PPACK (Synonyms: Pebac, D-Phenylalanyl-prolyl-arginyl Chloromethyl Ketone)

Kinetic Procedures(Inhibition of Thrombin with PPACK) ^[1]:

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.

Applications

PPACK is a potent selective and irreversible thrombin inhibitor that inhibits human alpha thrombin with a K_i value of 0.24 nM.

Cell experiment ^[2]:

Cell lines

Bovine aortic endothelial cells

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.

Reaction Conditions

0.2 mM;2h

Applications

Coincubation with PPACK reduced the increase in endothelial permeability induced by plasmin by 59%.

Cell experiment ^[3]:

Cell lines

Human osteoarthritis synovial fibroblasts (OASF)

Preparation method

Cell were treated with thrombin (3 U/ml), thrombin plus PPACK (30 nM) for 24 hours.

Reaction Conditions

24h; 30 nM

Applications

Pretreatment of cells with PPACK effectively antagonized the potentiating effect of thrombin on HO-1 expression.

Animal experiment ^[4]:

Animal models

Male rabbits

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.

Applications

PPACK pretreatment can inhibit thrombine-induced cerebral thromboembolism in rabbits.

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.