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

OBJECTIVETo study the anticoagulant constituents in dried leech (Whitmania pigra Whitman).

METHODThe plasma recalcification time (PRT) as index, the constituents with anticoagulant activity were isolated and purified by anion-exchange chromatography on Sephadex DEAE A-50, gel permeation chromatography on Sephadex G-25 and Sephadex LH-20 columns, and then reversed phase high-performance liquid chromatography successively.

RESULTThree anticoagulant polypeptides were isolated and purified. Compounds 1 and 2 can be translated each other in natural conditions, and their molecular weights are 7100 and 5531, respectively. Compound 3 was identified as a pure polypeptide by HPLC and SDS-PAGE, and its molecular weight was determined as 8 608 by MALDI-TOF-MS. The amino acid composition of compound 3 was also determined.

CONCLUSIONCompound 3 was inferred to be a novel anticoagulant, and named whitmanin.

Animals ; Anticoagulants ; analysis ; chemistry ; isolation & purification ; Leeches ; chemistry ; Molecular Weight

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

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

Immobilization of heparin onto the surfaces of biomaterials is an effective approach for improving their anticoagulant properties and biocompatibility. In this article are reviewed the relevant principle, experimental researches and applications. Finally, a prospect for heparin immobilization is made as well.
Animals ; Anticoagulants ; chemistry ; Coated Materials, Biocompatible ; chemistry ; Heparin ; chemistry ; Humans ; Surface Properties ; Tissue Engineering

In order to enhance the anticoagulant properties of decellularized biological materials as scaffolds for tissue engineering research via heparinized process, the decellularized porcine liver scaffolds were respectively immobilized with heparin through layer-by-layer self-assembly technique (LBL), multi-point attachment (MPA) or end-point attachment (EPA). The effects of heparinization and anticoagulant ability were tested. The results showed that the three different scaffolds had different contents of heparin. All the three kinds of heparinized scaffolds gained better performance of anticoagulant than that of the control scaffold. The thrombin time (TT), prothrombin time (PT) and activated partial thromboplastin time (APTT) of EPA scaffold group were longest in all the groups, and all the three times exceeded the measurement limit of the instrument. In addition, EPA scaffolds group showed the shortest prepared time, the slowest speed for heparin release and the longest recalcification time among all the groups. The decellularized biological materials for tissue engineering acquire the best effect of anticoagulant ability in vitro via EPA heparinized technique.
Animals ; Anticoagulants ; chemistry ; Biocompatible Materials ; chemistry ; Heparin ; chemistry ; Liver ; Swine ; Tissue Engineering ; Tissue Scaffolds

Blood compatibility is the main restriction of blood-contacting medical devices in clinical application, especially long-term blood-contacting medical devices will stimulate the immune defense mechanism of the host, resulting in thrombosis. Heparin anticoagulant coating links heparin molecules to the surface of medical device product materials, improves the compatibility between the material surface interface and the body, and reduces the host immune defense reactions. This study reviews the structure and biological properties of heparin, the market application status of heparin-coated medical products, the insufficiency and improvement of heparin coating, which can provide a reference for the application research of blood contact medical devices.
Humans ; Heparin/chemistry* ; Anticoagulants/chemistry* ; Thrombosis ; Coated Materials, Biocompatible/chemistry* ; Surface Properties

OBJECTIVETo establish a method for determining anticoagulation potency of Sparganii Rhizoma, and evaluate the effect of Sparganii Rhizoma herbs from different producing areas on promoting blood circulation and removing blood stasis; and study the material basis of Sparganii Rhizoma through the correlation analysis on its anticoagulation potency, ferulic acid and total flavonoid content.

METHODThe anticoagulation time of Sparganii Rhizoma from different producing areas with activeated partial thromboplastin time for their active extracts. Their biopotency was calculated by using the method of "parallel lines of dose effect" (3, 3). The degree of correlation between their anticoagulation potency and chemical constituents were calculated by using Pearson correlational analysis method.

RESULTSparganii Rhizoma and is control drugs had a good linear relationship between dose and effect (Y = 172.76X - 193.39, R2 = 0.9955). The method had better accuracy (RSD 4.7%), repeatability (RSD 2.3%) and intermediate precision (RSD 5.4%), finding that the biopotency of Sparganii Rhizoma from different producing areas ranged between 52.33-238.58 U x g(-1), and all of them passed the test on reliability. The results of correlation analysis showed no remarkable relationship between the anticoagulation potency of Sparganii Rhizoma and the contents of the two chemical constituents.

CONCLUSIONThis biopotency determination method established in the experiment can be used as one of approaches for qulaity evaluation on Sparganii Rhizoma.

Animals ; Anticoagulants ; chemistry ; pharmacology ; Blood Coagulation ; drug effects ; Drugs, Chinese Herbal ; chemistry ; pharmacology ; Rabbits ; Rhizome ; chemistry ; Typhaceae ; chemistry

OBJECTIVEApplying for the activity of enzyme in vitro,the research optimized the best preparation procedure for the anticoagulated blood region from Lumbricus.

METHODAll through our experiment, the content of protein and theactivity of enzyme were examined. The extraction process, the refining technology, concentration processes of Lumbricus were optimized with single factor checking and orthogonal design method.

RESULTAt 37 degrees C, the coarse powder of Lumbricus soaking with 15 fold of 0.9% sodium chloride and ultrasonic extracting 40 minites for three times was the best ultrasonic extraction. Utrafiltration membrane with molecular weights of 30 x 10(3) for refining and 10 x 10(3) for concentrating were selected.

CONCLUSIONUltrasonic extraction and membrane separation technology, to well improve the effect of purification for the anticoagulant site of Lumbricus, is conducive to further study.

Animals ; Anticoagulants ; chemistry ; isolation & purification ; Drug Compounding ; methods ; Oligochaeta ; chemistry ; enzymology ; Temperature ; Ultracentrifugation ; Ultrasonics

OBJECTIVETo evaluate the anticoagulant and antineoplastic activities of chemically modified low-molecular-weight heparin (LMWH).

METHODSLMWH obtained by splitting unfractionated heparin (UFH) with sodium periodate oxidation and sodium borohydride reduction was subjected to acetylation catalyzed by DCC and DMAP to produce acetylated LMWH (ALMWH). The anticoagulant activity of ALMWH was determined in mice, and its antiproliferative and anti-invasion activities was assessed in human breast cancer cells MDA-MB-231 and MFC-7.

RESULTSThe anticoagulant activity of LMWH was decreased significantly after acetylation. The concentrations of commercial LMWH* and ALMWH for doubling the coagulation time (CT) were 33.04 µmol/L and 223.56 µmol/L, respectively, and the IC(50) of ALMWH for doubling CT was 6 times of that of LMWH*. ALMWH and LMWH at 0.1, 0.3, 0.9, 2.7 and 8.1 mmol/L both significantly inhibited the proliferation of MDA-MB-231 and MCF-7 cells in a concentration-dependent manner, but ALMWH produced stronger inhibitory effects. The IC(50) of LMWH and ALMWH for inhibiting cell proliferation was 3168.4 µmol/L and 152.6 µmol/L in MCF-7 cells, and 12299.6 µmol/L and 22.2 µmol/L in MDA-MB-231 cells, respectively. ALMWH and LMWH all markedly suppressed the invasion of MDA-MB-231 cells with comparable effects.

CONCLUSIONChemical modification of structure can endow LMWH with a low anticoagulant and high antiproliferative activities.

Animals ; Anticoagulants ; chemistry ; pharmacology ; Antineoplastic Agents ; chemistry ; pharmacology ; Blood Coagulation ; Blood Coagulation Tests ; Cell Line, Tumor ; Heparin ; chemistry ; Heparin, Low-Molecular-Weight ; chemistry ; pharmacology ; Humans ; Mice