WO1993025220A1 - Test for quantitative thrombin time - Google Patents
Test for quantitative thrombin time Download PDFInfo
- Publication number
- WO1993025220A1 WO1993025220A1 PCT/US1993/005297 US9305297W WO9325220A1 WO 1993025220 A1 WO1993025220 A1 WO 1993025220A1 US 9305297 W US9305297 W US 9305297W WO 9325220 A1 WO9325220 A1 WO 9325220A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- thrombin
- fibrinogen
- plasma
- concentration
- buffer
- Prior art date
Links
- 108090000190 Thrombin Proteins 0.000 title claims abstract description 112
- 229960004072 thrombin Drugs 0.000 title claims abstract description 92
- 238000012360 testing method Methods 0.000 title abstract description 24
- 229940012952 fibrinogen Drugs 0.000 claims abstract description 57
- 108010049003 Fibrinogen Proteins 0.000 claims abstract description 56
- 102000008946 Fibrinogen Human genes 0.000 claims abstract description 56
- 238000000034 method Methods 0.000 claims abstract description 41
- 239000003112 inhibitor Substances 0.000 claims abstract description 37
- 206010053567 Coagulopathies Diseases 0.000 claims abstract description 26
- 230000000694 effects Effects 0.000 claims abstract description 24
- 230000036470 plasma concentration Effects 0.000 claims abstract description 11
- 229920000669 heparin Polymers 0.000 claims description 23
- 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 claims description 22
- 230000035602 clotting Effects 0.000 claims description 22
- 229960002897 heparin Drugs 0.000 claims description 22
- 230000001225 therapeutic effect Effects 0.000 claims description 20
- 239000000243 solution Substances 0.000 claims description 17
- 238000003556 assay Methods 0.000 claims description 15
- 239000000203 mixture Substances 0.000 claims description 15
- 229940106780 human fibrinogen Drugs 0.000 claims description 9
- 239000003795 chemical substances by application Substances 0.000 claims description 8
- 238000007865 diluting Methods 0.000 claims description 7
- 239000013610 patient sample Substances 0.000 claims description 7
- 102000007327 Protamines Human genes 0.000 claims description 6
- 108010007568 Protamines Proteins 0.000 claims description 6
- 229950008679 protamine sulfate Drugs 0.000 claims description 6
- 239000000523 sample Substances 0.000 claims description 6
- 238000002156 mixing Methods 0.000 claims description 5
- 238000013207 serial dilution Methods 0.000 claims description 4
- 230000003472 neutralizing effect Effects 0.000 claims description 3
- 230000035945 sensitivity Effects 0.000 claims description 3
- FGUUSXIOTUKUDN-IBGZPJMESA-N C1(=CC=CC=C1)N1C2=C(NC([C@H](C1)NC=1OC(=NN=1)C1=CC=CC=C1)=O)C=CC=C2 Chemical compound C1(=CC=CC=C1)N1C2=C(NC([C@H](C1)NC=1OC(=NN=1)C1=CC=CC=C1)=O)C=CC=C2 FGUUSXIOTUKUDN-IBGZPJMESA-N 0.000 claims 2
- GNFTZDOKVXKIBK-UHFFFAOYSA-N 3-(2-methoxyethoxy)benzohydrazide Chemical compound COCCOC1=CC=CC(C(=O)NN)=C1 GNFTZDOKVXKIBK-UHFFFAOYSA-N 0.000 claims 1
- 239000000376 reactant Substances 0.000 claims 1
- 238000010790 dilution Methods 0.000 abstract description 12
- 239000012895 dilution Substances 0.000 abstract description 12
- 230000015271 coagulation Effects 0.000 abstract description 7
- 238000005345 coagulation Methods 0.000 abstract description 7
- 238000004445 quantitative analysis Methods 0.000 abstract 1
- 210000002381 plasma Anatomy 0.000 description 87
- WQPDUTSPKFMPDP-OUMQNGNKSA-N hirudin Chemical compound C([C@@H](C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC=1C=CC(OS(O)(=O)=O)=CC=1)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(N)=O)C(O)=O)NC(=O)[C@H](CC(O)=O)NC(=O)CNC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CC=1NC=NC=1)NC(=O)[C@H](CO)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H]1N(CCC1)C(=O)[C@H](CCCCN)NC(=O)[C@H]1N(CCC1)C(=O)[C@@H](NC(=O)CNC(=O)[C@H](CCC(O)=O)NC(=O)CNC(=O)[C@@H](NC(=O)[C@@H](NC(=O)[C@H]1NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CCCCN)NC(=O)[C@H](CCC(O)=O)NC(=O)CNC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CO)NC(=O)CNC(=O)[C@H](CC(C)C)NC(=O)[C@H]([C@@H](C)CC)NC(=O)[C@@H]2CSSC[C@@H](C(=O)N[C@@H](CCC(O)=O)C(=O)NCC(=O)N[C@@H](CO)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@H](C(=O)N[C@H](C(NCC(=O)N[C@@H](CCC(N)=O)C(=O)NCC(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CCCCN)C(=O)N2)=O)CSSC1)C(C)C)NC(=O)[C@H](CC(C)C)NC(=O)[C@H]1NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CCC(N)=O)NC(=O)CNC(=O)[C@H](CO)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H]([C@@H](C)O)NC(=O)[C@@H](NC(=O)[C@H](CC(O)=O)NC(=O)[C@@H](NC(=O)[C@H](CC=2C=CC(O)=CC=2)NC(=O)[C@@H](NC(=O)[C@@H](N)C(C)C)C(C)C)[C@@H](C)O)CSSC1)C(C)C)[C@@H](C)O)[C@@H](C)O)C1=CC=CC=C1 WQPDUTSPKFMPDP-OUMQNGNKSA-N 0.000 description 56
- 230000002159 abnormal effect Effects 0.000 description 15
- 108010027612 Batroxobin Proteins 0.000 description 14
- 108010058861 Fibrin Fibrinogen Degradation Products Proteins 0.000 description 13
- 239000003146 anticoagulant agent Substances 0.000 description 13
- 239000000208 fibrin degradation product Substances 0.000 description 13
- 229940127219 anticoagulant drug Drugs 0.000 description 10
- 238000006243 chemical reaction Methods 0.000 description 8
- 239000000463 material Substances 0.000 description 8
- 102000007625 Hirudins Human genes 0.000 description 6
- 108010007267 Hirudins Proteins 0.000 description 6
- 210000004369 blood Anatomy 0.000 description 6
- 239000008280 blood Substances 0.000 description 6
- 229940006607 hirudin Drugs 0.000 description 6
- 238000002560 therapeutic procedure Methods 0.000 description 6
- 208000007536 Thrombosis Diseases 0.000 description 5
- 238000012544 monitoring process Methods 0.000 description 5
- 108010007948 Abnormal Fibrinogens Proteins 0.000 description 4
- 102000007376 Abnormal Fibrinogens Human genes 0.000 description 4
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 4
- 230000010100 anticoagulation Effects 0.000 description 4
- 239000001110 calcium chloride Substances 0.000 description 4
- 229910001628 calcium chloride Inorganic materials 0.000 description 4
- 239000003153 chemical reaction reagent Substances 0.000 description 4
- 239000002323 fibrinogen variant Substances 0.000 description 4
- 238000004128 high performance liquid chromatography Methods 0.000 description 4
- 238000002360 preparation method Methods 0.000 description 4
- 230000002035 prolonged effect Effects 0.000 description 4
- FTOAOBMCPZCFFF-UHFFFAOYSA-N 5,5-diethylbarbituric acid Chemical compound CCC1(CC)C(=O)NC(=O)NC1=O FTOAOBMCPZCFFF-UHFFFAOYSA-N 0.000 description 3
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 description 3
- 108010080865 Factor XII Proteins 0.000 description 3
- 102000000429 Factor XII Human genes 0.000 description 3
- 206010047249 Venous thrombosis Diseases 0.000 description 3
- 230000005856 abnormality Effects 0.000 description 3
- 208000015294 blood coagulation disease Diseases 0.000 description 3
- 206010025135 lupus erythematosus Diseases 0.000 description 3
- 239000011550 stock solution Substances 0.000 description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 3
- 206010003178 Arterial thrombosis Diseases 0.000 description 2
- 108010014173 Factor X Proteins 0.000 description 2
- 206010051125 Hypofibrinogenaemia Diseases 0.000 description 2
- 108010094028 Prothrombin Proteins 0.000 description 2
- 238000010171 animal model Methods 0.000 description 2
- 230000002785 anti-thrombosis Effects 0.000 description 2
- 239000004019 antithrombin Substances 0.000 description 2
- 229960002319 barbital Drugs 0.000 description 2
- 230000003247 decreasing effect Effects 0.000 description 2
- 230000007812 deficiency Effects 0.000 description 2
- 238000011156 evaluation Methods 0.000 description 2
- 208000027826 familial dysfibrinogenemia Diseases 0.000 description 2
- 150000003904 phospholipids Chemical class 0.000 description 2
- 230000002265 prevention Effects 0.000 description 2
- 108090000765 processed proteins & peptides Proteins 0.000 description 2
- 102000004169 proteins and genes Human genes 0.000 description 2
- 108090000623 proteins and genes Proteins 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 238000012216 screening Methods 0.000 description 2
- 239000011780 sodium chloride Substances 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 1
- 102000009027 Albumins Human genes 0.000 description 1
- 108010088751 Albumins Proteins 0.000 description 1
- 102000015081 Blood Coagulation Factors Human genes 0.000 description 1
- 108010039209 Blood Coagulation Factors Proteins 0.000 description 1
- 241000167854 Bourreria succulenta Species 0.000 description 1
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 description 1
- KRKNYBCHXYNGOX-UHFFFAOYSA-K Citrate Chemical compound [O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O KRKNYBCHXYNGOX-UHFFFAOYSA-K 0.000 description 1
- 206010051055 Deep vein thrombosis Diseases 0.000 description 1
- 241000237902 Hirudo medicinalis Species 0.000 description 1
- 241000282412 Homo Species 0.000 description 1
- 102000008100 Human Serum Albumin Human genes 0.000 description 1
- 108091006905 Human Serum Albumin Proteins 0.000 description 1
- 206010029164 Nephrotic syndrome Diseases 0.000 description 1
- 108090000778 Platelet factor 4 Proteins 0.000 description 1
- 102000004211 Platelet factor 4 Human genes 0.000 description 1
- 108010023197 Streptokinase Proteins 0.000 description 1
- 229940122388 Thrombin inhibitor Drugs 0.000 description 1
- 108010000499 Thromboplastin Proteins 0.000 description 1
- 102000002262 Thromboplastin Human genes 0.000 description 1
- 230000003024 amidolytic effect Effects 0.000 description 1
- 238000002399 angioplasty Methods 0.000 description 1
- 230000002429 anti-coagulating effect Effects 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 108010058143 atroxin Proteins 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 239000003114 blood coagulation factor Substances 0.000 description 1
- 239000011575 calcium Substances 0.000 description 1
- 229910052791 calcium Inorganic materials 0.000 description 1
- 235000019693 cherries Nutrition 0.000 description 1
- 239000000701 coagulant Substances 0.000 description 1
- AGVAZMGAQJOSFJ-WZHZPDAFSA-M cobalt(2+);[(2r,3s,4r,5s)-5-(5,6-dimethylbenzimidazol-1-yl)-4-hydroxy-2-(hydroxymethyl)oxolan-3-yl] [(2r)-1-[3-[(1r,2r,3r,4z,7s,9z,12s,13s,14z,17s,18s,19r)-2,13,18-tris(2-amino-2-oxoethyl)-7,12,17-tris(3-amino-3-oxopropyl)-3,5,8,8,13,15,18,19-octamethyl-2 Chemical compound [Co+2].N#[C-].[N-]([C@@H]1[C@H](CC(N)=O)[C@@]2(C)CCC(=O)NC[C@@H](C)OP(O)(=O)O[C@H]3[C@H]([C@H](O[C@@H]3CO)N3C4=CC(C)=C(C)C=C4N=C3)O)\C2=C(C)/C([C@H](C\2(C)C)CCC(N)=O)=N/C/2=C\C([C@H]([C@@]/2(CC(N)=O)C)CCC(N)=O)=N\C\2=C(C)/C2=N[C@]1(C)[C@@](C)(CC(N)=O)[C@@H]2CCC(N)=O AGVAZMGAQJOSFJ-WZHZPDAFSA-M 0.000 description 1
- 238000010835 comparative analysis Methods 0.000 description 1
- 210000004351 coronary vessel Anatomy 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- AAOVKJBEBIDNHE-UHFFFAOYSA-N diazepam Chemical compound N=1CC(=O)N(C)C2=CC=C(Cl)C=C2C=1C1=CC=CC=C1 AAOVKJBEBIDNHE-UHFFFAOYSA-N 0.000 description 1
- 239000003085 diluting agent Substances 0.000 description 1
- 238000010494 dissociation reaction Methods 0.000 description 1
- 230000005593 dissociations Effects 0.000 description 1
- 231100000673 dose–response relationship Toxicity 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 239000003527 fibrinolytic agent Substances 0.000 description 1
- 230000003480 fibrinolytic effect Effects 0.000 description 1
- 230000023597 hemostasis Effects 0.000 description 1
- ZFGMDIBRIDKWMY-PASTXAENSA-N heparin Chemical compound CC(O)=N[C@@H]1[C@@H](O)[C@H](O)[C@@H](COS(O)(=O)=O)O[C@@H]1O[C@@H]1[C@@H](C(O)=O)O[C@@H](O[C@H]2[C@@H]([C@@H](OS(O)(=O)=O)[C@@H](O[C@@H]3[C@@H](OC(O)[C@H](OS(O)(=O)=O)[C@H]3O)C(O)=O)O[C@@H]2O)CS(O)(=O)=O)[C@H](O)[C@H]1O ZFGMDIBRIDKWMY-PASTXAENSA-N 0.000 description 1
- 230000001900 immune effect Effects 0.000 description 1
- 230000002452 interceptive effect Effects 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 238000009533 lab test Methods 0.000 description 1
- 238000012417 linear regression Methods 0.000 description 1
- 208000019423 liver disease Diseases 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 229940045627 porcine heparin Drugs 0.000 description 1
- 102000004196 processed proteins & peptides Human genes 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 238000011002 quantification Methods 0.000 description 1
- 239000003381 stabilizer Substances 0.000 description 1
- 238000010561 standard procedure Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 229960003766 thrombin (human) Drugs 0.000 description 1
- 239000003868 thrombin inhibitor Substances 0.000 description 1
- 101150033948 tsf gene Proteins 0.000 description 1
- 238000010200 validation analysis Methods 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/56—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving blood clotting factors, e.g. involving thrombin, thromboplastin, fibrinogen
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/81—Protease inhibitors
- C07K14/815—Protease inhibitors from leeches, e.g. hirudin, eglin
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/81—Protease inhibitors
- G01N2333/815—Protease inhibitors from leeches, e.g. hirudin, eglin
-
- 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
- G01N2333/90—Enzymes; Proenzymes
- G01N2333/914—Hydrolases (3)
- G01N2333/948—Hydrolases (3) acting on peptide bonds (3.4)
- G01N2333/974—Thrombin
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2400/00—Assays, e.g. immunoassays or enzyme assays, involving carbohydrates
- G01N2400/10—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
- G01N2400/38—Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence, e.g. gluco- or galactomannans, e.g. Konjac gum, Locust bean gum, Guar gum
- G01N2400/40—Glycosaminoglycans, i.e. GAG or mucopolysaccharides, e.g. chondroitin sulfate, dermatan sulfate, hyaluronic acid, heparin, heparan sulfate, and related sulfated polysaccharides
Definitions
- This invention relates to laboratory testing useful in monitoring of anticoagulant therapy. More specifically, this invention relates to assays for the anticoagulants that are known to inhibit the enzyme thrombin.
- thrombin-specific inhibitors are in clinical trials as anticoagulant drugs for the treatment of arterial (after coronary artery angioplasty) and deep venous thrombosis.
- the specificity of these drugs is reflected in the dissociation constants ranging from 2 picomoles/L - 2 nanomoles/L.
- the thrombin specific inhibitors have demonstrated efficacy in animal models for both treatment and prevention of arterial and venous thrombosis.
- Recombinant hirudin derived from the medicinal leech Hirudo medicinalis. is the thrombin-specific inhibitor most studied. Effective doses and their range for therapeutic plasma levels in humans have been determined. The low and high ends of the range are used for venous and arterial thrombosis, respectively.
- U.S. patents have been issued on anti- thrombin agents for use as medicinals or bound to implantable materials.
- U.S. Patent 4,944,943 to Eschenfelder, et al. teaches use of hirudin and t-PA for treatment of thrombosis.
- U.S. Patent 4,952,562 to Klein, et al. discloses anti-thrombotic peptides and psuedopeptides.
- U.S. Patent 5,019,393 to Ito, et al. teaches use of implantable materials having a thrombogenesis inhibitor immobilized thereon.
- U.S. Patent 5,640,814 to Klein, et al. discloses an anti-thrombotic peptide for administration as a medicinal.
- U.S. Patent 5,087,613 to Courtney, et al. discloses hirudin variants for use as inhibitors of thrombin activity.
- U.S. Patent 5,095,092 discloses a process for isolation and purification of hirudin. None of the cited patents disclose a quantitative thrombin-time test as taught herein. Several methods are presently used to assess whether the patient taking the new thrombin-specific inhibitors is therapeutically anticoagulated.
- HPLC high performance liquid chromatography
- APTT activated partial thromboplastin time
- the APTT is a standard screening test of the coagulation system. This test is also used to follow the degree of anticoagulation in patients receiving the anticoagulant heparin.
- a variety of coagulopathies e.g. isolated or multiple factor deficiency, abnormal fibrinogen, decreased fibrinogen concentration or antiphospholipid antibodies
- Some patients with coagulopathies associated with the nephrotic syndrome or the lupus anticoagulant also develop thrombosis requiring treatment with anticoagulants. In these cases, the APTT is already prolonged and cannot be used to monitor anticoagulation with thrombin specific inhibitors.
- Another clotting assay is routinely used in a qualitative fashion in clinical laboratories to determine the presence of heparin and abnormal or low levels of fibrinogen.
- the standard thrombin time is not effective and has not been used to quantitate the concentrations of thrombin-specific inhibitors in the blood because of the inherent sensitivity of the standard thrombin time, as presently developed, to factors other than these inhibitors (Walenga JM et al. Seminars in Thrombosis Hemostasis 17:103, 1991).
- plasmas obtained from patients with liver disease, dysfibrinogenemia, hypofibrinogenemia, or who have received fibrinolytic therapy are likely to produce a prolonged standard thrombin time.
- TSI thrombin-specific inhibitor
- QTT quantitative thrombin time
- the object of the present invention is to provide a method to measure thrombin time that will not be affected by presence of abnormal plasmas which cause a prolongation of standard tests (APTT and standard thrombin time, vide supra), but will be sensitive to the presence of thrombin-specific inhibitors.
- Another object of the invention is to provide a method from which unknown plasma concentrations of thrombin-specific inhibitors can be determined by generating a standard curve for the therapeutic range of the inhibitor using the QTT.
- a related object is to provide a method which can be readily performed on laboratory instruments commonly used for measuring the standard thrombin time.
- Fig. 1 shows the effect of concentration of purified fibrinogen on the quantitative thrombin time in plasma and abnormal plasmas.
- Fig. 2 shows the effect of heparin concentration on the QTT.
- Fig. 3 is the standard curve for recombinant hirudin using the quantitative thrombin time.
- Fig. 4 shows the effect of recombinant hirudin concentration on the APTT.
- This invention provides a novel method for the quantitative determination of plasma levels of thrombin- specific inhibitors by specifically measuring thrombin time in the presence of exogenous fibrinogen and thrombin.
- Normal plasmas and plasmas with a coagulopathy have the same baseline for thrombin-specific coagulation time when tested by the QTT.
- the prolongation of the QTT will depend only on the presence of thrombin- specific anticoagulant present in the sample and will, therefore, provide a method for evaluation of the anticoagulant independent of other plasma abnormalities that could affect the thrombin time.
- the phospholipid based APTT was abandoned in favor of the thrombin-based assay which is independent of phospholipid and coagulation factors other than fibrinogen.
- the inhibitor exemplified in the testing disclosed herein was recombinant hirudin. Quantification was tested by adding recombinant hirudin to normal plasma at different concentrations within the known therapeutic range. The QTT was then tested with these plasmas and a linear relationship was shown between the log (QTT) and concentration of recombinant hirudin. This allowed the determination of concentrations of recombinant hirudin in plasma. The specificity of the QTT was assessed by adding a known concentration of recombinant hirudin to patient plasma samples demonstrating a coagulopathy (prolonged APTT and/or standard thrombin time).
- Plasmas were those submitted to the coagulation laboratory for evaluation of abnormal screening coagulation studies and from healthy volunteers who gave informed consent under a protocol approved by the Human Use
- Blood samples were collected into plastic tubes containing balanced citrate as the coagulant in a ratio of one part anticoagulant to nine parts blood. Each sample was centrifuged at 4°C and 10,000 x g for 20 minutes to obtain platelet-poor plasma which was then stored at -70°C until ready for use.
- the thrombin time was measured as the time to clot formation after adding 100 ⁇ L of a 5 U/mL solution of alpha thrombin to 100 ⁇ L of plasma and 100 ⁇ L VS buffer (vide infra). Either a dataclot-2 fibrometer (Helena Laboratories, Beaumont, TX) or an ST4 coagulation instrument (Diagnostica Stago, Parsippany, NJ) was used.
- Quantitative thrombin time Human alpha-thrombin was diluted to a concentration of 2.5-20 U/mL in veronal-saline-calcium (VSC) buffer (2.8 mM sodium diethyl-barbiturate, lOOmM sodium chloride, 25mM calcium chloride, pH 7.35) that also contained 1 % human serum albumin. Fibrinogen concentrations (0.1- 18 ⁇ M) were made in VS buffer (as VSC buffer except the buffer contained no calcium, and sodium chloride concentration was 144mM). Plasma samples were diluted 1:10 in VS buffer. The plasma dilutions, alpha-thrombin and fibrinogen solutions were incubated separately at 37°C for six minutes before using.
- VSC veronal-saline-calcium
- Equal volumes of plasma samples and fibrinogen were mixed and incubated at 37°C for one minute. 200 ⁇ L of the plasma-fibrinogen mixture were removed and placed in an assay well. The thrombin time was measured as the time to clot formation after adding 100 ⁇ L of the alpha- thrombin solution. Instruments used for the standard thrombin time were also used for the QTT.
- the APTT assay was done using a CP-8 photooptic coagulation profiler (BIODATA Corporation,
- the reptilase time was done using a dataclot-2 fibrometer. 100 ⁇ L of reconstituted reptilase was added to 200 ⁇ L of plasma and the clotting time was recorded. Preparation of plasma samples for standard curve determination:
- Recombinant hirudin (lyophilized preparation) was diluted in normal pooled plasma to a concentration of 50 ⁇ g/mL. Further dilutions in normal plasma were made to obtain concentrations within the therapeutic range (0.1 -
- Standard curve The log of the QTTs were calculated and a standard curve was generated after linear regression by plotting the log (QTT) vs concentration of recombinant hirudin ( ⁇ g/mL). From the standard curve, an unknown plasma level of recombinant hirudin can be determined.
- Heparin (initial concentration 1000 u/mL) was diluted in normal pooled plasma to a concentration of 10 U/mL. Further dilutions in plasma were made to obtain heparin concentrations of 0.1 - 8 U/mL.
- the effect of concentration of purified fibrinogen on the quantitative thrombin time in plasma and abnormal plasmas was determined. Different fibrinogen concentrations were used in the quantitative thrombin time to determine at which concentration the effects of the abnormal plasmas were eliminated.
- Plasmas were diluted 1 :10 in buffer; 100 ⁇ L of the diluted plasma was added to 100 ⁇ L 9 ⁇ M purified human fibrinogen and incubated for 30 seconds; 100 ⁇ L of 5 U/mL human alpha-thrombin was added to start the reaction; the clotting time was measured.
- heparin concentration on the QTT Normal plasma supplemented with heparin at various concentrations were tested in the QTT. Procedure: Plasma was supplemented with porcine heparin at different concentrations (0.1-10 U/mL); these plasmas were diluted 1:10 in buffer; 100 ⁇ L of the diluted plasma was added to 100 ⁇ L purified human fibrinogen (9 ⁇ M) and incubated for 30 seconds; 100 ⁇ L of 5 U/mL human alpha-thrombin was added to start the reaction; the clotting time was measured. The QTT became sensitive to heparin at 0.8 U/mL (Fig. 2). The therapeutic range for heparin is 0.3-0.5 U/mL.
- thrombin concentrations were tested in developing assays for recombinant hirudin.
- Low thrombin concentrations were too sensitive to high therapeutic plasma levels of recombinant hirudin whereas high thrombin concentrations were insensitive to low therapeutic levels.
- a 1 : 1 dilution in pooled plasma was required before the 1 :10 dilution in the assay.
- the optimal conditions for the QTT were: 100 ⁇ L 1 :10 plasma dilution, 100 ⁇ L fibrinogen (9 ⁇ M) and 100 ⁇ L alpha-thrombin (5 U/mL).
- the validity of the QTT in determining plasma recombinant hirudin levels was assessed by adding known concentrations of recombinant hirudin to abnormal and normal pooled plasma. The QTT was measured and the recombinant hirudin concentration determined from the standard curve described above. Table 3 demonstrates that a number of coagulopathies that may prolong the standard thrombin time do not interfere with the measurement of recombinant hirudin levels as determined by the QTT.
- Plasma was supplemented with purified recombinant hirudin at different concentrations (0.1 -1.75 ⁇ g/mL); these plasmas were diluted 1 :10 in buffer; 100 ⁇ L of the diluted plasma was added to 100 ⁇ L purified human fibrinogen (9 ⁇ M) and incubated for 30 seconds; 100 ⁇ L of 5U/mL human alpha-thrombin was added to start the reaction; the clotting time was measured.
- Table 3
- Recombinant hirudin was added to all plasma samples at a final concentration of 1.25 ⁇ g/mL.
- a Nexpected-measuredX x 100 ⁇ 1.25 ⁇ g/mL - measured ⁇ x 100 expected 1.25 ⁇ g/mL
- Antithrombin UI 54% (normal 88-140); c factor XII ⁇ 10% (normal 50-
- Plasma was supplemented with purified recombinant hirudin at different concentrations (0.1-4 ⁇ g mL); 100 ⁇ L of the APTT reagent was added to 100 ⁇ L of the plasma supplemented with recombinant hirudin and incubated for 6 minutes; 100 ⁇ L of 25 mM CaCl 2 was added to start the reaction; the clotting time was measured.
- Fig. 4 demonstrates that the APTT is not useful at the low end of the therapeutic ranges for recombinant hirudin.
- Plasma was supplemented with purified recombinant hirudin at different concentrations (0.1-4 ⁇ g/mL); 100 ⁇ L of the APTT reagent was added to 100 ⁇ L of the plasma supplemented with recombinant hirudin and incubated for 6 minutes; 100 ⁇ L of 25 mM CaCl 2 was added to start the reaction; the clotting time was measured.
- Table 4 conclusively shows that the presence of a lupus anticoagulant, abnormal fibrinogens, heparin and fibrin degradation products interfere with the APTT assay for the recombinant hirudin.
- Coagulopathies that interfere with the APTT interfere with the determination of plasma levels of recombinant hirudin as performed with the APTT.
- Recombinant hirudin was added to all plasma samples at a final concentration of 1.25 ⁇ g/mL.
- a - indicates value could not be determined.
- c fibrinogen 38 mg/dL (normal 200-400);
- d standard thrombin time 38 mg/dL (normal 200-400);
- the thrombin-specific inhibitor hirudin and its analogues have demonstrated efficacy in the prevention of thrombosis in preclinical studies.
- the anticoagulant effects have been monitored by the APTT because the standard thrombin time is too sensitive to the agents to be of clinical utility.
- the APTT has two major problems that limit its usefulness in monitoring recombinant hirudin: 1) patients with a prolonged baseline APTT cannot be followed (this also holds true for patients receiving heparin therapy); and 2) there is a nonlinear dose-response at low therapeutic levels of recombinant hirudin with regard to the APTT (Fig. 4).
- the clinical consequence of using the APTT to monitor therapy in the low therapeutic range is excessive anticoagulation for the specific clinical condition.
- the problems which the invention is designed to solve are the sensitivity of standard tests to therapeutic heparin levels, low levels of fibrinogen, abnormal fibrinogens and fibrin degradation products, lupus anticoagulant (antiphospholipid antibodies), and low plasma factor levels.
- the method is simple and can be done in any lab that performs thrombin times routinely (most hospitals). The procedure is specifically adapted for use on instruments presently available to clinical laboratories. The newer automated equipment and methods used in determination of thrombin time may be used for practice of the invention. The method is also quick and requires no additional expertise above that already available in the clinical laboratory. A small amount of patient material is needed (25-50 ⁇ L of plasma for 4 tests); where standard tests require 100 ⁇ L for a single test.
- the method allows quantitation of plasma levels of recombinant hirudin and other thrombin-specific inhibitors for use with other thrombin-specific inhibitors (e.g. synthetic or semisynthetic analogues of recombinant hirudin).
- the mean error is less than 10%.
- the standard curve is linear in the therapeutic range, where standard tests are either not linear in the low therapeutic range (APTT) or are too sensitive to recombinant hirudin (standard thrombin time).
- the method is more sensitive to thrombin-specific inhibitors in comparison to standard tests, as demonstrated by the response of the clotting times to recombinant hirudin concentrations.
- the test is inexpensive and fast in comparison to HPLC.
- mutant or normal thrombin from other sources may be used in the QTT so long as the alternate thrombin provides a reliable dosage curve when tested against the specific thrombin-specific inhibitor for which concentration is being determined.
- the fibrinogen can be from any source so long as the fibrinogen is functional in (normal) clot formation.
- a veronal based buffer was used in the development of the QTT, any buffer that provides a reliable dosage curve when tested against a TSI can be used.
- the materials for the QTT may be sold as kits.
- the following components could be provided in the kit:
- T J thrombin-specific inhibitor
- the contents of the vial(s) may be reconstituted with water so that the final concentration of the TSI is such that serial dilutions allow for TSI within the therapeutic range.
- the addition of the protamine sulfate may be used to effectively remove heparin from the reaction. If protamine sulfate is used, a preferred concentration in the final diluted TSI-containing solution would be about 120 ⁇ g/mL. However, in some instances the TSI may be available to laboratories as a stock solution(s).
- One or more vials of lyophilized alpha-thrombin which may be formulated with a stabilizing agent such as albumin, may contain buffer should be provided.
- the thrombin could be reconstituted with water to provide the desired concentration.
- the plasma may contain a neutralizing agent such as protamine sulfate.
- the kit can, if desired, hold a separate vial of protamine sulfate or another heparin neutralizing agent to be used with TSI and patient samples when a patient is on heparin therapy.
- Vials of buffer for diluting samples may be included.
- the standard curve may be developed to identify effective levels of TSI by the following method: a. Preparing serial dilutions of a thrombin- specific inhibitor in normal plasma in known concentrations to provide samples (though a kit containing varying amounts of TSI within the appropriate range will avoid this procedure). b. Dilute the samples in buffer to provide 1:10 dilutions. c. Mixing the dilutions with a 9-18 ⁇ M solution of purified human fibrinogen to provide samples. d. Adding a 5 U/mL solution of purified human alpha-thrombin to the samples of step c to provide solutions. e. Measuring the clotting times of the solutions in step d. f. Plotting a standard curve from the results obtained from step e.
- the level of TSI concentration in patient plasma is tested in the following manner to determine the TSI level in the patient: 1. Diluting in buffer a plasma sample from patients receiving a thrombin-specific inhibitor to provide a 1:10 plasma to buffer concentration.
- step 3 Measuring the clotting times of the solutions of step 3. 5. Determining the concentration of the inhibitor in the patient samples using the standard curve previously developed.
- the invention may also be practiced using whole blood rather than patient plasma. Vials containing varying amounts of the TSI required for generating the standard curve would be made by diluting the TSI to provide varying dilutions for use in determining the curve.
- the normal plasma is reconstituted by dilution with buffered water (or the buffer may be added into the diluent) to provide varying concentrations of TSI.
- a second set of containers containing only plasma is prepared for purposes of comparison.
- a third container is prepared containing 1 mL of patient plasma with unknown concentration of TSI. To each sample is added 475 ⁇ L of reconstituted fibrinogen solution and. 50 ⁇ L reconstituted thrombin. The clotting time is then measured.
- Steps a to f may be used as a research tool to evaluate activity of TSFs.
- the running of a standard to obtain the curve may be unnecessary. In such instances, the QTT alone without the comparison testing will be sufficient to allow practitioners to evaluate concentration of TSI in the blood.
- the kit may contain only fibrinogen and thrombin, whilst one or more stock solutions containing appropriate concentrations of TSI in plasma is maintained for use in the test. (It would also be possible to have one stock concentration at highest level to be tested, which would be diluted to provide lower concentrations for use to provide the appropriate curve.)
- the extent to which stock solutions are used or multiple vials of TSI containing differing amounts of TSI are provided will depend on the number of tests done in any particular health care center and the quality of staff available to provide testing services. All of the components, plasma, thrombin, and fibrinogen, should be used fresh or be frozen to avoid loss of potency. All of the agents used, including plasma with TSI, normal plasma, thrombin, and fibrinogen may be provided as lyophilized materials in vials for reconstitution. Once reconstituted, the active components are easily inactivated.
- the uses for which the invention is suitable include quantitative clinical monitoring of patients anticoagulated with the thrombin-specific agents and laboratory research in animal models of thrombosis.
Landscapes
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Wood Science & Technology (AREA)
- Biophysics (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Zoology (AREA)
- Molecular Biology (AREA)
- Biochemistry (AREA)
- Engineering & Computer Science (AREA)
- Analytical Chemistry (AREA)
- Microbiology (AREA)
- Physics & Mathematics (AREA)
- Hematology (AREA)
- Gastroenterology & Hepatology (AREA)
- Biotechnology (AREA)
- Immunology (AREA)
- Neurosurgery (AREA)
- Tropical Medicine & Parasitology (AREA)
- Medicinal Chemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Investigating Or Analysing Biological Materials (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
A quantitative method for determining the plasma levels of thrombin-specific inhibitors is based on the quantitative thrombin time using plasma dilutions, excess fibrinogen and thrombin. The plasma dilutions and excess fibrinogen act in concert to eliminate the effects that coagulopathies have on standard coagulation tests. The method is relatively simple and provides superior results to standard conventional tests. The method is suitable for performance in clinical hematology laboratories on a routine basis using commercially available instrumentation.
Description
Title: TEST FOR QUANTITATIVE THROMBIN TIME
Field of the Invention
This invention relates to laboratory testing useful in monitoring of anticoagulant therapy. More specifically, this invention relates to assays for the anticoagulants that are known to inhibit the enzyme thrombin.
Background of the Invention Presently, thrombin-specific inhibitors are in clinical trials as anticoagulant drugs for the treatment of arterial (after coronary artery angioplasty) and deep venous thrombosis. The specificity of these drugs is reflected in the dissociation constants ranging from 2 picomoles/L - 2 nanomoles/L. The thrombin specific inhibitors have demonstrated efficacy in animal models for both treatment and prevention of arterial and venous thrombosis. Recombinant hirudin, derived from the medicinal leech Hirudo medicinalis. is the thrombin-specific inhibitor most studied. Effective doses and their range for therapeutic plasma levels in humans have been determined. The low and high ends of the range are used for venous and arterial thrombosis, respectively.
Several U.S. patents have been issued on anti- thrombin agents for use as medicinals or bound to implantable materials. U.S. Patent 4,944,943 to Eschenfelder, et al.
teaches use of hirudin and t-PA for treatment of thrombosis. U.S. Patent 4,952,562 to Klein, et al. discloses anti-thrombotic peptides and psuedopeptides. U.S. Patent 5,019,393 to Ito, et al. teaches use of implantable materials having a thrombogenesis inhibitor immobilized thereon. U.S. Patent
5,053,453 to Ku discloses hirudin or hirudin derivatives covalently linked to support materials to avoid formation of thrombi. U.S. Patent 5,640,814 to Klein, et al. discloses an anti-thrombotic peptide for administration as a medicinal. U.S. Patent 5,087,613 to Courtney, et al. discloses hirudin variants for use as inhibitors of thrombin activity. U.S. Patent 5,095,092 discloses a process for isolation and purification of hirudin. None of the cited patents disclose a quantitative thrombin-time test as taught herein. Several methods are presently used to assess whether the patient taking the new thrombin-specific inhibitors is therapeutically anticoagulated. It is possible to measure plasma thrombin-specific inhibitors levels by high performance liquid chromatography (HPLC). Though accurate, HPLC is generally available only in large referral hospitals and commercial labs and requires a high level of staff expertise. Even when available in large referral hospitals, the technique is time consuming. Hence, there is delay in obtaining results. This delay is unacceptable when a clinical practitioner is attempting to closely monitor and control the progress of the patient.
Another approach is to monitor the prolongation of the activated partial thromboplastin time (APTT). The APTT is a standard screening test of the coagulation system. This test is also used to follow the degree of anticoagulation in patients receiving the anticoagulant heparin. However, in the absence of specific thrombin inhibitors a variety of coagulopathies (e.g. isolated or multiple factor deficiency, abnormal fibrinogen, decreased fibrinogen concentration or antiphospholipid antibodies) tend to prolong the APTT results.
Some patients with coagulopathies associated with the nephrotic syndrome or the lupus anticoagulant also develop thrombosis requiring treatment with anticoagulants. In these cases, the APTT is already prolonged and cannot be used to monitor anticoagulation with thrombin specific inhibitors.
Another clotting assay, the standard thrombin time, is routinely used in a qualitative fashion in clinical laboratories to determine the presence of heparin and abnormal or low levels of fibrinogen. The standard thrombin time is not effective and has not been used to quantitate the concentrations of thrombin-specific inhibitors in the blood because of the inherent sensitivity of the standard thrombin time, as presently developed, to factors other than these inhibitors (Walenga JM et al. Seminars in Thrombosis Hemostasis 17:103, 1991). In addition, plasmas obtained from patients with liver disease, dysfibrinogenemia, hypofibrinogenemia, or who have received fibrinolytic therapy are likely to produce a prolonged standard thrombin time. Besides HPLC and the APTT, further methods including amidolytic assays for thrombin and immunologic assays for the hirudin-thrombin complex have been suggested. However, the techniques are not easily applied to the clinical laboratory. Because of the potential therapeutic efficacy of the thrombin-specific inhibitors, a new and improved laboratory test for determining plasma levels that can be performed in clinical laboratories with presently available instrumentation is needed. Furthermore, such a new process capable of providing results that are easily interpreted by clinical practitioners would be of great utility.
Summary of the Invention
A novel clinically useful test for monitoring patients on thrombin-specific inhibitor (TSI) therapy, a quantitative thrombin time (QTT) for the management of
anticoagulation with thrombin-specific inhibitors, based on the specificity of the TSI for thrombin is disclosed and claimed herein.
The object of the present invention is to provide a method to measure thrombin time that will not be affected by presence of abnormal plasmas which cause a prolongation of standard tests (APTT and standard thrombin time, vide supra), but will be sensitive to the presence of thrombin-specific inhibitors. Another object of the invention is to provide a method from which unknown plasma concentrations of thrombin-specific inhibitors can be determined by generating a standard curve for the therapeutic range of the inhibitor using the QTT. A related object is to provide a method which can be readily performed on laboratory instruments commonly used for measuring the standard thrombin time.
Brief Description of the Drawings
Fig. 1 shows the effect of concentration of purified fibrinogen on the quantitative thrombin time in plasma and abnormal plasmas.
Fig. 2 shows the effect of heparin concentration on the QTT.
Fig. 3 is the standard curve for recombinant hirudin using the quantitative thrombin time. Fig. 4 shows the effect of recombinant hirudin concentration on the APTT.
Detailed Description of the Invention
This invention provides a novel method for the quantitative determination of plasma levels of thrombin- specific inhibitors by specifically measuring thrombin time in the presence of exogenous fibrinogen and thrombin. Normal plasmas and plasmas with a coagulopathy have the same baseline for thrombin-specific coagulation time when tested by the QTT. By eliminating the interfering effects of factors in
the plasmas from patients with coagulopathy, the prolongation of the QTT will depend only on the presence of thrombin- specific anticoagulant present in the sample and will, therefore, provide a method for evaluation of the anticoagulant independent of other plasma abnormalities that could affect the thrombin time. To eliminate the effect of abnormalities such as antiphospholipid antibodies, and factor deficiencies, the phospholipid based APTT was abandoned in favor of the thrombin-based assay which is independent of phospholipid and coagulation factors other than fibrinogen.
Only fibrin degradation products, heparin, and abnormal fibrinogens or a decreased fibrinogen potentially present in plasma could interfere with the assay. The effect of these abnormalities can be removed by diluting plasma 1 :10 in buffer and adding excess fibrinogen.
While the test developed has been shown to be effective when using other thrombin-specific inhibitors, the inhibitor exemplified in the testing disclosed herein was recombinant hirudin. Quantification was tested by adding recombinant hirudin to normal plasma at different concentrations within the known therapeutic range. The QTT was then tested with these plasmas and a linear relationship was shown between the log (QTT) and concentration of recombinant hirudin. This allowed the determination of concentrations of recombinant hirudin in plasma. The specificity of the QTT was assessed by adding a known concentration of recombinant hirudin to patient plasma samples demonstrating a coagulopathy (prolonged APTT and/or standard thrombin time). The expected and observed values of recombinant hirudin levels were then compared and shown to be compatible with use of the QTT in the clinical monitoring of patients on recombinant hirudin. It was shown, by comparative evaluation, that the APTT does not provide the specificity provided by the inventive method.
The technique was performed using two different commercially available instruments and equivalent results were obtained.
Methods and materials:
Materials:
Plasmas were those submitted to the coagulation laboratory for evaluation of abnormal screening coagulation studies and from healthy volunteers who gave informed consent under a protocol approved by the Human Use
Committee of the Walter Reed Army Institute of Research. Human alpha-thrombin (specific activity 2,000-4,000 NIH units/mg protein), recombinant hirudin (specific activity 8,500 units/mg protein) and reptilase (ATROXIN) were purchased from Sigma Chemical Company (St. Louis, MO). Human fibrinogen was supplied by KabiVitrum (Stockholm, Sweden). APTT reagent was purchased from Organon Teknika Corporation (Durham, NC). Sodium heparin (1000 U/mL) was purchased from Elkins-Sinn, Incorporated (Cherry Hill, NJ).
Blood sample preparation:
Blood samples were collected into plastic tubes containing balanced citrate as the coagulant in a ratio of one part anticoagulant to nine parts blood. Each sample was centrifuged at 4°C and 10,000 x g for 20 minutes to obtain platelet-poor plasma which was then stored at -70°C until ready for use.
Standard thrombin time:
The thrombin time was measured as the time to clot formation after adding 100 μL of a 5 U/mL solution of alpha thrombin to 100 μL of plasma and 100 μL VS buffer (vide infra). Either a dataclot-2 fibrometer (Helena
Laboratories, Beaumont, TX) or an ST4 coagulation instrument (Diagnostica Stago, Parsippany, NJ) was used.
Quantitative thrombin time (QTT : Human alpha-thrombin was diluted to a concentration of 2.5-20 U/mL in veronal-saline-calcium (VSC) buffer (2.8 mM sodium diethyl-barbiturate, lOOmM sodium chloride, 25mM calcium chloride, pH 7.35) that also contained 1 % human serum albumin. Fibrinogen concentrations (0.1- 18μM) were made in VS buffer (as VSC buffer except the buffer contained no calcium, and sodium chloride concentration was 144mM). Plasma samples were diluted 1:10 in VS buffer. The plasma dilutions, alpha-thrombin and fibrinogen solutions were incubated separately at 37°C for six minutes before using. Equal volumes of plasma samples and fibrinogen were mixed and incubated at 37°C for one minute. 200 μL of the plasma-fibrinogen mixture were removed and placed in an assay well. The thrombin time was measured as the time to clot formation after adding 100 μL of the alpha- thrombin solution. Instruments used for the standard thrombin time were also used for the QTT.
APTT assav:
The APTT assay was done using a CP-8 photooptic coagulation profiler (BIODATA Corporation,
Willow Grove, PA). 100 μL of plasma was incubated with 100 μL APTT reagent for six minutes. The APTT was measured as the time of clot formation after adding 100 μL 25 mM CaCb.
Reptilase time:
The reptilase time was done using a dataclot-2 fibrometer. 100 μL of reconstituted reptilase was added to 200 μL of plasma and the clotting time was recorded.
Preparation of plasma samples for standard curve determination:
Recombinant hirudin (lyophilized preparation) was diluted in normal pooled plasma to a concentration of 50 μg/mL. Further dilutions in normal plasma were made to obtain concentrations within the therapeutic range (0.1 -
3 μg/mL).
Standard curve: The log of the QTTs were calculated and a standard curve was generated after linear regression by plotting the log (QTT) vs concentration of recombinant hirudin (μg/mL). From the standard curve, an unknown plasma level of recombinant hirudin can be determined.
Preparation of plasma samples for determining effect of heparin concentration on the QTT:
Heparin (initial concentration 1000 u/mL) was diluted in normal pooled plasma to a concentration of 10 U/mL. Further dilutions in plasma were made to obtain heparin concentrations of 0.1 - 8 U/mL.
The QTT in normal plasma samples:
The results of the QTT from twenty normal individuals (ten male, ten female) are shown in Table 1 ; all had a normal standard thrombin time and APTT.
Table 1
Summary statistics based on plasmas from twenty normal individuals, ten male and ten female. All plasma samples had normal APTT and standard thrombin time, usinε standard procedures (see text).
a standard error of the mean; b standard deviation; c root mean square difference.
Effect of fibrinogen concentration on the QTT:
The effect of concentration of purified fibrinogen on the quantitative thrombin time in plasma and abnormal plasmas was determined. Different fibrinogen concentrations were used in the quantitative thrombin time to determine at which concentration the effects of the abnormal plasmas were eliminated.
Procedure:
Plasmas were diluted 1 :10 in buffer; 100 μL of the diluted plasma was added to 100 μL purified human fibrinogen at different concentrations (0.1 - 18 μM) and incubated for 30 seconds; 100 μL of 5U/mL human alpha- thrombin was added to start the reaction; the clotting time was measured. The addition of excess fibrinogen in the assay eliminated the effects of abnormal fibrinogens (Fig. 1) and low fibrinogen (Fig. 1, insert). Plasmas with an abnormal fibrinogen (dysfibrinogenemia: standard thrombin time = 45.2 and 18.5 sec. [normal 11-14], reptilase time = 22.6 and 15.5 sec Lnormal 8-11 ]), low fibrinogen (hypofibrinogenemia: fibrinogen = 38 mg/dL [normal 200-400]) and plasma with increased fibrin degradation products (FDP = 1024 μg/mL [normal <10 μg/mL]) and low fibrinogen (fibrinogen = 112
mg/dL) were evaluated. The optimal fibrinogen concentration was determined to be 9-18 μM. This concentration was more than sufficient to overcome the effects of abnormal plasma samples.
Effect of diluting plasma (in buffer) on the QTT:
Diluting the plasma samples 1 : 10 in buffer mitigated the effects of fibrin degradation products and therapeutic heparin levels (Table 2). The presence of antiphospholipid antibodies did not interfere with the assay.
Procedure:
Plasmas were diluted 1 :10 in buffer; 100 μL of the diluted plasma was added to 100 μL 9 μM purified human fibrinogen and incubated for 30 seconds; 100 μL of 5 U/mL human alpha-thrombin was added to start the reaction; the clotting time was measured.
Table 2. Coagulopathies that might otherwise interfere with standard clotting tests, do not interfere with the quantitative thrombin time.a
Table 2
a* Abnormal value. D Normal: 12.6-15.2 seconds; c normal: 1 1-14 seconds; d normal: 22-34 seconds. e reptilase time = 29.4 sec (normal 8- 11); f reptilase time = 22.6; S reptilase time = 15.5; -- reptilase time = 15.2; 1 fibrin degradation products = 128 μg/mL (normal < 10), fibrinogen = 225 mg/dL (normal 200-400); J fibrin degradation products = 1024 μg/mL, fibrinogen = 112 mg/dL; k factor II = 37%, factor X = 16% (normal 50-
150); • 0.5 U heparin/mL; m fibrinogen = 38 mg/dL; n factor XII < 10% (normal 50-150).
Effect of heparin concentration on the QTT: Normal plasma supplemented with heparin at various concentrations were tested in the QTT. Procedure: Plasma was supplemented with porcine heparin at different concentrations (0.1-10 U/mL); these plasmas were diluted 1:10 in buffer; 100 μL of the diluted plasma was added to 100 μL purified human fibrinogen (9 μM) and incubated for 30 seconds; 100 μL of 5 U/mL human alpha-thrombin was added to start the reaction; the clotting time was measured. The QTT became sensitive to heparin at 0.8 U/mL (Fig. 2). The therapeutic range for heparin is 0.3-0.5 U/mL.
Standard curve for recombinant hirudin using the QTT:
The quantitative thrombin time was determined for different known concentrations of recombinant hirudin in plasma. A standard curve was developed by plotting the LOG (quantitative thrombin time) vs concentration of recombinant hirudin. Procedure: Plasma was supplemented with purified recombinant hirudin at different concentrations (0.1 -1.75 μg/mL); these plasmas were diluted 1 :10 in buffer; 100 μL of the diluted plasma was added to 100 μL purified human fibrinogen (9 μM) and incubated for 30 seconds; 100 μL of human alpha-thrombin (5 U/mL) was added to start the reaction; the clotting time was measured.
Several thrombin concentrations were tested in developing assays for recombinant hirudin. Low thrombin concentrations were too sensitive to high therapeutic plasma levels of recombinant hirudin whereas high thrombin concentrations were insensitive to low therapeutic levels. There was no thrombin concentration that was effective over the entire therapeutic range of recombinant hirudin. Therefore, a thrombin concentration (5 U/mL) sensitive at the
low therapeutic range (0.1 -1.75 μg/mL) of recombinant hirudin was selected (Fig. 3). The hirudin-free plasma data point was not included in the standard curve. To assay hirudin concentrations greater than 1.75 μg/mL, a 1 : 1 dilution in pooled plasma was required before the 1 :10 dilution in the assay. The optimal conditions for the QTT were: 100 μL 1 :10 plasma dilution, 100 μL fibrinogen (9 μM) and 100 μL alpha-thrombin (5 U/mL).
Validity of the QTT in determining plasma concentrations of recombinant hirudin:
The validity of the QTT in determining plasma recombinant hirudin levels was assessed by adding known concentrations of recombinant hirudin to abnormal and normal pooled plasma. The QTT was measured and the recombinant hirudin concentration determined from the standard curve described above. Table 3 demonstrates that a number of coagulopathies that may prolong the standard thrombin time do not interfere with the measurement of recombinant hirudin levels as determined by the QTT.
Procedure:
Plasma was supplemented with purified recombinant hirudin at different concentrations (0.1 -1.75 μg/mL); these plasmas were diluted 1 :10 in buffer; 100 μL of the diluted plasma was added to 100 μL purified human fibrinogen (9 μM) and incubated for 30 seconds; 100 μL of 5U/mL human alpha-thrombin was added to start the reaction; the clotting time was measured.
Table 3
Recombinant hirudin was added to all plasma samples at a final concentration of 1.25 μg/mL. a Nexpected-measuredX x 100 =\1.25 μg/mL - measured\x 100 expected 1.25 μg/mL b Antithrombin UI = 54% (normal 88-140); c factor XII < 10% (normal 50-
150); standard thrombin time = 18.5 sec (normal 11-14), reptilase time =
15.5 sec (normal 8-11); e standard thrombin time = 45.2 sec, reptilase time
= 22.6 sec; *" standard thrombin time = 18.1 sec, reptilase time = 13.1 sec: S fibrin degradation products = 1024 μg/mL (normal <10), fibrinogen = 183 mg/dL (normal 200-400); n fibrin degradation products = 256 μg/mL, fibrinogen = 360 mg/dL; - fibrin degradation products = 1024 μg/mL, fibrinogen = 112 mg/dL; J fibrinogen = 38 mg/dL; ^0.5 U heparin/mL; - factor II = 37%, factor X = 16% (normal 50-150).
Standard curve for recombinant hirudin using the APTT:
The effect of recombinant hirudin concentrations on the APTT was measured. Normal plasma was supplemented with recombinant hirudin at various concentrations and the APTT was measured.
Procedure: Plasma was supplemented with purified recombinant hirudin at different concentrations (0.1-4 μg mL); 100 μL of the APTT reagent was added to 100 μL of the plasma supplemented with recombinant hirudin and incubated for 6 minutes; 100 μL of 25 mM CaCl2 was added to start the reaction; the clotting time was measured. Fig. 4 demonstrates that the APTT is not useful at the low end of the therapeutic ranges for recombinant hirudin.
Procedure: Plasma was supplemented with purified recombinant hirudin at different concentrations (0.1-4 μg/mL); 100 μL of the APTT reagent was added to 100 μL of the plasma supplemented with recombinant hirudin and incubated for 6 minutes; 100 μL of 25 mM CaCl2 was added to start the reaction; the clotting time was measured.
Table 4 conclusively shows that the presence of a lupus anticoagulant, abnormal fibrinogens, heparin and fibrin degradation products interfere with the APTT assay for the recombinant hirudin.
Table 4
Coagulopathies that interfere with the APTT, interfere with the determination of plasma levels of recombinant hirudin as performed with the APTT.a
Recombinant hirudin was added to all plasma samples at a final concentration of 1.25 μg/mL. a - indicates value could not be determined. b \expected-measured\ x 100 =\1.25 μg/mL - measured\ x 100 expected 1.25 μg/mL c fibrinogen = 38 mg/dL (normal 200-400); d standard thrombin time =
45.2 sec (normal 11-14), reptilase time = 22.6 sec (normal 8-11): e standard thrombin time = 18.5 sec, reptilase time = 15.5 sec; fibrin degradation products = 1024 μg/mL (normal < 10), fibrinogen = 112 mg/dL; S fibrin
degradation products = 256 μg/mL, fibrinogen = 360 mg/dL; n 0.5 U heparin/mL; - factor XII < 10% (normal 50-150).
Comparison of results from the QTT and APTT using abnormal plasmas supplemented with recombinant hirudin:
The mean errors for determining the concentration of recombinant hirudin in abnormal plasma using the QTT were less than 10%. In contrast, using the same plasmas, the measured concentration of recombinant hirudin using the standard APTT showed no agreement with the expected value (Table 5).
Table 5 Summary of Data for validation of the QTT in different clinical conditions.
The thrombin-specific inhibitor hirudin and its analogues have demonstrated efficacy in the prevention of thrombosis in preclinical studies. The anticoagulant effects have been monitored by the APTT because the standard thrombin time is too sensitive to the agents to be of clinical utility. The APTT has two major problems that limit its usefulness in monitoring recombinant hirudin: 1) patients with a prolonged baseline APTT cannot be followed (this also holds true for patients receiving heparin therapy); and 2) there is a nonlinear dose-response at low therapeutic levels of recombinant hirudin with regard to the APTT (Fig. 4). The clinical consequence of using the APTT to monitor therapy in
the low therapeutic range is excessive anticoagulation for the specific clinical condition. We have developed a quantitative thrombin time that eliminates the inherent difficulties associated with the APTT and the standard thrombin time. The problems which the invention is designed to solve are the sensitivity of standard tests to therapeutic heparin levels, low levels of fibrinogen, abnormal fibrinogens and fibrin degradation products, lupus anticoagulant (antiphospholipid antibodies), and low plasma factor levels. Some of the novel aspects of the invention include
1 ) the addition of fibrinogen in excess to compensate for abnormal or low fibrinogen and 2) the plasma samples are diluted 1 :10 in buffer which effectively prevents interference of fibrin degradation products, abnormal fibrinogen and heparin. There are several distinct advantages of the invention. The method is simple and can be done in any lab that performs thrombin times routinely (most hospitals). The procedure is specifically adapted for use on instruments presently available to clinical laboratories. The newer automated equipment and methods used in determination of thrombin time may be used for practice of the invention. The method is also quick and requires no additional expertise above that already available in the clinical laboratory. A small amount of patient material is needed (25-50 μL of plasma for 4 tests); where standard tests require 100 μL for a single test.
The method allows quantitation of plasma levels of recombinant hirudin and other thrombin-specific inhibitors for use with other thrombin-specific inhibitors (e.g. synthetic or semisynthetic analogues of recombinant hirudin). The mean error is less than 10%. The standard curve is linear in the therapeutic range, where standard tests are either not linear in the low therapeutic range (APTT) or are too sensitive to recombinant hirudin (standard thrombin time). The method is more sensitive to thrombin-specific inhibitors in comparison to standard tests, as demonstrated by the response of the
clotting times to recombinant hirudin concentrations. Finally, the test is inexpensive and fast in comparison to HPLC.
While normal human thrombin was used in the procedure as exemplified, mutant or normal thrombin from other sources may be used in the QTT so long as the alternate thrombin provides a reliable dosage curve when tested against the specific thrombin-specific inhibitor for which concentration is being determined. Similarly, the fibrinogen can be from any source so long as the fibrinogen is functional in (normal) clot formation. Although a veronal based buffer was used in the development of the QTT, any buffer that provides a reliable dosage curve when tested against a TSI can be used.
The materials for the QTT may be sold as kits. The following components could be provided in the kit:
1) One or more vials of lyophilized plasma taining a known amount of thrombin-specific inhibitor (T J which may contain protamine sulfate should be provided. The contents of the vial(s) may be reconstituted with water so that the final concentration of the TSI is such that serial dilutions allow for TSI within the therapeutic range. The addition of the protamine sulfate may be used to effectively remove heparin from the reaction. If protamine sulfate is used, a preferred concentration in the final diluted TSI-containing solution would be about 120 μg/mL. However, in some instances the TSI may be available to laboratories as a stock solution(s).
2) One or more vials of lyophilized alpha-thrombin which may be formulated with a stabilizing agent such as albumin, may contain buffer should be provided. The thrombin could be reconstituted with water to provide the desired concentration.
3) One or more vials of lyophilized fibrinogen to be reconstituted to a final concentration of about 4.75 μM (160 mg/dL). The plasma may contain a neutralizing agent such as protamine sulfate. The kit can, if desired, hold a separate vial of protamine sulfate or another heparin neutralizing agent to be used with TSI and patient samples when a patient is on heparin therapy.
4) Vials of buffer for diluting samples may be included.
Using the kit items, the following procedure may be followed. In a preferred embodiment the standard curve may be developed to identify effective levels of TSI by the following method: a. Preparing serial dilutions of a thrombin- specific inhibitor in normal plasma in known concentrations to provide samples (though a kit containing varying amounts of TSI within the appropriate range will avoid this procedure). b. Dilute the samples in buffer to provide 1:10 dilutions. c. Mixing the dilutions with a 9-18 μM solution of purified human fibrinogen to provide samples. d. Adding a 5 U/mL solution of purified human alpha-thrombin to the samples of step c to provide solutions. e. Measuring the clotting times of the solutions in step d. f. Plotting a standard curve from the results obtained from step e.
The level of TSI concentration in patient plasma is tested in the following manner to determine the TSI level in the patient:
1. Diluting in buffer a plasma sample from patients receiving a thrombin-specific inhibitor to provide a 1:10 plasma to buffer concentration.
2. Mixing the patient samples with a solution of fibrinogen.
3. Adding a thrombin solution to the patient samples prepared in step 2.
4. Measuring the clotting times of the solutions of step 3. 5. Determining the concentration of the inhibitor in the patient samples using the standard curve previously developed.
The invention may also be practiced using whole blood rather than patient plasma. Vials containing varying amounts of the TSI required for generating the standard curve would be made by diluting the TSI to provide varying dilutions for use in determining the curve. The normal plasma is reconstituted by dilution with buffered water (or the buffer may be added into the diluent) to provide varying concentrations of TSI. A second set of containers containing only plasma is prepared for purposes of comparison. A third container is prepared containing 1 mL of patient plasma with unknown concentration of TSI. To each sample is added 475 μL of reconstituted fibrinogen solution and. 50 μL reconstituted thrombin. The clotting time is then measured. Steps a to f may be used as a research tool to evaluate activity of TSFs. However, as more is known about the effect on QTT of various concentrations of a particular TSI, the running of a standard to obtain the curve may be unnecessary. In such instances, the QTT alone without the comparison testing will be sufficient to allow practitioners to evaluate concentration of TSI in the blood.
As an alternative, the kit may contain only fibrinogen and thrombin, whilst one or more stock solutions
containing appropriate concentrations of TSI in plasma is maintained for use in the test. (It would also be possible to have one stock concentration at highest level to be tested, which would be diluted to provide lower concentrations for use to provide the appropriate curve.) The extent to which stock solutions are used or multiple vials of TSI containing differing amounts of TSI are provided will depend on the number of tests done in any particular health care center and the quality of staff available to provide testing services. All of the components, plasma, thrombin, and fibrinogen, should be used fresh or be frozen to avoid loss of potency. All of the agents used, including plasma with TSI, normal plasma, thrombin, and fibrinogen may be provided as lyophilized materials in vials for reconstitution. Once reconstituted, the active components are easily inactivated.
Among the uses for which the invention is suitable include quantitative clinical monitoring of patients anticoagulated with the thrombin-specific agents and laboratory research in animal models of thrombosis.
Claims
1. A method for quantitating plasma levels of specific inhibitors of the enzyme thrombin, comprising the steps of: a. preparing serial dilutions of a thrombin-specific inhibitor in known concentrations to provide samples, b. mixing the samples prepared in step a with a purified human fibrinogen, c. adding purified human alpha-thrombin to the samples prepared in step b, d. measuring the clotting times of the solutions in step c, e. plotting a standard curve from the effect of TSI based on results obtained from step d, f . diluting a plasma sample from patients receiving a thrombin-specific inhibitor, g. mixing the patient samples with a solution of fibrinogen of step f, h. adding a thrombin solution to the patient samples prepared in step g, i. measuring the clotting times of the solutions of step h, j. determining the concentration of the inhibitor in the patient samples using the standard curve.
2. The method according to Claim 1, wherein step f comprises retaining sensitivity (graph linearity) at low therapeutic levels of the inhibitor.
3. The method according to Claim 1 , wherein, additionally, the effect of therapeutic heparin levels in the plasma is avoided by addition of a neutralizing agent.
4. A method of Claim 3 wherein the neutralizing agent is protamine sulfate.
5. A method of evaluating appropriate dosage of a thrombin-specific inhibitor comprising the steps of: a. preparing serial dilutions of a thrombin-specific inhibitor in known concentrations to provide samples, b. mixing the samples prepared in step a with purified human fibrinogen, c. adding purified human alpha-thrombin to the samples prepared in step b, d. measuring the clotting times of the solutions in step c, and e. plotting a standard curve from the effect of TSI based on results obtained from step d.
6. A kit comprising:
1. at least one vial of lyophilized fibrinogen, 2. at least one vial containing thrombin, and
3. instructions for evaluating QTT.
7. A kit of Claim 6 which, additionally, contains at least one vial of plasma.
8. A kit of Claim 6 containing, additionally, at least one vial of a thrombin-specific inhibitor.
9. A method of measuring quantitative thrombin time comprising the steps of:
(1) preparing a composition comprising plasma in buffer to obtain a concentration of about 1:10 plasma to buffer;
(2) preparing a fibrinogen mixture comprising fibrinogen concentration of about 0.1 to 18 μM fibrinogen in buffer;
(3) preparing a composition containing a concentration of about 2.5 to 20 U/mL alpha-thrombin in buffer;
(4) preparing a mixture comprising equal amounts of the compositions obtained in steps (1) and (2); (5) placing in an assay well an aliquot of the mixture prepared in step (4) and an aliquot of the mixture prepared in step (3); and (6) determining the amount of time required for clot formation.
10. A method of Claim 9 wherein the mixture prepared in step (2) is at a concentration of 9 to 18 μM fibrinogen in buffer.
11. A composition of matter comprising plasma, purified fibrinogen, and alpha-thrombin in a buffer.
12. A composition of Claim 11 wherein the alpha- thrombin is human alpha-thrombin.
13. A composition of Claim 11 wherein the fibrinogen is present in a concentration of 0.1 to 18 μM.
14. A composition of Claim 13 wherein the fibrinogen is present in a concentration of between 3 μM and 18 μM.
15. A method of Claim 9 wherein, in step (5), the ratio of amount of reactants is 2 parts of the mixture obtained in step (4) to 1 part of the mixture obtained in step (3).
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
AU44052/93A AU4405293A (en) | 1992-06-05 | 1993-06-02 | Test for quantitative thrombin time |
Applications Claiming Priority (4)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US89363192A | 1992-06-05 | 1992-06-05 | |
US893,631 | 1992-06-05 | ||
US021,033 | 1993-02-22 | ||
US08/021,033 US5476771A (en) | 1993-02-22 | 1993-02-22 | Test for quantitative thrombin time |
Publications (1)
Publication Number | Publication Date |
---|---|
WO1993025220A1 true WO1993025220A1 (en) | 1993-12-23 |
Family
ID=26694177
Family Applications (2)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/US1993/005297 WO1993025220A1 (en) | 1992-06-05 | 1993-06-02 | Test for quantitative thrombin time |
PCT/US1993/005315 WO1993025578A1 (en) | 1992-06-05 | 1993-06-03 | Test for quantitative thrombin time |
Family Applications After (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/US1993/005315 WO1993025578A1 (en) | 1992-06-05 | 1993-06-03 | Test for quantitative thrombin time |
Country Status (5)
Country | Link |
---|---|
EP (1) | EP0643727A4 (en) |
JP (1) | JPH08501682A (en) |
AU (2) | AU4405293A (en) |
CA (1) | CA2137342A1 (en) |
WO (2) | WO1993025220A1 (en) |
Cited By (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP0902091A2 (en) * | 1997-08-29 | 1999-03-17 | Kyoto Daiichi Kagaku Co., Ltd. | Method and kit for measuring the concentration or the activity of a protease inhibitor |
WO2012041334A1 (en) * | 2010-10-01 | 2012-04-05 | Rigshospitalet | Compounds capable of modulating/preserving endothelial integrity for use in prevention or treatment of acute traumatic coagulopathy and resuscitated cardiac arrest |
WO2013143548A1 (en) * | 2012-03-30 | 2013-10-03 | Rigshospitalet | Compounds capable of modulating/preserving endothelial integrity for use in prevention or treatment of acute traumatic coagulopathy and resuscitated cardiac arrest |
CN117741166A (en) * | 2024-02-19 | 2024-03-22 | 北京水木济衡生物技术有限公司 | Multi-project composite coagulation quality control product and preparation method thereof |
Families Citing this family (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
DE29723808U1 (en) * | 1996-04-30 | 1999-11-04 | Medtronic, Inc., Minneapolis, Minn. | Device for determining platelet inhibitor responsiveness |
US6221672B1 (en) | 1996-04-30 | 2001-04-24 | Medtronic, Inc. | Method for determining platelet inhibitor response |
Citations (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US4379142A (en) * | 1980-10-09 | 1983-04-05 | Boehringer Mannheim Gmbh | Thrombin inhibitor and preparation and use thereof |
US4496653A (en) * | 1980-10-09 | 1985-01-29 | Boehringer Mannheim Gmbh | Process for the determination of antithrombin-BM |
US4767742A (en) * | 1984-12-13 | 1988-08-30 | Plantorgan-Werk Heinrich G. F. Christensen, KG | Hirudin-PA and its derivatives, process for manufacturing these and the use of these substances |
US5187102A (en) * | 1990-02-14 | 1993-02-16 | Pentapharm Ag | Inhibitors for the anticoagulant pretreatment of blood samples |
US5196404A (en) * | 1989-08-18 | 1993-03-23 | Biogen, Inc. | Inhibitors of thrombin |
Family Cites Families (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5019393A (en) * | 1988-08-03 | 1991-05-28 | New England Deaconess Hospital Corporation | Biocompatible substance with thromboresistance |
US4952562A (en) * | 1989-09-29 | 1990-08-28 | Rorer Pharmaceutical Corporation | Anti-thrombotic peptides and pseudopeptides |
US5118790A (en) * | 1990-07-24 | 1992-06-02 | Sri International | Analogs of hirudin |
-
1993
- 1993-06-02 WO PCT/US1993/005297 patent/WO1993025220A1/en active Application Filing
- 1993-06-02 AU AU44052/93A patent/AU4405293A/en not_active Abandoned
- 1993-06-03 AU AU47681/93A patent/AU4768193A/en not_active Abandoned
- 1993-06-03 CA CA 2137342 patent/CA2137342A1/en not_active Abandoned
- 1993-06-03 JP JP6501586A patent/JPH08501682A/en active Pending
- 1993-06-03 EP EP93918119A patent/EP0643727A4/en not_active Withdrawn
- 1993-06-03 WO PCT/US1993/005315 patent/WO1993025578A1/en not_active Application Discontinuation
Patent Citations (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US4379142A (en) * | 1980-10-09 | 1983-04-05 | Boehringer Mannheim Gmbh | Thrombin inhibitor and preparation and use thereof |
US4496653A (en) * | 1980-10-09 | 1985-01-29 | Boehringer Mannheim Gmbh | Process for the determination of antithrombin-BM |
US4767742A (en) * | 1984-12-13 | 1988-08-30 | Plantorgan-Werk Heinrich G. F. Christensen, KG | Hirudin-PA and its derivatives, process for manufacturing these and the use of these substances |
US5196404A (en) * | 1989-08-18 | 1993-03-23 | Biogen, Inc. | Inhibitors of thrombin |
US5196404B1 (en) * | 1989-08-18 | 1996-09-10 | Biogen Inc | Inhibitors of thrombin |
US5187102A (en) * | 1990-02-14 | 1993-02-16 | Pentapharm Ag | Inhibitors for the anticoagulant pretreatment of blood samples |
Non-Patent Citations (2)
Title |
---|
AMERICAN JOURNAL OF PHYSIOLOGY, Volume 239, issued December 1980, T.L. MURPHY et al., "Endogenous Anticoagulation During Extracorporeal Perfusion: Generation of a Heparinlike Inhibitor", pages H742-50. * |
BLOOD VESSELS, Volume 21, No. 2, issued 1984, V.M. HAVER et al., "Characterization of the Thrombin-Induced Contraction of Vascular Smooth Muscle", pages 53-63. * |
Cited By (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP0902091A2 (en) * | 1997-08-29 | 1999-03-17 | Kyoto Daiichi Kagaku Co., Ltd. | Method and kit for measuring the concentration or the activity of a protease inhibitor |
EP0902091A3 (en) * | 1997-08-29 | 2001-01-10 | Kyoto Daiichi Kagaku Co., Ltd. | Method and kit for measuring the concentration or the activity of a protease inhibitor |
WO2012041334A1 (en) * | 2010-10-01 | 2012-04-05 | Rigshospitalet | Compounds capable of modulating/preserving endothelial integrity for use in prevention or treatment of acute traumatic coagulopathy and resuscitated cardiac arrest |
WO2013143548A1 (en) * | 2012-03-30 | 2013-10-03 | Rigshospitalet | Compounds capable of modulating/preserving endothelial integrity for use in prevention or treatment of acute traumatic coagulopathy and resuscitated cardiac arrest |
CN117741166A (en) * | 2024-02-19 | 2024-03-22 | 北京水木济衡生物技术有限公司 | Multi-project composite coagulation quality control product and preparation method thereof |
CN117741166B (en) * | 2024-02-19 | 2024-04-26 | 北京水木济衡生物技术有限公司 | Multi-project composite coagulation quality control product and preparation method thereof |
Also Published As
Publication number | Publication date |
---|---|
EP0643727A4 (en) | 1998-05-13 |
EP0643727A1 (en) | 1995-03-22 |
AU4405293A (en) | 1994-01-04 |
CA2137342A1 (en) | 1993-12-23 |
JPH08501682A (en) | 1996-02-27 |
AU4768193A (en) | 1994-01-04 |
WO1993025578A1 (en) | 1993-12-23 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
Yin et al. | Plasma heparin: a unique, practical, submicrogram-sensitive assay | |
US5525477A (en) | Method for diagnosing blood clotting disorders | |
Nilsson et al. | Two different mechanisms in patients with venous thrombosis and defective fibrinolysis: low concentration of plasminogen activator or increased concentration of plasminogen activator inhibitor. | |
d'Angelo et al. | Relationship between protein C antigen and anticoagulant activity during oral anticoagulation and in selected disease states. | |
US5051357A (en) | Method and assay using inactivation of factors Va and VIIIa by activated Protein C to diagnose thrombic disease or assay for Protein C and kit therefor | |
US5525478A (en) | Soluble thrombomodulin-based one-stage assay for vitamin-K dependent coagulation-inhibiting proteins | |
Lange et al. | Ecarin chromogenic assay–a new method for quantitative determination of direct thrombin inhibitors like hirudin | |
Bergström et al. | Determination of vitamin K sensitive coagulation factors in plasma. Studies on three methods using synthetic chromogenic substrates | |
JP5123496B2 (en) | Methods for standardization of blood coagulation tests | |
Goldenberg et al. | A new global assay of coagulation and fibrinolysis | |
JP3094165B2 (en) | Anticoagulant concentration measurement method | |
CA2333890A1 (en) | Corn trysin inhibitor stabilizes blood-derived plasma and improves sensitivity of plasma clotting assays | |
Walenga et al. | Newer avenues in the monitoring of antithrombotic therapy: The role of automation | |
Corrigan Jr et al. | Factor II antigen in liver disease and warfarin‐induced vitamin K deficiency: Correlation with coagulant activity using echis venom | |
US5476771A (en) | Test for quantitative thrombin time | |
US4948724A (en) | Composition, kit and method for assaying heparin and a method for making the composition | |
WO1993025220A1 (en) | Test for quantitative thrombin time | |
Stevens et al. | Evaluation of three methods for plasma fibrinogen determination | |
Weinstein et al. | Species specificity of the fibrinolytic effects of activated protein C | |
US5529905A (en) | Method of assaying plasma proteins with prothrombin fragments | |
CA2039173C (en) | Factor ix chromogenic assay | |
JPH0646898A (en) | Method and reagent for analyzing fibrinogen | |
Kandall et al. | Determinants of Prothrombinase Activity and Modification of Prothrombin Conversion by Thrombin‐Treated Factor V | |
EP0217768A2 (en) | Method and compositions for heparin assays | |
Duncan et al. | A clinical evaluation of automated chromogenic tests as substitutes for conventional prothrombin time and activated partial thromboplastin time tests. |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
AK | Designated states |
Kind code of ref document: A1 Designated state(s): AU CA JP KR |
|
AL | Designated countries for regional patents |
Kind code of ref document: A1 Designated state(s): AT BE CH DE DK ES FR GB GR IE IT LU MC NL PT SE |
|
121 | Ep: the epo has been informed by wipo that ep was designated in this application | ||
DFPE | Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101) | ||
122 | Ep: pct application non-entry in european phase | ||
NENP | Non-entry into the national phase |
Ref country code: CA |