The fine structure of enoxaparin sodium samples with different degree of 1,6-anhydro derivatives were analyzed with polyacrylamide gel electrophoresis, high performance liquid chromatography, ultraviolet spectroscopy, infrared spectroscopy and nuclear magnetic resonance spectroscopy. A further study of anticoagulation activity of enoxaparins was performed, including those on their inhibition activities of coagulation factor Xa (FXa) and thrombin (FIIa). The results showed that the anti-FXa and -FIIa activities of enoxaparins with different degree of 1,6-anhydro derivatives (20.0%-39.7%) with similar structure characteristics, had decreasing tendency when the degree of 1,6-anhydro derivatives increased. Especially, the anti-FXa activity was sensitive to the change of the degree of 1,6-anhydro derivatives.
Anticoagulants ; chemistry ; Enoxaparin ; chemistry ; Factor Xa Inhibitors ; chemistry ; Thrombin ; antagonists & inhibitors

This dissertation is to determine the biopotency of hemostat which processed in different places by establishing a bioassay method of Bletillae Rhizoma based on the thrombin time. Contrast test is the main methodology. Specifically, the reference substance of Bletillae Rhizoma is determined by comparing with the control substance of vitamin K1 using thrombin time, which is calibrated the Bletillae Rhizoma. The hemostatic biopotency is calculated by using the method of "parallel line assay method based on quantitative responses" (3.3) from different processed products. It indicates that there is a strong linear correlation between Bletillae Rhizoma and control drugs (Y = 66.332-23.913X, R2 = 0.995 3). The hemostatic biopotency of Bletillae Rhizoma from different processed products ranged between 821.93-1 187.53 U x g(-1) shown in the paper, and all of them can meet the requirements of the test. The methodology has an appropriate instrument precision (RSD 3.8%), intermediate precision (RSD 4.6%), repeatability (RSD 3.2%) and stability (RSD 3.7%). Therefore, it can be turned out that the methodology which established in the dissertation is good at determinating the hemostatic biopotency of Bletillae Rhizoma and it is reliable, simple and repeatable.
Animals ; Drugs, Chinese Herbal ; pharmacology ; standards ; Hemostatics ; pharmacology ; standards ; Orchidaceae ; chemistry ; Rats ; Rats, Sprague-Dawley ; Rhizome ; chemistry ; Thrombin Time

The effect of chitosan with different molecular weight and deacetylation degree on blood hemostasis was tested. The experiments found evident alteration of red blood cell morphology and unusual coagglutination between erythrocytes in the anticoagulant blood which was treated by chitosan acetic acid solution. The red blood cells clot formation experiments showed that chitosan with low deacetylation degree (60%-70%) caused more red blood cells to assemble when compared versus chitosan with other deacetylation degrees. The effect of molecular weight between 10(5)-10(6) was not obvious. The thrombin time (TT), prothrombin time (PT), activated partial thromboplastin time (APTT), and concentration of fibrinogen (FIB) of blood treated by chitosan acetic acid solution were measured. The results proved that the hemostasis property of chitosan acetic acid solution was independent of the platelets and the normal "Cascade-like" coagulation pathway.
Acetic Acid ; chemistry ; pharmacology ; Animals ; Chitosan ; chemistry ; pharmacology ; Fibrinogen ; analysis ; Hemostatics ; chemistry ; pharmacology ; Partial Thromboplastin Time ; Prothrombin Time ; Rabbits ; Thrombin Time

Aged ; Aged, 80 and over ; Blood Platelets ; chemistry ; Humans ; Receptor, PAR-1 ; blood ; Receptors, Thrombin ; blood ; Renal Dialysis ; Uremia ; blood ; therapy

Rat model of blood stasis syndrome was prepared by subcutaneous injecting of epinephrine hydrochlorid,then the model rats were administrated by Yunnan Baiyao for 15 days. Blood rheology,coagulation function and histopathology were chosen as indicators to evaluate the successful replication of blood stasis syndrome model and the treatment effect of Yunnan Baiyao. UPLC-Q-TOF-MS was used to rapidly analyze the serum samples of blood stasis syndrome rat after 15 days Yunnan Baiyao treatment,Progenesis QI software was employed to identify the alkaloids components. The results showed that Yunnan Baiyao reduced the plasma viscosity and whole blood viscosity of rats with blood stasis syndrome,prolonged thrombin and prothrombin time,reduced fibrinogen content,and effectively improved pathological state such as inflammatory cell infiltration,blood stasis,congestion and edema of various organs in rats with blood stasis syndrome. Seven alkaloids components from Aconitum kusnezoffii,including karacolidine,senbusine B,isotalatizidine,karakoline,denudatine,talatisamine and chasmanine were found in the rat serum after Yunnan Baiyao treatment. Based on the effectiveness of Yunnan Baiyao in the treatment of blood stasis syndrome induced by epinephrine hydrochloride in rats,alkaloids components from the root of A. kusnezoffii absorbed into blood after Yunnan Baiyao treatment were clarified rapidly and accurately with the help of UPLC-Q-TOF-MS. Karacolidine,senbusine B,isotalatizidine,karakoline,denudatine,talatisamine and chasmanine are the pharmacodynamic material basis of the root of A. kusnezoffii for activating blood circulation and removing blood stasis.
Aconitum ; chemistry ; Animals ; Blood Circulation ; drug effects ; Blood Viscosity ; Drugs, Chinese Herbal ; pharmacology ; Prothrombin Time ; Rats ; Thrombin Time

The changes of bioactive constituents were analyzed for Puhuang-Wulingzhi before and after compatibility and the antiplatelet and antithrombin activitives were evaluated in order to elucidate the scientific and reasonable of Puhuang-Wulingzhi compatibility. UPLC-QTOF-MA-Markerlynx, principal component analysis (PCA) and orthogonal partial least-squares discriminant analysis were used for data analysis and tracking changes of chemical composition during the decocting process. In vitro platelet aggregation induced by ADP, thrombin time(TT) and prothrombin time (PT) were investigated for Puhuang-Wulingzhi before and after compatibility. The results showed that significant differences were found between the mixed decoction and codecoction of Wulingzhi and Puhuang. Five compounds changed obviously were identified as typhaneoside, naringenin, isorhamnetin-3-O-ruinoside, quercetin-3-O-neohesperidoside, kaempferol-3-O-neohesperidoside. The codecoction, comparing with the single decoction, was more significant in antiplatelet aggregation and could prolong thrombin time. In the same crude drug dose, the thrombin time (TT) elongation were greater. These data could provide references for elucidation of bioactive components for this herb pair.
Animals ; Antithrombins ; chemistry ; pharmacology ; Blood Platelets ; drug effects ; physiology ; Drugs, Chinese Herbal ; chemistry ; pharmacology ; Female ; Humans ; Molecular Structure ; Platelet Aggregation ; drug effects ; Rabbits ; Thrombin Time

SHG was sulfated by chlorosulfonic acid-pyridine method, and six samples which we got were prepared in different reaction conditions. There is a characteristic absorption peak near 260 nm in UV spectra and there are two characteristic absorption peaks near 1240 cm(-1) and 810 cm(-1) in the FT-IR. Degree of sulfation (DS) was calculated by elemental analysis and turbidimetry. Under the same conditions the absorption peaks become strong with the DS increase. The anticoagulant activity of SHG and sulfated modification samples was evaluated by the classic coagulant assays of prothrombin time (PT), activated partial thrombin time (APTT) live enzymes, and plasma thrombin time (TT). Results show that sulfated SHG has a good anticoagulant activity in vitro, and DS increased activity within a certain range.
Animals ; Anticoagulants ; chemistry ; isolation & purification ; pharmacology ; Blood Coagulation Tests ; Fabaceae ; chemistry ; Glucans ; chemistry ; isolation & purification ; pharmacology ; Partial Thromboplastin Time ; Plants, Medicinal ; chemistry ; Prothrombin Time ; Rabbits ; Spectrophotometry, Ultraviolet ; Spectroscopy, Fourier Transform Infrared ; Sulfonic Acids ; chemistry ; Thrombin Time

OBJECTIVE: To develop methods of extraction and purification of Cterminal NUDT9 homology domain of human transient receptor potential melastatin 2 (TRPM2) channel. METHODS: After sonication and centrifuge of strain Rosetta (DE3) which was induced by isopropylthio-β-D-galactoside, GST-NUDT9-H was collected after the binding of supernatant with GST beads and eluted with reduced glutathione. Then the elution buffer containing fusion protein was purified by size exclusion chromatography after concentration and centrifuge. Finally, with the cleavage of thrombin and binding with the GST beads, NUDT9-H with high purity in supernatant was collected. RESULTS: The GST-NUDT9-H fusion protein was stabilized with lysis buffer containing 0.5% n-dodecyl -β-d-maltoside (DDM), and wash buffer containing 0.025% DDM in size-exclusion chromatography system, and finally the NUDT9-H with high purity was obtained after cleaved by thrombin (1 U/2 mg fusion protein) for 24 h. CONCLUSIONS Due to the poor stability of NUDT9-H, it is necessary to add DDM in extraction and purification buffer to stabilize the conformation of NUDT9-H, so as to increase its yields and purity.
Escherichia coli ; genetics ; Glucosides ; chemistry ; Humans ; Protein Domains ; Protein Stability ; Pyrophosphatases ; chemistry ; genetics ; isolation & purification ; Recombinant Fusion Proteins ; chemistry ; isolation & purification ; TRPM Cation Channels ; chemistry ; isolation & purification ; Thrombin ; metabolism

This study was designed to compare the effects of protease-activated receptor 1 (PAR-1) and protease-activated receptor 4 (PAR-4) to the expression of platelet surface GPIbalpha and cytoskeleton reorganization, then to investigate the role of PARs in platelet signal transmission. PAR1 (25 micromol/L) and PAR4 (250 micromol/L) were used to stimulate platelet at different time points (0 - 60 minutes), and the platelet surface GPIbalpha, actin and myosin and P-selectin were detected with flow cytometry, the alteration of GPIbalpha, actin and myosin in cytoskeleton was compared by Western blot, the membrane cytoskeleton followed GPIbalpha immunoprecipitation was analyzed. The results showed that an increase of P-selectin and reversible decrease of GPIbalpha expression were obtained after platelet activation by PAR1 o r PAR4, and a different kinetics of redistribution of GPIbalpha was found for the two peptides all over the time course (P < 0.05). PAR1 acted more potently and rapidly than PAR4, but the effect of PAR4 persisted longer in the course of platelet activation. Meanwhile, there was a transient change of actin, myosin and GPIbalpha in cytoskeleton proteins. Similar redistribution was also found in GPIbalpha/myosin and GPIbalpha/actin association. It is concluded that PAR1 and PAR4 possess an important role in platelet signal transmission. Either of the receptors can mediate platelet activation and GPIbalpha redistribution, which is correlated with cytoskeleton reorganization. PAR1 acts more rapidly, and effect of PAR4 persists longer.
Cytoskeleton ; chemistry ; Flow Cytometry ; Humans ; Myosins ; analysis ; P-Selectin ; analysis ; Platelet Activation ; Platelet Glycoprotein GPIb-IX Complex ; Receptor, PAR-1 ; physiology ; Receptors, Thrombin ; physiology

Fibrinogen is a key protein involved in coagulation and its deposition on blood vessel walls plays an important role in the pathology of atherosclerosis. Although the causes of fibrinogen (fibrin) deposition have been studied in depth, little is known about the relationship between fibrinogen deposition and reactive carbonyl compounds (RCCs), compounds which are produced and released into the blood and react with plasma protein especially under conditions of oxidative stress and inflammation. Here, we investigated the effect of glycolaldehyde on the activity and deposition of fibrinogen compared with the common RCCs acrolein, methylglyoxal, glyoxal and malondialdehyde. At the same concentration (1 mmol/L), glycolaldehyde and acrolein had a stronger suppressive effect on fibrinogen activation than the other three RCCs. Fibrinogen aggregated when it was respectively incubated with glycolaldehyde and the other RCCs, as demonstrated by SDS-PAGE, electron microscopy and intrinsic fluorescence intensity measurements. Staining with Congo Red showed that glycolaldehyde- and acrolein-fibrinogen distinctly formed amyloid-like aggregations. Furthermore, the five RCCs, particularly glycolaldehyde and acrolein, delayed human plasma coagulation. Only glycolaldehyde showed a markedly suppressive effect on fibrinogenesis, none did the other four RCCs when their physiological blood concentrations were employyed, respectively. Taken together, it is glycolaldehyde that suppresses fibrinogenesis and induces protein aggregation most effectively, suggesting a putative pathological process for fibrinogen (fibrin) deposition in the blood.
Acetaldehyde ; analogs & derivatives ; blood ; chemistry ; Acrolein ; blood ; chemistry ; Blood Coagulation ; Congo Red ; Electrophoresis, Polyacrylamide Gel ; Fibrinogen ; chemistry ; metabolism ; Glyoxal ; blood ; chemistry ; Humans ; Malondialdehyde ; chemistry ; Polymerization ; Protein Carbonylation ; Pyruvaldehyde ; blood ; chemistry ; Solutions ; Spectrometry, Fluorescence ; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization ; Thrombin ; chemistry