels and cloned into an appropriate vector.
  • the emphasis of this invention is on the substitution of native amino acids of the 6-EGF region.
  • the invention is not limited to alanine substitutions.
  • Alterations in the natural gene sequence of TM beyond the 43 residues identified can be introduced by the techniques of in vitro mutagenesis which include the single site mutation techniques as described herein, cassette mutagenesis whereby multiple bases are replaced and random mutagenesis where a part of the gene or cDNA is altered by insert of a randomly generated oligomuleotide.
  • the TM peptides described herein are secreted when expressed in eukaryotic cell culture. Secretion may be obtained by the use of the native signal sequence of the thrombomodulin gene. Alternatively, genes encoding the TM peptides of the present invention may be ligated in proper reading frame to a signal sequence other than that corresponding to the native thrombomodulin gene. For example, the signal sequence of t-PA, (see WO 89/00605 incorporated herein by reference) or of hypodermin A or B (see EP 326,419 which is incorporated hereby by reference) can be linked to the polypeptide (See Table 2) .
  • t-PA which contains the second intron of the human t-PA gene.
  • the inclusion of the intron enhances the productivity of the adjacent structural gene.
  • those portions of the gene encoding the transmembrane and cytoplasmic domains of the carboxyl terminal region of the native thrombomodulin gene are typically deleted. Therefore, it is necessary to add a stop codon so that translation will be terminated at the desired position. Alternatively, a stop codon can be provided by the desired expression plasmid.
  • a polyadenylation sequence is helpful to ensure proper processing of the mRNA in eukaryotic cells encoding the TM analog. Also, it may be necessary to provide an initiation codon, if one is not present, for expression of the TM peptides. Such sequences may be provided from the native gene or by the expression plasmid.
  • Preferred cloning vectors suitable for replication and integration in prokaryotes or eukaryotes and containing transcription and translation terminators, initiation sequences, and promoters useful for regulation of the expression of TM peptides are described herein.
  • the vectors are comprised of expression cassettes containing at least one independent terminator sequence, sequences permitting replication of the plasmid in both eukaryotes and prokaryotes, i.e., shuttle vectors, and selection markers for both prokaryotic and eukaryotic systems.
  • TM Peptides in Prokaryotic Cells
  • carbohydrate moieties of the mature protein are not essential for activity as a cofactor for the activation of protein C but do have an effect on the direct anticoagulant properties of the TM analogs as well as the molecule's half life in circulation.
  • Expression of thrombomodulin analogs in E. coli has provided a useful tool for analysis of this issue. It is possible to recover a therapeutically functional protein from E. coli transformed with an expression plasmid encoding a soluble TM analog.
  • cloned genes in bacteria are well known.
  • To obtain high level expression of a cloned gene in a prokaryotic system it is essential to construct expression vectors which contain, at the minimum, a strong promoter to direct mRNA transcription termination.
  • regulatory regions suitable for this purpose are the promoter and operator region of the E. coli ⁇ - galactosidase gene, the E. coli tryptophane biosynthetic pathway, or the leftward promoter from the phage lambda.
  • selection markers in D ⁇ A vectors transformed in E. coli are useful. Examples of such markers include the genes specifying resistance to ampicillin, tetracycline, or chloramphenicol.
  • p select is used as a vector for the subcloning and amplification of desired gene sequences.
  • the D ⁇ A sequence encoding a soluble TM analog can be ligated to various expression vectors for use in transforming host cell cultures.
  • the vectors typically contain marker genes and gene sequences to initiate transcription and translation of the heterologous gene.
  • the vectors preferably contain a marker gene to provide a phenotypic trait for selection of transformed host cells such as dihydrofolate reductase, metallothionein, hygromycin, or neomycin phosphotransferase.
  • the nuclear polyhedral viral protein from Autographa calif ornica is useful to screen transfected insect cell lines from Spodoptera frugiperda and Bombyx mori to identify recombinants.
  • mammalian cell lines include RPMI 7932, VERO and HeLa cells, Chinese hamster ovary (CHO) cell lines, WI38, BHK, COS-7, C127 or MDCK cell lines.
  • a preferred mammalian cell line is CHL-l. When CHL-l is used hygromycin is included as a eukaryotic selection marker.
  • CHL-l cells are derived from RPMI 7932 melanoma cells, a readily available human cell line.
  • the CHL-l cell line has been deposited with the ATCC according to the conditions of the Budapest Treaty and has been assigned #CRL 9446, deposited June 18, 1987.
  • Cells suitable for use in this invention are commercially available from the American Type Culture Collection.
  • Illustrative insect cell lines include Spodoptera frugiperda (fall Armyworm) and Bombyx mori (silkworm) .
  • the expression vector e.g., plasmid
  • the expression vector preferably contains gene sequences to initiate the transcription and sequences to control the translation of the TM peptide gene sequence. These sequences are referred to as expression control sequences.
  • illustrative expression control sequences include but are not limited to the following: the retroviral long terminal repeat promoters (Nature, 257:479-483, 1982), SV40 promoter (Science, 222:524-527, 1983); thymidine kinase promoter; ( Cell , 27:299-308, 1982), or the beta-globin promoter, ( Cell , 33:705-716, 1983) .
  • the recipient vector nucleic acid containing the expression control sequences is cleaved using restriction enzymes and adjusted in size as necessary or desirable. This segment is ligated to a DNA sequence encoding at the TM peptide by means well known in the art.
  • polyadenylation or transcription termination sequences need to be incorporated into the vector.
  • An example of a polyadenylation sequence is the polyadenylation sequence from SV40, which may also function as a transcription terminator.
  • Genes incorporated into the appropriate vectors can be used to direct synthesis of proteins in either transient expression systems or in stable clones. In the former case yields are low, but the experiments are quick. In the latter case it takes more time to isolate high producing clones. Different vectors may be used for the two different types of experiments.
  • sequences may be included within the plasmid that allow the plasmid to replicate to a high copy number within the cell. These sequences may be derived from virus such as SV40 (e.g. C. Doyle et al . , (1985) J. Cell Biol .
  • the vector for use in transient expression should also contain a strong promoter such as the SV40 early promoter (e.g., A. van Zonnenfeld et al . , (1987) Proc. Natl . Acad. Sci . USA. , 83:4670-4674) to control transcription of the gene of interest. While transient expression provides a rapid method for assay of gene products, the plasmid DNA is not incorporated into the host cell chromosome.
  • transient expression vectors does not provide stable transfected cell lines.
  • a description of a plasmid suitable for transient expression is provided by A. Aruffo & B. Seed, (1987) Proc. Natl . Acad. Sci . USA . , 84:8573-8577.
  • TM analogs may alternatively be produced in the insect cell lines described above using the baculovirus system.
  • This system has been described by V.A. Luckow and M.D. Summers (1988) Bio/Technology, 6:47-55.
  • this expression system provides for a level of expression higher than that provided by most mammalian systems.
  • the baculovirus infects the host insect cells, replicates its genome through numerous cycles, and then produces large amounts of polyhedron crystals.
  • the polyhedron gene can be replaced with a TM peptide gene.
  • the polyhedron promoter will then make large amounts of analog protein following infection of the culture host cell and replication of the baculovirus genome.
  • the non- secreted gene product is harvested from the host 3-7 days post infection.
  • the TM peptide may be secreted from the cells if appropriate signal sequences are present on the protein.
  • the host cells are competent or rendered competent for transfection by various means. There are several well- known methods of introducing DNA into animal cells. These include: calcium phosphate precipitation, DEAE-dextran technique, fusion of the recipient cells with bacterial protoplasts containing the DNA, treatment of the recipient cells with liposomes containing the DNA, electroporation and microinjection of the DNA directly into the cells. See, B. Perbal, "Practical Guide to Molecular Cloning, ⁇ 2nd edition, John Wiley & Sons, New York and Wigler, et al . , (1987) Cell , 16:777-785.
  • the host cell is capable of rapid cell culture and able to appropriately glycosylate expressed gene products.
  • Cells known to be suitable for dense growth in tissue culture are particularly desirable and a variety of invertebrate or vertebrate cells have been employed in the art, both normal and transformed cell lines.
  • the transfected cells are grown up by means well known in the art. For examples, see Biochemical Methods in
  • the expression products are harvested from the cell medium in those systems where the protein is secreted from the host cell or from the cell suspension after disruption of the host cell system by, e.g., mechanical or' enzymatic means, which are well known in the art.
  • the TM peptides of this invention be secreted by cultured recombinant eukaryotic cells.
  • the TM analogs are produced in serum-free or serum supplemented media and are secreted intact. If prokaryotic cells are used, the TM analogs may be deposited intracellularly. The peptides may be fully or partially glycosylated or non-glycosylated. Following the growth of the recombinant cells and concomitant secretion of TM analogs into the culture media, this "conditioned media" is harvested. The conditioned media is then clarified by centrifugation or filtration to remove cells and cell debris.
  • the proteins contained in the clarified media are concentrated by adsorption to any suitable resin such as, for example, Q Sepharose or metal chelators, or by use of ammonium sulfate fractionation, polyethylene glycol precipitation, or by ultrafiltration. Other means known in the art may be equally suitable.
  • Further purification of the TM analogs can be accomplished in the manner described in Galvin, J. B., et al . , (1987) J. Biol . Chem. , 262:2199-2205 and Salem, H.H. et al . , (1984) J. Biol . Chem. , 255:12246-12251 and in the manner described in the embodiment disclosed herein.
  • the purification of TM analogs secreted by cultured cells may require the additional use of, for example, affinity chromatography, ion exchange chromatography, sizing chromatography or other protein purification techniques.
  • Recombinant TM analogs may be produced in multiple conformational forms which are detectable under nonreducing chromatographic conditions. Removal of those species having a low specific activity is desirable and is achieved by a variety of chromatographic techniques including anion exchange or size exclusion chromatography. Recombinant TM analogs may be concentrated by pressure dialysis and buffer exchanged directly into volatile buffers (e.g., N-ethylmorpholine (NEM) , ammonium bicarbonate, ammonium acetate, and pyridine acetate) . In addition, samples can be directly freeze-dried from such volatile buffers resulting in a stable protein powder devoid of salt and detergents.
  • volatile buffers e.g., N-ethylmorpholine (NEM) , ammonium bicarbonate, ammonium acetate, and pyridine acetate
  • freeze-dried samples of recombinant analogs can be efficiently resolubilized before use in buffers compatible with infusion (e.g., phosphate buffered saline) .
  • buffers compatible with infusion e.g., phosphate buffered saline
  • suitable buffers might include hydrochloride, hydrobromide, sulphate acetate, benzoate, malate, citrate, glycine, glutamate, and aspartate.
  • Native thrombomodulin is susceptible to oxidation and when oxidized loses its ability to promote the activation of protein C.
  • Many of the disease conditions requiring anticoagulation are also associated with high levels of toxic oxygen radicals, which can inactivate biomolecules and cause significant tissue damage. Examples of these conditions are reperfusion injury associated with myocardial infarction, DIC associated with septicemia, and alveolar fibrosis associated with adult respiratory distress syndrome.
  • any wound such as occurring in surgical procedures, involves the influx of activated monocytes, polymorphonuclear leukocytes, etc. which can create toxic oxygen species as well as releasing a host of proteolytic enzymes, such as elastase.
  • Thrombomodulin is subject to inactivation by exposure to toxic oxygen species and that this is expected to have a significant role in many pathogenic states.
  • the test material (100 - 250 ⁇ g/ml) is first incubated with an oxidant such as, for example, chloramine-T, hydrogen peroxide at 5-lOmM chloramine-T or 200-1000 mM hydrogen peroxide in a buffer of 0.2% N-ethylmorpholine and 0.008% Tween 80 at pH 7.0 for 20 minutes at room temperature. After such oxidant exposure, the test material is evaluated using one of the bioactivity assays described below, specifically for the ability to act as a cofactor for the activation of protein C.
  • an oxidant such as, for example, chloramine-T, hydrogen peroxide at 5-lOmM chloramine-T or 200-1000 mM hydrogen peroxide in a buffer of 0.2% N-ethylmorpholine and 0.008% Tween 80 at pH 7.0 for 20 minutes at room temperature.
  • TM analogs that retain at least 60%, and preferably 90%, of activity they had prior to exposure to oxidants are considered to be oxidation resistant as compared to wild-type (non- mutant) TM analog or native thrombomodulin.
  • Protease activity has been a problem with recombinant production of TM.
  • the resulting TM has two chains.
  • Protease resistant species of TM are those designed to be resistant to protease cleavage at amino acid residues, 456/457. In the native numbering system, these residues are arginine and histidine. These residues are preferably altered to glycine and glutamine, although other substitutions are possible. This modification will result in the preparation of single chain TM.
  • Heterogeneity of the termini of recombinant TM is a further problem during production.
  • the amino terminus should be modified so that the processing enzyme of the host cell will generate a single N-terminus in the mature protein.
  • deleting the first three amino acids and beginning expression with the fourth amino acid (glu) provides for a fully functional analog having a homogeneous amino terminus.
  • Heterogeneity of the carboxy terminus is also a problem when producing TM from recombinantly altered cells. Ending the molecule at the pro-pro residues at positions 489 and 490, seven amino acids from the native TM carboxy terminus provides a preferred TM analog. This carboxy-terminus is particularly resistant to c-terminal exonucleases and provides a fully functional soluble TM.
  • Protein C cofactor activity can be measured in the assay described by Salem, et al . , (1984) J. Biol . Chem. 255(19) :12246-12251 and Galvin, et al., (1987) J. Biol . Chem. 262(5) :2199-2205.
  • this assay consists of two steps. The first is the incubation of the test TM analog with thrombin and protein C under defined conditions (see Examples below) .
  • the thrombin is inactivated with hirudin or antithrombin III and heparin, and the activity of the newly activated protein C is determined by the use of a chromogenic substrate, whereby the chromophore is released by the proteolytic activity of activated protein C.
  • This assay is carried out with purified reagents.
  • TM analog can be measured using plasma in clotting time assays such as the activated partial thromboplastin time (APTT) , thrombin clotting time (TCT) and/or prothrombin time (PT) .
  • APTT activated partial thromboplastin time
  • TCT thrombin clotting time
  • PT prothrombin time
  • thrombomodulin activities of native thrombomodulin such as inhibition of thrombin catalyzed formation of fibrin from fibrinogen (Jakubowski, et al . , (1986) J. Biol . Chem. 261 ( B ) :3876-3882) , inhibition of thrombin activation of Factor V (Esmon, et al . , (1982) J. Biol . Chem. 257:7944-7947) , accelerated inhibition of thrombin by antithrombin III and heparin cofactor II (Esmon, et al . , (1983) J. Biol . Chem.
  • TM analogs do not have all activities equal to that of native thrombomodulin.
  • Carbohydrate substituents on proteins can affect both biological"activity and circulating half-life.
  • O-linked glycosaminoglycan carbohydrate such as is found in the native thrombomodulin protein is eliminated.
  • a glycanase known to specifically degrade sulfated glycosaminoglycans, such as chondroitinase ABC or hyaluronidase. This method is described in Bourin, M, et al . , (1988) J " . Biol . Chem.
  • a second method for eliminating the 0-linked carbohydrate is by introducing site directed mutations into the protein.
  • the attachment of glycosaminoglycans is often directed by the consensus recognition sequence of amino acids X-serine-glycine-X-glycine-X (Bourdon, M.A. , et al . , (1987) PNAS, U. S.A . 84:3194-3198) where X is any amino acid.
  • the recognition sequence for other types of 0-linked sugars is threonine/serine-X-X-proline.
  • the 0-linked domain of thrombomodulin has one potential glycosaminoglycan addition site (aa 472) and three other potential 0-linked carbohydrate addition sites (aa 474, 480 and 486) . Any change introduced into the nucleotide sequence that removes or changes the identity of any one or more of the amino acids in this recognition sequence will eliminate the potential 0-linked carbohydrate attachment site. Methods of introducing site directed mutations into a nucleotide sequence are described above.
  • a preferred method of eliminating 0-linked carbohydrate from a TM analog is by making an analog peptide that does not include the amino acids that are considered to be the O-linked domain, i.e., amino acids 468 through 485 of the native thrombomodulin gene sequence as shown in Table l. Methods of accomplishing this are well known in the art and have been described above. The circulating half-life of a protein can be altered by the amount and composition of carbohydrate attached to it.
  • the TM analogs cf the present invention contain both 0-linked and N-linked carbohydrate. In addition to the potential glycosylation sites discussed above there are potential N-linked sites at amino acids 364, 391 and 393 and potential 0-linked sites at amino acids 319, 393 and 396.
  • Methods of altering carbohydrate composition in addition to those described above are: l) expression of the TM analog gene in bacteria such E. coli , which does not have the cellular mechanisms necessary to glycosylate mammalian proteins, 2) expression of the TM analog gene in various eukaryotic cells, as each has its own characteristic enzymes that are responsible for the addition of characteristic sugar residues, and 3) treatment with chemicals such as hydrofluoric acid.
  • Hydrofluoric acid for example, chemically digests acid and neutral pH sugars while leaving intact basic sugars such as N- acetyl glucosamines and, under certain conditions, galactosamines.
  • TM analogs described herein may be prepared in a lyophilized or liquid formulation.
  • the material is to be provided in a concentration suitable for pharmaceutical use as either an injectable or intravenous preparation, preferably in single dose for mutations.
  • TM can be administered alone or as mixtures with other physiologically acceptable active materials, such as antibiotics, other anti coagulants, one-chain t-PA, or inactive materials, or with suitable carriers such as, for example, water or normal saline.
  • suitable carriers such as, for example, water or normal saline.
  • the analogs can be administered parenterally, for example, by injection. Injection can be subcutaneous, intravenous or intramuscular.
  • salts such as acid addition salts.
  • Such salts can include, e.g., hydrochloride, hydrobromide, phosphate, sulphate, acetate, benzoate, malate, citrate, glycine, glutamate, and aspartate, among others.
  • the analogs described herein may display enhanced in vivo activity by incorporation into micelles. Methods for incorporation into ionic detergent micelles or phospholipid micelles are known.
  • An antithrombotic agent can be prepared using the soluble TM analogs described herein and can consist of a completely purified analog alone or in combination with a thrombolytic agent as described above.
  • Compounds of the present invention which are shown to have the above recited physiological effects can find use in numerous therapeutic applications such as, for example, the inhibition of blood clot formation.
  • these compounds can find use as therapeutic agents in the treatment of thrombotic disease 'and of various circulatory disorders, such as, for example, coronary or pulmonary embolism, strokes, as well as the prevention of reocclusion following thrombolytic therapy and enhancement of thrombolytic therapies.
  • These compounds also have utility in the cessation of further enlargement of a clot during an infarction incident.
  • the compounds disclosed can be useful for treatment of systemic coagulation disorders such as disseminated intravascular coagulation (DIC) , which is often associated with septicemia, certain cancers and toxemia of pregnancy.
  • DIC disseminated
  • Thrombomodulin mutants can be recombinantly produced by isolation of the critical EGF domain of human TM (amino acid 227 to 462) using polymerase chain reaction of human genomic DNA.
  • the following primers can be used: (Sequence ID No. 21 5'CCGGGATCCTCAACAGTCGGTGCCAATGTGGCG3* and Seq. ID. No. 22 5'CCGGGATCCTGCAGCGTGGAGAACGGCGGCTGC3' .
  • This fragment through a series of intermediate constructs, is then placed under the control of ⁇ -lactamase promoter and signal sequence in pKT279 purchased from Strategene, La Jolla, CA..
  • Plasmids coding for TM mutants were constructed using a site-directed mutagenesis procedure described by Kunkel et.al., (1987) Methods in Enzymology 154 , 367-382. Briefly, a single-stranded uracil DNA prepared from E. coli strain CJ236 with R408 helper phage was used as a template for the synthesis of the mutagenic strand in the presence of specific oligonucleotides using T4 DNA polymerase and T4 DNA ligase.
  • T4 DNA polymerase and E. coli strain CJ236 were from BioRad Laboratories, Richmond, CA.
  • a restriction enzyme recognition sequence in oligonucleotides was incorporated without changing the amino acid sequence, and resulting DH5 alpha tranformants were characterized by restriction digests of isolated plasmid DNAs.
  • Three independent positive clones were isolated for each mutant except Y368A, E411A, I414A and E408A mutants, for which only 2 positive clones were obtained. In cases where there were large discrepancies between cofactor activities of triplicates, plasmids were further characterized by dideoxy sequencing.
  • Example 3 Production of E. coli shockates containing TM mutants
  • DH5 alpha cells expressing TM mutants are grown overnight in 1.5 ml of Luria broth containing ampicillin (50 ⁇ g/ml) at 37°C. Cells are harvested by centrifuging at
  • shockates are obtained by centrifuging 5 minutes at full speed.
  • shockates are assayed immediately or less preferably stored at -70°C until ready for assaying (no longer than 30 days) .
  • TM mutants to act as cofactor for thrombin-mediated activation of protein C was assayed directly in the shockates.
  • Recombinant human protein C was from Dr. John McPherson, Genzyme Corp., Framingham, MA., and was purified as described (BioTechnology 8:655-661, 1990) . Twenty five ⁇ l of each shockate was mixed with equal volumes of recombinant human protein C (final concentration of 0.3 ⁇ M) and human alpha thrombin (Sigma Chemicals, St. Louis, MO, at a final concentration of InM) in a microtiter plate.
  • All assays contained triplicate shockate samples each of DH5 alpha cells transfected with either pSELECT-1 vector (no TM) , pTHR211 (wild type) or pMJM57 (pTHR211 with methionine at 388 altered to leucine) , as internal controls.
  • Cofactor activities of TM mutants were expressed as mean % of that obtained for pMJM57 and are provided in Fig. 1. Statistical Analysis Each mutant was assayed for activity at least twice
  • E. coli shockates were run in 10% Tris-tricine SDS PAGE under reduced conditions according to the manufacture's specifications (Novex Inc., San Diego, CA) . Reduced and alkylated samples were prepared by boiling shockates in sample buffer (62.5 mM Tris, pH6.8/2% SDS/10% glycerol/0.0025% bromophenol blue) containing 10 mM dithiothreitol for 10 minutes, followed by incubation with 50 mM iodoacetamide.
  • sample buffer (62.5 mM Tris, pH6.8/2% SDS/10% glycerol/0.0025% bromophenol blue) containing 10 mM dithiothreitol for 10 minutes, followed by incubation with 50 mM iodoacetamide.
  • Proteins were transferred to nitrocellulose filter in transfer buffer (192 mM glycine/25 mM Tris,pH8.3/20 % methanol) at 4°C.
  • transfer buffer 192 mM glycine/25 mM Tris,pH8.3/20 % methanol
  • the nitrocellulose filter was blocked with a blocking buffer (1% bovine serum albumin in 10 mM Tris,ph7.5/0.9 % NaCl/0.05 % NaN 3 ) , and then incubated with mouse polyclonal antiserum (raised against reduced and alkylated EGF domain of human thrombomodulin) in the blocking buffer.
  • the activity of selected alanine mutants was assayed as described above in Example D except that the final concentration of thrombin in the reaction mixture was varied from 1 to 60 nM and the reaction was terminated by the addition of 25 ⁇ l of 8000 units/ml hirudin.
  • Cofactor activity at each thrombin concentration was determined from the mOD/min in the presence of mutant minus the mOD/min in the absence of mutant (corrected mOD/min) . The reciprocal of the corrected mOD/min was then plotted against the reciprocal of the thrombin concentration.
  • Example 6 Results of alanine mutation experiments of the EGF domain of TM.
  • each residue between amino acid 333 to 462 was systematically replaced with alanine by site-directed mutagenesis, and the effect of substitutions on activity was determined directly in shockate samples.
  • Seventy seven mutants were constructed using M388L mutant as a template to increase the basal level of activity. Three independent clones were isolated for each mutant and assayed at least twice (or three assays for 4 mutants with 2 clones) . The results are expressed as mean percentages of
  • M388L mutant with standard deviations in error bars (Fig. 2) .
  • 22 mutations resulted in cofactor activity below 25 % of M388L mutant. They were D349A, E357A, Y358A, F376A, D398A, D400A, N402A, E408A, Y413A, I414A, L415A, D416A, D417A, D423A, I424A, D425A, E426A, N429A, N439A, L440A, F444A and D461A.
  • Other mutations produced proteins with activity ranging between 25 to 100 % of the control.
  • Asp423,Asp425 and Glu426) were found within a region spanning the third loop of the 5th repeat to the interdomain, the region known to play a key role in thrombin-binding.
  • Asn439 and Phe444 were part of the proposed consensus sequence -C-X-D/N-X-X-X-X-F/Y-X-C-X-C- for the 3-hydroxylation of Asp or Asn residue (Stemflow, J. , et al., PNAS 84:368-372, 1987) . While 3-hydroxylation of Asp or Asn has not shown to be obligatory for Ca 2+ binding, it could contribute to the higher affinity for Ca 2+ (Hanford, P.A., et al . , Nature 351:161-167, 1991).
  • Thrombomodulin peptides according to this invention have been expressed in malian cells, i.e., in HEK293 (human embryonic kidney) cells, and the resultant peptides demonstrated essentially the same alterations in specific activity when compared with wild-type and M388L as was found in those peptides expressed in E. coli .
  • Xaa is the natural amino acid for that position as specified in SEQ ID NO 1 or an aliphatic amino acid of the group comprising Gly, Ala , Val , Leu and He " .
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • SEQUENCE DESCRIPTION SEQ ID NO:22: CCGGGATCCT GCAGCGTGGA GAACGGCGGC TGC 33
  • MOLECULE TYPE DNA (genomic)
  • CAGCCCCTGA ACCAAACTAG CTACCTCTGC GTCTGCGCCG AGGGCTTCGC GCCCATTCCC 1140 CACGAGCCGC ACAGGTGCCA GATGTTTTGC AACCAGACTG CCTGTCCAGC CGACTGCGAC 1200

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Abstract

L'invention concerne l'identification de résidus critiques d'acides aminés dans la région du facteur de croissance épidermique 6 de la thrombomoduline (TM). La modification de ces résidus a un effet prévisible sur la capacité de la TM de modifier l'activité du cofacteur, par exemple la liaison à la thrombine et/ou la médiation de la potentialisation de l'activation de la protéine C au moyen de la thrombine.
PCT/US1993/005585 1992-06-10 1993-06-10 Mutants des domaines du facteur de croissance epidermique de la thrombomoduline humaine WO1993025675A1 (fr)

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU675422B2 (en) * 1992-02-05 1997-02-06 David Richard Light Protease-resistant thrombomodulin analogs
WO2001098352A3 (fr) * 2000-06-21 2002-08-01 Schering Ag Analogues de thrombomoduline et leur utilisation pharmaceutique
WO2010142461A2 (fr) 2009-06-12 2010-12-16 Paion Deutschland Gmbh Traitement de coagulopathie avec hyperfibrinolyse
WO2011157283A1 (fr) 2010-06-14 2011-12-22 Paion Deutschland Gmbh Traitement d'une coagulopathie avec hyperfibrinolyse
EP2754447A3 (fr) * 2006-12-12 2014-11-12 Indiana University Research and Technology Corporation Traitement de la défaillance rénale aigüe avec des variants de thrombomoduline solubles

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WO1990010081A1 (fr) * 1989-02-17 1990-09-07 Codon Analogues solubles de la thrombomoduline

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WO1990010081A1 (fr) * 1989-02-17 1990-09-07 Codon Analogues solubles de la thrombomoduline

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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, Volume 185, Number 2, issued 15 June 1992, J.F. PARKINSON et al., "Structure-Function Studies of the Epidermal Growth Factor Domains of Human Thrombomodulin", pages 567-576. *
JOURNAL OF BIOLOGICAL CHEMISTRY, Volume 266, Number 30, issued 25 October 1991, M. ZUSHI et al., "Aspartic Acid 349 in the Fourth Epidermal Growth Factor-Like Structure of Human Thrombomodulin Plays a Role in its Ca2+-Mediated Binding to Protein C", pages 19886-19889. *
JOURNAL OF BIOLOGICAL CHEMISTRY, Volume 267, Number 9, issued 25 March 1992, M. TSIANG et al., "Functional Domains of Membrane-Bound Human Thrombomodulin", pages 6164-6170. *
JOURNAL OF BIOLOGICAL CHEMISTRY, Volume 268, Number 4, issued 05 February 1993, M. NAGASHIMA et al., "Alanine-Scanning Mutagenesis of the Epidermal Growth Factor-Like Domains of Human Thrombomodulin Identifies Critical Residues for its Cofactor Activity", pages 2888-2893. *
JOURNAL OF BIOLOGICAL CHEMISTRY, Volume 268, Number 9, issued 25 March 1993, J.H. CLARKE et al., "The Short Loop Between Epidermal Growth Factor-Like Domains 4 and 5 is Critical for Human Thrombomodulin Function", pages 6309-6315. *

Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU675422B2 (en) * 1992-02-05 1997-02-06 David Richard Light Protease-resistant thrombomodulin analogs
WO2001098352A3 (fr) * 2000-06-21 2002-08-01 Schering Ag Analogues de thrombomoduline et leur utilisation pharmaceutique
JP4855627B2 (ja) * 2000-06-21 2012-01-18 バイエル ファーマ アクチエンゲゼルシャフト 医薬用途のためのトロンボモジュリン
EP2754447A3 (fr) * 2006-12-12 2014-11-12 Indiana University Research and Technology Corporation Traitement de la défaillance rénale aigüe avec des variants de thrombomoduline solubles
WO2010142461A2 (fr) 2009-06-12 2010-12-16 Paion Deutschland Gmbh Traitement de coagulopathie avec hyperfibrinolyse
WO2010142309A1 (fr) * 2009-06-12 2010-12-16 Paion Deutschland Gmbh Traitement de coagulopathie avec hyperfibrinolyse
WO2010142461A3 (fr) * 2009-06-12 2011-11-24 Paion Deutschland Gmbh Traitement de coagulopathie avec hyperfibrinolyse
WO2011157283A1 (fr) 2010-06-14 2011-12-22 Paion Deutschland Gmbh Traitement d'une coagulopathie avec hyperfibrinolyse
CN103037893A (zh) * 2010-06-14 2013-04-10 帕昂德国有限公司 具有纤溶亢进的凝血病的治疗
JP2013531651A (ja) * 2010-06-14 2013-08-08 パイオン ドイチュラント ゲーエムベーハー 線溶亢進を伴う凝固障害の処置

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