Assay of Functional Plasminogen in Rat Plasma Applicable to Experimental Studies of Thrombolysis

Summary

An improved sensitive, specific, precise and accurate assay of plasminogen in rat plasma was developed. It is performed in 96-well microtiter plates and can be completed within one hour. The assay is based on activation of plasminogen by human urokinase-type plasminogen activator (uPA) and simultaneous measurement of generated plasmin with the specific plasmin substrate H-D-Val-Phe-Lys-4-nitroanilide (S-2390), using purified native rat plasminogen for calibration. The concentration of S-2390 in the final reaction mixture during the whole reaction period is much greater than the K _m value (≈20 µM) for rat plasmin-cleavage of S-2390 ensuring that hydrolysis of substrate follows zero order kinetics and that the substrate produces a 20-35 fold decrease in rate of inhibition of plasmin by its target inhibitors in plasma. Analogous to the human system the target plasma inhibitors of rat plasmin are shown to be plasmin inhibitor and α-macroglobulins. Tranexamic acid (0.8 mM) is incorporated in the reaction mixture resulting in a 19-fold increase in the rate of plasminogen activation and presumably an about 50-fold decrease in the rate of inhibition of generated plasmin by plasmin inhibitor. The assay is suitable for accurate measurement of plasminogen in samples obtained from animals containing pharmacological concentrations of uPA or tissue-type plasminogen activator (tPA) in their plasma when in vitro plasminogen activation is blocked at pH 5 by collecting blood in acidic anticoagulant. Judged from in vitro experiments formation of catalytic active plasmin-α-macroglobulin complexes during massive activation of plasminogen in vivo does not interfere with the assay.

Key words

Rat - plasminogen - assay

• References

• 1 Wiman B, Hamsten A. The fibrinolytic enzyme system and its role in the etiology of thromboembolic disease. Semin Thromb Hemost 1990; 16: 207-16.
• 2 Lijnen HR, Bachmann F, Collen D, Ellis V, Pannekoek H, Rijken DC, Thorsen S. Mechanisms of plasminogen activation. J Intern Med 1994; 236: 415-24.
• 3 Chapman HA. Plasminogen activators, integrins, and the coordinated regulation of cell adhesion and migration. Curr Opin Cell Biol 1997; 09: 714-24.
• 4 Blasi F. uPA, uPAR, PAI-1: key intersection of proteolytic, adhesive and chemotactic highways?. Immunol Today 1997; 18: 415-7.
• 5 Thorsen S, Müllertz S, Suenson E, Kok P. Sequence of formation of molecular forms of plasminogen and plasmin-inhibitor complexes in plasma activated by urokinase or tissue-type plasminogen activator. Biochem J 1984; 223: 179-87.
• 6 Degen SJF, Bell SM, Schaefer LA, Elliott RW. Characterization of the cDNA coding for mouse plasminogen and localization of the gene to mouse chromosome 17. Genomics 1990; 08: 49-61.
• 7 Bangert K. Rattus norvegicus mRNA for plasminogen protein. [AJ242649]. EMBL Nucleotide Sequence Database.. 1999
• 8 Petersen LC, Brender J, Suenson E. Zymogen-activation kinetics. Modulatory effects of trans-4-(aminomethyl)cyclohexane-1-carboxylic acid and poly-D-lysine on plasminogen activation. Biochem J 1985; 225: 149-58.
• 9 Markus G. Conformational changes in plasminogen, their effect on activation, and the agents that modulate activation rates – A review. Fibrinolysis 1996; 10: 75-85.
• 10 Thorsen S, Kok P, Astrup T. Reversible and irreversible alterations of human plasminogen indicated by changes in susceptibility to plasminogen activators and in response to epsilon-aminocaproic acid. Thromb Diath Haemorrh 1974; 32: 325-40.
• 11 Kulseth MA, Helgeland L. A highly sensitive chromogenic microplate assay for quantification of rat and human plasminogen. Anal Biochem 1993; 210: 314-7.
• 12 Owens MR, Cimino CD. Biosynthesis of plasminogen by the perfused rat liver. J Lab Clin Med 1985; 105: 368-73.
• 13 Mussoni L, Raczka E, Chmielewska J, Donati MB, Latallo ZS. Plasminogen assay in rabbit, rat and mouse plasma using the chromogenic substrate S-2251. Thromb Res 1979; 15: 341-9.
• 14 Gram J, Jespersen J. A functional plasminogen assay utilizing the potentiating effect of fibrinogen to correct for the overestimation of plasminogen in pathological plasma samples. Thromb Haemost 1985; 53: 255-9.
• 15 Baillie AJ, Sim AK. Activation of the fibrinolytic enzyme system in laboratory animals and in man. Thromb Diath Haemorrh 1971; 25: 499-506.
• 16 Bangert K, Johnsen AH, Thorsen S. Characterization and assay of rat plasminogen (PG). Thromb Haemost 1997; Supplement: 560-0.
• 17 Philips M, Juul A-G, Selmer J, Lind B, Thorsen S. A specific immunologic assay for functional plasminogen activator inhibitor 1 in plasma – standardized measurements of the inhibitor and related parameters in patients with venous thromboembolic disease. Thromb Haemost 1992; 68: 486-94.
• 18 Kyhse-Andersen J. Semidry Electroblotting – Transfer Using Equipment Without Buffer Vessel. In: CRC Handbook of Immunoblotting of Proteins. Bjerrum OJ, Heegaard NHH. eds. Boca Raton, Florida: CRC Press; 1988: 79-85.
• 19 Bangert K, Johnsen AH, Christensen U, Thorsen S. Different N-terminal forms of α₂-plasmin inhibitor in human plasma. Biochem J 1993; 291: 623-5.
• 20 Thorsen S, Clemmensen I, Sottrup-Jensen L, Magnusson S. Adsorption to fibrin of native fragments of known primary structure from human plasminogen. Biochim Biophys Acta 1981; 668: 377-87.
• 21 Müllertz S, Thorsen S, Sottrup-Jensen L. Identification of molecular forms of plasminogen and plasmin-inhibitor complexes in urokinase-activated human plasma. Biochem J 1984; 223: 169-77.
• 22 Ipsen H-H, Christensen U. Kinetic studies of urokinase-catalysed hydrolysis of 5-oxo-l-prolylglycyl-l-arginine 4-nitroanilide. Biochim Biophys Acta 1980; 613: 476-81.
• 23 Petersen LC, Boel E, Johannessen M, Foster D. Quenching of the amidolytic activity of one-chain tissue-type plasminogen activator by mutation of lysine-416. Biochemistry 1990; 29: 3451-7.
• 24 Thorsen S, Müllertz S. Rate of activation and electrophoretic mobility of unmodified and partially degraded plasminogen. Scand J Clin Lab Invest 1974; 34: 167-76.
• 25 Ganrot PO. On the determination of molar concentrations of plasmin and plasmin inhibitors. Acta Chem Scand 1967; 21: 595-601.
• 26 Collen D, Van Hoef B, Schlott B, Hartmann M, Gührs K-H, Lijnen HR. Mechanisms of activation of mammalian plasma fibrinolytic systems with streptokinase and with recombinant staphylokinase. Eur J Biochem 1993; 216: 307-14.
• 27 Christensen U, Ipsen H-H. Steady-state kinetics of plasmin-and trypsincatalysed hydrolysis of a number of tripeptide-p-nitroanilides. Biochim Biophys Acta 1979; 569: 177-83.
• 28 Lottenberg R, Christensen U, Jackson CM, Coleman PL. Assay of coagulation proteases using peptide chromogenic and fluorogenic substrates. Methods Enzymol 1981; 80: 341-61.
• 29 Kiss I, Aurell L, Pozsgay M, Elodi P. Investigation on the substrate specificity of human plasmin using tripeptidyl-p-nitroanilide substrates. Biochem Biophys Res Commun 1985; 131: 928-34.
• 30 Lijnen HR, Van Hoef B, Collen D. Influence of cyanogen-bromide-digested fibrinogen on the kinetics of plasminogen activation by urokinase. Eur J Biochem 1984; 144: 541-4.
• 31 Cantor CR, Schimmel PR. Absorption Spectroscopy. In: Biophysical Chemistry. San Franciso, CA: W. H. Freeman and Company; 1980: 374-85.
• 32 Wallén P, Wiman B. Characterization of human plasminogen. I. On the relationship between different molecular forms of plasminogen demonstrated in plasma and found in purified preparations. Biochim Biophys Acta 1970; 221: 20-30.
• 33 Cummings HS, Castellino FJ. Interaction of human plasmin with human α_{2 -}macroglobulin. Biochemistry 1984; 23: 105-11.
• 34 Lonberg-Holm K, Reed DL, Roberts RC, Damato-McCabe D. Three high molecular weight protease inhibitors of rat plasma. Reactions with trypsin. J Biol Chem 1987; 262: 4844-53.
• 35 Webb DJ, Crookston KP, Figler NL, LaMarre J, Gonias SL. Differences in the binding of transforming growth factor β1 to the acute-phase reactant and constitutively synthesized α-macroglobulins of rat. Biochem J 1995; 312: 579-86.
• 36 Menoud PA, Sappino N, Boudal-Khoshbeen M, Vassalli JD, Sappino AP. The kidney is a major site of α₂-antiplasmin production. J Clin Invest 1996; 97: 2478-84.
• 37 Petersen LC, Clemmensen I. Kinetics of plasmin inhibition in the presence of a synthetic tripeptide substrate. The reaction with pancreatic trypsin inhibitor and two forms of α₂-plasmin inhibitor. Biochem J 1981; 199: 121-7.
• 38 Peterson GL. Determination of total protein. Methods Enzymol 1983; 91: 95-119.
• 39 Sugiyama N, Sasaki T, Iwamoto M, Abiko Y. Binding site of α₂-plasmin inhibitor to plasminogen. Biochim Biophys Acta 1988; 952: 1-7.
• 40 Weitz JI, Leslie B, Ginsberg J. Soluble fibrin degradation products potentiate tissue plasminogen activator-induced fibrinogen proteolysis. J Clin Invest 1991; 87: 1082-90.
• 41 Suenson E, Lützen O, Thorsen S. Initial plasmin-degradation of fibrin as the basis of a positive feed-back mechanism in fibrinolysis. Eur J Biochem 1984; 140: 513-22.
• 42 Suenson E, Petersen LC. Fibrin and plasminogen structures essential to stimulation of plasmin formation by tissue-type plasminogen activator. Biochim Biophys Acta 1986; 870: 510-9.
• 43 Suenson E, Thorsen S. The course and prerequisites of lys-plasminogen formation during fibrinolysis. Biochemistry 1988; 27: 2435-43.
• 44 Stewart RJ, Fredenburgh JC, Weitz JI. Characterization of the interactions of plasminogen and tissue and vampire bat plasminogen activators with fibrinogen, fibrin, and the complex of D-dimer noncovalently linked to fragment E. J Biol Chem 1998; 273: 18292-9.
• 45 Thorsen S, Müllertz S. Reaction Sequences and the Mechanism of Plasminogen Activation in Urokinase-Activated Plasma. Effects of Fibrin and 6-Aminohexanoic Acid. In: Progress in Chemical Fibrinolysis and Thrombolysis. Davidson JF, Cepelák V, Samama MM, Desnoyers PC. eds. Edinburgh: Churchill Livingstone; 1979: 462-8.