The blood eanticoagulant factor (G. E.) in garlic was isolated. This substance was precipitated at a neutral pH as the calcium salt in water, and then the calcium salt was dissolved at a pH of 3.0. Calcium was removed by adding sodium oxalate. Then G. E. was precipitated by adding two volumes of 95% ethanol. The effect of G. E. on blood coagulation was studied; prothrombin time, blood clotting time, antithrombin, anti-Ac-globulin and fibrinolysis. A half mg of G. E. completely inhibited one ml of blood from coagulating. The blood specimen containing G.E. showed a prolongation of the prothrombin time. As the calcium ion concentration increased, the prothrombin time of the plasma containing G. E. was reduced, but not to that of the control(oxalated plasma). This indicated that G. E. inhibited the prothrombin time by precipitating calcium ions, and, in addition to this calcium precipitation, another means of G. E. inhibition may be present. G. E. showed fibrinolytic effects and, in the prothrombin time tests, the plasma containing G. E. always showed less fibrin formation than was shown with oxalated plasma. G. E. showed inhibition of fibrin formation in experiments on its antithrombic effect. But this action may not be due to the antithrombin effect of G. E. but to the fibrinolytic effect of G. E. In in-vivo experiments G. E. did not show any anticoagulant effect. From these facts, it may be said that G. E. has an anticoagulant effect in at least two ways in vitro; first by precipitating calcium ions and secondly by causing fibrinolysis.
Anticoagulants/*pharmacology ; Chemistry ; *Garlic ; In Vitro ; *Plants, Medicinal

G. E. as prepared in our laboratory is a non-volatile white substance, which is odorless and water soluble. Only in vivo does it have a hypotensive effect, while both in vivo and in vitro it has a hypo-calcemic effect. We determined the chemical analysis, toxicity, lethal dose, and the effect on isolated intestinal and auricular movements of rabbits of G. E. The sodium salt of G. E. contains 18.7% Phosphorus and l5.7% Sodium. It contains inositol and a small amount of sulfur and nitrogen. The ratio of inositol: phosphorus: sodium is 1:6:6.7. Also G. E. may contain phytic acid and other mat erials which have not been identified. Toxicity tests of G. E. done on mice. The first symptoms of toxicity in mice began with irritability and unstable walking, which were followed by dyspnea and sluggish movement, and finally by coma. Mice LD 50 was 222mg/kg. As the dose of G. E. was increased in successive injections in the rabbits, the rabbits died, when the total dose reached 100-200 mg%. Probably G. E. is not destroyed quickly nor excreted rapidly. The blood pressure in the rabbits continued to fall at each injection indicating no development of tachyphylaxis. If 70mg. of G. E. was injected intravenously, as one dose, the rabbit died with muscular hyperactivity. On post mortem examination, we found G. E. had a hypocalcemic effect. However if the calcium salt of G. E. was injected no muscular hyperactivity developed, but severe hypotension was observed. The hypocalcemic effect of G. E. is due to the combining of G. E. with the blood calcium and the muscular activity may be secondary to hypocalcemic. The G. E. hypotensive effect in atropinized rabbits and in ganglionic blocked rabbits (Hexamethonium) was the same as the effect found in rabbits which had not been drugged. Epinephrine also did not change the hypotensive effect of G. E., G. E. itself showed no effect on the isolated intestinal and auricular movements of a rabbit as long as there were enough calcium ions in the solution. Hence we can not say that the hypotension of G. E. is due to vagus stimulation and or to paralysis of sympathetic nerve endings. The mechanism of the hypotensive effect of G. E. is not yet clear.
Animals ; Anticoagulants/*pharmacology ; Chemistry, Analytical ; *Garlic ; In Vitro ; *Plants, Medicinal ; Rabbits

Anticoagulants ; pharmacology ; therapeutic use ; Cardiovascular Diseases ; drug therapy ; Humans

Whitmania pigra is the most widely distributed species of leeches in the market. In this study, the effect of heavy metal lead pollution on the anticoagulant activity of Wh. pigra was studied and the potential mechanism was explored. Pb(NO_3)_2 was used to contaminate the breeding soil which was then used to rear Wh. pigra for 50 days(lead-contaminated group, LC group), and meanwhile the blank control group(CG group) was set. Proteins were extracted from the obtained leech samples, and the differentially expressed proteins between LC and CG groups were analyzed by label-free proteomics technology. In this study, a total of 152 differentially expressed proteins were screened out, of which 93 proteins were up-regulated and 59 proteins were down-regulated in LC group. Bioinformatics analysis showed that the biological processes enriched with the differentially expressed proteins were mainly vesicle-mediated transport and transport positive regulation; the enriched cell components were mainly endocytosis vesicles and apical plasma membrane; the enriched molecular functions mainly included carbohydrate binding. The differentially expressed proteins were enriched in 76 KEGG pathways, which mainly involved metabolic pathways, biosynthesis of secondary metabolites, and bacterial invasion of epithelial cells. In this study, two differentially expressed proteins with Antistasin domain were presumed, which provides reference for further exploring the regulatory mechanism and signal transduction underlying the effect of lead pollution on the anticoagulant activity of leech.
Animals ; Anticoagulants/pharmacology* ; Environmental Pollution ; Leeches ; Metals, Heavy ; Proteomics

This study first optimized the processing technology for Zhangbang vinegar-processed Olibanum and investigated its in vitro anticoagulant activity. A multi-index-response surface methodology was used, with yield, powder yield, and the relative percentage of the content of six non-volatile components [11-keto-boswellic acid(KBA), 3-acetyl-11-keto-boswellic acid(AKBA), β-elemonic acid, α-boswellic acid(α-BA), β-boswellic acid(β-BA), and α-acetyl-boswellic acid(α-BA)] and three volatile components(octyl acetate, incensole, and incensole acetate) as evaluation indicators. Analytical hierarchy process(AHP) combined with coefficient of variation method was used to calculate the weight of each indicator and calculate the comprehensive score(OD). Furthermore, response surface methodology was used to investigate the effects of frying temperature(A), burning time(B), rice vinegar dosage(C), and steaming time(D) on the processing technology of vinegar-processed Olibanum. Vinegar-steamed Olibanum was prepared according to the optimal processing technology for in vitro anticoagulant experiments. The results showed that the weights of octyl acetate, incensole, incensole acetate, KBA, AKBA, β-elemonic acid, α-BA, β-BA, α-ABA, yield, and powder yield were 0.358 2, 0.104 5, 0.146 4, 0.032 9, 0.123 7, 0.044 4, 0.022 1, 0.042 2, 0.110 1, 0.012 2, and 0.0032, respectively. The optimal processing technology for Zhangbang vinegar-processed Olibanum was as follows. Olibanum(50 g) with a particle size of 1-5 mm was continuously stir-fried at a low heat of 150-180 ℃ until in a gel-like state, ignited for burning for 15 s, sprayed with 7.5 g of rice vinegar(15%), and steamed for 3 min without fire. Subsequently, the cover was removed, and the product was continuously stir-fried at 150-180 ℃ until in a soft lump shape, removed, cooled, and crushed. The results of the in vitro anticoagulant experiments showed that compared with the blank group, both Olibanum and vinegar-processed Olibanum significantly prolonged the activated partial thromboplastin time(APTT), thrombin time(TT), and prothrombin time(PT) of rat platelet-poor plasma(PPP), and the effect of vinegar-processed Olibanum was significantly better than that of Olibanum(P<0.05). The optimized processing technology for Zhangbang vinegar-processed Olibanum is stable, feasible, and beneficial for the further development and utilization of Olibanum slices. At the same time, using the content of volatile and non-volatile components, yield, and powder yield as indicators, and verifying through pharmacological experiments, the obtained results are more reasonable and credible, and have positive guiding significance for the clinical application of characteristic processed Olibanum products.
Rats ; Animals ; Frankincense ; Acetic Acid ; Powders ; Triterpenes ; Anticoagulants/pharmacology* ; Technology

BACKGROUND: The identification of in vitro hemolysis (IVH) using a hematology analyzer is challenging because centrifugation of the specimens cannot be performed for cell counts. In the present study, we aimed to develop a scoring system to help identify the presence of hemolysis in anticoagulated blood specimens. METHODS: Thirty-seven potassium EDTA anticoagulated blood specimens were obtained, and each specimen was divided into 3 aliquots (A, B, and C). Aliquots B and C were mechanically hemolyzed by aspirating 2 and 5 times, respectively, using a 27-gauge needle and then tested; aliquot A was analyzed immediately without any hemolysis. After the cells were counted, aliquots B and C were centrifuged and the supernatants were tested for the hemolytic index and lactate dehydrogenase levels. RESULTS: The 4 hematologic parameters were selected and scored from 0 to 3 as follows:< 34.0, 34.0-36.2, 36.3-38.4, and > or =38.5 for mean cell hemoglobin concentration (MCHC, g/dL); <0.02, 0.02, 0.03, and > or =0.04 for red blood cell ghosts (10(12)/L); <0.13, 0.13-0.38, 0.39-1.30, and > or =1.31 for difference value (g/dL) of measured hemoglobin and calculated hemoglobin; and <0.26, 0.26-0.95, 0.96-3.34, and > or =3.35 for difference value (g/dL) of MCHC and cell hemoglobin concentration mean. The hemolysis score was calculated by adding all the scores from the 4 parameters. At the cutoff hemolysis score of 3, the IVH of aliquots B and C were detected as 64.9% and 91.9%, respectively. CONCLUSIONS: The scoring system might provide effective screening for detecting spurious IVH.
Anticoagulants/*pharmacology ; *Blood Specimen Collection ; Edetic Acid/pharmacology ; Hemoglobins/analysis ; Hemolysis/drug effects ; Humans

Anticoagulants ; adverse effects ; pharmacology ; Blood Coagulation Disorders ; chemically induced ; Drug Interactions ; Humans ; Warfarin ; adverse effects ; pharmacology

This study explored the anticoagulant material basis and mechanism of Trichosanthis Semen and its shell and kernel based on spectrum-effect relationship-integrated molecular docking. High performance liquid chromatography(HPLC) fingerprints of Trichosanthis Semen and its shell and kernel were established. Prothrombin time(PT) and activated partial thromboplastin time(APTT) in mice in the low-and high-dose(5, 30 g·kg~(-1), respectively) Trichosanthis Semen, the shell, and kernel groups were determined as the coagulation markers. The spectrum-effect relationship and anticoagulant material basis of Trichosanthis Semen and its shell and kernel were analyzed with mean value calculation method of Deng's correlation degree(MATLAB) and the common effective component cluster was obtained. Then the common targets of the component cluster and coagulation were retrieved from TCMSP, Swiss-TargetPrediction, GenCLiP3, GeneCards, and DAVID, followed by Gene Ontology(GO) term enrichment and Kyoto Encyclopedia of Genes and Genomes(KEGG) pathway enrichment of the targets. The main anticoagulant molecular mechanism of the component cluster was verified by SYBYL-X 2.1.1. The spectrum-effect relationship of Trichosanthis Semen and its shell and kernel was in positive correlation with the dosage. The contribution of each component to anticoagulation was not the same, suggesting that the material basis for anticoagulation was different, but they have common effective components(i.e. common material basis), such as adenine(peak 3), uracil(peak 4), hypoxanthine(peak 6), xanthine(peak 9), and adenosine(peak 11). Network pharmacology showed that these components can act on multiple target proteins such as NOS3, KDR, and PTGS2, and exert anticoagulant effect through multiple pathways such as VEGF signaling pathway. They involved the biological functions such as proteolysis, cell component such as cytosol, and molecular functions. The results of molecular docking showed that the binding free energy of these components with NOS3(PDB ID: 1 D0 C), KDR(PDB ID: 5 AMN), and PTGS2(PDB ID: 4 COX) was ≤-5 kJ·mol~(-1), and the docking conformations were stable. Spectrum-effect relationship-integrated molecular docking can be used for the optimization, virtual screening, and verification of complex chemical and biological information of Chinese medicine. Trichosanthis Semen and its shell and kernel have the common material basis for anticoagulation and they exert the anticoagulant through multiple targets and pathways.
Animals ; Anticoagulants/pharmacology* ; Drugs, Chinese Herbal/pharmacology* ; Gene Ontology ; Mice ; Molecular Docking Simulation ; Semen

Angelicae Sinensis Radix excels in activating blood, but the scientific mechanism has not been systematically analyzed, thus limiting the development of the medicinal. This study employed the computer-aided drug design methods, such as structural similarity-based target reverse prediction, complex network analysis, molecular docking, binding free energy calculation, cluster analysis, and ADMET(absorption, distribution, metabolism, excretion, toxicity) calculation, and enzyme activity assay in vitro, to explore the components and mechanism of Angelicae Sinensis Radix in activating blood. Target reverse prediction and complex network analysis yielded 40 potential anticoagulant targets of the medicinal. Gene Ontology(GO) term enrichment and Kyoto Encyclopedia of Genes and Genomes(KEGG) pathway enrichment analysis indicated that the targets mainly acted on the complement and coagulation cascade signaling pathway to exert the anticoagulant function. Among them, the key enzymes thrombin(THR) and coagulation factor Xa(FXa) in coagulation cascade and thrombosis were the drug targets for thromboembolic diseases. At the same time, molecular docking and cluster analysis showed that the medicinal had high selectivity for FXa. According to binding free energy score, 8 potential active components were selected for enzyme activity assay in vitro. The results demonstrated that 8 components inhibited THR and FXa, and the inhibition was stronger on FXa than on THR. The pharmacophore model of 8 active compounds was constructed, which suggested that the components had the common pharmacophore AAHH. The ADMET calculation result indicated that they had good pharmacokinetic properties and were safe. Based on target reverse prediction, complex network analysis, molecular docking and binding free energy calculation, anticoagulant activity in vitro, spatial binding conformation of molecules and targets, pharmacophore model construction, and ADMET calculation, this study preliminarily clarified the material basis and molecular mechanism of Angelicae Sinensis Radix in activating blood from the perspective of big data, and calculated the pharmacology and toxicology parameters of the active components. Our study, for the first time, revealed that the medicinal had obvious selectivity and pertinence for different coagulation proteins, reflecting the unique effect of different Chinese medicinals and the biological basis. Therefore, this study can provide clues for precision application of Angelicae Sinensis Radix and the development of the blood-activating components with modern technology.
Anticoagulants/pharmacology* ; Blood Coagulation ; Drug Design ; Drugs, Chinese Herbal/pharmacology* ; Molecular Docking Simulation

Lung cancer is the first leading cause of morbidity and mortality in the world. Venous thromboembolism (VTE) is a recognized complication in patients with lung cancer, which is one of the leading cause of death in lung cancer patients. The cancer-related, patient-related and treatment-related factors are the main causes of VTE in lung cancer patients. Malignant cells can directly activate blood coagulation by producing tissue factor (TF), cancer procoagulance (CP), inflammatory factors and cytokines; And the one of predominant mechanisms in cancer-related thrombosis is the overexpression of TF. The 10th edition of the antithrombotic therapy guidelines for VTE with cancer patients (AT-10) published in 2016 by American College of Chest Physicians (APCC) recommended that anticoagulant therapy is the basic treatment for patients with lung cancer complicated with VTE; And low molecular-weight-heparin (LMWH) is preferred as an anticoagulant drug, but can be use with caution due to increasing risk of bleeding.
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Anticoagulants ; pharmacology ; therapeutic use ; Humans ; Lung Neoplasms ; complications ; Risk Factors ; Venous Thromboembolism ; complications ; drug therapy