PPACK Dihydrochloride
(Synonyms: PPACK Dihydrochloride) 目录号 : GC10277
PPACK是一种有效的选择性和不可逆凝血酶抑制剂，能够抑制人α凝血酶，Ki值为0.24 nM。
Sample solution is provided at 25 µL, 10mM.
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PPACK is a potent selective and irreversible thrombin inhibitor that inhibits human alpha thrombin with a Ki value of 0.24 nM. PPACK inhibit the binding of rt-PA to plasma protease inhibitors in vitro[1-3]. It forms a covalent bond to the thrombin active site Ser and cross-links with His57 at the active site to form a tetrahedral PPack-thrombin complex. PPACK completely inhibited changes in fibrin degradation products, plasminogen and alpha 2-antiplasmin[4-6].
Coincubation with PPACK(0.2 mM;2h) reduced the increase in endothelial permeability induced by plasmin by 59% in bovine aortic endothelial cells[7]. Heme oxygenase (HO)-1 is a stress-inducible rate-limiting enzyme in heme degradation that confers cytoprotection against oxidative injury. Pretreatment of cells with PPACK(24h; 30 nM) effectively antagonized the potentiating effect of thrombin on HO-1 expression[8].
PPACK(bolus dose of 10-100 μg/kg or infusion of 0.25-1.0 μg/kg/min) pretreatment can inhibit thrombine-induced cerebral thromboembolism in rabbits[9].
References:
[1]. Mohler MA, Refino CJ, et,al. D-Phe-Pro-Arg-chloromethylketone: its potential use in inhibiting the formation of in vitro artifacts in blood collected during tissue-type plasminogen activator thrombolytic therapy. Thromb Haemost. 1986 Oct 21;56(2):160-4. PMID: 2433785.
[2]. Bode W, Mayr I, et,al. The refined 1.9 A crystal structure of human alpha-thrombin: interaction with D-Phe-Pro-Arg chloromethylketone and significance of the Tyr-Pro-Pro-Trp insertion segment. EMBO J. 1989 Nov;8(11):3467-75. doi: 10.1002/j.1460-2075.1989.tb08511.x. PMID: 2583108; PMCID: PMC401503.
[3]. Lyon ME, Fine JS, et,al. D-phenylalanyl-L-prolyl-L-arginine chloromethyl ketone (PPACK): alternative anticoagulant to heparin salts for blood gas and electrolyte specimens. Clin Chem. 1995 Jul;41(7):1038-41. PMID: 7600685.
[4]. Kovach IM, Kelley P, et,al. Proton bridging in the interactions of thrombin with small inhibitors. Biochemistry. 2009 Aug 4;48(30):7296-304. doi: 10.1021/bi900098s. PMID: 19530705; PMCID: PMC2800789.
[5]. Kettner C, Shaw E. D-Phe-Pro-ArgCH2C1-A selective affinity label for thrombin. Thromb Res. 1979;14(6):969-73. doi: 10.1016/0049-3848(79)90014-8. PMID: 473131.
[6]. Nienaber VL, Mersinger LJ, et,al. Structure-based understanding of ligand affinity using human thrombin as a model system. Biochemistry. 1996 Jul 30;35(30):9690-9. doi: 10.1021/bi952164b. PMID: 8703940.
[7]. Rabbani LE, Johnstone MT, et,al. PPACK attenuates plasmin-induced changes in endothelial integrity. Thromb Res. 1993 Jun 15;70(6):425-36. doi: 10.1016/0049-3848(93)90085-3. PMID: 8362368.
[8].Liu JF, Hou SM, et,al. Thrombin induces heme oxygenase-1 expression in human synovial fibroblasts through protease-activated receptor signaling pathways. Arthritis Res Ther. 2012 Apr 27;14(2):R91. doi: 10.1186/ar3815. PMID: 22541814; PMCID: PMC3446465.
[9]. Liu JT, Paul W, et,al. Thrombin inhibitors and anti-coagulants on thrombin-induced embolisation in rabbit cranial vasculature. Eur J Pharmacol. 1994 Oct 24;264(2):183-90. doi: 10.1016/0014-2999(94)00464-1. PMID: 7851481.
PPACK是一种有效的选择性和不可逆凝血酶抑制剂，能够抑制人α凝血酶，Ki值为0.24 nM。PPACK在体外抑制rt-PA与血浆蛋白酶抑制剂的结合[1-3]。PPACK可以与凝血酶活性位点Ser形成共价键，并在活性位点与His57交联形成四面体PPACK-凝血酶复合物。PPACK完全抑制纤维蛋白降解产物、纤溶酶原和2α-antiplasmin的变化[4-6]。
与PPACK共孵育(0.2 mM;2h)可使纤溶酶诱导的牛主动脉内皮细胞通透性增加减少59%[7]。血红素加氧酶(HO)-1是一种应激诱导的血红素降解限速酶，可保护细胞免受氧化损伤。用PPACK预处理(24h; 30 nM)细胞可有效拮抗凝血酶对HO-1表达的增强作用[8]。
PPACK(bolus dose of 10-100 μg/kg or infusion of 0.25-1.0 μg/kg/min)预处理可抑制凝血素诱导的家兔脑血栓栓塞[9]。
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Kinetic Procedures(Inhibition of Thrombin with PPACK) [1]: |
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Preparation method |
The thrombin stock solution was diluted in the appropriate buffer, resulting in a 20 mL solution with a concentration of 3-5×10-10 M. An aliquot of 980 μL was incubated in a cuvette in the temperature controlled cell compartment of the spectrophotometer to reach thermal equilibrium. Thrombin inhibition was initiated by injecting 10 μL of a thermally equilibrated 1.1 × 10-7 to 3.65 × 10-7 M PPACK solution and mixing. The reaction mixture was promptly incubated for a time interval between 15 and 200 s, after which 10 μL of a 3-5×10-3 M solution of substrate in DMSO was quickly added to measure the remaining enzyme activity. Pseudo-first-order rate constants were calculated by fitting the exponential function to the remaining enzyme activity versus time of inhibition for 10 successive incubation times. The thrombin activity remained constant for the duration of the experiment in the 288-308 K temperature range. |
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Applications |
PPACK is a potent selective and irreversible thrombin inhibitor that inhibits human alpha thrombin with a Ki value of 0.24 nM. |
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Cell experiment [2]: |
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Cell lines |
Bovine aortic endothelial cells |
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Preparation method |
Confluent bovine aortic endothelial cell monolayers were obtained three to five days after plating on the polycarbonate membrane cluster plates. Cells were treated with plasminogen and a plasminogen activator. PPACK were added to the upper compartments. The monolayers were subsequently incubated for two hours in a 5% CO2 / 95% O2 atmosphere. |
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Reaction Conditions |
0.2 mM;2h |
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Applications |
Coincubation with PPACK reduced the increase in endothelial permeability induced by plasmin by 59%. |
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Cell experiment [3]: |
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Cell lines |
Human osteoarthritis synovial fibroblasts (OASF) |
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Preparation method |
Cell were treated with thrombin (3 U/ml), thrombin plus PPACK (30 nM) for 24 hours. |
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Reaction Conditions |
24h; 30 nM |
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Applications |
Pretreatment of cells with PPACK effectively antagonized the potentiating effect of thrombin on HO-1 expression. |
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Animal experiment [4]: |
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Animal models |
Male rabbits |
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Preparation method |
PPACK was prepared in saline and administered as a bolus dose of 10-100 μg/kg or as an infusion of 0.25-1.0 μg/kg/min. Rabbits were treated with PPACK bolus 1 min either before or after the administration of thrombin or were infused with PPACK commencing 30 min prior to the administration of thrombin and continued for 1 h at a rate of 0.1 mL/min. |
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Applications |
PPACK pretreatment can inhibit thrombine-induced cerebral thromboembolism in rabbits. |
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References: [1]. Kovach IM, Kelley P, et,al. Proton bridging in the interactions of thrombin with small inhibitors. Biochemistry. 2009 Aug 4;48(30):7296-304. doi: 10.1021/bi900098s. PMID: 19530705; PMCID: PMC2800789. [2]. Rabbani LE, Johnstone MT, et,al. PPACK attenuates plasmin-induced changes in endothelial integrity. Thromb Res. 1993 Jun 15;70(6):425-36. doi: 10.1016/0049-3848(93)90085-3. PMID: 8362368. [3].Liu JF, Hou SM, et,al. Thrombin induces heme oxygenase-1 expression in human synovial fibroblasts through protease-activated receptor signaling pathways. Arthritis Res Ther. 2012 Apr 27;14(2):R91. doi: 10.1186/ar3815. PMID: 22541814; PMCID: PMC3446465. [4]. Liu JT, Paul W, et,al. Thrombin inhibitors and anti-coagulants on thrombin-induced embolisation in rabbit cranial vasculature. Eur J Pharmacol. 1994 Oct 24;264(2):183-90. doi: 10.1016/0014-2999(94)00464-1. PMID: 7851481. |
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| Cas No. | SDF | ||
| 别名 | PPACK Dihydrochloride | ||
| 化学名 | N-(2-amino-3-phenylpropanoyl)-1-[1-chloro-6-(diaminomethylideneamino)-2-oxohexan-3-yl]pyrrolidine-2-carboxamide | ||
| Canonical SMILES | C1CC(N(C1)C(CCCN=C(N)N)C(=O)CCl)C(=O)NC(=O)C(CC2=CC=CC=C2)N | ||
| 分子式 | C21H31ClN6O3.2HCl | 分子量 | 523.88 |
| 溶解度 | 20mg/mL in ethanol, 33mg/mL in DMSO, or in DMF | 储存条件 | Store at -20°C |
| 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 | 1.9088 mL | 9.5442 mL | 19.0883 mL |
| 5 mM | 0.3818 mL | 1.9088 mL | 3.8177 mL |
| 10 mM | 0.1909 mL | 0.9544 mL | 1.9088 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。
Quality Control & SDS
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- Purity: >98.00%
- COA (Certificate Of Analysis)
- SDS (Safety Data Sheet)
- Datasheet