Thrombin (MW 37kDa)
(Synonyms: 凝血酶) 目录号 : GC61495
凝血酶 (MW 37kDa) 是一种 Na+- 激活的变构丝氨酸蛋白酶。
Cas No.:9002-04-4
Sample solution is provided at 25 µL, 10mM.
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Quality Control & SDS
- View current batch:
- Purity: >98.00%
- COA (Certificate Of Analysis)
- SDS (Safety Data Sheet)
- Datasheet
| Cell experiment [1]: | |
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Cell lines |
N27 cells derived from E12 rat mesencephalic tissue |
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Preparation Method |
AA and lipid hydroperoxides content was assayed from the N27 cells 24 h after thrombin (0.5 U/mL) treatment. |
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Reaction Conditions |
24 h ,thrombin (0.5 U/ml) |
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Applications |
Thrombin dose-dependently induced N27 neuronal cell death, which accompanied elevated AA and lipid hydroperoxides, and smaller mitochondria with increased membrane density. Thrombin did not affect the intracellular level of ferrous iron |
| Animal experiment [2]: | |
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Animal models |
Male Sprague-Dawley rats, each weighing between 250 and 350 g |
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Preparation Method |
Solutions containing 10 U of thrombin in 10 μl saline were infused into the brain over a period of 1 minute using a Harvard pump. |
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Dosage form |
10 U of thrombin in 10 μl saline for 1 minute |
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Applications |
AIB(a marker of BBB opening) has increased BBB permeability. Thrombin causes disruption of the BBB in the ipsilateral hemisphere |
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References: [1]. Tuo QZ, Liu Y, et,al. Thrombin induces ACSL4-dependent ferroptosis during cerebral ischemia/reperfusion. Signal Transduct Target Ther. 2022 Feb 23;7(1):59. doi: 10.1038/s41392-022-00917-z. PMID: 35197442; PMCID: PMC8866433. [2]. Lee KR, Kawai N, et,al. Mechanisms of edema formation after intracerebral hemorrhage: effects of thrombin on cerebral blood flow, blood-brain barrier permeability, and cell survival in a rat model. J Neurosurg. 1997 Feb;86(2):272-8. doi: 10.3171/jns.1997.86.2.0272. PMID: 9010429. |
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Thrombin (MW 37kDa) is a Na+-activated, allosteric serine protease. Thrombin induces the activation of ERK1 and ERK2[1]. Thrombin recognition sequence and can be used to digest GST-tagged proteins[2].Thrombin could activate the immune system by directly cleaving pro-interleukin-1α to the active form (IL-1α) [3]. Thrombin may be “a gas pedal” driving the innate immune system[4].
Thrombin induces neuronal ferroptosis, in N27 cells, Thrombin dose-dependently induced N27 neuronal cell death, which accompanied elevated AA and lipid hydroperoxides, and smaller mitochondria with increased membrane density. Thrombin did not affect the intracellular level of ferrous iron[5]. Thrombin elicits rapid and full activation of cPLA2 not only by promoting a rise in cytosolic free Ca2+ but also by inducing phosphorylation of cPLA2 thereby improving its catalytic activity[6].Low concentrations of thrombin and thrombin preconditioning yield the potential of rescuing cells and to induce survival of neurons and astrocytes exposed to various ischemic insults[7].Thrombin also has roles in brain cell death and survival as well as neuroinflammation, predominantly via the cellular protease-activated receptor (PAR) activation and downstream signaling pathways [5]
In vivo，thrombin induces BBB disruption as well as death of parenchymal cells, whereas CBF and vasoreactivity are not altered. Cell toxicity and BBB disruption by thrombin are triggering mechanisms for the edema formation that follows intracerebral hemorrhage[9].In the primary hemostatic processis. Thrombin activates platelets, and also in the secondary hemostasis, it mediates the conversion of fibrinogen to fibrin. Thus, thrombin contributes to thrombus formation that stops bleeding and hematoma formation after blood enters the brain parenchyma [10]. Intracerebral administration of exogenous thrombin (at a dose that is non-toxic to normal brain), markedly exacerbated brain edema after transient focal cerebral ischemia. Extravascular thrombin inhibition may be a new therapeutic target for cerebral ischemia[6]
References:
[1]. Kramer RM, Roberts EF, et,al. Differential activation of cytosolic phospholipase A2 (cPLA2) by thrombin and thrombin receptor agonist peptide in human platelets. Evidence for activation of cPLA2 independent of the mitogen-activated protein kinases ERK1/2. J Biol Chem. 1995 Jun 16;270(24):14816-23. doi: 10.1074/jbc.270.24.14816. PMID: 7782348.
[2]. Golderman V, Shavit-Stein E, et,al. Thrombin and the Protease-Activated Receptor-1 in Organophosphate-Induced Status Epilepticus. J Mol Neurosci. 2019 Feb;67(2):227-234. doi: 10.1007/s12031-018-1228-6. Epub 2018 Dec 4. PMID: 30515700.
[3]. Burzynski LC, Humphry M, et,al. The Coagulation and Immune Systems Are Directly Linked through the Activation of Interleukin-1α by Thrombin. Immunity. 2019 Apr 16;50(4):1033-1042.e6. doi: 10.1016/j.immuni.2019.03.003. Epub 2019 Mar 26. PMID: 30926232; PMCID: PMC6476404.
[4]. Petzold T, Massberg S. Thrombin: A Gas Pedal Driving Innate Immunity. Immunity. 2019 Apr 16;50(4):1024-1026. doi: 10.1016/j.immuni.2019.03.006. PMID: 30995493.
[5]. Tuo QZ, Liu Y, et,al. Thrombin induces ACSL4-dependent ferroptosis during cerebral ischemia/reperfusion. Signal Transduct Target Ther. 2022 Feb 23;7(1):59. doi: 10.1038/s41392-022-00917-z. PMID: 35197442; PMCID: PMC8866433.
[6]. Kramer RM, Roberts EF, et,al. Thrombin-induced phosphorylation and activation of Ca(2+)-sensitive cytosolic phospholipase A2 in human platelets. J Biol Chem. 1993 Dec 15;268(35):26796-804. PMID: 8253817.
[7]. Donovan FM, Pike CJ, et,al. Thrombin induces apoptosis in cultured neurons and astrocytes via a pathway requiring tyrosine kinase and RhoA activities. J Neurosci. 1997 Jul 15;17(14):5316-26. doi: 10.1523/JNEUROSCI.17-14-05316.1997. PMID: 9204916; PMCID: PMC6793831.
[8]. Delvaeye M, Conway EM. Coagulation and innate immune responses: can we view them separately? Blood. 2009 Sep 17;114(12):2367-74. doi: 10.1182/blood-2009-05-199208. Epub 2009 Jul 7. PMID: 19584396.
[9]. Lee KR, Kawai N, et,al. Mechanisms of edema formation after intracerebral hemorrhage: effects of thrombin on cerebral blood flow, blood-brain barrier permeability, and cell survival in a rat model. J Neurosurg. 1997 Feb;86(2):272-8. doi: 10.3171/jns.1997.86.2.0272. PMID: 9010429.
[10]. Goldsack NR, Chambers RC, et,al. Thrombin. Int J Biochem Cell Biol. 1998 Jun;30(6):641-6. doi: 10.1016/s1357-2725(98)00011-9. PMID: 9695019.
[11]. Hua Y, Wu J, et,al. Thrombin exacerbates brain edema in focal cerebral ischemia. Acta Neurochir Suppl. 2003;86:163-6. doi: 10.1007/978-3-7091-0651-8_34. PMID: 14753426.
凝血酶 (MW 37kDa) 是一种 Na+- 激活的变构丝氨酸蛋白酶。凝血酶诱导 ERK1 和 ERK2[1] 的激活。凝血酶识别序列，可用于消化 GST 标签蛋白[2]。凝血酶可通过直接将 pro-interleukin-1α 裂解为活性形式 (IL-1α) 来激活免疫系统 [3].凝血酶可能是驱动先天免疫系统的"油门踏板"[4]。
凝血酶诱导神经元铁死亡，在 N27 细胞中，凝血酶剂量依赖性地诱导 N27 神经元细胞死亡，伴随着 AA 和脂质氢过氧化物的升高，以及线粒体变小和膜密度增加。凝血酶不影响细胞内亚铁水平[5]。凝血酶不仅通过促进胞质游离 Ca2+ 的升高，而且通过诱导 cPLA2 的磷酸化从而提高其催化活性[6]，从而引起 cPLA2 的快速和完全激活。低凝血酶浓度和凝血酶预处理产生拯救细胞的潜力，并诱导暴露于各种缺血性损伤的神经元和星形胶质细胞存活[7]。凝血酶还在脑细胞死亡和存活以及神经炎症中发挥作用，主要通过细胞蛋白酶-激活受体 (PAR) 激活和下游信号通路 [5]
在体内，凝血酶诱导 BBB 破坏以及实质细胞死亡，而 CBF 和血管反应性没有改变。细胞毒性和凝血酶破坏血脑屏障是脑出血后水肿形成的触发机制[9]。在原发性止血过程中。凝血酶激活血小板，还在二次止血中，介导纤维蛋白原向纤维蛋白的转化。因此，凝血酶有助于血栓形成，从而在血液进入脑实质后止血和血肿形成 [10]。外源性凝血酶的脑内给药（对正常大脑无毒的剂量）显着加剧了短暂性局灶性脑缺血后的脑水肿。血管外凝血酶抑制或成为脑缺血治疗的新靶点[6]
| Cas No. | 9002-04-4 | SDF | |
| 别名 | 凝血酶 | ||
| Canonical SMILES | [Thrombin] | ||
| 分子式 | 分子量 | 37000(Average) | |
| 溶解度 | 储存条件 | 4°C, protect from light | |
| General tips | 请根据产品在不同溶剂中的溶解度选择合适的溶剂配制储备液；一旦配成溶液，请分装保存，避免反复冻融造成的产品失效。 储备液的保存方式和期限：-80°C 储存时，请在 6 个月内使用，-20°C 储存时，请在 1 个月内使用。 为了提高溶解度，请将管子加热至37℃，然后在超声波浴中震荡一段时间。 |
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| Shipping Condition | 评估样品解决方案：配备蓝冰进行发货。所有其他可用尺寸：配备RT，或根据请求配备蓝冰。 | ||
| 制备储备液 | |||
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1 mg | 5 mg | 10 mg |
| 1 mM | 0.027 mL | 0.1351 mL | 0.2703 mL |
| 5 mM | 0.0054 mL | 0.027 mL | 0.0541 mL |
| 10 mM | 0.0027 mL | 0.0135 mL | 0.027 mL |
| 第一步：请输入基本实验信息（考虑到实验过程中的损耗，建议多配一只动物的药量） | ||||||||||
| 给药剂量 | mg/kg |  |
动物平均体重 | g | |
每只动物给药体积 | ul | |
动物数量 | 只 |
| 第二步：请输入动物体内配方组成（配方适用于不溶于水的药物；不同批次药物配方比例不同，请联系GLPBIO为您提供正确的澄清溶液配方） | ||||||||||
| % DMSO % % Tween 80 % saline | ||||||||||
| 计算重置 | ||||||||||
计算结果:
工作液浓度： mg/ml；
DMSO母液配制方法： mg 药物溶于 μL DMSO溶液（母液浓度 mg/mL，
体内配方配制方法：取 μL DMSO母液，加入 μL PEG300，混匀澄清后加入μL Tween 80，混匀澄清后加入 μL saline，混匀澄清。
1. 首先保证母液是澄清的；
2.
一定要按照顺序依次将溶剂加入，进行下一步操作之前必须保证上一步操作得到的是澄清的溶液，可采用涡旋、超声或水浴加热等物理方法助溶。
3. 以上所有助溶剂都可在 GlpBio 网站选购。
An overview of the structure and function of thrombin
Semin Thromb Hemost2006 Apr;32 Suppl 1:3-15.PMID: 16673262DOI: 10.1055/s-2006-939550
The fundamental importance of thrombin in biology and medicine has made it one of the most extensively studied of all proteases. Thrombin performs essential functions in vertebrate biology as the central enzyme involved in blood coagulation and platelet aggregation, and as a mitogen and secretagogue for a variety of cell types. Thrombin is synthesized in the liver and secreted into the general circulation in an inactive zymogen form (prothrombin), a complex multidomain glycoprotein that is activated to yield thrombin at sites of vascular injury by limited proteolysis following upstream activation of the coagulation cascade. Thrombin shares its general architecture and catalytic mechanism with those of pancreatic trypsin, the prototypical digestive serine protease. However, the specificity of thrombin toward substrates and cofactors, as well as its spatiotemporal regulation by effectors and inhibitors, is directed by features of the molecule that distinguish it from relatively nonspecific serine proteases like trypsin. Structural and functional studies have demonstrated the presence of surface loops that partially occlude the active site and make specific contacts with residues adjacent to the scissile bond of substrates. Specificity toward macromolecular substrates and cofactors is additionally enhanced by anion-binding exosites that are spatially distinct from the active site. More than five decades of multidisciplinary research on thrombin have produced an abundance of functional and structural information and provided a robust framework for understanding the role of thrombin in vertebrate biology.
Thrombin
Mol Aspects Med2008 Aug;29(4):203-54.PMID: 18329094DOI: 10.1016/j.mam.2008.01.001
Thrombin is a Na+-activated, allosteric serine protease that plays opposing functional roles in blood coagulation. Binding of Na+ is the major driving force behind the procoagulant, prothrombotic and signaling functions of the enzyme, but is dispensable for cleavage of the anticoagulant protein C. The anticoagulant function of thrombin is under the allosteric control of the cofactor thrombomodulin. Much has been learned on the mechanism of Na+ binding and recognition of natural substrates by thrombin. Recent structural advances have shed light on the remarkable molecular plasticity of this enzyme and the molecular underpinnings of thrombin allostery mediated by binding to exosite I and the Na+ site. This review summarizes our current understanding of the molecular basis of thrombin function and allosteric regulation. The basic information emerging from recent structural, mutagenesis and kinetic investigation of this important enzyme is that thrombin exists in three forms, E*, E and E:Na+, that interconvert under the influence of ligand binding to distinct domains. The transition between the Na+ -free slow from E and the Na+ -bound fast form E:Na+ involves the structure of the enzyme as a whole, and so does the interconversion between the two Na+ -free forms E* and E. E* is most likely an inactive form of thrombin, unable to interact with Na + and substrate. The complexity of thrombin function and regulation has gained this enzyme pre-eminence as the prototypic allosteric serine protease. Thrombin is now looked upon as a model system for the quantitative analysis of biologically important enzymes.
Thrombin plasticity
Biochim Biophys Acta2012 Jan;1824(1):246-52.PMID: 21782041DOI: 10.1016/j.bbapap.2011.07.005
Thrombin is the final protease generated in the blood coagulation cascade. It has multiple substrates and cofactors, and serves both pro- and anti-coagulant functions. How thrombin activity is directed throughout the evolution of a clot and the role of conformational change in determining thrombin specificity are issues that lie at the heart of the haemostatic balance. Over the last 20 years there have been a great number of studies supporting the idea that thrombin is an allosteric enzyme that can exist in two conformations differing in activity and specificity. However, recent work has shown that thrombin in its unliganded state is inherently flexible in regions that are important for activity. The effect of flexibility on activity is discussed in this review in context of the zymogen-to-protease conformational transition. Understanding thrombin function in terms of 'plasticity' provides a new conceptual framework for understanding regulation of enzyme activity in general. This article is part of a Special Issue entitled: Proteolysis 50 years after the discovery of lysosome.
Thrombin
Int J Biochem Cell Biol1998 Jun;30(6):641-6.PMID: 9695019DOI: 10.1016/s1357-2725(98)00011-9
Thrombin is a multifunctional serine protease which plays a central role in haemostasis by regulating platelet aggregation and blood coagulation. It is formed from its precursor prothrombin following tissue injury and converts fibrinogen to fibrin in the final step of the clotting cascade. It also promotes numerous cellular effects including chemotaxis, proliferation, extracellular matrix turnover and release of cytokines. These actions of thrombin on cells have been implicated in tissue repair processes and in the pathogenesis of inflammatory and fibroproliferative disorders such as pulmonary fibrosis and atherosclerosis. Thrombin mediates its cellular effects by proteolytically activating cell surface receptors. Presently, two such receptors have been described and their roles in regulation of these functions are currently being investigated. The discovery of multiple thrombin receptors creates the possibility of selective receptor blockade of specific thrombin mediated events. New drugs with these actions should add to our current repertoire of thrombin inhibitors used to treat thrombotic diseases.
Directing thrombin
Blood2005 Oct 15;106(8):2605-12.PMID: 15994286DOI: 10.1182/blood-2005-04-1710
Following initiation of coagulation as part of the hemostatic response to injury, thrombin is generated from its inactive precursor prothrombin by factor Xa as part of the prothrombinase complex. Thrombin then has multiple roles. The way in which thrombin interacts with its many substrates has been carefully scrutinized in the past decades, but until recently there has been little consideration of how its many functions are coordinated or directed. Any understanding of how it is directed requires knowledge of its structure, how it interacts with its substrates, and the role of any cofactors for its interaction with substrates. Recently, many of the interactions of thrombin have been clarified by crystal structure and site-directed mutagenesis analyses. These analyses have revealed common residues used for recognition of some substrates and overlapping surface exosites used for recognition by cofactors. As many of its downstream reactions are cofactor driven, competition between cofactors for exosites must be a dominant mechanism that determines the fate of thrombin. This review draws together much recent work that has helped clarify structure function relationships of thrombin. It then attempts to provide a cogent proposal to explain how thrombin activity is directed during the hemostatic response.