Melting Point | 47.7 °C |
Solubility | H2O: 1mL/vial clear to slightly hazy, colorless solution |
Appearance | powder |
Color | white |
Merck | 13,9461 |
Storage Condition | -20°C |
MDL | MFCD00166053 |
Physical and Chemical Properties | Thrombin is prothrombin extracted from bovine blood or pig blood. It is a sterile freeze-dried product of thrombin obtained by activation. It is a white or white-like freeze-dried block or powder. The dry powder is stable in storage at 2 ~ 80C, the dry powder is easily soluble in water, and the 0.9% sodium chloride solution is turbid and insoluble in organic solvents. Inactivation within 8h of aqueous solution at room temperature. Therefore, it must be prepared freshly when it is used, and the vitality of heating, acid, alkali, metal, etc. is reduced. |
Use | Local hemostatic for local bleeding and gastrointestinal bleeding |
Risk Codes | R36/37/38 - Irritating to eyes, respiratory system and skin. R42 - May cause sensitization by inhalation |
Safety Description | S22 - Do not breathe dust. S24/25 - Avoid contact with skin and eyes. S36/37 - Wear suitable protective clothing and gloves. S26 - In case of contact with eyes, rinse immediately with plenty of water and seek medical advice. S24 - Avoid contact with skin. |
WGK Germany | 2 |
RTECS | XO8950000 |
TSCA | Yes |
HS Code | 30021099 |
2kg of pig blood was collected, 200ml of sodium citrate solution (38g of sodium citrate was contained) was added, mixed well, and centrifuged. The supernatant plasma was taken, diluted with distilled water, adjusted to pH 5.3 with ml of 1% acetic acid, and the precipitate was collected to obtain prothrombin. The prothrombin was dissolved by adding sodium chloride solution at 25-30 ° C., and then 15g of calcium chloride was added to each liter of the solution. After sufficient stirring, th was left to stand in the cold room and centrifuged. The supernatant was added with frozen acetone, and the mixture was stirred uniformly and then allowed to stand overnight. The precipitate was filtered, washed with ether and dried to give crude thrombin. The crude product was dissolved by adding sodium chloride solution, placed at 0 ° C. For 6h, and filtered. The filtrate was adjusted to pH 5.5 with acetic acid, and centrifuged. Then, the supernatant was added to 2 times the amount of frozen acetone, and allowed to stand for 2 hours. The precipitate was washed with anhydrous ethanol and ether respectively, and dried under vacuum to prepare thrombin.
EPA chemical substance information | information provided by: ofmpeb.epa.gov (external link) |
Overview | thrombin, a protease that hydrolyzes fibrinogen to fibrin, consists of 308 amino acid residues, and is A double-stranded molecule, containing A chain and B chain, the two chains are connected by disulfide bonds, wherein the chain contains 49 amino acid residues, also known as light chain, B chain consists of 259 amino acid residues, also known as heavy chain; This product exists in animal blood, usually in the form of inactive zymogen, can directly act on the final link of the coagulation process, make fibrinogen into fibrin, accelerate blood coagulation, play a role in rapid hemostasis; Can activate the coagulation factor VIII, so that the soluble fibrin into poorly soluble fibrin and the formation of blood clots, to achieve the purpose of hemostasis; Can enhance the activity of coagulation factor VIII and V, with the participation of calcium and phospholipids, promote thrombin factor Xa, so that prothrombin into thrombin; it can promote irreversible platelet aggregation and platelet release reaction, and accelerate blood coagulation; It can also promote the mitosis of epithelial cells and shorten the wound healing time by 30% ~ 50%. Therefore, it is often used to ligation and stop bleeding of small blood vessels, capillaries and substantive organs, can also be used for trauma, surgery, stomatology, otolaryngology, urology, obstetrics and other parts of the digestive tract bleeding. |
Structural composition | A protein consisting of 308 amino acid residues it is A double-stranded molecule containing A and B chains, the two chains are linked by disulfide bonds. The A chain contains 49 amino acid residues, also known as the light chain; The B chain consists of 259 amino acid residues, also known as the heavy chain. The amino terminal amino acid of the chain is threonine (Thr); The amino terminal amino acid of the B chain is isoleucine (Ile), its first 4 amino acid residues are isoleucine-L-glutamic acid-glycine (Ile-Val-Glu-Gly. |
pharmacological effects | thrombin exists in the form of prothrombin in the body, the prothrombin complex activates it and converts it into thrombin with serine protein-like hydrolytic activity. The formation of thrombin plays a central role in the coagulation process. The role of thrombin after formation includes at least the following five aspects:(1) start: once the formation of thrombin, it can quickly start the coagulation process in the later stage, including the activation of platelets as a coagulant, endothelial cells, and activation of Factor V, VII, VIII. (2) amplification; The amplification of the coagulation reaction is achieved by the synthesis of the coagulase complex on the surface of the subcutaneous matrix and peripheral blood cells. Among them, the amplification of coagulation reaction is mainly completed by the activation of α-thrombin, at the same time, the amplification reaction phase of coagulation can promote the generation of more α-thrombin, A stable thrombus is formed at the site of the vascular lesion, thereby terminating the loss of blood. (3) termination of clot formation: the termination of clot formation involves at least two successive inhibition processes and one kinetic inhibition process. The former is mainly a protease inhibitor AT-Ⅲ system and a tissue factor pathway inhibitor (TFPI) system. The kinetic inhibition system is carried out through the protein C pathway. In the protein C pathway, the first is the generation of APC, and the formation of APC is the complex formed by α-thrombin and thrombomodulin (TM), that is, α-thrombin-TM changes PC to APC. By means of this kinetic inhibition system, the prothrombinase and the endogenous thrombin component can be inactivated, It is primarily the activity of cofactor proteins such as factor Va and factor IIa that are involved in stopping the formation of blood clots. (4) removal of blood clots: the removal of blood clots is a basic step in the process of tissue repair, and requires the participation of plasmin. Thrombin regulates this process indirectly by affecting fibrinolysis. TAFIa generated by the TM-α-THROMBIN COMPLEX reduces the rate of activation of glutamate plasminogen by tPA and fibrin, protecting fibrin from degradation. In the presence of APC, the survival of α-thrombin can be significantly reduced, while reducing the activation of TAFIa. (5) repair: repair of damaged tissue is the last step in the coagulation process. This process requires plasmin to activate metalloproteinases, which degrade the damaged extracellular matrix and promote the migration of new cells to the site of injury. In this process, thrombin is a potent growth factor and chemotactic attractant that has an activating effect on fibroblasts, macrophages and smooth muscle cells; Α-thrombin can activate platelets, release vasoactive substances and growth factors from platelets, and promote the proliferation and growth of different types of cells. Figure 1 is Lyophilizing Thrombin Powder |
separation and purification | 1. Separation of crude thrombin extraction of crude thrombin is to remove the plasma fibrinogen and other miscellaneous proteins, while retaining the coagulation factor, is conducive to the activation of prothrombin. Prothrombin itself has no activity and cannot convert fibrinogen into fibrin. Only when it is activated to thrombin can it play a role in the coagulation cascade. (1) extraction of prothrombin: the extraction of prothrombin usually adopts three methods: isoelectric point precipitation method, barium citrate adsorption method and magnesium hydroxide adsorption method. Isoelectric point precipitation method according to the principle that the solubility of protein is the lowest under the condition that the pH of the solution is the isoelectric point, the plasma pH after appropriate dilution is adjusted to the isoelectric point of thrombin between 5.0 and 5.5 with 1% acetic acid, after static stratification, centrifugation, the buffer solution was dissolved and precipitated to obtain a crude prothrombin solution. Barium citrate adsorption law according to the characteristics that barium citrate can adsorb the coagulation factor dependent on vitamin K, barium chloride is added to the plasma obtained by sodium citrate anticoagulation to produce barium citrate, prothrombin and other factors were separated by precipitation, and the precipitate was desorbed with EDTA, and then centrifuged to obtain a crude prothrombin solution. The magnesium hydroxide adsorption method was first reported by Seeger. Based on the isoelectric point precipitation method, the precipitated precipitate was adsorbed with magnesium hydroxide, the prothrombin was eluted with carbon dioxide, and the prothrombin was precipitated with ammonium sulfate to 65% saturation. Compared with the above three methods, the isoelectric point precipitation method is easier to operate and has more applications, but the crude thrombin activity obtained by the latter two methods is not high, but the operation is more complicated. (2) prothrombin activation: under physiological conditions, thrombin is generated by activation of prothrombin under the action of coagulation factor Xa, coagulation factor V, Ca2 + and phospholipids. In the absence of certain activators, prothrombin can also be converted to thrombin, But the conversion rate is very low. Activation of prothrombin is a critical step in the extraction of thrombin from plasma. Using Ca2 + activation alone, that is, adding a certain concentration of CaCl2 solution to the crude prothrombin solution, and placing it at room temperature for 1.5~2 h, the thrombin activity reaches the highest, the residual fibrinogen is converted into fibrin and precipitated, and the activated crude thrombin solution is obtained after filtration. 2. There are many methods of purification of crude thrombin, the traditional physical and chemical methods of acetone precipitation and ammonium sulfate fractionation method, these two methods are simple, low cost, however, the purity of the obtained thrombin product is not high, the recovery rate is low, and the application is limited. Compared with the traditional physical and chemical methods, the separation of plasma proteins by chromatography has the advantages of high specificity and high selectivity, and can be used for the separation of some trace substances in plasma, in order to obtain high purity plasma products suitable for clinical needs, and compared with the precipitation method is easier to automate; At the same time, chromatography purification process can also play a role in the removal of pathogens in plasma products, so as to improve the safety of clinical use of blood products. At present, gel filtration chromatography, ion exchange chromatography and heparin affinity chromatography are mainly used to purify thrombin. (1) gel filtration chromatography: gel filtration chromatography is a technology that uses gel filtration media to separate substances of different sizes, it can be used for the separation and purification of substances with relative molecular masses ranging from several hundred to 10 current 6 orders of magnitude. This technology is commonly used in the fractionation and desalination of biological macromolecules such as proteins. The molecular mass of thrombin is 36 ku, and the appropriate filter medium can effectively remove other impurity proteins. (2) ion exchange chromatography: Ion exchange chromatography is the use of proteins with ion or surface charge difference, the target protein adsorption to DEAE, A method of removing foreign proteins on a CM plasma exchange column. Ion exchange chromatography is widely used in the preparation of therapeutic plasma protein preparations. Under the condition of pH> 5.5, the thrombin molecule is negatively charged, It can therefore be purified using an anion exchange column. (3) heparin affinity chromatography: Affinity chromatography is a liquid chromatography method in which the target product is separated and purified by affinity adsorption medium using a coupled affinity ligand as a stationary phase. There is a specific interaction between thrombin and low molecular weight heparin. By using the specific affinity between them, the low molecular weight heparin can be immobilized on the carrier, and the affinity chromatography of thrombin can be purified. |
drug interaction | 1. This product reacts with acid, alkali and heavy metals to reduce the effect. 2. In order to improve the hemostatic effect of upper gastrointestinal bleeding, it is advisable to take a certain amount of antacids and gastric acid after oral administration of this product, or at the same time, intravenous acid inhibitor. 3. This product can also be dissolved in phosphate buffer (pH7.6) or cold milk. Such as the use of Arabic gum, gelatin, fructose gum, honey and other formulations into a Latex solution, can improve the hemostatic effect of thrombin, and can appropriately reduce the amount of this product. (2015-11-16) |
Clinical application | 1. For patients with acute gastrointestinal bleeding, thrombin can be taken orally to stop bleeding site rapidly. For intractable postpartum hemorrhage, thrombin can be given by liquid spraying or gauze pressing. In urology, hemorrhagic cystitis can be treated by catheter infusion of thrombin solution and antibiotics, which can simultaneously achieve hemostasis and promote regeneration and repair of damaged epithelial tissue. 4. In the past, the doctor felt that Head Pain of the ear nose and throat diseases and related surgical bleeding were treated by direct infusion of thrombin or gelatin sponge soaked with thrombin solution, which could obtain satisfactory hemostatic effect, relieve the pain of the patient. It not only has toxic effect in cerebral hemorrhage and cerebral ischemia, but also plays an important role in normal development and injury protection of central nervous system. |
dosage | oral or Infusion: for gastrointestinal bleeding, use warm boiled water or normal saline (no more than 37 ℃) dissolved into 10 ~ 100U/ml solution, each time 1000 ~ 4000U, can also be appropriate according to the bleeding site and degree of concentration, frequency. Local medication: ① The General dose is to dissolve the product into 50 ~ 200U/ml solution with sterilized physiological saline and spray the wound surface. (2) when there is a large amount of bleeding, you can use the amine sponge gauze or oxidized cellulose to dip the solution in the wound or spray the dry powder on the wound surface. (3) plastic surgery, tooth extraction, skin transplantation, heparin in patients with puncture site Bleeding, commonly used this product 100U/ml solution to stop bleeding. ④Thrombin solution 1000 ~ 2000U/ml was used to stop bleeding due to rupture of liver and spleen. |
adverse reactions | 1. Occasionally, it may cause allergic reactions. Blood clotting (thrombosis) can be life-threatening as a result of thrombin misplacement into blood vessels. 3. The application of thrombin in surgical hemostasis has been reported to cause low thermal reaction. |
note | 1. This product must be in direct contact with the wound to stop bleeding; It should be freshly prepared for use. Intravascular, intramuscular or subcutaneous injection is strictly prohibited to prevent local necrosis and even the formation of thrombosis and life-threatening. 3. Heating, acid, alkali or heavy metal salts can make its vitality decline and lose its effect. 4. If symptoms of allergic reactions should be discontinued. 5. Pregnancy only with obvious indications, the condition can be used when necessary. |
Usage | thrombin is a local hemostatic agent with no side effects. Use must be directly in contact with the wound, in order to play a hemostatic effect. However, this product is strictly prohibited injection, not with acid, alkali and heavy metal compatibility. Animal test organic antigen body reaction, allergic reaction. If this symptom occurs, the drug should be discontinued immediately. Sodium chloride injection for topical hemostasis is dissolved into a solution or dry powder containing 50-250 units/ml and sprayed or sprayed on the wound surface. Gastrointestinal hemostasis, with warm water (≤ 370) dissolved into 10~100 units/ml solution, clothing or local perfusion, according to the bleeding site and degree of appropriate increase or decrease concentration, frequency. Local hemostatic for local bleeding and gastrointestinal bleeding Biochemical Study |
production method | raw material treatment, precipitation 2kg of pig blood was taken and sodium citrate (38g/L or 3.8%) was added. Solution 200ml anticoagulation, mixing, separation, remove the supernatant plasma, add 10 times the amount of plasma in the plasma diluted with distilled water, with 300ml acetic acid (1%) to adjust the Ph to 5.3, after complete precipitation, centrifuge in the centrifuge for 5~10min, discard the Centrifugal liquid, collect the precipitate, get prothrombin. Animal blood sodium citrate → raw material treatment plasma distilled water: HAc;Ph5.3 → precipitation prothrombin separation, precipitation, washing to take prothrombin, at 25~30C0, add sodium chloride (0.9%) solution 700ml, dissolve it, then add 15g of calcium chloride to each liter of solution, fully stir for 10min, dissolve it, let the cold room stand for 1H, centrifuge, take the supernatant and add the same amount of frozen acetone, stir evenly, left standing overnight. Centrifugal separation, precipitation and frozen acetone grinding, cold room placed 2~3 days, filtered, the precipitate was washed with ether, vacuum dried, that is, crude thrombin, about 10g. Prothrombin NaCl;CaCl2; Acetone → separation; Precipitation of ether → washing crude thrombin in addition to impurities, precipitation, drying 200ml of sodium chloride solution (9g/L) was added to the crude thrombin to dissolve, after dissolving, the mixture was placed at 00C for 6h, filtered, and the precipitate was taken and 150ml of sodium chloride solution (9g/L) was added. The above operation was repeated once, and the filtrate was combined twice. The Ph was adjusted to 5.5 with acetic acid (1%). The supernatant was added to 2 times the amount of frozen acetone in the supernatant, left for 2H, until the precipitation was complete, centrifuged, the precipitate was ground with frozen acetone, and left for 24h, the solution was filtered off. The precipitates were washed with anhydrous ethanol and ether, respectively, and dried under vacuum to obtain refined thrombin, about 0.4g. Crude thrombin NaCl;HAc;^ 0<0C;pH5.5 → filtrate acetone → precipitation drying refined product |