Effect of Fibrin-Like Stimulators on the Activation of Plasminogen by Tissue-Type Plasminogen Activator (t-PA) - Studies with Active Site Mutagenized Plasminogen and Plasmin Resistant t-PA

H R Lijnen; B Van Hoet; F De Cock; D Collen

doi:10.1055/s-0038-1647254

Thrombosis and Haemostasis, Table of Contents

Thromb Haemost 1990; 64(01): 061-068
DOI: 10.1055/s-0038-1647254

Original Article

Schattauer GmbH Stuttgart

Effect of Fibrin-Like Stimulators on the Activation of Plasminogen by Tissue-Type Plasminogen Activator (t-PA) - Studies with Active Site Mutagenized Plasminogen and Plasmin Resistant t-PA^[*]

H R Lijnen

The Centerfor Thrombosis and Vascular Research, University of Leuven, Leuven, Belgium

,

B Van Hoet

The Centerfor Thrombosis and Vascular Research, University of Leuven, Leuven, Belgium

,

F De Cock

The Centerfor Thrombosis and Vascular Research, University of Leuven, Leuven, Belgium

,

D Collen

The Centerfor Thrombosis and Vascular Research, University of Leuven, Leuven, Belgium

› Author Affiliations

Abstract

PDF Download

Summary

The activation of plasminogen by t-PA was measured in the presence and absence of fibrin stimulation, using natural human plasminogen (nPlg) and rPlg-Ala⁷⁴⁰, a recombinant plasminogen with the active site Ser⁷⁴⁰ mutagenaed to Ala. Recombinant wild type t-PA (rt-PA) was used as well as rt-PA -Glul²⁷⁵, a recombinant single chain t-PA in which the Arg of the plasmin sensitiv e Arg²⁷⁵- Ile²⁷⁶ peptide bond was substituted with Glu. Conversion of ¹²⁵I-labeled single chain plasminogen to two-chain plasmin by wild-type or mutant t-PA, was quantitated by SDS gel electrophoresis and radioisotope counting of gel slices, and expressed as initial activation rates (v₀ in pM s⁻¹) per 1 μM enzyme. In the absence of fibrin stimulation, the vs for the activation of nPlg and rPlg-Ala⁷⁴⁰ with the single chain forms of both t-PAs were comparable (0.6 to 2.7 pM s⁻¹) but were lower than with the corresponding two-chain forms (5.3 to 23 pM s⁻¹). In the presence of 1 μM soluble fibrin monomer (desAAfibrin), the v₀ for nPlg and rPlg-Ala⁷⁴⁰ by single chain rt-PA was also comparable (24 and, 33 pM s-1 respectively), whereas with 1 pM CNBr-digested fibrinogen, the vs for nPlg with single chain rt-PA was about 20-fold higher than that of rPlg-Ala⁷⁴⁰ (135 and 7.5 pM s⁻¹ respectively). In contrast, the vs for nPlg and rPlg-Ala⁷⁴⁰ by single chain rt-PA- G1u²⁷⁵, two-chain rt-PA-G1u²⁷⁵ or two-chain rt-PA were comparable in the presence of either desAAfibrin or CNBr-digested fibrinogen.

These findings confirm and establish: 1) that single chain t-PA is an active enzyme both in the presence and absence of fibrin stimulator; 2) that, in a system devoid of plasmin activity (rPlg- Ala⁷⁴⁰), the two-chain form of t-PA is about L5 times more active than the single chain form in the absence of fibrin but equipotent in the presence of desAAfibrin; and 3) that the mechanism of stimulation of plasminogen activation with single chain t-PA by CNBr-digested fibrinogen is different from that by soluble fibrin.

PDF (705 kb)

References

References
1 Collen D. On the regulation and control of fibrinolysis. Thromb Haemostas 1980; 43: 77-89
2 Wallen P, Wiman B. Characterization of human plasminogen. II. Separation and partial characterization of different molecular forms of human plasminogen. Biochim Biophys Acta 1972; 257: 122-134
3 Sottrup-Jensen L, Petersen TE, Magnusson S. Atlas of Protein Sequence and Structure. Vol 5, Suppl 3 Dayhoff MO. (ed) National Biomedical Research Foundation; Washington DC: 1978: 91
4 Robbins KC, Summaria L, Hsieh B, Shah RJ. The peptide chains of human plasmin. Mechanism of activation of human plasminogen to plasmin. J Biol Chem 1967; 242: 2333-2342
5 Holvoet P, Lijnen HR, Collen D. A monoclonal antibody specific for Lys-plasminogen. Application to the study of the activation pathways of plasminogen in vivo. J Biol Chem 1985; 260: 12106-12111
6 Summaria L, Arzadon L, Bemabe P, Robbins KC. The activation of plasminogen to plasmin by urokinase in the presence of the plasmin inhibitor trasylol. The preparation of plasmin with the same NH2-terminal heavy (A) chain sequence as the parent zymogen. J Biol Chem 1975; 250: 3988-3995
7 Pennica D, Holmes WE, Kohr WJ, Harkins RN, Vehar GA, Ward CA, Bennett WF, Yelverton E, Seeburg PH, Heyneker HL, Goeddel DV, Collen D. Cloning and expression of human tissue-type plasminogen activator cDNA in E. coli. Nature 1983; 301: 214-221
8 Camiolo SM, Thorsen S, Astrup T. Fibrinogenolysis and fibrinolysis with tissue plasminogen activator, urokinase, streptokinase-activated human globulin, and plasmin. Proc Soc Exp Biol Med 1971; 138: 277-280
9 Hoylaerts M, Rijken DC, Lijnen HR, Collen D. Kinetics of the activation of plasminogen by human tissue plasminogen activator. Role of fibrin. J Biol Chem 1982; 257: 2912-2919
10 Rijken DC, Hoylaerts M, Collen D. Fibrinolytic properties of one-chain and two-chain human extrinsic (tissue-type) plasminogen activator. J Biol Chem 1982; 257: 2920-2925
11 Higgins DL, Vehar GA. Interaction of one-chain and two-chain tissue plasminogen activator with intact and plasmin-degraded fibrin. Biochemistry 1987; 26: 7786-7791
12 Husain SS, Hasan A AK, Budzynski AZ. Differences between binding of one-chain and two-chain tissue plasminogen activators to non-cross-linked and cross-linked fibrin clots. Blood 1989; 74: 999-1006
13 Suenson E, Luetzen O, Thorsen S. Initial plasmin-degradation of fibrin as the basis of a positive feed-back mechanism in fibrinolysis. Eur J Biochem 1984; 140: 513-522
14 Tate KM, Higgins DL, Holmes WE, Winkler ME, Heyneker HL, Vehar GA. Functional role of proteolytic cleavage at Arginine-275 of human tissue plasminogen activator as assessed by site-directed mutagenesis. Biochemistry 1987; 26: 338-343
15 Busby SJ, Rao D, Heipel M, Markson M, Loubin P, Mulvihill E. Expression of human plasminogen in baby hamster kidney cells. Fibrinolysis 1988; 2 (Suppl. 01) 64 (abstract 147)
16 Verheijen JH, Mullaart E, Chang G TG, Kluft C, Wijngaards F. A simple, sensitive spectrophotometric assay for extrinsic (tissue-type) plasminogen activator applicable to measurements in plasma. Thromb Haemostas 1982; 48: 266-269
17 Zamarron C, Lijnen HR, Collen D. Kinetics of the activation of plasminogen by natural and recombinant tissue-type plasminogen activator. J Biol Chem 1984; 259: 2080-2083
18 Lijnen HR, Uytterhoeven M, Collen D. Inhibition of trypsin-like serine proteinases by tripeptide arginyl and lysyl chloromethyl-ketones. Thromb Res 1984; 34: 431-437
19 Collen D, Lijnen HR, De Cock F, Durieux JP, Loffet A. Kinetic properties of tripeptide lysyl chloromethyl ketone and lysyl p-nitroanilide derivatives towards trypsin-like serine proteinases. Biochim Biophys Acta 1980; 165: 158-166
20 Shapiro RA, Scherer NM, Habecker BA, Subers EM, Nathanson NM. Isolation, sequence and functional expression of the mouse Ml muscarinic acetylcholine receptor gene. J Biol Chem 1988; 263: 18397-18403
21 Nelles L, Lijnen HR, Collen D, Holmes WE. Characterization of recombinant human single chain urokinase-type plasminogen activator mutants produced by site-specific mutagenesis of lysine 158. J Biol Chem 1987; 262: 5682-5689
22 Deutsch DG, Mertz ET. Plasminogen: purification from human plasma by affinity chromatography. Science 1970; 170: 1095-1096
23 Holmes WE, Lijnen HR, Collen D. Characterization of recombinant human α₂-antiplasmin and of mutants obtained by site-directed mutagenesis of the reactive site. Biochemistry 1987; 26: 5133-5140
24 Fraker PJ, Speck Jr JC. Protein and cell membrane iodinations with a sparingly soluble chloroamide 1,3,4,6-tetrachloro-3a,6a-diphenylgly-coluril. Biochem Biophys Res Commun 1978; 80: 849-857
25 Nelles L, Lijnen HR, Collen D, Holmes WE. Characterization of a fusion protein consisting of amino acids 1 to 263 of tissue-type plasminogen activator and amino acids 144 to 411 of urokinase-type plasminogen activator. J Biol Chem 1987; 262: 10855-10862
26 Holvoet P, Lijnen HR, Collen D. Characterization of functional domains in human tissue-type plasminogen activator with the use of monoclonal antibodies. Eur J Biochem 1986; 158: 173-177
27 Wallen 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
28 Holvoet P, Cleemput H, Collen D. Assay of human tissue-type plasminogen activator (t-PA) with an enzyme-linked immunosorbent assay (ELISA) based on three murine monoclonal antibodies to t-PA. Thromb Haemostas 1985; 54: 684-687
29 Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 1976; 72: 248-254
30 Blomback B, Blomback M. Purification of human and bovine fibrinogen. Arkiv Kemi 1956; 10: 415-443
31 Astrup T, Muellertz S. Fibrin plate method for estimating fibrinolytic activity. Arch Biochem Biophys 1952; 40: 346-351
32 Gaffney PJ, Curtis AD. A collaborative study to establish the 2nd international standard for tissue plasminogen activator (t-PA). Thromb Haemostas 1987; 58: 1085-1087
33 Lijnen HR, Van Hoef B, De Cock F, Collen D. The mechanism of plasminogen activation and fibrin dissolution by single chain urokinase-type plasminogen activator in a plasma milieu in vitro. Blood 1989; 73: 1864-1872
34 Ranby M, Bergsdorf N, Norrman B, Suenson E, Wallen P. Tissue plasminogen activator kinetics. In: Progress in Fibrinolysis, Vol 6 Davidson JF, Bachman F, Wallen P, Bouvier CA, Kruithof EK O. (eds). Churchill Livingstone, Edinburgh: 1983: 182-184
35 Johannessen M, Nielsen F, Petersen LC. Plasmin-catalysed cleavage of single chain tissue-type plasminogen activator in fibrin clots. Fibrinolysis 1989; 3: 215-220
36 Ranby M. Studies on the kinetics of plasminogen activation by tissue plasminogen activator. Biochim Biophys Acta 1982; 704: 461-469
37 Nieuwenhuizen W, Voskuilen M, Vermond A, Hoegee-de Nobel B, Traas DW. The influence of fibrin(ogen) fragments on the kinetic parameters of the tissue-type plasminogen-activator-mediated activation of different forms of plasminogen. Eur J Biochem 1988; 174: 163-169
38 Boose JA, Kuismanen E, Gerard R, Sambrook J, Gething MJ. The single-chain form of tissue-type plasminogen activator has catalytic activity: studies with a mutant enzyme that lacks the cleavage site. Biochemistry 1989; 28: 635-643
39 Andreasen PA, Nielsen LS, Grondahl-Hansen J, Skriver L, Zeuthen J, Stephens RW, Dano K. Inactive proenzyme to tissue-type plasminogen activator from human melanoma cells, identified after affinity purification with a monoclonal antibody. EMBO J 1984; 3: 51-56
40 Petersen LC, Boel E, Johannessen M, Foster D. Possible involvement of a lysine residue in establishing the charge-relay system responsible for one-chain t-PA activity. Thromb Haemostas 1989; 62: 322 (Abstr)

Effect of Fibrin-Like Stimulators on the Activation of Plasminogen by Tissue-Type Plasminogen Activator (t-PA) - Studies with Active Site Mutagenized Plasminogen and Plasmin Resistant t-PA[*]

Effect of Fibrin-Like Stimulators on the Activation of Plasminogen by Tissue-Type Plasminogen Activator (t-PA) - Studies with Active Site Mutagenized Plasminogen and Plasmin Resistant t-PA^[*]