PPACK
(Synonyms: H-D-苯丙酰氨-脯酰氨-精氨酸-氯甲基酮三氟乙酸,Pebac, D-Phenylalanyl-prolyl-arginyl Chloromethyl Ketone) 目录号 : GC44671
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。
Cas No.:71142-71-7
Sample solution is provided at 25 µL, 10mM.
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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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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]。
| Cas No. | 71142-71-7 | SDF | |
| 别名 | H-D-苯丙酰氨-脯酰氨-精氨酸-氯甲基酮三氟乙酸,Pebac, D-Phenylalanyl-prolyl-arginyl Chloromethyl Ketone | ||
| 化学名 | D-phenylalanyl-N-[(1S)-4-[(aminoiminomethyl)amino]-1-(2-chloroacetyl)butyl]-L-prolinamide, trifluoroacetate salt | ||
| Canonical SMILES | O=C([C@H](N)CC1=CC=CC=C1)N2[C@H](C(N[C@H](C(CCl)=O)CCCNC(N)=N)=O)CCC2 | ||
| 分子式 | C21H31ClN6O3 | 分子量 | 451 |
| 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 | 2.2173 mL | 11.0865 mL | 22.1729 mL |
| 5 mM | 0.4435 mL | 2.2173 mL | 4.4346 mL |
| 10 mM | 0.2217 mL | 1.1086 mL | 2.2173 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。
PPACK attenuates plasmin-induced changes in endothelial integrity
Thromb Res 1993 Jun 15;70(6):425-36.PMID:8362368DOI:10.1016/0049-3848(93)90085-3.
In order to determine whether plasmin affects endothelial cell integrity directly, confluent bovine aortic endothelial cells were treated with plasminogen and a plasminogen activator. The permeability of the monolayer to [125I]-albumin was shown to be increased significantly (P < 0.01) with a concomitant decrease in viability. Plasmin activity correlated significantly with endothelial cell permeability (p < 0.004; r = 0.82). Coincubation with D-phenylalanyl-L-prolyl-L-arginyl chloromethylketone, a tripeptide inhibitor of plasmin, reduced the increase in endothelial permeability induced by plasmin by 59% (p = 0.033). Monolayers studied in parallel were stained with rhodamine-phalloidin to visualize F-actin. There were significant morphologic changes in the endothelial monolayers exposed to plasmin compared to control monolayers, and these changes could be attenuated by coincubation with D-phenylalanyl-L-prolyl-L-arginyl chloromethylketone. These studies show that: 1) plasmin induces significant increases in endothelial cell permeability with accompanying morphologic changes; and 2) these deleterious functional and morphologic effects can be attenuated by coincubation with the plasmin inhibitor, D-phenylalanyl-L-prolyl-L-arginyl chloromethylketone.
PPACK-thrombin inhibits thrombin-induced platelet aggregation and cytoplasmic acidification but does not inhibit platelet shape change
Blood 1990 May 15;75(10):1989-90.PMID:2337670doi
We have re-evaluated the previously reported ability of TLCK-thrombin (N alpha-tosyl-L-lysine chloromethyl ketone-treated alpha-thrombin) and PPACK-thrombin (D-phenylalanyl-L-prolyl-L-arginine chloromethyl ketone-treated alpha-thrombin) to inhibit alpha-thrombin-induced platelet activation (Harmon JT, Jamieson GA: J Biol Chem 261:15928, 1986; and Harmon JT, Jamieson GA: Biochemistry 27:2151, 1988). Despite several cycles of derivatization with TLCK (10,000-fold molar excess), preparations of TLCK-thrombin have been found to contain about 4% residual alpha-thrombin activity, suggesting that these preparations are an equilibrium mixture of TLCK-thrombin and alpha-thrombin and cannot be used for evaluating competition between these two agents. In contrast, alpha-thrombin activity was completely inhibited by PPACK at 15-fold molar excess. PPACK-thrombin, free of unreacted PPACK and devoid of residual alpha-thrombin activity, did not markedly affect platelet shape change at concentrations as high as 1 mumol/L, but inhibited aggregation and secretion in intact platelets activated with the minimal concentration of alpha-thrombin causing a full response (0.3 to 0.5 nmol/L) and yielded a 50% inhibition constant (IC50) for inhibition of aggregation by PPACK-thrombin of 110 nmol/L. This inhibition was specific for alpha-thrombin-induced platelet activation, and no inhibition was seen with activation induced by ADP, collagen, epinephrine, ristocetin, or arachidonate. At these low alpha-thrombin concentrations (approximately 0.4 nmol/L), a persistent cytoplasmic acidification was observed of -0.062 +/- 0.016 pH units, although alkalinization was observed at higher alpha-thrombin concentrations (greater than 1 nmol/L). While inhibition of aggregation and secretion occurred when alpha-thrombin and PPACK-thrombin were added simultaneously, inhibition of cytoplasmic acidification and of the elevation of cytoplasmic [Ca2+] induced by low concentrations of alpha-thrombin (0.4 nmol/L) occurred only if platelets were preincubated with PPACK-thrombin for 5 minutes before the addition of alpha-thrombin. In platelets treated with Serratia marcescens protease to remove glycoprotein lb (GPlb), alpha-thrombin-induced shape change was attenuated but persisted in the presence of a high concentration (2 mumol/L) of PPACK-thrombin, although aggregation and secretion were inhibited, as seen in intact platelets. The IC50 value for inhibition of aggregation by PPACK-thrombin was approximately 1 mumol/L at the higher alpha-thrombin concentrations (5 nmol/L) required for full activation in this case. These results suggest that PPACK-thrombin may be a useful probe of platelet function since it specifically blocks platelet aggregation and secretion induced by alpha-thrombin.(ABSTRACT TRUNCATED AT 400 WORDS)
Inhibition of Thrombin With PPACK-Nanoparticles Restores Disrupted Endothelial Barriers and Attenuates Thrombotic Risk in Experimental Atherosclerosis
Arterioscler Thromb Vasc Biol 2016 Mar;36(3):446-55.PMID:26769047DOI:10.1161/ATVBAHA.115.306697.
Objective: A role for thrombin in the pathogenesis of atherosclerosis has been suggested through clinical and experimental studies revealing a critical link between the coagulation system and inflammation. Although approved drugs for inhibition of thrombin and thrombin-related signaling have demonstrated efficacy, their clinical application to this end may be limited because of significant potential for bleeding side effects. Thus, we sought to implement a plaque-localizing nanoparticle-based approach to interdict thrombin-induced inflammation and hypercoagulability in atherosclerosis. Approach and results: We deployed a novel magnetic resonance spectroscopic method to quantify the severity of endothelial damage for correlation with traditional metrics of vessel procoagulant activity after dye-laser injury in fat-fed apolipoprotein E-null mice. We demonstrate that a 1-month course of treatment with antithrombin nanoparticles carrying the potent thrombin inhibitor PPACK (d-phenylalanyl-l-prolyl-l-arginyl chloromethylketone) nanoparticle (1) reduces the expression and secretion of proinflammatory and procoagulant molecules, (2) diminishes plaque procoagulant activity without the need for systemic anticoagulation, (3) rapidly restores disrupted vascular endothelial barriers, and (4) retards plaque progression in lesion-prone areas. Conclusions: These observations illustrate the role of thrombin as a pleiotropic atherogenic molecule under conditions of hypercholesterolemia and suggest the utility of its inhibition with locally acting antithrombin nanoparticle therapeutics as a rapid-acting anti-inflammatory strategy in atherosclerosis to reduce thrombotic risk.
Markers of coagulation and fibrinolysis in blood drawn into citrate with and without D-Phe-Pro-Arg-Chloromethylketone (PPACK)
Thromb Res 1994 Mar 1;73(5):279-84.PMID:8016814DOI:10.1016/0049-3848(94)90024-8.
In order to compare levels of Prothrombin Fragment 1 + 2 (F1 + 2), Thrombin-antithrombin III complex (TAT), Fibrinogen Degradation Products (FgDP) and Fibrin Degradation Products (FbDP) in plasma from blood drawn into sodium citrate with and without the protease inhibitor D-Phe-Pro-Arg-Chloromethylketone (PPACK), blood samples were collected from 41 patients on the first day after elective gastric surgery. Levels of F1 + 2, TAT and FbDP were not significantly different in plasma with and without PPACK. FgDP levels were significantly higher in plasma with PPACK. Our results did not support previous suggestions that PPACK should be used as a routine anticoagulant for measurement of haemostatic activation products.
Differential effects of citrate versus PPACK anticoagulation on measured platelet inhibition by abciximab, eptifibatide and tirofiban
J Thromb Thrombolysis 2001 Oct;12(2):123-7.PMID:11729363DOI:10.1023/a:1012991303381.
Background: High levels of glycoprotein (GP) IIb/IIIa receptor inhibition are required to prevent arterial thrombosis following percutaneous coronary intervention. Ex-vivo turbidometric platelet aggregation in citrate anticoagulated blood samples has been the primary method previously utilized to derive dose regimens for administering platelet GP IIb/IIIa inhibitors. Enhanced GP IIb/IIIa binding and inhibition of platelet aggregation for eptifibatide secondary to citrate induced reduction of ionized plasma calcium concentrations has been reported. Methods/results: We evaluated the differential effects of citrate versus PPACK anticoagulation on turbidometric platelet inhibition in normal volunteers by eptifibatide, tirofiban or abciximab. The decrease in ionized calcium afforded by citrate was associated with enhanced in vitro platelet inhibition for all three GP IIb/IIIa inhibitors, including abciximab. The magnitude of citrate effect was greatest for eptifibatide. Both tirofiban and abciximab have similar citrate calcium chelation associated enhancement of measured platelet inhibition. Conclusion: Accurate assessment and comparison of platelet inhibition by GP IIb/IIIa inhibitors may require avoidance of calcium chelating anticoagulants.