WO2022145475A1 - 血液凝固能の評価方法および血栓症リスクの検査方法 - Google Patents
血液凝固能の評価方法および血栓症リスクの検査方法 Download PDFInfo
- Publication number
- WO2022145475A1 WO2022145475A1 PCT/JP2021/048977 JP2021048977W WO2022145475A1 WO 2022145475 A1 WO2022145475 A1 WO 2022145475A1 JP 2021048977 W JP2021048977 W JP 2021048977W WO 2022145475 A1 WO2022145475 A1 WO 2022145475A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- inhibitor
- blood coagulation
- final concentration
- blood
- factor
- Prior art date
Links
- 230000023555 blood coagulation Effects 0.000 title claims abstract description 178
- 238000000034 method Methods 0.000 title claims abstract description 28
- 208000007536 Thrombosis Diseases 0.000 title description 20
- 239000003112 inhibitor Substances 0.000 claims abstract description 169
- 210000004369 blood Anatomy 0.000 claims abstract description 115
- 239000008280 blood Substances 0.000 claims abstract description 115
- 102100030951 Tissue factor pathway inhibitor Human genes 0.000 claims abstract description 67
- 108010013555 lipoprotein-associated coagulation inhibitor Proteins 0.000 claims abstract description 67
- 239000003130 blood coagulation factor inhibitor Substances 0.000 claims abstract description 22
- 238000000338 in vitro Methods 0.000 claims abstract description 11
- 239000003153 chemical reaction reagent Substances 0.000 claims description 47
- 108010000499 Thromboplastin Proteins 0.000 claims description 44
- 102000002262 Thromboplastin Human genes 0.000 claims description 44
- 101000975003 Homo sapiens Kallistatin Proteins 0.000 claims description 27
- 101001077723 Homo sapiens Serine protease inhibitor Kazal-type 6 Proteins 0.000 claims description 27
- 229940122920 Kallikrein inhibitor Drugs 0.000 claims description 27
- 102100023012 Kallistatin Human genes 0.000 claims description 27
- 206010028980 Neoplasm Diseases 0.000 claims description 21
- 238000013169 thromboelastometry Methods 0.000 claims description 21
- 201000011510 cancer Diseases 0.000 claims description 19
- 238000004458 analytical method Methods 0.000 claims description 18
- 102000015081 Blood Coagulation Factors Human genes 0.000 claims description 13
- 108010039209 Blood Coagulation Factors Proteins 0.000 claims description 13
- 239000012190 activator Substances 0.000 claims description 13
- 239000003114 blood coagulation factor Substances 0.000 claims description 13
- 108010085662 ecarin Proteins 0.000 claims description 11
- 229940019700 blood coagulation factors Drugs 0.000 claims description 10
- 238000011156 evaluation Methods 0.000 claims description 6
- 238000005259 measurement Methods 0.000 claims description 5
- 230000003213 activating effect Effects 0.000 claims description 4
- 102000004190 Enzymes Human genes 0.000 claims description 3
- 108090000790 Enzymes Proteins 0.000 claims description 3
- 101001038021 Lonomia obliqua Lopap Proteins 0.000 claims description 3
- 108010080865 Factor XII Proteins 0.000 claims description 2
- 102000000429 Factor XII Human genes 0.000 claims description 2
- 108010074864 Factor XI Proteins 0.000 claims 2
- 239000003998 snake venom Substances 0.000 claims 1
- 230000002401 inhibitory effect Effects 0.000 abstract description 9
- 239000003795 chemical substances by application Substances 0.000 abstract description 7
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 description 59
- 239000001110 calcium chloride Substances 0.000 description 59
- 229910001628 calcium chloride Inorganic materials 0.000 description 59
- 108010059382 Zea mays trypsin inhibitor Proteins 0.000 description 55
- NAXNMKUKSBHHMP-QCXPCDNBSA-N 2-[4-[[(2s)-2-[[4-(aminomethyl)cyclohexanecarbonyl]amino]-3-phenylpropanoyl]amino]phenyl]acetic acid;hydrochloride Chemical compound Cl.C1CC(CN)CCC1C(=O)N[C@H](C(=O)NC=1C=CC(CC(O)=O)=CC=1)CC1=CC=CC=C1 NAXNMKUKSBHHMP-QCXPCDNBSA-N 0.000 description 51
- 230000015271 coagulation Effects 0.000 description 25
- 238000005345 coagulation Methods 0.000 description 25
- 210000001808 exosome Anatomy 0.000 description 25
- 229920001971 elastomer Polymers 0.000 description 22
- 238000007792 addition Methods 0.000 description 20
- 239000002158 endotoxin Substances 0.000 description 20
- 229920006008 lipopolysaccharide Polymers 0.000 description 20
- 239000001509 sodium citrate Substances 0.000 description 20
- NLJMYIDDQXHKNR-UHFFFAOYSA-K sodium citrate Chemical compound O.O.[Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O NLJMYIDDQXHKNR-UHFFFAOYSA-K 0.000 description 20
- 229940126214 compound 3 Drugs 0.000 description 19
- PHEDXBVPIONUQT-RGYGYFBISA-N phorbol 13-acetate 12-myristate Chemical compound C([C@]1(O)C(=O)C(C)=C[C@H]1[C@@]1(O)[C@H](C)[C@H]2OC(=O)CCCCCCCCCCCCC)C(CO)=C[C@H]1[C@H]1[C@]2(OC(C)=O)C1(C)C PHEDXBVPIONUQT-RGYGYFBISA-N 0.000 description 17
- 230000004913 activation Effects 0.000 description 14
- 238000012360 testing method Methods 0.000 description 13
- 102100030563 Coagulation factor XI Human genes 0.000 description 12
- 210000004027 cell Anatomy 0.000 description 12
- 239000003146 anticoagulant agent Substances 0.000 description 10
- 229940127219 anticoagulant drug Drugs 0.000 description 10
- 239000011859 microparticle Substances 0.000 description 10
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 description 9
- 239000000126 substance Substances 0.000 description 9
- 238000004904 shortening Methods 0.000 description 8
- 210000001519 tissue Anatomy 0.000 description 8
- 101001067880 Homo sapiens Histone H4 Proteins 0.000 description 7
- 206010020608 Hypercoagulation Diseases 0.000 description 7
- 208000035250 cutaneous malignant susceptibility to 1 melanoma Diseases 0.000 description 7
- 230000003027 hypercoagulation Effects 0.000 description 7
- 201000001441 melanoma Diseases 0.000 description 7
- 108090000765 processed proteins & peptides Proteins 0.000 description 7
- 229920002971 Heparan sulfate Polymers 0.000 description 6
- 108010033040 Histones Proteins 0.000 description 6
- 241000283973 Oryctolagus cuniculus Species 0.000 description 6
- 230000002378 acidificating effect Effects 0.000 description 6
- 210000004556 brain Anatomy 0.000 description 6
- 201000010099 disease Diseases 0.000 description 6
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 6
- 230000000694 effects Effects 0.000 description 6
- 150000003904 phospholipids Chemical class 0.000 description 6
- 230000010118 platelet activation Effects 0.000 description 6
- HTTJABKRGRZYRN-UHFFFAOYSA-N Heparin Chemical compound OC1C(NC(=O)C)C(O)OC(COS(O)(=O)=O)C1OC1C(OS(O)(=O)=O)C(O)C(OC2C(C(OS(O)(=O)=O)C(OC3C(C(O)C(O)C(O3)C(O)=O)OS(O)(=O)=O)C(CO)O2)NS(O)(=O)=O)C(C(O)=O)O1 HTTJABKRGRZYRN-UHFFFAOYSA-N 0.000 description 5
- 108090000190 Thrombin Proteins 0.000 description 5
- 229960002897 heparin Drugs 0.000 description 5
- 229920000669 heparin Polymers 0.000 description 5
- 230000001737 promoting effect Effects 0.000 description 5
- 230000002441 reversible effect Effects 0.000 description 5
- 229960004072 thrombin Drugs 0.000 description 5
- 201000005665 thrombophilia Diseases 0.000 description 5
- 210000004881 tumor cell Anatomy 0.000 description 5
- 108091008102 DNA aptamers Proteins 0.000 description 4
- 241000588724 Escherichia coli Species 0.000 description 4
- 210000003556 vascular endothelial cell Anatomy 0.000 description 4
- 208000035473 Communicable disease Diseases 0.000 description 3
- 102000006947 Histones Human genes 0.000 description 3
- 229940127379 Kallikrein Inhibitors Drugs 0.000 description 3
- 230000015572 biosynthetic process Effects 0.000 description 3
- 238000006243 chemical reaction Methods 0.000 description 3
- 206010012601 diabetes mellitus Diseases 0.000 description 3
- 210000002889 endothelial cell Anatomy 0.000 description 3
- 230000005764 inhibitory process Effects 0.000 description 3
- 210000000265 leukocyte Anatomy 0.000 description 3
- 210000002381 plasma Anatomy 0.000 description 3
- -1 small molecule compound Chemical class 0.000 description 3
- 150000003384 small molecules Chemical class 0.000 description 3
- 230000009261 transgenic effect Effects 0.000 description 3
- 210000003606 umbilical vein Anatomy 0.000 description 3
- WDJHHCAKBRKCLW-IBGZPJMESA-N (2S)-2-[[2-[3-(6-carbamimidoyl-1H-benzimidazol-2-yl)-4-hydroxy-5-(2-hydroxy-5-sulfamoylphenyl)phenyl]acetyl]amino]butanedioic acid Chemical compound N=1C2=CC(C(=N)N)=CC=C2NC=1C(C=1O)=CC(CC(=O)N[C@@H](CC(O)=O)C(O)=O)=CC=1C1=CC(S(N)(=O)=O)=CC=C1O WDJHHCAKBRKCLW-IBGZPJMESA-N 0.000 description 2
- PGOHTUIFYSHAQG-LJSDBVFPSA-N (2S)-6-amino-2-[[(2S)-5-amino-2-[[(2S)-2-[[(2S)-2-[[(2S)-2-[[(2S)-4-amino-2-[[(2S)-2-[[(2S)-2-[[(2S)-2-[[(2S)-2-[[(2S)-5-amino-2-[[(2S)-5-amino-2-[[(2S)-2-[[(2S)-2-[[(2S)-2-[[(2S,3R)-2-[[(2S)-5-amino-2-[[(2S)-2-[[(2S)-2-[[(2S,3R)-2-[[(2S)-2-[[(2S)-2-[[(2S)-2-[[(2S)-2-[[(2S)-5-amino-2-[[(2S)-1-[(2S,3R)-2-[[(2S)-2-[[(2S)-2-[[(2R)-2-[[(2S)-2-[[(2S)-2-[[2-[[(2S)-2-[[(2S)-2-[[(2S)-2-[[(2S)-1-[(2S)-2-[[(2S)-2-[[(2S)-2-[[(2S)-2-amino-4-methylsulfanylbutanoyl]amino]-3-(1H-indol-3-yl)propanoyl]amino]-5-carbamimidamidopentanoyl]amino]propanoyl]pyrrolidine-2-carbonyl]amino]-3-methylbutanoyl]amino]-4-methylpentanoyl]amino]-4-methylpentanoyl]amino]acetyl]amino]-3-hydroxypropanoyl]amino]-4-methylpentanoyl]amino]-3-sulfanylpropanoyl]amino]-4-methylsulfanylbutanoyl]amino]-5-carbamimidamidopentanoyl]amino]-3-hydroxybutanoyl]pyrrolidine-2-carbonyl]amino]-5-oxopentanoyl]amino]-3-hydroxypropanoyl]amino]-3-hydroxypropanoyl]amino]-3-(1H-imidazol-5-yl)propanoyl]amino]-4-methylpentanoyl]amino]-3-hydroxybutanoyl]amino]-3-(1H-indol-3-yl)propanoyl]amino]-5-carbamimidamidopentanoyl]amino]-5-oxopentanoyl]amino]-3-hydroxybutanoyl]amino]-3-hydroxypropanoyl]amino]-3-carboxypropanoyl]amino]-3-hydroxypropanoyl]amino]-5-oxopentanoyl]amino]-5-oxopentanoyl]amino]-3-phenylpropanoyl]amino]-5-carbamimidamidopentanoyl]amino]-3-methylbutanoyl]amino]-4-methylpentanoyl]amino]-4-oxobutanoyl]amino]-5-carbamimidamidopentanoyl]amino]-3-(1H-indol-3-yl)propanoyl]amino]-4-carboxybutanoyl]amino]-5-oxopentanoyl]amino]hexanoic acid Chemical compound CSCC[C@H](N)C(=O)N[C@@H](Cc1c[nH]c2ccccc12)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](C)C(=O)N1CCC[C@H]1C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(C)C)C(=O)NCC(=O)N[C@@H](CO)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CS)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H]([C@@H](C)O)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CO)C(=O)N[C@@H](CO)C(=O)N[C@@H](Cc1cnc[nH]1)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](Cc1c[nH]c2ccccc12)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](Cc1ccccc1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](Cc1c[nH]c2ccccc12)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCCCN)C(O)=O PGOHTUIFYSHAQG-LJSDBVFPSA-N 0.000 description 2
- WYFCZWSWFGJODV-MIANJLSGSA-N 4-[[(1s)-2-[(e)-3-[3-chloro-2-fluoro-6-(tetrazol-1-yl)phenyl]prop-2-enoyl]-5-(4-methyl-2-oxopiperazin-1-yl)-3,4-dihydro-1h-isoquinoline-1-carbonyl]amino]benzoic acid Chemical compound O=C1CN(C)CCN1C1=CC=CC2=C1CCN(C(=O)\C=C\C=1C(=CC=C(Cl)C=1F)N1N=NN=C1)[C@@H]2C(=O)NC1=CC=C(C(O)=O)C=C1 WYFCZWSWFGJODV-MIANJLSGSA-N 0.000 description 2
- 108010039627 Aprotinin Proteins 0.000 description 2
- 108010094028 Prothrombin Proteins 0.000 description 2
- 102100027378 Prothrombin Human genes 0.000 description 2
- 108091008103 RNA aptamers Proteins 0.000 description 2
- 108090000040 Russellysin Proteins 0.000 description 2
- 229960004405 aprotinin Drugs 0.000 description 2
- 125000002619 bicyclic group Chemical group 0.000 description 2
- 230000001413 cellular effect Effects 0.000 description 2
- 230000003111 delayed effect Effects 0.000 description 2
- 229940088598 enzyme Drugs 0.000 description 2
- 238000011534 incubation Methods 0.000 description 2
- ZPNFWUPYTFPOJU-LPYSRVMUSA-N iniprol Chemical compound C([C@H]1C(=O)NCC(=O)NCC(=O)N[C@H]2CSSC[C@H]3C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](C)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@H](C(N[C@H](C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC=4C=CC(O)=CC=4)C(=O)N[C@@H](CC=4C=CC=CC=4)C(=O)N[C@@H](CC=4C=CC(O)=CC=4)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](C)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](C)C(=O)NCC(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CSSC[C@H](NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](C)NC(=O)[C@H](CO)NC(=O)[C@H](CCCCN)NC(=O)[C@H](CC=4C=CC=CC=4)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CCCCN)NC(=O)[C@H](C)NC(=O)[C@H](CCCNC(N)=N)NC2=O)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CSSC[C@H](NC(=O)[C@H](CC=2C=CC=CC=2)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H]2N(CCC2)C(=O)[C@@H](N)CCCNC(N)=N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(O)=O)C(=O)N2[C@@H](CCC2)C(=O)N2[C@@H](CCC2)C(=O)N[C@@H](CC=2C=CC(O)=CC=2)C(=O)N[C@@H]([C@@H](C)O)C(=O)NCC(=O)N2[C@@H](CCC2)C(=O)N3)C(=O)NCC(=O)NCC(=O)N[C@@H](C)C(O)=O)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@H](C(=O)N[C@@H](CC=2C=CC=CC=2)C(=O)N[C@H](C(=O)N1)C(C)C)[C@@H](C)O)[C@@H](C)CC)=O)[C@@H](C)CC)C1=CC=C(O)C=C1 ZPNFWUPYTFPOJU-LPYSRVMUSA-N 0.000 description 2
- 210000000440 neutrophil Anatomy 0.000 description 2
- 230000036961 partial effect Effects 0.000 description 2
- 210000004623 platelet-rich plasma Anatomy 0.000 description 2
- 230000002035 prolonged effect Effects 0.000 description 2
- 229940039716 prothrombin Drugs 0.000 description 2
- 230000002829 reductive effect Effects 0.000 description 2
- 230000001732 thrombotic effect Effects 0.000 description 2
- 108020004491 Antisense DNA Proteins 0.000 description 1
- 108091023037 Aptamer Proteins 0.000 description 1
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 description 1
- 102100026735 Coagulation factor VIII Human genes 0.000 description 1
- 108020004414 DNA Proteins 0.000 description 1
- 108010080805 Factor XIa Proteins 0.000 description 1
- 102000003886 Glycoproteins Human genes 0.000 description 1
- 108090000288 Glycoproteins Proteins 0.000 description 1
- 208000031220 Hemophilia Diseases 0.000 description 1
- 208000009292 Hemophilia A Diseases 0.000 description 1
- 208000032843 Hemorrhage Diseases 0.000 description 1
- 108010022901 Heparin Lyase Proteins 0.000 description 1
- 101000911390 Homo sapiens Coagulation factor VIII Proteins 0.000 description 1
- 102000001399 Kallikrein Human genes 0.000 description 1
- 108060005987 Kallikrein Proteins 0.000 description 1
- 102100037265 Podoplanin Human genes 0.000 description 1
- 101710118150 Podoplanin Proteins 0.000 description 1
- 102000007327 Protamines Human genes 0.000 description 1
- 108010007568 Protamines Proteins 0.000 description 1
- 102000008233 Toll-Like Receptor 4 Human genes 0.000 description 1
- 108010060804 Toll-Like Receptor 4 Proteins 0.000 description 1
- 206010047249 Venous thrombosis Diseases 0.000 description 1
- 239000007825 activation reagent Substances 0.000 description 1
- WYTGDNHDOZPMIW-RCBQFDQVSA-N alstonine Natural products C1=CC2=C3C=CC=CC3=NC2=C2N1C[C@H]1[C@H](C)OC=C(C(=O)OC)[C@H]1C2 WYTGDNHDOZPMIW-RCBQFDQVSA-N 0.000 description 1
- 230000033115 angiogenesis Effects 0.000 description 1
- 230000010100 anticoagulation Effects 0.000 description 1
- 239000002246 antineoplastic agent Substances 0.000 description 1
- 239000003816 antisense DNA Substances 0.000 description 1
- 230000000740 bleeding effect Effects 0.000 description 1
- 229920005549 butyl rubber Polymers 0.000 description 1
- 239000011575 calcium Substances 0.000 description 1
- 229910052791 calcium Inorganic materials 0.000 description 1
- 238000012512 characterization method Methods 0.000 description 1
- 208000037976 chronic inflammation Diseases 0.000 description 1
- 208000037893 chronic inflammatory disorder Diseases 0.000 description 1
- 229940126051 coagulation factor XIa Drugs 0.000 description 1
- 230000000052 comparative effect Effects 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 230000003511 endothelial effect Effects 0.000 description 1
- 210000003989 endothelium vascular Anatomy 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- 210000003743 erythrocyte Anatomy 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 230000023597 hemostasis Effects 0.000 description 1
- 230000028993 immune response Effects 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 239000003055 low molecular weight heparin Substances 0.000 description 1
- 229940127215 low-molecular weight heparin Drugs 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 210000001616 monocyte Anatomy 0.000 description 1
- 230000001575 pathological effect Effects 0.000 description 1
- 230000007170 pathology Effects 0.000 description 1
- 230000020971 positive regulation of blood coagulation Effects 0.000 description 1
- 102000004196 processed proteins & peptides Human genes 0.000 description 1
- 229940048914 protamine Drugs 0.000 description 1
- 229940121649 protein inhibitor Drugs 0.000 description 1
- 239000012268 protein inhibitor Substances 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 229940124597 therapeutic agent Drugs 0.000 description 1
- 230000002792 vascular Effects 0.000 description 1
- 239000002525 vasculotropin inhibitor Substances 0.000 description 1
- 239000002821 viper venom Substances 0.000 description 1
Images
Classifications
-
- 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
-
- 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
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
- G01N2333/4609—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates from reptiles
- G01N2333/4613—Snake venom
- G01N2333/4616—Snake venom from Russell's viper
-
- 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
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
- G01N2333/4609—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates from reptiles
- G01N2333/4613—Snake venom
- G01N2333/4633—Snake venom from Echis carinatus; Ecarin
Definitions
- the present invention relates to a method for evaluating blood coagulation ability and a method for testing the risk of thrombosis.
- thrombosis cancer-related thrombosis
- thrombootic tendencies due to cancer, diabetes and infectious diseases are associated with microparticles derived from vascular endothelial cells and platelets, acidic phospholipids on the surface of activated platelets, and tissue factors in the blood, but especially cancer-related thrombosis.
- the main cause of thrombosis is the activation of extrinsic coagulation by the expression of tissue factors in the surface of tumor cells and in tumor cells and tissue factors in monospheres and vascular endothelial cells due to an immune response to the tumor cells. It is considered to be one (Non-Patent Document 1).
- blood coagulation tests such as APTT (activated partial thromboplastin time), PT (prothrombin time), ROTEM (trademark: rotational thromboelastometry), and TEG (thromboelastography) show deficiency of blood coagulation factors such as hemophilia and perioperative period.
- the main purpose is to evaluate the blood-stopping function (proneness of bleeding), and it is not used to evaluate the degree of shortening of coagulation time caused by the tendency of blood clots caused by cancer or the like.
- An object of the present invention is to provide a method capable of appropriately evaluating a pathological condition of hypercoagulation caused by extrinsic coagulation, microparticles, platelet activation, etc. in a blood coagulation test.
- the present inventor has made diligent studies to solve the above problems.
- the blood coagulation ability was evaluated by adding a contact factor inhibitor (one or more of FXI inhibitor, FXII inhibitor, calicrane inhibitor, etc.) and an inhibitor against Tissue Factor Pathway Inhibitor (TFPI) to the blood sample.
- a contact factor inhibitor one or more of FXI inhibitor, FXII inhibitor, calicrane inhibitor, etc.
- TFPI Tissue Factor Pathway Inhibitor
- the present invention is characterized in that blood coagulation ability is measured by adding an inhibitor (TFPI inhibitor) to a contact factor inhibitor and a tissue factor pathway inhibitor (TFPI) to a blood sample.
- TFPI inhibitor an inhibitor
- TFPI tissue factor pathway inhibitor
- the present invention is also a method for evaluating an intrinsic blood coagulation ability in vitro, which comprises adding both a contact factor inhibitor and an extrinsic blood coagulation inhibitor to a blood sample to measure the blood coagulation ability. I will provide a.
- the present invention also provides reagents for measuring extrinsic blood coagulation in vitro, including contact factor inhibitors and TFPI inhibitors.
- the present invention also provides reagents for measuring endogenous blood coagulation ability in vitro, including a contact factor inhibitor and an extrinsic blood coagulation inhibitor.
- the contact factor inhibitor can be an FXI inhibitor, an FXII inhibitor and / or a kallikrein inhibitor. Measurements can be made by ROTEM and / or TEG.
- the blood sample can be a blood sample of a cancer patient or a patient suspected of having cancer.
- a contact factor inhibitor FXI inhibitor, FXII inhibitor and / or kallikrein inhibitor, etc.
- a TFPI inhibitor a trace amount of tissue factor, microparticles, platelet activation, etc. It is possible to analyze the blood coagulation ability that sensitively reflects the hypercoagulable state caused by the above. Furthermore, by comparing the blood coagulation time with the addition of contact factor inhibitors and TFPI inhibitors with the blood coagulation time with the addition of extrinsic blood coagulation inhibitors (FVII inhibitors, tissue factor inhibitors, etc.), blood coagulation is enhanced.
- FVII inhibitors, tissue factor inhibitors, etc. extrinsic blood coagulation inhibitors
- tissue factor in the blood or other factors eg, acidic phospholipids such as microparticles and activated platelets.
- tissue factor in the blood or other factors eg, acidic phospholipids such as microparticles and activated platelets.
- FIG. 1 The figure which shows the result of Example 1.
- FIG. 2 The figure which shows the result of Example 2.
- FIG. 3 The figure which shows the result of Example 3.
- the method for analyzing blood coagulation of the present invention is It is characterized in that blood coagulation ability is measured by adding an inhibitor against a contact factor inhibitor and a tissue factor pathway inhibitor (TFPI) to a blood sample.
- TFPI tissue factor pathway inhibitor
- the blood sample is preferably a whole blood sample, and may be a blood sample that has been anticoagulated with citric acid or the like. In that case, the anticoagulant treatment can be canceled with calcium or the like as described later, and the blood coagulation reaction can be started.
- the blood sample is preferably a blood sample of a patient having a disease such as cancer or a patient suspected of having a disease such as cancer.
- the contact factor inhibitor FXI (blood coagulation factor 11) inhibitor, FXII (blood coagulation factor 12) inhibitor, kallikrein inhibitor and the like are used, and two or more of these inhibitors are used. Is preferably mixed and added. In particular, by combining an FXII inhibitor and a kallikrein inhibitor, it is possible to satisfactorily evaluate the degree of shortening due to the enhancement of blood coagulation.
- the inhibitor of the contact factor a small molecule compound, a peptide / protein, or the like can be used as the inhibitor of the contact factor.
- the FXI inhibitor is not particularly limited as long as it is a substance having FXI inhibitory activity, but BMS-962212 (J Med Chem. 2017 Dec 14; 60 (23): 9703-9723) and FXI inhibitor aptamer (Sci Rep. 2017 May). 18; 7 (1): 2102. Selection and characterization of a DNA aptamer inhibiting coagulation factor XIa) and the like can be used.
- the FXI inhibitor is preferably added at a final concentration of 100 nM to 100 ⁇ M.
- the FXII inhibitor is not particularly limited as long as it is a substance having FXII inhibitory activity, but is a corntrypsin inhibitor (CTI) or a peptide inhibitor (for example, bicyclic peptide 61 (BP-61) or bicyclic peptide 73 (BP-73); J Med Chem. 2017 Feb 9; 60 (3): 1151-1158) etc. can be used.
- CTI corntrypsin inhibitor
- a peptide inhibitor for example, bicyclic peptide 61 (BP-61) or bicyclic peptide 73 (BP-73); J Med Chem. 2017 Feb 9; 60 (3): 1151-1158
- the FXII inhibitor is preferably added at a final concentration of 10 ⁇ g / mL to 200 ⁇ g / mL.
- BP61 and BP73 it is preferable to add them at a final concentration of 1 to 100 ⁇ g / mL.
- the kallikrein inhibitor is not particularly limited, but a synthetic kallikrein inhibitor (for example, PKSI-527 (CAS No .: 128837-71-8) Abcam) or aprotinin is desirable. If it is PKSI-527, it is preferable to add it at a final concentration of 1 ⁇ M to 1 mM. If it is aprotinin, it is preferable to add it at a final concentration of 10 to 1000 KIU / mL.
- the addition of both the FXII inhibitor and the kallikrein inhibitor sufficiently suppresses the blood coagulation reaction caused by the activation of the contact factor in whole blood, and it is possible to identify the enhancement of extrinsic coagulation, which is preferable.
- an inhibitor of the activation of the unactivated coagulation factor FXII, FXI
- an inhibitor of the enzyme activity of the activated coagulation factor FXIIa, FXIa
- TFPI inhibitor a small molecule and a peptide / protein inhibitor can be used, and is not particularly limited as long as it is a substance capable of inhibiting the function of TFPI, but specifically, a TFPI inhibitor of a small molecule peptide (for example, , Compound 3; J Biol Chem. 2014 Jan 17; 289 (3): 1732-41).
- a TFPI inhibitor of a small molecule peptide for example, Compound 3; J Biol Chem. 2014 Jan 17; 289 (3): 1732-41).
- the TFPI inhibitor is added at a final concentration of 1 ⁇ g / mL to 200 ⁇ g / mL.
- compound 3 Ac-FQSKpNVHVDGYFERL-Aib-AKL-NH 2
- RNA aptamer inhibitors can also be used as other TFPI inhibitors.
- BAX499 J Thromb Haemost. 2012 Aug; 10 (8): 1581-90.
- BAX499 J Thromb Haemost. 2012 Aug; 10 (8): 1581-90.
- BAX499 J Thromb Haemost. 2012 Aug; 10 (8): 1581-90.
- BAX499 it is added at a final concentration of 1 nM to 1000 nM.
- an anti-TFPI antibody having a TFPI inhibitory activity can also be used.
- the method for analyzing blood coagulation is not particularly limited, and is used for whole blood such as ROTEM (Rotational Thromboelastometry), TEG (Thromboelastography), ClotPro (Hemonetics) SONOCLOT (Scienko), ACTAS (Fujimori Kogyo Co., Ltd .: WO2018 / 043420).
- a device capable of evaluating blood coagulation is desirable. By using whole blood as a sample, it becomes possible to satisfactorily evaluate the hypercoagulation caused by cellular components (leukocytes / erythrocytes).
- a contact factor inhibitor and a TFPI inhibitor are added to the blood of a healthy person and the blood of a patient with hypercoagulation in advance, and a blood coagulation test is performed to calculate the coagulation time and coagulation waveform. By looking at which one is closer to, it is possible to investigate whether a hypercoagulable state exists.
- the results of the blood coagulation test conducted by adding the contact factor inhibitor and the TFPI inhibitor to the blood sample are obtained when the TFPI inhibitor is not added (in the absence of the TFPI inhibitor), that is, the contact factor inhibition is performed on the blood sample.
- the hypercoagulation state may be mainly due to tissue factors in the blood or other (for example, microparticles or platelet activation). It is also possible to evaluate whether it is caused.
- FVII inhibitors also called FVIIa inhibitors
- tissue factor inhibitors tissue factor inhibitors
- inactivity It is also possible to analyze the factors of the enhancement of blood coagulation by comparing with the results of the endogenous blood coagulation test conducted in the presence of FVIIa, etc.).
- extrinsic blood coagulation inhibitor used in the endogenous blood coagulation test examples include FVII (blood coagulation factor 7) inhibitor, tissue factor inhibitor, inactivated FVIIa and the like.
- FVII inhibitor an inhibitor for the activation of FVII or an inhibitor for the enzymatic activity of FVIIa can be used.
- FVII inhibitors include synthetic peptides (A-183X; J Biol Chem. 2003 Jun 13; 278 (24): 21823-30) and synthetic small molecules (PCI-27483; 2019; 96 (4): 217-222). .Doi: 10.1159 / 000495988. Epub 2019 Mar 7.) and inactivated FVIIa (Eur J Vasc Endovasc Sur. 1998 Jun; 15 (6): 515-20. Doi: 10.1016 / s1078-5884 (98) 80112- 3.) and antibodies against tissue factors can be used.
- FVII inhibitor for example, A183-X can be added at a final concentration of 1 nM to 1 ⁇ M.
- PCI-27483 can be added at a final concentration of 10 nM to 10 ⁇ M.
- the inactivated FVIIa can be added at a final concentration of 10 ng / mL to 100 ⁇ g / mL.
- blood coagulation it is preferable to use whole blood for the analysis of blood coagulation performed by adding an extrinsic blood coagulation inhibitor.
- blood coagulation can be analyzed by adding an extrinsic blood coagulation inhibitor and calcium chloride.
- contact factor inhibitors FXI inhibitors, FXII inhibitors, kallikrein inhibitors
- FXI inhibitors a contact factor inhibitor
- FXII inhibitors a contact factor inhibitor
- kallikrein inhibitors a contact factor inhibitor
- the above-mentioned FXI (blood coagulation factor 11) inhibitor, FXII (blood coagulation factor 12) inhibitor, kallikrein inhibitor and the like are used, and the preferred concentration range thereof is also the same.
- a trace amount of the blood coagulation activator may be further added as an activation reagent for the endogenous blood coagulation.
- Trace amounts of blood coagulation activators include activated coagulation factors (FXa and thrombin) and FX (blood coagulation factor 10) activating enzymes (RVV-X; J Biol Chem) derived from Russel's viper venom (RVV). Using FX activators such as 1994 Apr 8; 269 (14): 10644-50.) And prothrombin activators such as Ecarin (Am J Hematol. 2020 Jul; 95 (7): 863-869.). Can be done.
- RVV-X In the case of RVV-X, it can be used at a final concentration of 0.1 ng / mL to 1000 ng / mL. In the case of ecarin, it can be used at a final concentration of 0.1 mU to 100 mU.
- These blood coagulation activators are not inhibited by contact factor inhibitors or extrinsic coagulation inhibitors. Further, it is desirable that the blood coagulation activator is added to the blood of a healthy person so that the blood coagulation time is 20 to 60 minutes. If the blood coagulation time in a healthy person is 20 minutes or more, it is possible to detect a shortening of the blood coagulation time in the blood of a patient with hypercoagulation.
- an extrinsic blood coagulation inhibitor and a kallikrein inhibitor may be combined.
- a kallikrein inhibitor is used alone as a contact factor inhibitor in addition to an extrinsic blood coagulation inhibitor, FXII is not directly inhibited, but FXIIa production is suppressed by suppressing the mutual activation of FXIIa and kallikrein. Is suppressed, so that activation in healthy subjects is extended to 20 minutes or more, and it is possible to detect a tendency for shortening of coagulation time in patients with hypercoagulability.
- tumor cells may directly express tissue factor or emit microparticles that express tissue factor.
- certain tumor cells express a glycoprotein called podoplanin, which directly activates platelets.
- podoplanin which directly activates platelets.
- the acidic phospholipids on the surface of activated platelets promote blood coagulation. It releases microparticles derived from vascular endothelium due to vascular endothelial damage caused by treatment with various anticancer agents.
- VEGF inhibitor angiogenesis inhibition
- the blood sample may be measured for blood coagulation by adding a contact factor inhibitor and a TFPI inhibitor immediately after collection, but it may be difficult to carry out a blood coagulation test immediately after blood collection. Therefore, the blood is collected in a blood collection container containing a reversible anticoagulant treatment agent, and at the start of measurement, a reagent containing an anticoagulant treatment release agent, a contact factor inhibitor and a TFPI inhibitor contained in the blood collection container is added, and blood coagulation is performed. It is desirable to analyze. For example, when blood is collected in a container containing sodium citrate as a reversible anticoagulant, calcium chloride, a contact factor inhibitor, and a TFPI inhibitor are added to analyze blood coagulation.
- Such a combination of a reversible anticoagulant and a release agent includes heparin and a heparin neutralizer (hepalinase, polybrain, protamine, etc.), thrombin-inhibiting DNA aptamer, and thrombin-inhibiting DNA aptamer antisense DNA. can give.
- citric acid When citric acid is used as a reversible anticoagulant, it is convenient to use a commercially available blood collection tube (or blood collection container) containing sodium citrate. However, when a vacuum blood collection tube containing sodium citrate having a rubber stopper made of butyl rubber is used, blood coagulation may be activated by contact with the rubber stopper and the coagulation time may be shortened. Therefore, it is preferable to use a blood collection tube containing citric acid that does not use a rubber stopper. For example, as a blood collection tube that does not use a rubber stopper, a Venoject blood collection tube (Terumo Corporation) sealed with a laminated film can be used.
- a Venoject blood collection tube Teumo Corporation
- Anticoagulants such as small amounts of heparin-like substances (heparin, low molecular weight heparin, pentasaccharides, heparan sulfate) may be added (WO2017 / 119508).
- heparin-like substances heparin, low molecular weight heparin, pentasaccharides, heparan sulfate
- the activation of the contact factor by the rubber stopper is suppressed by using the blood collection tube to which sodium citrate and an FXII inhibitor have been previously added to the blood collection tube. It is possible.
- the present invention also provides a reagent kit for in vitro measurement of extrinsic blood coagulation ability, which comprises a contact factor inhibitor and a TFPI inhibitor.
- the types of contact factor inhibitors and TFPI inhibitors are as described above, which are diluted at the time of use and added at a final concentration suitable for measuring blood coagulation ability (preferable final concentration range is as described above).
- the present invention also provides a reagent kit for measuring endogenous blood coagulation ability in vitro, which comprises a contact factor inhibitor and an extrinsic blood coagulation inhibitor.
- contact factor inhibitors and extrinsic blood coagulation inhibitors are as described above, and these are diluted at the time of use and added at a final concentration suitable for measuring blood coagulation ability (preferable final concentration range is as described above). Will be done.
- the blood sample of this example is collected from a healthy human.
- Example 1 ROTEM (Instrumentation Laboratory (IL)) was used for blood coagulation analysis. Blood was collected using a blood collection tube (5 mL) containing 3.2% sodium citrate manufactured by Terumo Corporation. This blood collection tube is sealed with a laminated film and no rubber stopper is used. The following reagents were added to 300 ⁇ L of whole blood and the blood coagulation waveform was analyzed.
- IL Intra Laboratory
- the results are shown in FIG.
- the coagulation time (CT) of (1) to which only calcium chloride was added was 1336 seconds, whereas the CT of (2) to which an inhibitor for calcium chloride and TFPI was added was shortened to 801 seconds. Furthermore, in (3) and (4), CT was shortened to 452 and 371 seconds by the addition of a trace amount of tissue thromboplastin. The blood coagulation promoting effect of the TFPI inhibitor in the presence of trace tissue thromboplastin was shown.
- Example 2 ROTEM (IL) was used for blood coagulation analysis. Blood was collected using a blood collection tube (5 mL) containing 3.2% sodium citrate manufactured by Terumo Corporation. This blood collection tube is sealed with a laminated film and no rubber stopper is used. The following reagents were added to 300 ⁇ L of whole blood and the blood coagulation waveform was analyzed.
- the coagulation time (CT) of (2) was extended to 2245 seconds. Furthermore, the CT of (3) to which the trace tissue thromboplastin was added was shortened to 1304 seconds, and the CT of (4) to which the trace tissue thromboplastin and the TFPI inhibitor was added was shortened to 489 seconds.
- the addition of the FXII inhibitor and the kallikrein inhibitor extended the coagulation time when the trace tissue thromboplastin was not added, and the addition of the trace tissue thromboplastin and the TFPI inhibitor significantly shortened the coagulation time. That is, the presence of FXII inhibitor, kallikrein inhibitor, and TFPI inhibitor made it possible to evaluate the effect of microtissue thromboplastin on promoting thrombus formation.
- Example 2 Compared with Example 1, in Example 2 to which the contact factor inhibitor and the kallikrein inhibitor were added, blood coagulation was delayed in the absence of the trace tissue factor, but when the trace tissue thromboplastin and the TFPI inhibitor were added, the blood coagulation was delayed.
- the blood coagulation time has been significantly reduced. That is, by adding a contact factor inhibitor and a TFPI inhibitor, it becomes easy to evaluate the thrombotic property of the trace tissue thromboplastin.
- Example 3 ROTEM (IL) was used for blood coagulation analysis. Blood was collected using a blood collection tube (2 mL) containing 3.2% sodium citrate manufactured by Becton Dickinson. A rubber stopper is used for this blood collection tube. The following reagents were added to whole blood and the blood coagulation waveform was analyzed.
- Example 4 ROTEM (IL) was used for blood coagulation analysis. Blood was collected using a blood collection tube containing 3.2% sodium citrate manufactured by Terumo Corporation. This blood collection tube is sealed with a laminated film and no rubber stopper is used. The following reagents were added to 300 ⁇ L of whole blood and the blood coagulation waveform was analyzed.
- the addition of both (3) increased the degree of prolongation of the coagulation time (CT).
- CT degree of prolongation of the coagulation time
- the coagulation times (5), (6) and (7) when a small amount of Dadeinobin was added were equivalent to the CT values of around 600 seconds. Therefore, by adding both the FXII inhibitor and the kallikrein inhibitor, the reduction in the coagulation time due to the addition of the trace tissue factor becomes large, and it becomes easy to identify the hypercoagulation.
- the reduction in the coagulation time of the non-additional contact factor inhibitors (4) and (8) was even smaller, indicating that it is not suitable for evaluation of hypercoagulation.
- Example 5 ROTEM (IL) was used for blood coagulation analysis. Blood was collected using a blood collection tube (5 mL) containing 3.2% sodium citrate manufactured by Terumo Corporation. This blood collection tube is sealed with a laminated film and no rubber stopper is used. The following reagents were added to 300 ⁇ L of whole blood and the blood coagulation waveform was analyzed.
- exosomes derived from vascular endothelial cells and cancer cells promote blood coagulation by having tissue factors and acidic phospholipids on the surface and cause thrombosis. It is also known that histones promote thrombus formation by activating platelets.
- Example 6 ROTEM (IL) was used for blood coagulation analysis. Blood was collected using a blood collection tube (5 mL) containing 3.2% sodium citrate manufactured by Terumo Corporation. This blood collection tube is sealed with a laminated film and no rubber stopper is used. Escherichia coli-derived lipopolysaccharide (LPS; manufactured by Merk) (final concentration 1 ⁇ g / mL) or phorbol myristate acetate (PMA; manufactured by Merk) (final concentration 50 nM) or human histone H4 (manufactured by ABCAM) in 300 ⁇ L of whole blood. After adding (final concentration 2 ⁇ M) and incubating at 37 ° C.
- LPS Escherichia coli-derived lipopolysaccharide
- PMA phorbol myristate acetate
- human histone H4 manufactured by ABCAM
- Reagents for analyzing blood coagulation by adding (a) (b) (c) after adding histone H4 to blood and incubating at 37 ° C for 4 hours (10) Startem reagent (IL) (20 ⁇ L) and human histone H4 (manufactured by ABCAM) (final concentration 2 ⁇ M) (11) Calcium chloride (final concentration 12 mM), CTI (final concentration 20 ⁇ g / mL), PKSI-527 (final concentration 40 ⁇ M) and human histone H4 (manufactured by ABCAM) (final concentration 2 ⁇ M) (12) Calcium chloride (final concentration 12 mM), CTI (final concentration 20 ⁇ g / mL), PKSI-527 (final concentration 40 ⁇ M), TFPI inhibitor (compound3) (final concentration 50 ⁇ M), and human histone H4 (manufactured by ABCAM) ( Final concentration 2 ⁇ M)
- LPS is known to promote and renew blood coagulation by expressing tissue factor on the monocyte surface via Toll-like receptor 4. It is also known that PMA acts on neutrophils and promotes thrombus formation by inducing extracellular traps (NETs) of neutrophils.
- NETs extracellular traps
- ROTEM ROTEM
- Plasma and platelet-rich plasma were obtained by centrifuging the obtained blood at 3000 rpm (15 minutes) and 2400 rpm (7 minutes).
- the same reagents (LPS, PMA, histone) as in Example 6 are added to these plasmas, and after incubation at 37 ° C. for 4 hours, the same reagents as in (1) to (12) of Example 6 are added. Blood coagulation waveforms were analyzed.
- Example 7 ROTEM (IL) was used for blood coagulation analysis. Blood was collected using a blood collection tube (5 mL) containing 3.2% sodium citrate manufactured by Terumo Corporation. This blood collection tube is sealed with a laminated film and no rubber stopper is used. The following reagents were added to 300 ⁇ L of whole blood, and the blood coagulation waveform was analyzed.
- CTI CTI, kallikrein inhibitor, FVIIa inhibitor, and a small amount are used for the purpose of evaluating the enhanced state of intrinsic blood coagulation in comparison with the evaluation of the enhanced state of extrinsic blood coagulation (2).
- Blood coagulation was analyzed when RVV-FX or Ecarin was added (4) and (5), and when kallikrein inhibitor and FVIIa inhibitor were added (6).
- RVV RVV activator
- ecarin prothrombin activator
- Thrombin produced by blood coagulation with trace amounts of ecarin or RVV-FX amplifies endogenous blood coagulation by activating FXI, FVIII, FV.
- blood coagulation time due to activation of endogenous blood coagulation originating from FIXa produced by contact factor activation in a partial contact factor inhibition environment by adding an FVIIa inhibitor and a kallikrein inhibitor. Is being evaluated.
- the A375-derived exosome contains a large amount of tissue factor.
- the shortening of blood coagulation time was limited. This is because the shortening of blood coagulation by A375-derived exosomes is mainly due to the activation of extrinsic blood coagulation caused by tissue factors present on the surface of exosomes, and the activity of endogenous blood coagulation caused by acidic phospholipids and the like. The conversion is considered to be limited and minor.
- Example 8 ROTEM (IL) was used for blood coagulation analysis. Blood was collected using a blood collection tube (5 mL) containing 3.2% sodium citrate manufactured by Terumo Corporation. This blood collection tube is sealed with a laminated film and no rubber stopper is used. Additive-free or LPS (final concentration 1 ⁇ g / mL) and PMA (final concentration 50 nM) were added to 300 ⁇ L of whole blood and incubated at 37 ° C. for 4 hours, and then in Examples 02 (1) to (6). Reagents were added and blood coagulation time was analyzed.
- LPS final concentration 1 ⁇ g / mL
- PMA final concentration 50 nM
- Example 9 Clotpro (Haemonetics) was used for blood coagulation analysis. Blood was collected using a blood collection tube (5 mL) containing 3.2% sodium citrate manufactured by Terumo Corporation. This blood collection tube is sealed with a laminated film and no rubber stopper is used. After adding the reagents (1) to (6) or less to the collected whole blood (340 ⁇ L), any of the reagents (7) to (12) was added and the blood coagulation time was analyzed.
- Clotpro (Haemonetics) was used for blood coagulation analysis. Blood was collected using a blood collection tube (5 mL) containing 3.2% sodium citrate manufactured by Terumo Corporation. This blood collection tube is sealed with a laminated film and no rubber stopper is used. The following reagents were added to whole blood and the blood coagulation waveform was analyzed.
- Example 11 ROTEM (IL) was used for blood coagulation analysis. Blood was collected using a blood collection tube (5 mL) containing 3.2% sodium citrate manufactured by Terumo Corporation. This blood collection tube is sealed with a laminated film and no rubber stopper is used. The following reagents were added to whole blood and the blood coagulation waveform was analyzed.
- coagulation promotion in the disease is due to factors other than tissue factor, such as microparticles. If it can be predicted that it is due to factors other than the extrinsic system such as platelet activation and there is a clear difference in the ROTEM waveforms when TFPI is added and when it is not added, the promotion of coagulation in the disease is due to the increase / enhancement of tissue factor. Can be predicted.
- extrinsic coagulation with FXII and / or kallikrein inhibitors and TFPI inhibitors added
- endogenous blood coagulation including factor XII and FVII inhibitors
- Factors that promote blood coagulation can be analyzed.
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Hematology (AREA)
- Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Biomedical Technology (AREA)
- Chemical & Material Sciences (AREA)
- Immunology (AREA)
- Urology & Nephrology (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Cell Biology (AREA)
- Food Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
Description
一方、APTT(活性化部分トロンボプラスチン時間)、PT(プロトロンビン時間)、ROTEM(商標:rotational thromboelastometry)、TEG(thromboelastography)などの血液凝固検査は、血友病などの血液凝固因子の欠損や周術期の止血機能(出血傾向)の評価を主な目的としており、癌等に起因する血栓傾向によって生じる凝固時間の短縮度合いを評価するのには使用されていない。
本発明は、血液凝固試験において、外因系凝固、マイクロパーティクル、血小板活性化などに起因する血液凝固亢進病態を適切に評価できる方法を提供することを課題とする。
本発明はまた、血液試料に、接触因子阻害剤と外因系血液凝固阻害剤の両方を添加して、血液凝固能の測定を行うことを特徴とする、インビトロにおける内因系血液凝固能の評価方法を提供する。
本発明はまた、接触因子阻害剤およびTFPI阻害剤を含む、インビトロにおける外因系血液凝固能測定のための試薬を提供する。
本発明はまた、接触因子阻害剤と外因系血液凝固阻害剤を含む、インビトロにおける内因系血液凝固能測定のための試薬を提供する。
ここで、接触因子阻害剤はFXI阻害剤、FXII阻害剤および/またはカリクレイン阻害剤であることができる。測定はROTEMおよび/またはTEGによって行うことができる。
また、血液試料は癌患者又は癌が疑われる患者の血液試料であることができる。
さらに、接触因子阻害剤とTFPI阻害剤を添加した血液凝固時間と外因系血液凝固阻害剤(FVII阻害剤、組織因子阻害剤等)を添加した血液凝固時間を対比することで、血液凝固の亢進状態が血中の微量の組織因子に起因するか、その他の要因(例えば、マイクロパーティクルや活性化血小板等の酸性リン脂質)に起因するかを解析することが可能となる。
血液試料に、接触因子阻害剤と外因系血液凝固阻害剤の両方を添加して、血液凝固能の測定を行うことで、血中の組織因子の影響を除外し、マイクロパーティクルや活性化血小板等の酸性リン脂質等に起因する内因系血液凝固の亢進状態を評価することが可能である。
血液試料に、接触因子阻害剤および組織因子経路阻害剤(TFPI)に対する阻害剤を添加して血液凝固能の測定を行うことを特徴とする。
血液試料は、癌などの疾患患者又は癌などの疾患が疑われる患者の血液試料であることが好ましい。
特に、FXII阻害剤とカリクレイン阻害剤を組み合わせることで、血液凝固の亢進による短縮度合いを良好に評価することが可能となる。
接触因子の阻害剤は、低分子合成物やペプチド/蛋白などを用いることができる。
BP61及びBP73であれば、終濃度1~100μg/mLで添加されることが好ましい。
RNAアプタマー阻害剤としてBAX499(J Thromb Haemost. 2012 Aug;10(8):1581-90. )が挙げられる。例えば、BAX499であれば、1nM~1000nMの終濃度で添加される。
また、TFPI阻害活性を有する抗TFPI抗体などを使用することもできる。
検体に全血を用いることで、細胞成分(白血球・赤血球)に起因する血液凝固亢進を良好に評価することが可能となる。
または、PCI-27483であれば、10nM~10μMの終濃度で添加されることができる。
又は、不活性化FVIIaであれば、10ng/mL~100μg/mLの終濃度で添加されることができる。
接触因子阻害剤としては、上述したFXI(血液凝固第11因子)阻害剤、FXII(血液凝固第12因子)阻害剤、カリクレイン阻害剤などが用いられ、その好ましい濃度範囲も同様である。
RVV-Xの場合には、0.1ng/mL~1000ng/mLの終濃度で使用することが可能である。
エカリンの場合には、0.1mU~100mUの終濃度で使用することが可能である。
これら血液凝固活性剤は、接触因子阻害剤や外因系凝固阻害剤による阻害を受けない。
また、血液凝固活性化剤は、健常者の血液において血液凝固時間が20分~60分になるように添加されることが望ましい。健常者における血液凝固時間が20分以上であれば、血液凝固亢進患者の血液における血液凝固時間の短縮を検出することが可能である。
外因系血液凝固阻害剤に加え接触因子阻害剤としてカリクレイン阻害剤を単独で用いた場合には、FXIIは直接的には阻害されないが、FXIIaとカリクレインの相互活性化が抑制されることでFXIIa産生が抑制されるため、健常者における活性化が20分以上にまで延長され、血液凝固亢進の患者における凝固時間の短縮傾向を検出することが可能である。
例えば、腫瘍細胞が直接的に組織因子を発現する、または組織因子を発現したマイクロパーティクルを放出することがある。また、ある種の腫瘍細胞はポドプラニンという糖タンパクを発現しており、血小板を直接的に活性化する。活性化した血小板表面の酸性リン脂質により血液凝固を促進する。種々の抗がん剤の治療による血管内皮障害による血管内皮由来のマイクロパーティクルなどを放出する。特に血管新生阻害(VEGF阻害薬)による血管内皮細胞の障害による静脈血栓症発症は多く報告されている。
このように、腫瘍に関連する血液凝固亢進病態の要因は複数あるが、組織因子の影響が強い場合は、TFPI阻害剤の存在下と非存在下の血液凝固の差または前述の外因系凝固と内因系凝固の解析結果の差、が大きくなり、反対に、マイクロパーティクルや血小板活性化による影響が強い場合は、差分は小さくなる。
このようにTFPIに対する阻害剤の存在下と非存在下の血液凝固を対比、または外因系及び内因系の血液凝固を対比することで、血液凝固亢進の度合いや要因を解析することが可能である。
よって、可逆的な抗凝固処理剤を含む採血容器で採取し、測定開始時に、採血容器に含まれる抗凝固処理の解除剤と接触因子阻害剤とTFPI阻害剤を含む試薬を添加し、血液凝固の解析を行うことが望ましい。
例えば、可逆的な抗凝固剤としてクエン酸ナトリウムを含む容器で血液を採取した場合には、塩化カルシウムと接触因子阻害剤とTFPI阻害剤を添加して血液凝固を解析する。
ただし、ブチルゴム製のゴム栓を有するクエン酸ナトリウムを含む真空採血管を用いた場合には、ゴム栓との接触により血液凝固が活性化され、凝固時間が短縮されることがある。
よって、ゴム栓を使用しないクエン酸を含む採血管を用いることが好ましい。
例えばゴム栓を使用しない採血管としてラミネートフィルムでシールされたベノジェクト採血管(テルモ社)などが使用できる。
ゴム栓のクエン酸を含む採血管を用いて採取した血液で検査する場合には、血液凝固時間を延長するために、血液凝固の検査時に塩化カルシウムと接触因子阻害剤とTFPI阻害剤に加えて、少量のヘパリン様物質(ヘパリン、低分子ヘパリン、ペンタサッカライド、ヘパラン硫酸)などの抗凝固物質を添加してもよい(WO2017/119508)。
この場合、健常人におけるROTEMやClotPro(enicor GmbH)などの装置を用いた血液凝固時間は、20-60分程度になるように調整されることが望ましい。
本発明はまた、接触因子阻害剤と外因系血液凝固阻害剤を含む、インビトロにおける内因系血液凝固能測定のための試薬キットを提供する。接触因子阻害剤および外因系血液凝固阻害剤の種類は上述したとおりであり、これらは、使用時に希釈されて血液凝固能測定に適した終濃度(好ましい終濃度の範囲は上述のとおり)で添加される。
血液凝固の解析にROTEM(Instrumentation Laboratory(IL)社)を用いた。
血液はテルモ社製の3.2%クエン酸ナトリウムを含む採血管(5mL)を用い採取した。
本採血管はラミネートフィルムでシールされ、ゴム栓が使用されていない。
全血 300μLに、以下の試薬を添加して血液凝固波形を解析した。
(1)塩化カルシウム試薬であるStartem試薬(IL社)20μL(終濃度12mM)
(2)Startem試薬(IL社)20μL(終濃度12mM)とTFPI阻害剤(compound3)(終濃度100μg/mL)
(3)Startem試薬(IL社)20μL(終濃度12mM)とTFPI阻害剤(compound3)(終濃度100μg/mL)とウサギ脳由来組織トロンボプラスチン(終濃 0.45ng/mL)
(4)Startem試薬(IL社)20μL(終濃度12mM)とTFPI阻害剤(compound3)(終濃度100μg/mL)とウサギ脳由来組織トロンボプラスチン(終濃度 0.9ng/mL)
TFPI阻害剤による微量組織トロンボプラスチン存在下の血液凝固促進作用が示された。
血液凝固の解析にROTEM(IL社)を用いた。
血液はテルモ社製の3.2%クエン酸ナトリウムを含む採血管(5mL)を用い採取した。
本採血管はラミネートフィルムでシールされ、ゴム栓が使用されていない。
全血 300μLに、以下の試薬を添加して血液凝固波形を解析した。
(1)塩化カルシウム(終濃度12mM)とCTI(終濃度50μg/mL)
(2)塩化カルシウム(終濃度12mM)とCTI(終濃度20μg/mL)とPKSI-527(終濃度40μM)
(3)塩化カルシウム(終濃度12mM)とCTI(終濃度20μg/mL)とPKSI-527(終濃度40μM)とウサギ脳由来組織トロンボプラスチン(終濃度 0.45ng/mL)
(4)塩化カルシウム(終濃度12mM)とCTI(終濃度20μg/mL)とPKSI-527(終濃度40μM)とウサギ脳由来組織トロンボプラスチン(終濃度 0.45ng/mL)とTFPI阻害剤(compound3)(終濃度100μg/mL)
すなわち、FXII阻害剤とカリクレイン阻害剤とTFPI阻害剤の存在により、微量組織トロンボプラスチンによる血栓形成の促進効果の評価が可能になった。
血液凝固の解析にROTEM(IL社)を用いた。
血液はベクトンディッキンソン社製の3.2%クエン酸ナトリウムを含む採血管(2mL)を用い採取した。本採血管はゴム栓が使用されている。
全血に、以下の試薬を添加して血液凝固波形を解析した。
(1)塩化カルシウム(終濃度12mM)とCTI(終濃度50μg/mL)
(2)塩化カルシウム(終濃度12mM)とCTI(終濃度20μg/mL)とPKSI-527(終濃度40μM)とヘパラン硫酸(終濃度1μg/mL)
(3)塩化カルシウム(終濃度12mM)とCTI(終濃度20μg/mL)とPKSI-527(終濃度40μM)とヘパラン硫酸(終濃度1μg/mL)とウサギ脳由来組織トロンボプラスチン(終濃度 0.9ng/mL)
(4)塩化カルシウム(終濃度12mM)とCTI(終濃度20μg/mL)とPKSI-527(終濃度40μM)とヘパラン硫酸(終濃度1μg/mL)とウサギ脳由来組織トロンボプラスチン(終濃度 0.9ng/mL)とTFPI阻害剤(compound3)(終濃度100μg/mL)
さらに、微量の組織トロンボプラスチンを添加した(3)のCTは1451秒であったが、さらにTFPI阻害剤を添加した(4)はCTが440秒に短縮された。
接触因子阻害剤とヘパラン硫酸とTFPI阻害剤を添加することで、微量の組織因子による血液凝固促進効果を評価することが可能であった。
血液凝固の解析にROTEM(IL社)を用いた。
血液はテルモ社製の3.2%クエン酸ナトリウムを含む採血管を用い採取した。
本採血管はラミネートフィルムでシールされ、ゴム栓が使用されていない。
全血300μLに、以下の試薬を添加して血液凝固波形を解析した。
(1)塩化カルシウム(終濃度12mM)とCTI(終濃度20μg/mL)
(2)塩化カルシウム(終濃度12mM)とPKSI-527(終濃度40μM)
(3)塩化カルシウム(終濃度12mM)とCTI(終濃度20μg/mL)とPKSI-527(終濃度40μM)
(4)Startem試薬(IL社)(20μL)
(5)塩化カルシウム(終濃度12mM)とCTI(終濃度20μg/mL)とヒト遺伝子組み換え組織因子(デイドイノビン;シーメンス社)(1000倍希釈を1%添加)
(6)塩化カルシウム(終濃度12mM)とPKSI-527(終濃度40μM)とヒト遺伝子組み換え組織因子(デイドイノビン;シーメンス社)(1000倍希釈を1%添加)
(7)塩化カルシウム(終濃度12mM)とCTI(終濃度20μg/mL)とPKSI-527(終濃度40μM) とヒト遺伝子組み換え組織因子(デイドイノビン;シーメンス社)(1000倍希釈を1%添加)
(8)Startem試薬(IL社)とヒト遺伝子組み換え組織因子(デイドイノビン;シーメンス社)(1000倍希釈を1%添加)
一方、デイドイノビンを微量添加した場合の凝固時間(5)(6)(7)は、CT値が600秒前後と同等であった。よって、FXII阻害剤とカリクレイン阻害剤の両方を加えることで微量組織因子添加による凝固時間の短縮幅が大きくなり、凝固亢進の識別が容易になる。一方、接触因子阻害剤の非添加(4)(8)の凝固時間の短縮幅はさらに小さく、凝固亢進の評価としては適さないことが示された。
血液凝固の解析にROTEM(IL社)を用いた。
血液はテルモ社製の3.2%クエン酸ナトリウムを含む採血管(5mL)を用い採取した。
本採血管はラミネートフィルムでシールされ、ゴム栓が使用されていない。
全血300μLに、以下の試薬を添加して血液凝固波形を解析した。
(1)Startem試薬(IL社)(20μL)
(2)塩化カルシウム(終濃度12mM)とCTI(終濃度20μg/mL)とPKSI-527(終濃度40μM)
(3)塩化カルシウム(終濃度12mM)とCTI(終濃度20μg/mL)とPKSI-527(終濃度40μM)及びTFPI阻害剤(compound3)(終濃度50μM)
(4)Startem試薬(IL社)(20μL)とヒト臍帯静脈内皮細胞由来エクソソーム(終濃度 1μg/mL)
(5)塩化カルシウム(終濃度12mM)とCTI(終濃度20μg/mL)とPKSI-527(終濃度40μM)とヒト臍帯静脈内皮細胞(HUVEC)由来エクソソーム(終濃度 1μg/mL)
(6)塩化カルシウム(終濃度12mM)とCTI(終濃度20μg/mL)とPKSI-527(終濃度40μM)及びTFPI阻害剤(compound3)(終濃度50μM)とヒト臍帯静脈内皮細胞(HUVEC)由来エクソソーム(終濃度 1μg/mL)
(7)Startem試薬(IL社)(20μL)とヒト悪性黒色腫A375細胞由来エクソソーム(終濃度1μg/mL)
(8)塩化カルシウム(終濃度12mM)とCTI(終濃度20μg/mL)とPKSI-527(終濃度40μM)と ヒト悪性黒色腫A375細胞由来エクソソーム(終濃度1μg/mL)
(9)塩化カルシウム(終濃度12mM)とCTI(終濃度20μg/mL)とPKSI-527(終濃度40μM)及びTFPI阻害剤(compound3)(終濃度50μM)とヒト悪性黒色腫A375細胞由来エクソソーム(終濃度1μg/mL)
(10)Startem試薬(IL社)(20μL) とヒト ヒストンH4(ABCAM社製)(終濃度2μM)
(11)塩化カルシウム(終濃度12mM)とCTI(終濃度20μg/mL)とPKSI-527(終濃度40μM)とヒト ヒストンH4(ABCAM社製)(終濃度2μM)
(12)塩化カルシウム(終濃度12mM)とCTI(終濃度20μg/mL)とPKSI-527(終濃度40μM)及びTFPI阻害剤(compound3)(終濃度50μg/mL)とヒト ヒストンH4(ABCAM社製)(終濃度2μM)
また、ヒストンは血小板を活性化することで血栓形成を促進することが知られる。
CTIとカリクレイン阻害剤とTFPI阻害剤を添加した場合、A375細胞由来エクソソームとヒストンH4添加の両方の血液凝固時間が短縮した。(9)(12)
さらに、癌細胞(A375)由来のエクソソームでは正常細胞(HUVEC)由来のエクソソームに比べて血液凝固の亢進度が大きいことが確認された。
血液凝固の解析にROTEM(IL社)を用いた。
血液はテルモ社製の3.2%クエン酸ナトリウムを含む採血管(5mL)を用い採取した。
本採血管はラミネートフィルムでシールされ、ゴム栓が使用されていない。
全血300μLに、大腸菌由来リポポリサッカライド(LPS;Merk社製)(終濃度1μg/mL)または、phorbol myristate acetate (PMA;Merk社製)(終濃度50nM)または ヒト ヒストンH4(ABCAM社製)(終濃度2μM)を添加して、4時間37℃でインキュベーションした後に、さらに(a)~(c)の試薬を添加して血液凝固波形を解析した。
(a)Startem試薬(IL社)(20μL)
(b)塩化カルシウム(終濃度12mM)とCTI(終濃度20μg/mL)とPKSI-527(終濃度40μM)
(c)塩化カルシウム(終濃度12mM)とCTI(終濃度20μg/mL)とPKSI-527(終濃度40μM)及びTFPI阻害剤(compound3)(終濃度50μM)
血液に何も添加せず4時間37℃インキュベーションした後に、(a)(b)(c)を添加して血液凝固を解析した場合
(1)Startem試薬(IL社)(20μL)
(2)塩化カルシウム(終濃度12mM)とCTI(終濃度20μg/mL)とPKSI-527(終濃度40μM)
(3)塩化カルシウム(終濃度12mM)とCTI(終濃度20μg/mL)とPKSI-527(終濃度40μM)とTFPI阻害剤(compound3)(終濃度50μM)
(4)Startem試薬(IL社)(20μL)と大腸菌由来リポポリサッカライド(LPS;Merk社製)(終濃度1μg/mL)
(5)塩化カルシウム(終濃度12mM)とCTI(終濃度20μg/mL)とPKSI-527(終濃度40μM)と大腸菌由来リポポリサッカライド(LPS;Merk社製)(終濃度1μg/mL)
(6)塩化カルシウム(終濃度12mM)とCTI(終濃度20μg/mL)とPKSI-527(終濃度40μM)及びTFPI阻害剤(compound3)(終濃度50μM)と大腸菌由来リポポリサッカライド(LPS;Merk社製)(終濃度1μg/mL)
(7)Startem試薬(IL社)(20μL)とphorbol myristate acetate (PMA;Merk社製)(終濃度50nM)
(8)塩化カルシウム(終濃度12mM)とCTI(終濃度20μg/mL)とPKSI-527(終濃度40μM)と ヒト悪性黒色腫A375細胞由来エクソソーム(終濃度1μg/mL)とphorbol myristate acetate (PMA;Merk社製)(終濃度50nM)
(9)塩化カルシウム(終濃度12mM)とCTI(終濃度20μg/mL)とPKSI-527(終濃度40μM)とTFPI阻害剤(compound3)(終濃度50μM)とヒト悪性黒色腫A375細胞由来エクソソーム(終濃度1μg/mL)とphorbol myristate acetate (PMA;Merk社製)(終濃度50nM)
(10)Startem試薬(IL社)(20μL) とヒト ヒストンH4(ABCAM社製)(終濃度2μM)
(11)塩化カルシウム(終濃度12mM)とCTI(終濃度20μg/mL)とPKSI-527(終濃度40μM)とヒト ヒストンH4(ABCAM社製)(終濃度2μM)
(12)塩化カルシウム(終濃度12mM)とCTI(終濃度20μg/mL)とPKSI-527(終濃度40μM)とTFPI阻害剤(compound3)(終濃度50μM)とヒト ヒストンH4(ABCAM社製)(終濃度2μM)
血液凝固の解析にROTEM(IL社)を用いた。
血液はテルモ社製の3.2%クエン酸ナトリウムを含む採血管(5mL)を用い採取した。
本採血管はラミネートフィルムでシールされ、ゴム栓が使用されていない。
得られた血液を3000rpm(15分)及び2400rpm(7分)遠心することで血漿と多血小板血漿を得た。これら血漿に実施例6と同様の試薬(LPS、PMA、ヒストン)を添加して、4時間37℃でインキュベーションした後に、実施例6の(1)~(12)と同様の試薬を添加して血液凝固波形を解析した。
血液凝固の解析にROTEM(IL社)を用いた。
血液はテルモ社製の3.2%クエン酸ナトリウムを含む採血管(5mL)を用い採取した。
本採血管はラミネートフィルムでシールされ、ゴム栓が使用されていない。
全血300μLに、以下の試薬を添加して、血液凝固波形を解析した。
(1)Startem試薬(IL社)(20μL)
(2)塩化カルシウム(終濃度12mM)とCTI(終濃度20μg/mL)とPKSI-527(終濃度40μM)
(3)塩化カルシウム(終濃度12mM)とCTI(終濃度20μg/mL)とPKSI-527(終濃度40μM)とFVIIa阻害剤(A-183X)(終濃度 100nM)
(4)塩化カルシウム(終濃度12mM)とCTI(終濃度20μg/mL)とPKSI-527(終濃度40μM)とFVIIa阻害剤(A-183X)(終濃度 100nM)とラッセル蛇毒FX活性化酵素(RVV-FX)(終濃度 5ng/ml)
(5)塩化カルシウム(終濃度12mM)とCTI(終濃度20μg/mL)とPKSI-527(終濃度40μM)とFVIIa阻害剤(A-183X)(終濃度 100nM)とエカリン(終濃度 4mU/ml単位)
(6)塩化カルシウム(終濃度12mM)とPKSI-527(終濃度40μM)とFVIIa阻害剤(A-183X)(終濃度 100nM)
(4)と(5)では、接触因子活性化とFVIIaの活性を抑制した状態で、微量なRVV(FX活性化剤)又はエカリン(プロトロンビン活性化剤)を添加することで、内因系の血液凝固の評価をしている。微量のエカリンまたはRVV-FXで血液凝固により産生されたトロンビンは、FXI, FVIII,FVを活性化することで、内因系血液凝固を増幅する。
(6)では、FVIIa阻害剤とカリクレイン阻害剤を添加し、部分的な接触因子の阻害環境下において接触因子活性化により産生されるFIXaを起点とする内因系血液凝固の活性化による血液凝固時間を評価している。
これは、健常人の血液中の組織因子が非常に少なく血液凝固に与える影響が軽微であることを示す。
(2)に対してさらにヒト悪性黒色腫A375細胞由来エクソソームを添加した場合に、血液凝固時間は大きく短縮するが、(3)に対してA375由来エクソソームを添加した場合の血液凝固時間の短縮は限定的である。これによりA375由来エクソソームには、多量の組織因子が含まれていると考えられる。
一方、(4)(5)(6)に対してA375由来エクソソームを添加した場合の血液凝固時間の短縮は限定的であった。
これは、A375由来エクソソームによる血液凝固短縮は、主にエクソソーム表面に存在する組織因子に起因した外因系血液凝固の活性化によるものであり、酸性リン脂質等に起因する内因系の血液凝固の活性化は限定的軽微であると考えられる。
血液凝固の解析にROTEM(IL社)を用いた。
血液はテルモ社製の3.2%クエン酸ナトリウムを含む採血管(5mL)を用い採取した。
本採血管はラミネートフィルムでシールされ、ゴム栓が使用されていない。
全血300μLに、添加剤無し又はLPS(終濃度1μg/mL)及びPMA(終濃度 50nM)を添加して、4時間37℃でインキュベーションした後に、実施例02の(1)~(6)の試薬を添加して血液凝固時間を解析した。
このことより、LPSの添加によって、組織因子の発現による外因系凝固の活性化に加え、内因系の血液凝固の活性化も引き起こされていると考えられる。
この結果より、PMAの添加では、LPSに比べ弱い外因系の血液凝固の活性化が起きていると考えられる。
血液凝固の解析にclotpro(Haemonetics社)を用いた。
血液はテルモ社製の3.2%クエン酸ナトリウムを含む採血管(5mL)を用い採取した。
本採血管はラミネートフィルムでシールされ、ゴム栓が使用されていない。
採血した全血(340μL)に、(1)~(6)以下の試薬を添加した後に、(7)~(12)のいずれかの試薬を添加して血液凝固時間を解析した。
(2) BP73(FXII阻害剤) 終濃度 5μg/mL
(3) BP61(FXII阻害剤) 終濃度5μg/mLとPKSI-527(終濃度40μM)
(4) BP73(FXII阻害剤) 終濃度 5μg/mLとPKSI-527(終濃度40μM)
(5) CTI終濃度20μg/mL
(6) CTI終濃度 20μg/mLとPKSI-527(終濃度40μM)
(7) 塩化カルシウム(終濃度12mM)
(8) 塩化カルシウム(終濃度12mM)、CTI(終濃度 20μg/mL)及びPKSI-527(終濃度40μM)
(9) 塩化カルシウム(終濃度12mM)、BP61(終濃度 10μg/mL)及びPKSI-527(終濃度40μM)
(10)塩化カルシウム(終濃度12mM)、BP61(終濃度 20μg/mL)及びPKSI-527(終濃度40μM)
(11)塩化カルシウム(終濃度12mM)、BP73(終濃度 10μg/mL)及びPKSI-527(終濃度40μM)
(12)塩化カルシウム(終濃度12mM)、BP73(終濃度 20μg/mL)及びPKSI-527(終濃度40μM)
血液凝固の解析にclotpro(Haemonetics社)を用いた。
血液はテルモ社製の3.2%クエン酸ナトリウムを含む採血管(5mL)を用い採取した。
本採血管はラミネートフィルムでシールされ、ゴム栓が使用されていない。
全血に、以下の試薬を添加して、血液凝固波形を解析した。
(1)Startem試薬(IL社)(20μL)
(2)塩化カルシウム(終濃度12mM)とCTI(終濃度20μg/mL)とPKSI-527(終濃度40μM)
(3)塩化カルシウム(終濃度12mM)とCTI(終濃度20μg/mL)とPKSI-527(終濃度40μM)及びTFPI阻害剤(compound3)(終濃度50μM)
(4)Startem試薬(IL社)(20μL)とA375由来エクソソーム(終濃度1μg/mL)
(5)塩化カルシウム(終濃度12mM)とCTI(終濃度20μg/mL)とPKSI-527(終濃度40μM) とA375由来エクソソーム(終濃度1μg/mL)
(6)塩化カルシウム(終濃度12mM)とCTI(終濃度20μg/mL)とPKSI-527(終濃度40μM)及びTFPI阻害剤(compound3)(終濃度50μM)とA375由来エクソソーム(終濃度1μg/mL)
血液凝固の解析にROTEM(IL社)を用いた。
血液はテルモ社製の3.2%クエン酸ナトリウムを含む採血管(5mL)を用い採取した。
本採血管はラミネートフィルムでシールされ、ゴム栓が使用されていない。
全血に、以下の試薬を添加して、血液凝固波形を解析した。
(1)塩化カルシウム(終濃度12mM)とCTI(終濃度20μg/mL)とPKSI-527(終濃度40μM)とFVIIa阻害剤(A-183X)(終濃度 100nM)とエカリン(終濃度 1mU/ml単位)
(2)塩化カルシウム(終濃度12mM)とCTI(終濃度20μg/mL)とPKSI-527(終濃度40μM)とFVIIa阻害剤(A-183X)(終濃度 100nM)とエカリン(終濃度 2mU/ml単位)
(3)塩化カルシウム(終濃度12mM)とCTI(終濃度20μg/mL)とPKSI-527(終濃度40μM)とFVIIa阻害剤(A-183X)(終濃度 100nM)とエカリン(終濃度 4mU/ml単位)
Claims (16)
- 血液試料に、接触因子阻害剤および組織因子経路阻害剤(TFPI)に対する阻害剤を添加して血液凝固能の測定を行うことを特徴とする、インビトロにおける外因系血液凝固能の評価方法。
- 接触因子阻害剤がFXI(血液凝固第11因子)阻害剤、FXII(血液凝固第12因子)阻害剤および/またはカリクレイン阻害剤である、請求項1に記載の方法。
- 接触因子阻害剤がFXII阻害剤とカリクレイン阻害剤の両方である、請求項1に記載の方法。
- TFPI阻害剤非存在下の血液凝固能の測定結果と比較を行う、請求項1~3のいずれか一項に記載の血液凝固能の解析方法。
- 血液試料に、接触因子阻害剤と外因系血液凝固阻害剤の両方を添加して、血液凝固能の測定を行うことを特徴とする、インビトロにおける内因系血液凝固能の評価方法。
- 外因系血液凝固阻害剤が、FVII(血液凝固第7因子)阻害剤、組織因子阻害剤、および不活性化FVIIaのいずれか1種以上である請求項5に記載の方法。
- さらに、血液凝固活性化剤としてプロトロンビン活性化剤および/または血液凝固第10因子活性化剤を含む、請求項5または6に記載の方法。
- プロトロンビン活性化剤がエカリンである、請求項7に記載の方法。
- 血液凝固第10因子活性化剤がラッセル蛇毒由来FX活性化酵素(RVV-FX)である請求項7に記載の方法。
- 血液試料が全血である、請求項1~9のいずれか一項に記載の方法。
- 請求項1~4のいずれか一項に記載の外因系血液凝固の評価と請求項5~9のいずれか一項に記載の内因系血液凝固の評価との比較を行うことを特徴とする、血液凝固能の解析方法。
- 測定がROTEM(Rotational Thromboelastometry)および/またはTEG(Thromboelastography)による、請求項1~11のいずれか一項に記載の方法。
- 前記血液試料が癌患者又は癌が疑われる患者の血液試料である、請求項1~12のいずれか一項に記載の方法。
- 接触因子阻害剤およびTFPI阻害剤を含む、インビトロにおける外因系血液凝固能測定のための試薬。
- 接触因子阻害剤と外因系血液凝固阻害剤を含む、インビトロにおける内因系血液凝固能測定のための試薬。
- さらに血液凝固活性化剤を含む、請求項15に記載の試薬。
Priority Applications (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US18/269,860 US20240044921A1 (en) | 2020-12-28 | 2021-12-28 | Method for evaluating blood coagulation performance, and method for inspecting risk of thrombosis |
EP21915333.5A EP4270010A1 (en) | 2020-12-28 | 2021-12-28 | Method for evaluating blood coagulation performance, and method for inspecting risk of thrombosis |
JP2022573120A JPWO2022145475A1 (ja) | 2020-12-28 | 2021-12-28 |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP2020219401 | 2020-12-28 | ||
JP2020-219401 | 2020-12-28 |
Publications (1)
Publication Number | Publication Date |
---|---|
WO2022145475A1 true WO2022145475A1 (ja) | 2022-07-07 |
Family
ID=82260820
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/JP2021/048977 WO2022145475A1 (ja) | 2020-12-28 | 2021-12-28 | 血液凝固能の評価方法および血栓症リスクの検査方法 |
Country Status (4)
Country | Link |
---|---|
US (1) | US20240044921A1 (ja) |
EP (1) | EP4270010A1 (ja) |
JP (1) | JPWO2022145475A1 (ja) |
WO (1) | WO2022145475A1 (ja) |
Citations (18)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JPH06324048A (ja) * | 1993-05-15 | 1994-11-25 | Shimizu Seiyaku Kk | 血液凝固能検査用試薬及び血液凝固能の検査方法 |
JP2003505678A (ja) * | 1999-07-23 | 2003-02-12 | ザ スクリップス リサーチ インスティテュート | 全血の凝固因子活性を測定する方法 |
JP2003517610A (ja) * | 1999-12-15 | 2003-05-27 | ペンタファルム・リミテッド | 血液学的アッセイ及び試薬 |
JP2008191171A (ja) * | 1997-07-23 | 2008-08-21 | Tokuyama Corp | 凝固パラメーターを決定するためのキット |
JP2008241621A (ja) * | 2007-03-28 | 2008-10-09 | Sysmex Corp | 血液凝固測定用試薬及び組織因子安定化方法 |
US20090042217A1 (en) * | 2006-01-27 | 2009-02-12 | Rappaport Family Institute For Research In The Medical Sciences | Methods and Kits for Determining Blood Coagulation |
JP2010085411A (ja) * | 2008-10-02 | 2010-04-15 | Siemens Healthcare Diagnostics Products Gmbh | 血液凝固アッセイ |
JP2011527897A (ja) * | 2008-07-17 | 2011-11-10 | ディアグノスチカ・スタゴ | 循環組織因子のインビトロアッセイ方法及び凝固疾患の検出における使用 |
WO2014116275A1 (en) * | 2013-01-24 | 2014-07-31 | Portola Pharmaceuticals, Inc. | INHIBITION OF TISSUE FACTOR PATHWAY INHIBITOR WITH FACTOR Xa DERIVATIVES |
JP2014521952A (ja) * | 2011-07-26 | 2014-08-28 | オブシェストヴォ エス オグラニチェノイ オトヴェトストヴェノスチュ“ゲマトロジチェスカヤ コーポラティシヤ” | 不均一系(ばらつき)におけるタンパク質分解酵素活性の空間分布および時間分布を求めるための方法、これを実現するための装置、不均一系におけるタンパク質分解酵素活性の空間分布および時間分布の変化をもとに、止血系の欠陥を診断するための方法 |
JP2014531413A (ja) * | 2011-08-23 | 2014-11-27 | シナプス・ビー.ブイ.Synapse B.V. | 異物表面との接触による血液凝固系の活性化の熱安定性阻害剤 |
JP2015509923A (ja) * | 2012-01-30 | 2015-04-02 | バクスター・インターナショナル・インコーポレイテッドBaxter International Incorp0Rated | 非抗凝固性の硫酸化またはスルホン酸化多糖 |
WO2015156322A1 (ja) * | 2014-04-08 | 2015-10-15 | 藤森工業株式会社 | 血液性状検査用マイクロチップおよび血液性状検査用装置 |
WO2017119508A1 (ja) | 2016-01-07 | 2017-07-13 | 藤森工業株式会社 | 採血管、試薬及びそれらを利用した血液性状分析方法 |
JP2017530349A (ja) * | 2014-09-09 | 2017-10-12 | ペロスフィア インコーポレイテッド | マイクロ流体チップベースの一般的な凝固アッセイ |
WO2018043420A1 (ja) | 2016-08-29 | 2018-03-08 | 藤森工業株式会社 | 血液凝固検査装置及び血液凝固検査方法 |
WO2018128002A1 (ja) * | 2017-01-06 | 2018-07-12 | ソニー株式会社 | 血液凝固系解析装置、血液凝固系解析システム、血液凝固系解析方法、及び血液凝固系解析用プログラム、並びに、出血量予測装置、出血量予測システム、出血量予測方法、及び出血量予測用プログラム |
JP2019521324A (ja) * | 2016-05-13 | 2019-07-25 | ザ・スクリップス・リサーチ・インスティテュートThe Scripps Research Institute | 抗血栓療法および止血療法のための組成物および方法 |
-
2021
- 2021-12-28 US US18/269,860 patent/US20240044921A1/en active Pending
- 2021-12-28 JP JP2022573120A patent/JPWO2022145475A1/ja active Pending
- 2021-12-28 EP EP21915333.5A patent/EP4270010A1/en active Pending
- 2021-12-28 WO PCT/JP2021/048977 patent/WO2022145475A1/ja active Application Filing
Patent Citations (18)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JPH06324048A (ja) * | 1993-05-15 | 1994-11-25 | Shimizu Seiyaku Kk | 血液凝固能検査用試薬及び血液凝固能の検査方法 |
JP2008191171A (ja) * | 1997-07-23 | 2008-08-21 | Tokuyama Corp | 凝固パラメーターを決定するためのキット |
JP2003505678A (ja) * | 1999-07-23 | 2003-02-12 | ザ スクリップス リサーチ インスティテュート | 全血の凝固因子活性を測定する方法 |
JP2003517610A (ja) * | 1999-12-15 | 2003-05-27 | ペンタファルム・リミテッド | 血液学的アッセイ及び試薬 |
US20090042217A1 (en) * | 2006-01-27 | 2009-02-12 | Rappaport Family Institute For Research In The Medical Sciences | Methods and Kits for Determining Blood Coagulation |
JP2008241621A (ja) * | 2007-03-28 | 2008-10-09 | Sysmex Corp | 血液凝固測定用試薬及び組織因子安定化方法 |
JP2011527897A (ja) * | 2008-07-17 | 2011-11-10 | ディアグノスチカ・スタゴ | 循環組織因子のインビトロアッセイ方法及び凝固疾患の検出における使用 |
JP2010085411A (ja) * | 2008-10-02 | 2010-04-15 | Siemens Healthcare Diagnostics Products Gmbh | 血液凝固アッセイ |
JP2014521952A (ja) * | 2011-07-26 | 2014-08-28 | オブシェストヴォ エス オグラニチェノイ オトヴェトストヴェノスチュ“ゲマトロジチェスカヤ コーポラティシヤ” | 不均一系(ばらつき)におけるタンパク質分解酵素活性の空間分布および時間分布を求めるための方法、これを実現するための装置、不均一系におけるタンパク質分解酵素活性の空間分布および時間分布の変化をもとに、止血系の欠陥を診断するための方法 |
JP2014531413A (ja) * | 2011-08-23 | 2014-11-27 | シナプス・ビー.ブイ.Synapse B.V. | 異物表面との接触による血液凝固系の活性化の熱安定性阻害剤 |
JP2015509923A (ja) * | 2012-01-30 | 2015-04-02 | バクスター・インターナショナル・インコーポレイテッドBaxter International Incorp0Rated | 非抗凝固性の硫酸化またはスルホン酸化多糖 |
WO2014116275A1 (en) * | 2013-01-24 | 2014-07-31 | Portola Pharmaceuticals, Inc. | INHIBITION OF TISSUE FACTOR PATHWAY INHIBITOR WITH FACTOR Xa DERIVATIVES |
WO2015156322A1 (ja) * | 2014-04-08 | 2015-10-15 | 藤森工業株式会社 | 血液性状検査用マイクロチップおよび血液性状検査用装置 |
JP2017530349A (ja) * | 2014-09-09 | 2017-10-12 | ペロスフィア インコーポレイテッド | マイクロ流体チップベースの一般的な凝固アッセイ |
WO2017119508A1 (ja) | 2016-01-07 | 2017-07-13 | 藤森工業株式会社 | 採血管、試薬及びそれらを利用した血液性状分析方法 |
JP2019521324A (ja) * | 2016-05-13 | 2019-07-25 | ザ・スクリップス・リサーチ・インスティテュートThe Scripps Research Institute | 抗血栓療法および止血療法のための組成物および方法 |
WO2018043420A1 (ja) | 2016-08-29 | 2018-03-08 | 藤森工業株式会社 | 血液凝固検査装置及び血液凝固検査方法 |
WO2018128002A1 (ja) * | 2017-01-06 | 2018-07-12 | ソニー株式会社 | 血液凝固系解析装置、血液凝固系解析システム、血液凝固系解析方法、及び血液凝固系解析用プログラム、並びに、出血量予測装置、出血量予測システム、出血量予測方法、及び出血量予測用プログラム |
Non-Patent Citations (16)
Title |
---|
AM J HEMATOL, vol. 95, no. 7, July 2020 (2020-07-01), pages 863 - 869 |
CHOWDARY PRATIMA: "Anti-tissue factor pathway inhibitor (TFPI) therapy: a novel approach to the treatment of haemophilia", INTERNATIONAL JOURNAL OF HEMATOLOGY, vol. 111, no. 1, 9 October 2018 (2018-10-09), NL , pages 42 - 50, XP036979117, ISSN: 0925-5710, DOI: 10.1007/s12185-018-2548-6 * |
CRISTINA PUY, TUCKER ERIK I, MATAFONOV ANTON, CHENG QIUFANG, ZIENTEK KEITH D, GAILANI DAVE, AS GRUBER AND, MCCARTY OWEN J T: "Activated factor XI increases the procoagulant activity of the extrinsic pathway by inactivating tissue factor pathway inhibitor", BLOOD, vol. 125, no. 9, 13 January 2015 (2015-01-13), pages 1488 - 1496, XP055293619, DOI: 10.1182/blood-2014-10-604587 * |
EUR JVASC ENDOVASC SUR, vol. 15, no. 6, June 1998 (1998-06-01), pages 515 - 20 |
HATO, TAKAAKI: "Evaluation of Anti-thrombotic Drugs in View of Recent Mechanism for Venous Thrombus Formation", PHLEBOLOGY, vol. 26, no. 1, 25 February 2015 (2015-02-25), JP , pages 1 - 8, XP009537970, ISSN: 0915-7395, DOI: 10.7134/phlebol.14-26 * |
J BIOL CHEM., vol. 269, no. 14, 8 April 1994 (1994-04-08), pages 10644 - 50 |
J BIOL CHEM., vol. 278, no. 24, 13 June 2003 (2003-06-13), pages 21823 - 30 |
J BIOL CHEM., vol. 289, no. 3, 17 January 2014 (2014-01-17), pages 1732 - 41 |
J MED CHEM, vol. 60, no. 23, 14 December 2017 (2017-12-14), pages 9703 - 9723 |
J MED CHEM., vol. 60, no. 3, 9 February 2017 (2017-02-09), pages 1151 - 1158 |
J THROMB HAEMOST, vol. 10, no. 8, August 2012 (2012-08-01), pages 1581 - 90 |
NAZAKAWA, NOZOMI; YOSHIHARA, SAORI; KAGA, RIEKO; AOKI, YOSHIKAZU; IGARASHI, SUMIKO: "Current status of coagulation test and examination of pre-measurement variable factors", ZENNINKAI RESEARCH ANNUAL REPORT, no. 33, 1 April 2012 (2012-04-01), pages 76 - 80, XP009537971, ISSN: 0916-8826 * |
OSAMU TAKAMIYA: "Discrepancies between coagulation test results and clinical symptoms-a new way of thinking about coagulation mechanisms", vol. 9, no. 1, 29 February 2008 (2008-02-29), pages 60 - 68, XP009537958 * |
SCI REP, vol. 7, no. 1, 18 May 2017 (2017-05-18), pages 2102 |
TAKASHI MORITA: "A new way of thinking about the blood coagulation mechanism", JAPANESE JOURNAL OF PEDIATRIC HEMATOLOGY, vol. 17, no. 2, 1 January 2003 (2003-01-01), JP , pages 49 - 57, XP009537960, ISSN: 0913-8706, DOI: 10.11412/jjph1987.17.49 * |
YUJIRO ASADA: "Issues with new oral anticoagulants Coagulation factors that do not contribute to bleeding and their inhibition Factors XI and XII", MEDICINA, vol. 49, no. 6, 10 June 2012 (2012-06-10), JP , pages 1046 - 1048, XP009537968, ISSN: 0025-7699 * |
Also Published As
Publication number | Publication date |
---|---|
US20240044921A1 (en) | 2024-02-08 |
JPWO2022145475A1 (ja) | 2022-07-07 |
EP4270010A1 (en) | 2023-11-01 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
Agarwal et al. | Evaluation of coagulation abnormalities in acute liver failure | |
von Meijenfeldt et al. | Prothrombotic changes in patients with COVID‐19 are associated with disease severity and mortality | |
Potze et al. | Preserved hemostatic status in patients with non-alcoholic fatty liver disease | |
Paar et al. | Anticoagulant action of low, physiologic, and high albumin levels in whole blood | |
Iba et al. | Potential diagnostic markers for disseminated intravascular coagulation of sepsis | |
Senzolo et al. | Increased anticoagulant response to low‐molecular‐weight heparin in plasma from patients with advanced cirrhosis | |
Habib et al. | Evidence of rebalanced coagulation in acute liver injury and acute liver failure as measured by thrombin generation | |
Cardenas et al. | Measuring thrombin generation as a tool for predicting hemostatic potential and transfusion requirements following trauma | |
Kim et al. | Circulating levels of DNA-histone complex and dsDNA are independent prognostic factors of disseminated intravascular coagulation | |
Uemura et al. | Comprehensive analysis of ADAMTS13 in patients with liver cirrhosis | |
Santucci et al. | Measurement of tissue factor activity in whole blood | |
Agarwal et al. | Hemostasis in patients with acute kidney injury secondary to acute liver failure | |
Ollivier et al. | Detection of endogenous tissue factor levels in plasma using the calibrated automated thrombogram assay | |
Raffa et al. | Hypercoagulability in patients with chronic noncirrhotic portal vein thrombosis | |
Curnow et al. | Reduced fibrinolysis and increased fibrin generation can be detected in hypercoagulable patients using the overall hemostatic potential assay | |
Hansson et al. | The effect of corn trypsin inhibitor and inhibiting antibodies for FXIa and FXIIa on coagulation of plasma and whole blood | |
Reddel et al. | Detection of hypofibrinolysis in stable coronary artery disease using the overall haemostatic potential assay | |
Andersen et al. | Thromboelastometry as a supplementary tool for evaluation of hemostasis in severe sepsis and septic shock | |
Debaugnies et al. | Evaluation of the procoagulant activity in the plasma of cancer patients using a thrombin generation assay | |
Hisada et al. | Circulating tissue factor‐positive extracellular vesicles and their association with thrombosis in different diseases | |
Sayyadi et al. | Status of major hemostatic components in the setting of COVID-19: The effect on endothelium, platelets, coagulation factors, fibrinolytic system, and complement | |
Hellum et al. | Microparticle-associated tissue factor activity measured with the Zymuphen MP-TF kit and the calibrated automated thrombogram assay | |
Cibor et al. | Levels and activities of von Willebrand factor and metalloproteinase with thrombospondin type-1 motif, number 13 in inflammatory bowel diseases | |
Boknäs et al. | Associations between hemostatic markers and mortality in COVID-19–Compounding effects of D-dimer, antithrombin and PAP complex | |
Peyvandi et al. | No changes of parameters nor coagulation activation in healthy subjects vaccinated for SARS-Cov-2 |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
121 | Ep: the epo has been informed by wipo that ep was designated in this application |
Ref document number: 21915333 Country of ref document: EP Kind code of ref document: A1 |
|
ENP | Entry into the national phase |
Ref document number: 2022573120 Country of ref document: JP Kind code of ref document: A |
|
WWE | Wipo information: entry into national phase |
Ref document number: 18269860 Country of ref document: US |
|
WWE | Wipo information: entry into national phase |
Ref document number: 2021915333 Country of ref document: EP |
|
NENP | Non-entry into the national phase |
Ref country code: DE |
|
ENP | Entry into the national phase |
Ref document number: 2021915333 Country of ref document: EP Effective date: 20230728 |