1. A chromogenic enzymatic assay capable of detecting prourokinase-like material in plasma
P R Kebabian, J Henkin Thromb Res . 1992 Feb 1;65(3):401-7. doi: 10.1016/0049-3848(92)90170-f.
We report a functional assay capable of quantifying prourokinase (ProUK)- like material in plasma where urokinase (UK) is also present. The assay involves inactivation of urokinase with a specific, active site directed irreversible inhibitor, dansyl-glutamyl glycyl arginine chloromethylketone (dansyl- GGACK). Excess inhibitor is subsequently quenched with dithiothreitol (DTT). The ProUK-like material in plasma is then converted to active urokinase with thermolysin, a proteolytic enzyme of bacterial origin. Alpha 2-macroglobulin in plasma inhibits thermolysin; however alpha 2-macroglobulin is inactivated with methylamine. The assay can detect as little as 20 ng of ProUK and is linear from 20 to 120 ng. The assay was applied to quantify the amount of ProUK-like material in plasma obtained from dog at various times after i.v. administration of 100,000 or 75,000 U/kg, of pro-urokinase.
2. Differential reactivity of Glu-Gly-Arg-CH2Cl, a synthetic urokinase inhibitor, with single-chain and two-chain forms of urokinase-type plasminogen activator
D Collen, B Van Hoef, H R Lijnen Eur J Biochem . 1987 Jan 15;162(2):351-6. doi: 10.1111/j.1432-1033.1987.tb10608.x.
Glu-Gly-Arg-CH2Cl, a synthetic inhibitor which alkylates the active-site histidine of urokinase with an apparent second-order rate constant of approximately 10(4) M-1 s-1, reacts differently with single-chain and two-chain forms of urokinase-type plasminogen activators (scu-PA and tcu-PA). Kinetic analysis indicated that the plasminogen activating potential of tcu-PA is irreversibly inhibited in a two-step reaction according to (formula; see text) with Ki = 5.0 X 10(-6) M and k2 = 0.05 s-1. The plasminogen-activating potential of scu-PA, however, is competitively inhibited by Glu-Gly-Arg-CH2Cl with Ki = 1.3 X 10(-6) M. Reversibility of the interaction of Glu-Gly-Arg-CH2Cl with scu-PA was confirmed by the restoration of full enzymatic activity after removal of inhibitor. The differential interaction of Glu-Gly-Arg-CH2Cl with scu-PA and tcu-PA supports the hypothesis that these molecular forms of urokinase-type PA have intrinsically different enzymatic properties.
3. Evaluation of the thrombin inhibitor D-phenylalanyl-L-prolyl-L-arginine chloromethylketone (PPACK) with the factor Xa inhibitor 1,5-dansyl-L-glutamyl-L-glycyl-L-arginine chloromethylketone (GGACK) as anticoagulants for critical care clinical chemistry specimens
A W Lyon, M E Lyon, D W Drobot, S R Harding Clin Chim Acta . 1999 Feb;280(1-2):91-9. doi: 10.1016/s0009-8981(98)00176-4.
The objective of this study was to determine whether a thrombin inhibitor (PPACK) and a factor Xa inhibitor (GGACK) either alone or in combination can anticoagulate whole blood without biasing the analysis of several critical care analytes. Whole blood clot time was used to assess anticoagulant efficacy. The analytical biases mediated by the anticoagulants on glucose, urea, creatinine, electrolytes, amylase, lactate dehydrogenase, creatine kinase, ionized calcium and pH were assessed. The protease inhibitor mixture (100 micrommol/l PPACK + 500 micromol/l GGACK) was more a potent anticoagulant than the individual agents at the same concentrations. Both PPACK and GGACK, alone and in combination, reduced the activity of creatine kinase and amylase by 3-10% while the remaining critical care analytes were less affected. In conclusion, PPACK and GGACK mixtures can effectively anticoagulate whole blood, but the mixtures exert pre-analytical influences that limit the analytical versatility of these novel plasma-matrices.
