All blood tests were implemented within 24 hours before operation. The conventional coagulation testing, including APTT, PT, thrombin time (TT), and fibrinogen (FIB), was performed on the patients. Those parameters were determined by CA1500®（Sysmex co.,ltd. Japan）. All the reagents were manufactured by Siemens co.,ltd.: APTT（Actin, 0.025 mol/L CaCl2）, PT(Thromborel S), TT（Test Thrombin Reagent）, FIB(Thrombin Reagent 100NIH).Similarly, a complete blood cell analysis was also conducted, where attention was given to the platelet count and neutrolphil‐to‐lymphocyte ratio (NLR). Conventional TEG analysis was performed with the TEG 5000® (Haemoscope co.,ltd.). Five TEG parameters were measured as a presentation of coagulation status: R (reaction time, normal range: 3‐8 minutes); K time (normal range: 1‐3 minutes); α (alpha angle, normal range: 55° to 78°); MA (maximum amplitude, normal range: 51‐69 mm), and CI (coagulation index, normal range: −3 to 3).
Test thrombin reagent
Manufactured by Siemens
Sourced in Germany
The Test Thrombin Reagent is a laboratory equipment product designed to detect and analyze the presence of thrombin, a key enzyme involved in the blood coagulation process. The reagent provides a reliable and standardized method for assessing thrombin levels, which is essential for various medical and research applications.
Automatically generated - may contain errors
Lab products found in correlation
Chloral hydrate, by Tianjin Damao (3 mentions)
Dade actin activated cephaloplastin reagent, by Siemens (2 mentions)
Ca 50, by Sysmex (2 mentions)
Actin fs activated ptt reagent, by Siemens (2 mentions)
Thromborel s, by Siemens (2 mentions)
Ca 1500, by Sysmex (1 mentions)
T4 polynucleotide kinase, by Merck Group (1 mentions)
32p labeled atp, by PerkinElmer (1 mentions)
Origin 8, by OriginLab (1 mentions)
Dade actin fs activated ptt reagent, by Siemens (1 mentions)
Thromborel s reagent, by Siemens (1 mentions)
Calcium chloride solution, by Siemens (1 mentions)
Dade fibrinogen determination reagents, by Siemens (1 mentions)
6 protocols using test thrombin reagent
1
Comprehensive Coagulation Assessment in Patients
2
Evaluating Antithrombogenicity of Stimuli-Responsive Hemicellulose Microgels
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To evaluate the antithrombogenicity of
the stimuli-responsive hemicellulose microgels, activated partial
thromboplastin time (APTT) and thrombin time (TT) were measured by
an automated blood coagulation analyzer CA-50 (Sysmex Corporation,
Kobe, Japan) according to the method described in a previous report.41 Healthy human fresh blood was collected in vacuum
tubes containing sodium citrate as the anticoagulant (anticoagulant
to blood ratio, 1:9 v/v), and the platelet-poor plasma (PPP) was obtained
after centrifuging at 4000 rpm for 15 min. Stimuli-responsive hemicellulose
microgels (10 mg/mL) were prepared in buffer solution (pH = 7.4),
5 μL suspensions and 100 μL of PPP were incubated at 37
°C for 0.5 h, and 50 μL of incubated solution was then
added to the test cup, followed by the addition of 50 μL of
APTT agent (Dade Actin Activated Cephaloplastin Reagent, Siemens;
incubated 10 min before use). The solution was incubated at 37 °C
for another 3 min, and 50 μL of 0.025 M CaCl2 was
subsequently added. The APPT was recorded by an automated blood coagulation
analyzer CA-50. Two independent measurements were averaged to reach
a reliable value. The TT test was performed in a process similar to
that of the APTT test. The only difference is the APTT agent replaced
by Test Thrombin Reagent (Siemens; incubated 10 min before use).
the stimuli-responsive hemicellulose microgels, activated partial
thromboplastin time (APTT) and thrombin time (TT) were measured by
an automated blood coagulation analyzer CA-50 (Sysmex Corporation,
Kobe, Japan) according to the method described in a previous report.41 Healthy human fresh blood was collected in vacuum
tubes containing sodium citrate as the anticoagulant (anticoagulant
to blood ratio, 1:9 v/v), and the platelet-poor plasma (PPP) was obtained
after centrifuging at 4000 rpm for 15 min. Stimuli-responsive hemicellulose
microgels (10 mg/mL) were prepared in buffer solution (pH = 7.4),
5 μL suspensions and 100 μL of PPP were incubated at 37
°C for 0.5 h, and 50 μL of incubated solution was then
added to the test cup, followed by the addition of 50 μL of
APTT agent (Dade Actin Activated Cephaloplastin Reagent, Siemens;
incubated 10 min before use). The solution was incubated at 37 °C
for another 3 min, and 50 μL of 0.025 M CaCl2 was
subsequently added. The APPT was recorded by an automated blood coagulation
analyzer CA-50. Two independent measurements were averaged to reach
a reliable value. The TT test was performed in a process similar to
that of the APTT test. The only difference is the APTT agent replaced
by Test Thrombin Reagent (Siemens; incubated 10 min before use).
3
Aptamer-Based Anticoagulant Formulation
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DNA aptamer RA-36, dGGTTGGTGTGGTTGGTGGTTGGTGTGGTTGG⋅2 KCl, was synthesized and purified up to pharmaceutical grade for intravenous injections at ‘APTO-PHARM,’ Ltd. Russian Federation. The drug formulation for intravenous injections contained 10 mg/ml of the active substance in 0.9% sodium chloride solution; to prepare that formulation, the aptamer stock solution was diluted with 0.9% sodium chloride immediately before the intravenous injection, when proper dosing is needed.
Lyophilized bivalirudin (Angiox®, lot# PL2097) was purchased from Medicines Company, United Kingdom. The powder was dissolved in 0.9% sodium chloride at a concentration of 5 mg/ml according to the manufacturer’s guidelines. The stock solution was diluted with 0.9% sodium chloride immediately before intravenous injection, when proper dosing is needed.
Standard human plasma, Test Thrombin Reagent, Thromborel® S and Actin FS Activated PTT Reagent were purchased from Siemens, Germany. 32P-labeled ATP with beta radiation activity of 20 MBq was purchased from Perkin-Elmer, United States. T4 polynucleotide kinase (Sigma–Aldrich, United States).
Lyophilized bivalirudin (Angiox®, lot# PL2097) was purchased from Medicines Company, United Kingdom. The powder was dissolved in 0.9% sodium chloride at a concentration of 5 mg/ml according to the manufacturer’s guidelines. The stock solution was diluted with 0.9% sodium chloride immediately before intravenous injection, when proper dosing is needed.
Standard human plasma, Test Thrombin Reagent, Thromborel® S and Actin FS Activated PTT Reagent were purchased from Siemens, Germany. 32P-labeled ATP with beta radiation activity of 20 MBq was purchased from Perkin-Elmer, United States. T4 polynucleotide kinase (Sigma–Aldrich, United States).
4
Coagulation Assay for Thrombin Inhibitors
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Samples of citrated blood were centrifuged for 15 min at 1400 rpm to separate plasma from blood cells. Coagulation of blood plasma was estimated with coagulometer CA-50 (Sysmex, Japan) using the reagent sets from Siemens, Germany: Test Thrombin Reagent for thrombin time (TT) determination, Thromborel® S for prothrombin time (PT) determination, Actin FS Activated PTT Reagent for activated partial thromboplastin time (APTT) determination. Standard Human Plasma was used to test the functional activity of the reagents. Aptamer RA-36 and bivalirudin prolong coagulation time of blood plasma due to thrombin inhibiting. The data were treated with Origin 8.1 (OriginLab, United States). Descriptive statistics was performed, including mean values, geometrical mean values, standard deviations, coefficients of variation, medians, lower 90% CI of mean and upper 90% CI of mean.
5
Coagulation Assays for Plasma Interactions
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The SF, Hep/SF films, and PTFE sheets were pre-incubated under PBS at 37 °C for 5 min. The PBS was replaced with fresh frozen plasma (FFP) that was already thawed at 37 °C and incubated at 37 °C for 1 h. After that, activated partial thromboplastin time (APTT), prothrombin time (PT), and thrombin time (TT) were measured with Dade Actin FS Activated PTT Reagent, Thromborel® S Reagent, and Test Thrombin Reagent, respectively, following the manufacturer’s protocols (Siemens Healthcare Diagnostics, Marburg, Germany). The clotting time was read and recorded using a semi-automated blood coagulation analyzer (Sysmex® CA-50, Sysmex Corporation, Kobe, Japan). The experiments were performed in triplicate for each sample.
6
Characterization of Moutan Cortex Charcoal Compounds
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Reagents All the chemicals used in the experiments of this study were of reagent grade and obtained from commercial suppliers: Dade Actin Activated Cephaloplastin Reagent, Calcium Chloride Solution, Dade Fibrinogen Determination Reagents, Test Thrombin Reagent and Detergent for Fully Automated Blood Coagulation Analyzer (all from Siemens Healthcare Diagnostics Products GmbH, Germany); Disposable Vacuum Blood Vessels (SANLI, China); Chloral Hydrate (Damao Chemical Reagent Factory, Tianjin, China).
New Zealand white rabbits purchased from Laboratory Animal Center, Guangzhou University of Chinese Medicine (License No: SCXK (YUE) 2016-0041, Guangzhou, China); Compounds (M3, M9, M10, M11, M12, M15, M24, M31) were extracted, isolated and purified from Moutan Cortex Charcoal in our laboratory. The purity of the isolated components was shown to be higher than 96% by HPLC, and their structures were elucidated by comparing their spectroscopic data (electrospray ionization (ESI)-MS, 1 H-NMR and 13 (link) C-NMR, heteronuclear multiple bond connectivity (HMBC), 1 H-detected heteronuclear multiple quantum coherence (HMQC) and H-H correlation spectroscopy (COSY)) in literature. 29, 30) Structures of the eight compounds are shown in Fig. 2.
New Zealand white rabbits purchased from Laboratory Animal Center, Guangzhou University of Chinese Medicine (License No: SCXK (YUE) 2016-0041, Guangzhou, China); Compounds (M3, M9, M10, M11, M12, M15, M24, M31) were extracted, isolated and purified from Moutan Cortex Charcoal in our laboratory. The purity of the isolated components was shown to be higher than 96% by HPLC, and their structures were elucidated by comparing their spectroscopic data (electrospray ionization (ESI)-MS, 1 H-NMR and 13 (link) C-NMR, heteronuclear multiple bond connectivity (HMBC), 1 H-detected heteronuclear multiple quantum coherence (HMQC) and H-H correlation spectroscopy (COSY)) in literature. 29, 30) Structures of the eight compounds are shown in Fig. 2.
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