WO2022145477A1 - 血液凝固検査方法 - Google Patents

血液凝固検査方法 Download PDF

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WO2022145477A1
WO2022145477A1 PCT/JP2021/048980 JP2021048980W WO2022145477A1 WO 2022145477 A1 WO2022145477 A1 WO 2022145477A1 JP 2021048980 W JP2021048980 W JP 2021048980W WO 2022145477 A1 WO2022145477 A1 WO 2022145477A1
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final concentration
blood coagulation
blood
thrombomodulin
heparin
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French (fr)
Japanese (ja)
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和也 細川
朋香 永里
寿代 金子
千秋 小山田
朋子 和田
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Zacros Corp
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Fujimori Kogyo Co Ltd
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Priority to EP21915335.0A priority Critical patent/EP4269605A4/en
Priority to US18/269,835 priority patent/US20240060999A1/en
Priority to JP2022573121A priority patent/JPWO2022145477A1/ja
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    • 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
    • G01N2333/745—Assays involving non-enzymic blood coagulation factors
    • G01N2333/7452—Thrombomodulin
    • 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, Konjac gum, Locust bean gum or 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

  • the present invention relates to a blood coagulation test method.
  • Thrombomodulin is a glycoprotein with anticoagulant ability expressed in vascular endothelial cells.
  • thrombomodulin When thrombin produced by activating blood coagulation binds to thrombomodulin, the substrate specificity of thrombin changes, and thrombin fibrinogen. Is reduced in its ability to convert to fibrin, effectively activating protein C (PC) and producing activated PC (APC).
  • PC protein C
  • APC activated PC
  • APC activated PC
  • APC activated PC
  • APC activated PC
  • APC activated PC
  • APC activated PC
  • APC activated PC
  • APC activated PC
  • APC activated PC
  • APC activated PC
  • APC activated PC
  • APC activated PC
  • APC activated PC
  • APC activated PC
  • APC activated PC
  • APC activated PC
  • FVa activated blood coagulation factor 5
  • FVIIIa activated blood coagulation factor VIII
  • PS protein
  • FVa FVa cause diseases such as thrombosis.
  • This is a resistance mutation to APC, and by inhibiting the inactivation of FVa by APC, anti-TM / APC pathways are used. Coagulation is impaired. It is called the FV Leiden mutation and is known as a major thrombophilia in Western Caucasians.
  • the present inventor has made diligent studies to solve the above problems.
  • the blood coagulation ability was evaluated by adding an extrinsic blood coagulation activation reagent and thrombomodulin and heparin-like substances to the blood sample, thereby evaluating the blood coagulation ability reflecting the functions of protein C and protein S.
  • One aspect of the present invention is a method for analyzing blood coagulation in vitro, which comprises adding an extrinsic blood coagulation activating reagent and a thrombomodulin and heparin-like substance to a blood sample to evaluate the blood coagulation ability.
  • the extrinsic blood coagulation activation reagent can be tissue factor or tissue thromboplastin.
  • the heparin-like substance can be one or more of the sulfated polysaccharides selected from the group consisting of unfractionated heparin, low molecular weight heparin, fondaparinux sodium and heparan sulfate and dextran sulfate.
  • the final concentration of thrombomodulin can be 9nM-200nM.
  • Thrombomodulin can be soluble thrombomodulin.
  • the function of protein C and protein S is evaluated by adding a protein C inhibitor to evaluate the blood coagulation ability and comparing the results when the protein C inhibitor is not added. It can be carried out.
  • the blood sample is a blood sample anticoagulated with citric acid, and blood coagulation is performed by adding calcium, an extrinsic blood coagulation activating reagent, and thrombomodulin and heparin-like substances. Can be evaluated.
  • the blood coagulation ability can be evaluated by adding an extrinsic blood coagulation activating reagent and a contact factor inhibitor.
  • the contact factor inhibitor can be either an FXII inhibitor or a kallikrein inhibitor.
  • the functions of protein C and protein S can be evaluated by comparing the analysis result of blood coagulation with the blood coagulation analysis result when thrombomodulin is not added.
  • the blood coagulation time is set to be 60 seconds to 700 minutes without the addition of thrombomodulin (ie, blood with tissue factor or tissue thrombomodulin and heparin-like substances). Blood coagulation can be evaluated by adding thrombomodulin to the blood.
  • the method of the present invention is an analysis of blood coagulation of an extrinsic system, and by adding thrombomodulin and a heparin-like substance, the effect of prolonging the blood coagulation time by thrombomodulin is enhanced, and the function of thrombomodulin is satisfactorily improved. It is possible to evaluate the functions of PC and PS. It is preferable to add a heparin-like substance because it is possible to analyze the prolongation of blood coagulation time with a small amount of thrombomodulin.
  • the method for analyzing blood coagulation of the present invention is It is characterized in that an extrinsic blood coagulation activating reagent and thrombomodulin and heparin-like substances are added to a blood sample to evaluate the blood coagulation ability.
  • an extrinsic blood coagulation activating reagent and thrombomodulin and heparin-like substances are added to a blood sample to evaluate the blood coagulation ability.
  • a whole blood sample or platelet-rich plasma is preferably used, and a blood sample that has been anticoagulated with citric acid or the like may be used.
  • the anticoagulant treatment can be canceled with calcium or the like to start the blood coagulation reaction.
  • thrombomodulin is preferably added in a final concentration in the range of 9 nM to 200 nM.
  • thrombomodulin is 9 nM or less, the anticoagulant effect of thrombomodulin may not be clearly shown even when a heparin-like substance is added to tissue factor or tissue thromboplastin.
  • thrombomodulin can exhibit anticoagulant ability even without the addition of a heparin-like substance, but thrombomodulin is expensive and poses a problem in use for inspection applications.
  • thrombomodulin it is preferable to use soluble thrombomodulin excluding the transmembrane domain.
  • Soluble thrombomodulin is produced by purification from blood or by genetically modified technology.
  • lycomodulin (Asahi Kasei Pharma) can be used as the soluble thrombomodulin for gene recombination.
  • Extrinsic blood coagulation activating reagents include tissue factor and tissue thromboplastin. Tissue factor and tissue thromboplastin have different specific activities, but are preferably added so that, for example, the blood coagulation time when added to whole blood is 50-600 seconds. Analysis of blood coagulation in the presence of low concentrations of tissue factor or tissue thromboplastin, heparan sulfate and thrombomodulin is preferred as it is close to the in vivo blood coagulation reaction.
  • an extrinsic blood coagulation activating reagent such as low-concentration tissue factor or tissue thromboplastin, a contact factor inhibitor, and thrombomodulin and heparin-like substances to the blood sample to evaluate the blood coagulation ability.
  • an extrinsic blood coagulation activating reagent such as low-concentration tissue factor or tissue thromboplastin, a contact factor inhibitor, and thrombomodulin and heparin-like substances to the blood sample to evaluate the blood coagulation ability.
  • examples of the contact factor inhibitor include a blood coagulation factor 12 (FXII) inhibitor, a blood coagulation factor 11 (FXI) inhibitor, and a kallikrein inhibitor.
  • FXII inhibitors and FXI inhibitors may be inhibitors for the activation of FXII and FXI or inhibitors for the enzymatic activity of activated FXII (FXIIa) and activated FXI (FXIa).
  • the FXII inhibitor is not particularly limited as long as it is a substance having FXII inhibitory activity, but a corntrypsin inhibitor (CTI), a peptide inhibitor (J Med Chem. 2017 Feb 9; 60 (3): 1151-1158), etc. are used. can do.
  • the FXII inhibitor is preferably added at a final concentration of 10 to 100 ⁇ g / mL.
  • the kallikrein inhibitor is not particularly limited, but a synthetic kallikrein inhibitor (PKSI-527: Fujifilm Wako Pure Chemical Industries, Ltd.) or aprotinin is desirable. If it is a kallikrein inhibitor (PKSI-527), it is preferable to add it at a final concentration of 0.1 ⁇ M to 100 ⁇ M.
  • heparin-like substances examples include unfractionated heparin, low molecular weight heparin (for example, heparin having a mass average molecular weight of 4500 to 6500), and fondaparinux sodium (five-sugar structure in heparin that promotes Xa inhibition of antithrombin: product example Alixtra). ), Or heparin sulfate (eg, danaparoid sodium: product example Olgaran), sulfated polysaccharides such as dextran sulfate. Heparan sulfate is present on vascular endothelial cells and is responsible for the physiological anticoagulant action in blood vessels.
  • heparin sulfate eg, danaparoid sodium: product example Olgaran
  • sulfated polysaccharides such as dextran sulfate.
  • Heparan sulfate is present on vascular endothelial cells and is responsible for the physiological anticoagulant action in blood vessels.
  • Heparan sulfate as a heparin-like substance because physiological blood coagulation can be reproduced. Heparan sulfate should be added at a final concentration of 0.4-15 ug / mL.
  • Basic substances such as protamine and polybrain are used as neutralizers for unfractionated heparin, but fondaparinux sodium is not neutralized with protamine and polybrain. Therefore, when fondaparinux sodium is used as the heparin-like substance, only unfractionated heparin can be specifically neutralized by protamine or polybrain.
  • Heparinase is used to decompose heparin and small molecule heparin, but dextran sulfate is not decomposed by heparinase. Therefore, when dextran sulfate is used as the heparin-like substance, only unfractionated heparin and small molecule heparin can be specifically decomposed by heparinase.
  • the blood when testing a blood sample containing heparin and low molecular weight heparin such as blood of a patient being treated with heparin, by using Fondaparinux sodium or dextran sulfate as a heparin-like substance, the blood can be contained in the blood. It becomes possible to specifically neutralize or decompose the contained heparin and low molecular weight heparin.
  • the final concentration is 0.01 to 1 U / mL for unfractionated heparin, 0.01 to 2 U / mL for low molecular weight heparin, and 0.1 to 10 ⁇ g / mL for fondaparinux sodium. It is desirable to add in.
  • heparin-like substances be added together with tissue factor or tissue thromboplastin and a contact factor inhibitor so that the blood coagulation time is 60-700 seconds.
  • the coagulation time is 60-700 seconds, when thrombomodulin (9-200 nM) is added, the blood coagulation time is appropriately extended, and the anticoagulant ability of thrombomodulin can be evaluated.
  • the method for analyzing blood coagulation is not particularly limited, and is ROTEM (Rotational Thromboelastometry: Instrumentation Laboratory (IL)), TEG (Thromboelastography), SONOCLOT (Scienko), variable angle thromboelastography and graph analyzer (blood coagulation). It is desirable to use a device that can evaluate the viscous elasticity of whole blood, such as a test device and blood coagulation test method; WO2018 / 043420), or a device that can analyze blood coagulation in plasma in detail, such as thromboelastography.
  • a blood coagulation test is performed by adding an extrinsic blood coagulation activation reagent and a thrombomodulin and a heparin-like substance to the blood of a healthy person and the blood of a patient with abnormal blood coagulation, respectively, and the coagulation time and coagulation waveform are calculated. By observing which coagulation time or coagulation waveform is closer to, it is possible to investigate whether or not a blood coagulation abnormality is present in the sample.
  • the results of the blood coagulation test conducted by adding an extrinsic blood coagulation activation reagent and a thrombomodulin and heparin-like substance to the blood sample are obtained when thrombomodulin is not added (in the absence of thrombomodulin), that is, the blood sample is extrinsic.
  • the functions of protein C and protein S can also be evaluated by comparing the results of blood coagulation tests conducted by adding a blood coagulation activating reagent and a heparin-like substance of the system. For example, by comparing the blood coagulation time with and without thrombomodulin and the waveform of ROTEM / TEG and evaluating the degree of prolongation, it is possible to evaluate the functions of protein C and protein S.
  • FV Leiden causes thrombosis in an abnormality in which FVa is not inactivated by APC (activated protein C) due to a mutation in the FV gene, but it is not reflected in the measurement of coagulation time in the absence of thrombomodulin.
  • APC activated protein C
  • the difference in blood coagulation time in the presence and absence of soluble thrombomodulin is reduced.
  • protein S has both free PS, complement regulator C4BP and complex (PS / C4BP complex) in blood.
  • Free PS acts as a coenzyme for APC.
  • C4BP increases due to inflammation etc.
  • free PS decreases and the anticoagulant ability of APC decreases.
  • the difference in blood coagulation time in the presence and absence of soluble thrombomodulin becomes small.
  • the start time of blood coagulation in the absence of thrombomodulin may be adjusted to be between 60 seconds and 700 seconds. desirable.
  • the coagulation time in the absence of thrombomodulin is within 60 seconds, the anticoagulation effect mediated by APC due to the addition of thrombomodulin is exhibited because the blood coagulation occurs immediately by the large amount of thrombin produced in a short time. Since the blood coagulation reaction is completed before, it becomes difficult to analyze PC and PS.
  • the anticoagulant ability of thrombomodulin includes the anticoagulant effect due to the anticoagulant effect by directly suppressing the fibrin production of thrombin and the ability to activate FVIII and FV, and the activation protein C (PC) to the activated protein C (APC).
  • PC activation protein C
  • APC activated protein C
  • the APC inhibitor includes a substance that inhibits the activation of PC, a substance that inhibits the enzyme activity of APC, and the like, and is not particularly limited.
  • an APC-inhibiting DNA aptamer is used. can do.
  • APC-inhibiting aptamers include HS02-44G (Chemistry & Biology Volume 16, Issue 4, 24 April 2009, Pages 442-451), but are not particularly limited.
  • the present invention also provides an extrinsic blood coagulation activating reagent and a blood coagulation measuring reagent containing thrombomodulin and heparin-like substances.
  • Tissue factor or tissue thromboplastin is preferred as the extrinsic blood coagulation activation reagent.
  • the heparin-like substance is preferably unfractionated heparin, low molecular weight heparin, fondapalinux sodium (Alixtra), and heparan sulfate and dextran sulfate.
  • the blood coagulation measurement reagent may further contain a contact factor inhibitor such as an FXII inhibitor and a kallikrein inhibitor.
  • the blood coagulation measurement reagent may further contain a protein C inhibitor.
  • the preferable concentration (final concentration) of each component at the time of use is as described above, and is diluted to a preferable final concentration at the time of use (when a blood sample is added) before use.
  • Each component may be mixed in advance, but it is preferable that each component is included as a separate reagent component in the blood coagulation measurement reagent kit so that each component is mixed at a concentration suitable for measurement at the time of use.
  • the blood coagulation measurement reagent may include an instruction manual for blood coagulation measurement.
  • Example 1 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. 20 ⁇ L of Extem reagent (IL), which is a 50-fold diluted extrinsic activation reagent, 20 ⁇ L (final concentration 12 mM) of Startem reagent (IL), which is a calcium chloride reagent, and soluble thrombomodulin (rTM; trade name lycomodulin) in 300 ⁇ L of whole blood. Asahi Kasei Pharma Co., Ltd.) was added so that the final concentrations were 0, 20 nM, 50 nM, and 200 nM, and blood coagulation was analyzed. The results are shown in FIG.
  • IL Extem reagent
  • rTM soluble thrombomodulin
  • Example 2 ROTEM was used for blood coagulation analysis. Blood was collected using a blood collection tube containing 3.2% sodium citrate manufactured by Terumo Corporation. The same blood coagulation analysis as in Example 1 was performed except that a 50-fold diluted rabbit ability-derived tissue thromboplastin (TP) reagent (final concentration 0.9 ⁇ g / mL) was added to the blood instead of the 50-fold diluted EXTEM reagent. rice field. The results are shown in FIG.
  • TP rabbit ability-derived tissue thromboplastin
  • the coagulation time (CT) of blood to which soluble thrombomodulin 0, 20, 50, 200 nM was added was 157, 198, 243, and 428 seconds, respectively. Even when a rabbit brain-derived thromboplastin reagent was used for extrinsic coagulation activation, the effect of soluble thrombomodulin 20nM on prolonging blood coagulation time was limited. Blood coagulation was significantly prolonged at 200 nM soluble thrombomodulin.
  • Example 3 ROTEM was used for blood coagulation analysis. Blood was collected using a blood collection tube containing 3.2% sodium citrate manufactured by Terumo Corporation. In the same experiment as in Example 2, heparan sulfate (iduron, average molecular weight 5500) was added to the rabbit brain-derived tissue thromboplastin (TP: final concentration 0.9 ⁇ g / mL) and soluble thrombomodulin (rTM: final concentration 20 nM). Blood coagulation waveforms were analyzed by adding 1 ⁇ g / mL, 1.5 ⁇ g / mL and 2 ⁇ g / mL. The results are shown in FIG.
  • the coagulation time (CT) of blood without soluble thrombomodulin was 157 seconds, whereas it was 198 seconds with the addition of soluble thrombomodulin 20 nM. Furthermore, the CT of blood supplemented with soluble thrombomodulin 20 nM and heparan sulfate 1 ⁇ g / mL was 319 seconds, and the effect of soluble thrombomodulin was significantly enhanced in the presence of heparan sulfate. Furthermore, when heparan sulfate was added at 2 ⁇ g / mL, a small amount of soluble thrombomodulin significantly delayed the coagulation time and at the same time suppressed the strength of the clot.
  • Example 4 In the same experiment as in Example 3, rabbit brain-derived tissue thromboplastin (TP; final concentration 0.9 ⁇ g / mL), heparan sulfate (final concentration 1 ⁇ g / mL), and FXII inhibitor (CTI; final concentration) as a contact factor inhibitor. 20 ⁇ g / mL) and a calicrane inhibitor (PKSI-527; final concentration 40 ⁇ M) were added to analyze blood coagulation in the presence and absence of soluble thrombomodulin (rTM; final concentration 20 nM). 20 ⁇ L (Ca; final concentration 12 mM) of calcium chloride solution was used instead of the Startem reagent. The results are shown in FIG.
  • CT Blood coagulation time
  • FXII inhibitor CRI
  • PHSI-527 kallikrein inhibitor
  • 20 nM of soluble thrombomodulin was added. It was shown that the addition of a low concentration (20 nM) of soluble thrombomodulin to blood supplemented with diluted rabbit brain-derived tissue thromboplastin, heparan sulfate and a contact factor inhibitor markedly prolonged coagulation time.
  • Example 5 In the same experiment as in Example 3, unfractionated heparin (Mochida Pharmaceutical Co., Ltd .; final concentration 0.1 U / mL) was added instead of heparan sulfate, and soluble thrombomodulin (rTM) final concentration 0 nM, 20 nM and 30 nM blood. The coagulation waveform was analyzed. The results are shown in FIG.
  • Example 6 In the same experiment as in Example 5, low-molecular-weight heparin (trade name: Fragmin Kissei Yakuhin Kogyo Co., Ltd. (sales), Pfizer Co., Ltd. (manufacturer / distributor) final concentration 0.2 U / mL) was used instead of unfractionated heparin. Blood coagulation was analyzed. The results are shown in FIG.
  • Example 7 In the same experiment as in Example 5, blood coagulation was analyzed by adding Olgalan (manufactured by Kyowa CritiCare Co., Ltd .; final concentration 0.2 U / mL) instead of unfractionated heparin.
  • Olgalan manufactured by Kyowa CritiCare Co., Ltd .; final concentration 0.2 U / mL
  • the results are shown in FIG. (1) TP (0.9 ⁇ g / mL) + Startem + Organan 0.2U / mL (2)
  • Example 8 In the same experiment as in Example 5, blood coagulation was analyzed by adding Alixtra (manufactured by Aspen Japan K.K.; 0.5 ⁇ g / mL) instead of unfractionated heparin. The results are shown in FIG. (1) TP (0.9 ⁇ g / mL) + Startem + Alixtra 0.5 ⁇ g / mL (2) TP (0.9 ⁇ g / mL) + Startem + Alixtra 0.5 ⁇ g / mL + rTM20nM (3) TP (0.9 ⁇ g / mL) + Startem + Alixtra 0.5 ⁇ g / mL + rTM30nM
  • Example 9 ROTEM was used for blood coagulation analysis. Blood was collected using a blood collection tube containing 3.2% sodium citrate manufactured by Terumo Corporation. Rabbit brain-derived tissue thromboplastin reagent (TP; final concentration 0.9 ⁇ g / mL), Startem reagent (calcium chloride: final concentration 12 mM), and heparan sulfate (1 ⁇ g / mL) were added for blood coagulation analysis.
  • TP brain-derived tissue thromboplastin reagent
  • Startem reagent calcium chloride: final concentration 12 mM
  • heparan sulfate (1 ⁇ g / mL
  • rabbit brain-derived tissue thromboplastin reagent TP; 0.9 ⁇ g / mL
  • Startem reagent calcium chloride: final concentration 12 mM
  • heparan sulfate final concentration 1 ⁇ g / mL
  • soluble thrombomodulin rTM; final concentration 20 nM
  • soluble thrombomodulin rTM; final concentration 20 nM
  • soluble thrombomodulin soluble thrombomodulin
  • Blood coagulation was analyzed by adding (rTM; final concentration 20 nM) and APC-inhibiting aptamer (HS02-44G; final concentration 1 ⁇ M; Chemistry & Biology Volume 16, Issue 4, 24 April 2009, Pages 442-451). The results are shown in FIG.
  • the coagulation time was extended from 154 seconds to 319 seconds by further adding soluble thrombomodulin (20 nM) to the blood supplemented with tissue thromboplastin and heparan sulfate.
  • the CT was 208 seconds when soluble thrombomodulin (20 nM) and APC-inhibiting aptamer were further added to blood supplemented with tissue thrombomodulin and heparan sulfate. From this result, it is considered that the delay of CT from 154 seconds to 208 seconds is due to the antithrombin effect of soluble thrombomodulin.
  • the reduction from 319 seconds to 208 seconds is thought to be due to the anticoagulant effect mediated by activated protein C. Therefore, it is possible to evaluate the function of PC / PS by comparing the coagulation time with and without the addition of APC-inhibiting aptamer.
  • Example 10 ROTEM was used for blood coagulation analysis. Blood was collected using a blood collection tube containing 3.2% sodium citrate manufactured by Terumo Corporation. Blood coagulation analysis was performed by adding 100-fold diluted rabbit brain-derived tissue thromboplastin reagent (TP; final concentration 0.45 ⁇ g / mL), Startem reagent (calcium chloride; final concentration 12 mM), and heparan sulfate (final concentration 1 ⁇ g / mL). .
  • rabbit brain-derived tissue thromboplastin reagent (0.9 ⁇ g / mL), Startem reagent (calcium chloride; final concentration 12 mM) and heparan sulfate (final concentration 1 ⁇ g / mL) soluble in soluble thrombomodulin (rTM; final concentration 100 nM) or soluble thrombomodulin (rTM).
  • Blood coagulation was analyzed by adding (final concentration 100 nM) and APC-inhibiting aptamer (HS02-44G; final concentration 1 ⁇ M). The results are shown in FIG.
  • the coagulation time increased from 251 seconds to 593 seconds, a 2.36-fold increase. It is considered to be due to both "anticoagulant effect by antithrombin" and "anticoagulant effect through APC production” by thrombomodulin.
  • the CT was 387 seconds when soluble thrombomodulin (100 nM) and APC-inhibiting aptamer were further added to blood supplemented with tissue thromboplastin and heparan sulfate. It specifically reflects the anticoagulant effect mediated by APC.
  • Example 11 ROTEM was used for blood coagulation analysis. Blood was collected using a blood collection tube containing 3.2% sodium citrate manufactured by Terumo Corporation. Rabbit brain-derived tissue thromboplastin reagent (TP; final concentration 16.6 ⁇ g / mL), calcium chloride (Ca; final concentration 12 mM) and heparan sulfate (final concentration 0, 1, 3, 10, 15, 20, 30 ⁇ g / mL) Blood coagulation analysis was performed with the addition of CTI (final concentration 20 ⁇ g / mL) and PKSI-527 (final concentration 40 ⁇ M), with and without the addition of soluble thrombomodulin (rTM; final concentration 50 nM).
  • TP brain-derived tissue thromboplastin reagent
  • Ca calcium chloride
  • heparan sulfate final concentration 0, 1, 3, 10, 15, 20, 30 ⁇ g / mL
  • Table 1 shows the CT (clotting time) and CFT value (clot formation time).
  • CT clotting time
  • CFT value clot formation time
  • Example 12 ROTEM was used for blood coagulation analysis. Blood was collected using a blood collection tube containing 3.2% sodium citrate manufactured by Terumo Corporation. Rabbit brain-derived tissue thromboplastin reagent (TP; final concentration 16.6 ⁇ g / mL), calcium chloride (Ca; final concentration 12 mM), heparan sulfate (final concentration 0.5 ⁇ g / mL), CTI (final concentration 20 ⁇ g / mL) and PKSI- Blood coagulation analysis was performed with the addition of 527 (final concentration 40 ⁇ M) and with and without the addition of soluble thrombomodulin (rTM; final concentration 100 nM).
  • TP brain-derived tissue thromboplastin reagent
  • Ca calcium chloride
  • heparan sulfate final concentration 0.5 ⁇ g / mL
  • CTI final concentration 20 ⁇ g / mL
  • PKSI- Blood coagulation analysis was performed with the addition of 527 (final concentration 40 ⁇
  • Table 2 shows the CT (clotting time) and CFT value (clot formation time). At 5 ⁇ g / mL of heparan sulfate, it was confirmed that the coagulation time was extended by 1.5 times or more by the addition of soluble thrombomodulin.
  • Example 13 ROTEM was used for blood coagulation analysis. Blood was collected using a blood collection tube containing 3.2% sodium citrate manufactured by Terumo Corporation. Rabbit brain-derived tissue thromboplastin reagent (TP; final concentration 0.033 ⁇ g / mL), calcium chloride (Ca; final concentration 12 mM), heparan sulfate (final concentration 0, 0.5 ⁇ g / mL), CTI (final concentration 20 ⁇ g / mL) and PKSI Blood coagulation analysis was performed with the addition of -527 (final concentration 40 ⁇ M) and with and without the addition of soluble thrombomodulin (rTM; final concentration 9 nM).
  • TP brain-derived tissue thromboplastin reagent
  • Ca calcium chloride
  • heparan sulfate final concentration 0, 0.5 ⁇ g / mL
  • CTI final concentration 20 ⁇ g / mL
  • PKSI Blood coagulation analysis was performed with the addition of -527 (final concentration
  • CT clotting time
  • CFT value clot formation time
  • Example 14 ROTEM was used for blood coagulation analysis. Blood was collected using a blood collection tube containing 3.2% sodium citrate manufactured by Terumo Corporation. Rabbit brain-derived tissue thromboplastin reagent (TP; final concentration 0.033 ⁇ g / mL), calcium chloride (Ca; final concentration 12 mM), heparan sulfate (final concentration 0,0.1,0.2,0.3,0.4,0.5 ⁇ g / mL) and CTI (final concentration 0,0.1,0.2,0.3,0.4,0.5 ⁇ g / mL) Final concentration 20 ⁇ g / mL) and PKSI-527 (final concentration 40 ⁇ M) were added, and blood coagulation was analyzed with and without soluble thrombomodulin (rTM; final concentration 10 nM).
  • TP brain-derived tissue thromboplastin reagent
  • Ca calcium chloride
  • CTI final concentration 0,0.1,0.2,0.3,0.4,0.5 ⁇ g / mL
  • rTM final concentration 10 nM
  • the CT (clotting time) and CFT value (clot formation time) are shown in Table 4.
  • 10 nM of soluble thrombomodulin was added, the prolongation of coagulation time was limited when heparan sulfate was 0.3 ⁇ g / mL or less, but when heparan sulfate was added 0.4 ⁇ g / mL or more, the coagulation time was 1.5. It was extended more than twice.
  • Example 15 ROTEM was used for blood coagulation analysis. Blood was collected using a blood collection tube containing 3.2% sodium citrate manufactured by Terumo Corporation. Rabbit brain-derived tissue thromboplastin reagent (TP; final concentration 0.033 ⁇ g / mL), calcium chloride (Ca; final concentration 12 mM), heparan sulfate (final concentration 0,0.1,0.2,0.3,0.4,0.5 ⁇ g / mL) and CTI (final concentration 0,0.1,0.2,0.3,0.4,0.5 ⁇ g / mL) A final concentration of 20 ⁇ g / mL) and PKSI-517 (final concentration of 40 ⁇ M) were added, and blood coagulation analysis was performed with and without the addition of soluble thrombomodulin (rTM; final concentration of 5 nM).
  • TP brain-derived tissue thromboplastin reagent
  • Ca calcium chloride
  • CTI final concentration 0,0.1,0.2,0.3,0.4,0.5 ⁇ g / mL
  • rTM final
  • the CT (clotting time) and CFT value (clot formation time) are shown in Table 5.
  • the coagulation time was not extended more than 1.5-fold even when heparan sulfate was added.
  • Example 16 ROTEM was used for blood coagulation analysis. Blood was collected using a blood collection tube containing 3.2% sodium citrate manufactured by Terumo Corporation. Rabbit brain-derived tissue thromboplastin reagent (TP; final concentration 16.6 ⁇ g / mL), calcium chloride (Ca; final concentration 12 mM), CTI (final concentration 20 ⁇ g / mL) and PKSI-527 (final concentration 40 ⁇ M) were added, and further. Blood coagulation analysis was performed with and without the addition of soluble thrombomodulin (rTM; final concentration 100, 200, 300, 400, 500 nM).
  • TP brain-derived tissue thromboplastin reagent
  • Ca final concentration 12 mM
  • CTI final concentration 20 ⁇ g / mL
  • PKSI-527 final concentration 40 ⁇ M
  • CT clotting time
  • CFT value clot formation time
  • Example 17 ROTEM was used for blood coagulation analysis. Blood was collected using a blood collection tube containing 3.2% sodium citrate manufactured by Terumo Corporation. Rabbit brain-derived tissue thromboplastin reagent (TP; final concentration 0.011 ⁇ g / mL), calcium chloride (Ca; final concentration 12 mM), heparan sulfate (final concentration 0,0.2,0.3, 0.5 ⁇ g / mL) and CTI (final concentration 20 ⁇ g / mL) mL) and PKSI-527 (final concentration 40 ⁇ M) were added, and blood coagulation was analyzed with and without soluble thrombomodulin (rTM; final concentration 10 nM).
  • TP brain-derived tissue thromboplastin reagent
  • Ca calcium chloride
  • heparan sulfate final concentration 0,0.2,0.3, 0.5 ⁇ g / mL
  • CTI final concentration 20 ⁇ g / mL
  • PKSI-527 final concentration 40 ⁇ M
  • the CT (clotting time) and CFT value (clot formation time) are shown in Table 7.
  • thromboplastin 0.011 ⁇ g / mL and heparan sulfate (0.2 ⁇ g / mL or more) were added, when soluble thrombomodulin 10 nM was added, the blood coagulation time became 1 hour or more, and no CT value was obtained.
  • Example 18 ROTEM was used for blood coagulation analysis. Blood was collected using a blood collection tube containing 3.2% sodium citrate manufactured by Terumo Corporation. Rabbit brain-derived tissue thromboplastin reagent (TP; final concentration 0.0166 ⁇ g / mL), calcium chloride (Ca; final concentration 12 mM), heparan sulfate (final concentration 0.3, 0.5, 1 ⁇ g / mL) and CTI (final concentration 20 ⁇ g / mL) Blood coagulation analysis was performed with and without the addition of soluble thrombomodulin (rTM; final concentration 10 nM) with the addition of PKSI-527 (final concentration 40 ⁇ M).
  • TP brain-derived tissue thromboplastin reagent
  • Ca calcium chloride
  • heparan sulfate final concentration 0.3, 0.5, 1 ⁇ g / mL
  • CTI final concentration 20 ⁇ g / mL
  • the CT (clotting time) and CFT value (clot formation time) are shown in Table 8.
  • thromboplastin 0.0166 ⁇ g / mL was added, no increase in CT value of 1.5 times or more was confirmed even when soluble thrombomodulin 10 nM was added.
  • heparan sulfate 1.0 ⁇ g / mL was added, the blood coagulation time was 1 hour or more and no CT value was obtained.
  • Example 19 ROTEM was used for blood coagulation analysis. Blood was collected using a blood collection tube containing 3.2% sodium citrate manufactured by Terumo Corporation. Rabbit brain-derived tissue thromboplastin reagent (TP; final concentration 16.6 ⁇ g / mL), calcium chloride (Ca; final concentration 12 mM) and unfractionated heparin (Mochida Pharmaceutical) (final concentration 0, 0.1, 0.2, 0.3 U / mL) Blood coagulation analysis was performed with the addition of CTI (final concentration 20 ⁇ g / mL) and PKSI-527 (final concentration 40 ⁇ M), with and without the addition of soluble thrombomodulin (rTM; final concentration 100 nM).
  • TP brain-derived tissue thromboplastin reagent
  • Ca calcium chloride
  • rTM final concentration 100 nM
  • the CT (clotting time) and CFT value (clot formation time) are shown in Table 9.
  • the CT values for unfractionated heparin (0 and 0.1 U / mL) at 16.6 ⁇ g / mL of thromboplastin were 46 and 53 seconds. Furthermore, no significant prolongation of CT values was confirmed even when soluble thrombomodulin 100 nM was added.
  • the CT value when thromboplastin 16.6 ⁇ g / mL and unfractionated heparin (0 and 0.3 U / mL) were added was 69 seconds. Furthermore, a significant prolongation of CT values was confirmed when soluble thrombomodulin 100 nM was added.
  • Example 20 ROTEM was used for blood coagulation analysis. Blood was collected using a blood collection tube containing 3.2% sodium citrate manufactured by Terumo Corporation. Rabbit brain-derived tissue thromboplastin reagent (TP; final concentration 16.6 ⁇ g / mL), calcium chloride (Ca; final concentration 12 mM) and Fonda parinux sodium (trade name; Alixtra; final concentration 0, 0.5, 2, 3, 4, 5 ⁇ g) / mL), CTI (final concentration 20 ⁇ g / mL) and PKSI-527 (final concentration 40 ⁇ M) were added, and blood coagulation analysis was performed with and without the addition of soluble thrombomodulin (rTM; final concentration 100 nM).
  • TP brain-derived tissue thromboplastin reagent
  • Ca calcium chloride
  • Fonda parinux sodium trade name; Alixtra; final concentration 0, 0.5, 2, 3, 4, 5 ⁇ g) / mL
  • CTI final concentration 20 ⁇ g / mL
  • the CT (clotting time) and CFT value (clot formation time) are shown in Table 10.
  • the CT values of thromboplastin 16.6 ⁇ g / mL and Alixtra (0,0.5 ⁇ g / mL) were both less than 1 minute, and a significant prolongation of CT values was confirmed when soluble thrombomodulin 100 nM was added. There wasn't.
  • the CT values when thromboplastin 16.6 ⁇ g / mL and Alixtra (2, 3, 4, 5 ⁇ g / mL) were added were both 64 seconds to 78 seconds, and when soluble thrombomodulin 100 nM was added, both were performed. It was confirmed that the CT value was extended by 1.5 times or more.
  • Example 21 ROTEM was used for blood coagulation analysis. Blood was collected using a blood collection tube containing 3.2% sodium citrate manufactured by Terumo Corporation. Rabbit brain-derived tissue thromboplastin reagent (TP; final concentration 0.64 ⁇ g / mL), calcium chloride (Ca; final concentration 12 mM), Alixtra (final concentration 0 and 0.5 ⁇ g / mL), CTI (final concentration 20 ⁇ g / mL) and PKSI- Blood coagulation analysis was performed with the addition of 527 (final concentration 40 ⁇ M) and with and without the addition of soluble thrombomodulin (rTM; final concentration 30 nM).
  • TP brain-derived tissue thromboplastin reagent
  • Ca calcium chloride
  • Alixtra final concentration 0 and 0.5 ⁇ g / mL
  • CTI final concentration 20 ⁇ g / mL
  • PKSI- Blood coagulation analysis was performed with the addition of 527 (final concentration 40 ⁇ M) and with and without
  • CT clotting time
  • CFT value clot formation time
  • Example 22 ROTEM was used for blood coagulation analysis. Blood was collected using a blood collection tube containing 3.2% sodium citrate manufactured by Terumo Corporation. Rabbit brain-derived tissue thromboplastin reagent (TP; final concentration 0.055 ⁇ g / mL), calcium chloride (Ca; final concentration 12 mM), Alixtra (final concentration 0, 0.05, 0.2 ⁇ g / mL) and CTI (final concentration 20 ⁇ g / mL) Blood coagulation analysis was performed with and without the addition of soluble thrombomodulin (rTM; final concentration 20 nM) with the addition of PKSI-517 (final concentration 40 ⁇ M).
  • TP brain-derived tissue thromboplastin reagent
  • Ca calcium chloride
  • Alixtra final concentration 0, 0.05, 0.2 ⁇ g / mL
  • CTI final concentration 20 ⁇ g / mL
  • the CT (clotting time) and CFT value (clot formation time) are shown in Table 12.
  • the CT value of thromboplastin at 0.055 ⁇ g / mL without the addition of Alixtra was 575 seconds. Furthermore, no significant prolongation of CT values was confirmed even when soluble thrombomodulin 20 nM was added.
  • the CT values when thromboplastin 0.055 ⁇ g / mL and Alixtra (0.05 ⁇ g / mL) were added were both 619 seconds. Furthermore, when soluble thrombomodulin 20nM was added, it was confirmed that the CT value was extended by about 1.7 times.
  • CT values when thromboplastin 0.055 ⁇ g / mL and Alixtra (0.2 ⁇ g / mL) were added were both 799 seconds. Furthermore, when soluble thrombomodulin 20 nM was added, the blood coagulation time was 1 hour or more, and no CT value was obtained.
  • Example 23 ROTEM was used for blood coagulation analysis. Blood was collected using a blood collection tube containing 3.2% sodium citrate manufactured by Terumo Corporation. Rabbit brain-derived tissue thromboplastin reagent (TP; final concentration 16.6 ⁇ g / mL), calcium chloride (Ca; final concentration 12 mM) and low-molecular-weight heparin (trade name Fragmin; Physer) (final concentration 0, 0.2, 1 U / mL) And CTI (final concentration 20 ⁇ g / mL) and PKSI-527 (final concentration 40 ⁇ M) were added, and blood coagulation analysis was performed with and without the addition of soluble thrombomodulin (final concentration 100 nM).
  • TP brain-derived tissue thromboplastin reagent
  • Ca calcium chloride
  • Fragmin trade name Fragmin
  • PKSI-527 final concentration 40 ⁇ M
  • the CT (clotting time) and CFT value (clot formation time) are shown in Table 13.
  • the CT values for thromboplastin (16.6 ⁇ g / mL) and fragmin (0 and 0.2 U / mL) were 46 and 50 seconds. Furthermore, no significant prolongation of CT values was confirmed even when soluble thrombomodulin 100 nM was added. On the other hand, the CT value when thromboplastin 16.6 ⁇ g / mL and fragmin (1 U / mL) were added was 83 seconds. Furthermore, when soluble thrombomodulin 100 nM was added, it was confirmed that the CT value was extended by about 1.5 times.
  • Example 24 ROTEM was used for blood coagulation analysis. Blood was collected using a blood collection tube containing 3.2% sodium citrate manufactured by Terumo Corporation. Rabbit ability-derived tissue Thromboplastin reagent (TP; final concentration 0.64 ⁇ g / mL), calcium chloride (Ca; final concentration 12 mM), low-molecular-weight heparin (Flagmin; Physer) (final concentration 0 and 0.2 U / mL) and CTI (final concentration 0 and 0.2 U / mL) Blood coagulation analysis was performed with and without the addition of soluble thrombomodulin (rTM; final concentration 30 nM) with the addition of PKSI-527 (final concentration 40 ⁇ M) and PKSI-527 (concentration 20 ⁇ g / mL).
  • TP tissue Thromboplastin reagent
  • Ca calcium chloride
  • Flagmin low-molecular-weight heparin
  • Flagmin low-molecular-weight heparin
  • CT clotting time
  • CFT value clot formation time
  • Example 25 ROTEM was used for blood coagulation analysis. Blood was collected using a blood collection tube containing 3.2% sodium citrate manufactured by Terumo Corporation. Rabbit brain-derived tissue thromboplastin reagent (TP; final concentration 0.055 ⁇ g / mL), calcium chloride (Ca; final concentration 12 mM), low-molecular-weight heparin (Flagmin; Physer) (final concentration 0, 0.05, 0.15 units / mL) and CTI Blood coagulation analysis was performed with the addition of (final concentration 20 ⁇ g / mL) and PKSI-527 (final concentration 40 ⁇ M), with and without the addition of soluble thrombomodulin (rTM; final concentration 20 nM).
  • TP brain-derived tissue thromboplastin reagent
  • Ca calcium chloride
  • Flagmin low-molecular-weight heparin
  • rTM final concentration 40 ⁇ M
  • the CT (clotting time) and CFT value (clot formation time) are shown in Table 15.
  • the CT values of thromboplastin 0.055 ⁇ g / mL and fragmin (0, 0.05, 0.15 units / mL) were 575, 614, 886 seconds.
  • soluble thrombomodulin 20 nM was added, no significant prolongation of CT values was confirmed when no fragmin was added, but by adding 0.05 unit / mL of fragmin, the blood increased about 1.6 times after the addition of soluble thrombomodulin. Coagulation was prolonged.
  • 0.15 unit / mL of fragmin was added, the blood coagulation time was 1 hour or more and no CT value was obtained when soluble thrombomodulin was added.
  • Example 26 ROTEM was used for blood coagulation analysis. Blood was collected using a blood collection tube containing 3.2% sodium citrate manufactured by Terumo Corporation. Dade innovin (recombinant tissue factor: Sysmex Co., Ltd.) was diluted 50-fold after dialysis with 4% (final concentration 0.569 ⁇ g / mL), calcium chloride (Ca; final concentration 12 mM) and heparan sulfate (final concentration 0 or 2 ⁇ g / mL).
  • Dade innovin recombinant tissue factor: Sysmex Co., Ltd.
  • APC-inhibiting aptamer (HS02-44G; final concentration 1 ⁇ M) was added for blood coagulation analysis.
  • the CT (clotting time) is shown in Table 16.
  • the addition of heparan sulfate significantly enhanced the anticoagulant capacity of soluble thrombomodulin.
  • the coagulation time was shortened by inhibiting activated protein C. The shortening range is considered to reflect the anticoagulant ability mediated by protein C and protein S.
  • Example 27 ROTEM was used for blood coagulation analysis. Blood was collected using a blood collection tube containing 3.2% sodium citrate manufactured by Terumo Corporation. 4% (final concentration 0.569 ⁇ g / mL), calcium chloride (Ca; final concentration 12 mM) and low molecular weight heparan (trade name Fragmin; Physer) (final concentration 0 or 0.2 U / mL) obtained by diluting Dadeinobin 50 times after dialysis.
  • the CT (clotting time) is shown in Table 17.
  • the addition of fragmine markedly enhanced the anticoagulant capacity of soluble thrombomodulin.
  • the coagulation time was shortened by inhibiting activated protein C. The shortening range is considered to reflect the anticoagulant ability mediated by protein C and protein S.
  • Example 28 ROTEM was used for the analysis of coagulation. Blood was collected using a blood collection tube containing 3.2% sodium citrate manufactured by Terumo Corporation. 4% (final concentration 0.569 ⁇ g / mL), calcium chloride (Ca; final concentration 12 mM), Alixtra (final concentration 0 or 0.5 ⁇ / mL) and CTI (final concentration 20 ⁇ g / mL) obtained by diluting Dadeinobin 50-fold after dialysis ) And PKSI-527 (final concentration 40 ⁇ M), with and without soluble thrombomodulin (rTM; final concentration 30 nM), and with soluble thrombomodulin 30 nM, in addition to the APC-inhibiting aptamer (HS02-44G; Blood coagulation analysis was performed when a final concentration of 1 ⁇ M) was added.
  • 4% final concentration 0.569 ⁇ g / mL
  • Ca calcium chloride
  • Alixtra final concentration 0 or 0.5 ⁇ / mL
  • the CT (clotting time) is shown in Table 18.
  • the addition of Alixtra markedly enhanced the anticoagulant capacity of soluble thrombomodulin.
  • the coagulation time was shortened by inhibiting activated protein C.
  • the shortening range is considered to reflect the anticoagulant ability mediated by protein C and protein S.
  • Example 29 Measurement of PT values of rabbit brain-derived tissues thromboplastin and daidoinobin Using a semi-automatic blood coagulation measuring device CA104 (Sysmex Corporation), the PT values of the tissue factors used in the examples were measured.
  • Rabbit brain-derived tissue thromboplastin (415 ⁇ g / mL) was diluted with Buffer (5 mM Hepes, 0.15 M NaCl (pH 7.4)) containing 100 ⁇ g / mL of human albumin.
  • Dade innovin was diluted in distilled water after dialysis (711.3 ⁇ g / mL) with Buffer containing 100 ⁇ g / mL of human albumin (5 mM Hepes, 0.15 M NaCl (pH 7.4)). After incubating 50 ⁇ L of standard plasma at 37 ° C. for 1 minute, the mixture was mixed with 50 ⁇ L of a rabbit brain-derived tissue thromboplastin or deidoinobin solution diluted with 50 ⁇ L of calcium chloride solution (25 mM), and the coagulation time (PT value) was measured.
  • PT value coagulation time
  • tissue thromboplastin or tissue recombinant tissue factor which has a prothrombin time of 19 to 105 seconds in standard plasma, as the extrinsic blood coagulation activation reagent to the blood. Shown.
  • Example 30 ROTEM was used for blood coagulation analysis. Blood was collected using a blood collection tube containing 3.2% sodium citrate manufactured by Terumo Corporation. Blood coagulation was added by adding the following reagents (1) to (4).
  • Rabbit brain-derived tissue thromboplastin reagent (TP; final concentration 0.664 ⁇ g / mL), calcium chloride (final concentration 12 mM) and heparan sulfate (final concentration 1 ⁇ g / mL)
  • Rabbit brain-derived tissue thromboplastin reagent (TP; final concentration 0.664 ⁇ g / mL), calcium chloride (Ca; final concentration 12 mM), heparan sulfate (final concentration 1 ⁇ g / mL), and CTI (final concentration 20 ⁇ g / mL)
  • Rabbit brain-derived tissue thromboplastin reagent (TP; final concentration 0.664 ⁇ g / mL), calcium chloride (Ca; final concentration 12 mM), heparan sulfate (final concentration 1 ⁇ g / mL) and PKSI-527 (final concentration 40 ⁇ M)
  • Rabbit brain-derived tissue thromboplastin reagent (TP; final concentration
  • the results are shown below.
  • the CT (clotting time) is shown in Table 21.
  • Example 31 ROTEM was used for blood coagulation analysis. Blood was collected using a blood collection tube containing 3.2% sodium citrate manufactured by Terumo Corporation. Blood coagulation was added by adding the following reagents (1) to (4).
  • Rabbit brain-derived tissue thromboplastin reagent (TP; final concentration 0.664 ⁇ g / mL), calcium chloride (Ca; final concentration 12 mM) and heparan sulfate (final concentration 1 ⁇ g / mL)
  • Rabbit brain-derived tissue thromboplastin reagent (TP; final concentration 0.664 ⁇ g / mL), calcium chloride (Ca; final concentration 12 mM), heparan sulfate (final concentration 1 ⁇ g / mL), and CTI (final concentration 20 ⁇ g / mL)
  • Rabbit brain-derived tissue thromboplastin reagent (TP; final concentration 0.664 ⁇ g / mL), calcium chloride (Ca; final concentration 12 mM), heparan sulfate (final concentration 1 ⁇ g / mL) and PKSI-527 (final concentration 40 ⁇ M)
  • Rabbit brain-derived tissue thromboplastin reagent (TP
  • CT clotting time
  • Example 32 ROTEM was used for blood coagulation analysis. Blood was collected using a blood collection tube containing 3.2% sodium citrate manufactured by Terumo Corporation. Blood coagulation was added by adding the following reagents (1) to (4).
  • TP rabbit brain-derived tissue thromboplastin reagent
  • Ca calcium chloride
  • CTI final concentration 20 ⁇ g / mL
  • PKSI-527 final concentration 40 ⁇ M
  • dextran sulfate dextran sulfate.
  • TP Rabbit brain-derived tissue thromboplastin reagent
  • Ca calcium chloride
  • CTI final concentration 20 ⁇ g / mL
  • PKSI-527 final concentration 40 ⁇ M
  • Dextran Sulfuric Acid (Meishu Sangyo Dextran Sulfate Ester Sodium Sulfur 18 Average Molecular Weight 5000, Final Concentration 60 ⁇ g / mL), Soluble Thrombomodulin (rTM; Final Concentration 40 nM) and Unfractionated Heparin (Final Concentration 0.5 U / mL) (6)
  • Rabbit brain-derived tissue thromboplastin reagent TP; final concentration 0.664 ⁇ g / mL
  • calcium chloride Ca
  • CTI final concentration 20 ⁇ g / mL
  • PKSI-527 final concentration 40 ⁇ M
  • dextran sulfate (Meishu Sangyo Dextran Sulfate Ester Sodium Sulfur 18 Average Molecular Weight 5000, Final Concentration 60 ⁇ g / mL), Soluble Thrombomodulin (rTM; Final Concentration 40 nM) and Unfractionated Heparin (Final Concentration 0.5 U /
  • CT clotting time
  • dextran sulfate As a heparin-like substance, a good prolonging effect of TM can be obtained. Furthermore, it was possible to evaluate the prolonging effect of soluble thrombomodulin even when unfractionated heparin and heparinase were added. From this, it is considered that the anticoagulant ability via PS / PC can be evaluated by using dextran sulfate as a heparin-like substance when evaluating the blood of a heparin-administered patient.

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SHINOZAWA, KEIKO; NOGAMI, KEIJI: "Novel factor V mutation (W1920R, FV Nara) associated with serious deep vein thrombosis and APC resistance: The plot thickens", JAPANESE JOURNAL OF THROMBOSIS AND HEMOSTASIS, vol. 25, no. 4, 1 January 2014 (2014-01-01), JP , pages 482 - 493, XP009537924, ISSN: 0915-7441, DOI: 10.2491/jjsth.25.482 *

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