The Role of the Finger Domain of Tissue-type Plasminogen Activator (t-PA) and Plasminogen Activator Inhibitor 1 (PAI-1) in Initiation and Progression of Thrombolysis

Harold A R Stringer; Peter van Swieten; Anton J G Horrevoets; Annelies Smilde; Hans Pannekoek

doi:10.1055/s-0038-1648981

Thrombosis and Haemostasis, Table of Contents

Thromb Haemost 1994; 72(06): 900-905
DOI: 10.1055/s-0038-1648981

Original Article

Schattauer GmbH Stuttgart

The Role of the Finger Domain of Tissue-type Plasminogen Activator (t-PA) and Plasminogen Activator Inhibitor 1 (PAI-1) in Initiation and Progression of Thrombolysis

Harold A R Stringer

The Department of Biochemistry, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands

,

Peter van Swieten

The Department of Biochemistry, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands

,

Anton J G Horrevoets

The Department of Biochemistry, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands

,

Annelies Smilde

The Department of Biochemistry, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands

,

Hans Pannekoek

The Department of Biochemistry, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands

› Author Affiliations

Abstract

PDF Download

Summary

We further investigated the role of the finger (F) and the kringle-2 (K2) domains of tissue-type plasminogen activator (t-PA) in fibrin-stimulated plasminogen activation. To that end, the action of purified (wt) t-PA or of variants lacking F (del.F) or K2 (del.K2) was assessed either in a static, human whole blood clot-lysis system or in whole blood thrombi generated in the “Chandler loop”. In both clot-lysis systems, significant differences were observed for the initiation of thrombolysis with equimolar concentrations of the t-PA variants. A relatively minor “lag phase” occurred in thrombolysis mediated by wt t-PA, whereas a 6.4-fold and 1.6-fold extension is found for del.F and del.K2, respectively. We observed identical lag-times, characteristic for each t-PA variant, in platelet-rich heads and in platelet-poor tails of thrombi. Since plasminogen activator inhibitor 1 (PAI-1) is preferentially retained in the platelet-rich heads, we conclude that the inhibitor does not interfere with the initial stage of thrombolysis but exerts its action in later stages, resulting in a reduction of the rate of clot lysis. A complementation clot-lysis assay was devised to study a potential interplay of del.F and del.K2. Accordingly, clot lysis was determined with combinations of del.F and del.K2 that were inversely varied in relation to equipotent dosage to distinguish between additive, antagonistic or synergistic effects of these variants. The isobole for combinations of del.F and del.K2 shows an independent, additive action of del.F and del.K2 in clot lysis. Under the conditions employed, namely a relatively high concentration of fibrin and Glu-plasminogen and a low concentration of t-PA variant, our data show: i) the crucial role of the F domain and the lack of effect of PAI-1 in initiation of thrombolysis, ii) the lack of importance of the fibrimbinding domains of t-PA and the regulatory role of PAI-1 in advanced stages of thrombolysis.

PDF (505 kb)

References

References
1 Collen D. On the regulation and control of fibrinolysis. Thromb Haemost 1980; 43: 77-89
2 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. coli. Nature 1983; 301: 214-221
3 Ny T, Elgh F, Lund B. The structure of the human tissue-type plasminogen activator gene: correlation of intron and exon structures to functional and structural domains. Proc Natl Acad Sci USA. 1984; 81: 5355-5359
4 Van Zonneveld A-J, Veerman H, Pannekoek H. Autonomous functions of structural domains on human tissue-type plasminogen activator. Proc Natl Acad Sci USA 1986; 83: 4670-4674
5 Van Zonneveld A-J, Veerman H, Pannekoek H. On the interaction of the finger and the kringle-2 domain of tissue-type plasminogen activator with fibrin. J Biol Chem 1986; 261: 14214-14218
6 Pannekoek H, de Vries C, van Zonneveld A-J. Mutants of human tissue-type plasminogen activator (t-PA): structural aspects and functional properties. Fibrinolysis 1988; 2: 123-132
7 Horrevoets AJG, Smilde A, de Vries C, Pannekoek H. The specific role of . finger and kringle-2 domains of tissue-type plasminogen activator during invitro fibrinolysis. J Biol Chem 1994; 269: 12639-12644
8 Higgins DL, Vehar GA. Interaction of one-chain and two-chain t-PA with intact and plasmin-degraded fibrin. Biochemistry 1987; 26: 7786-7791
9 Wilhelm OG, Jaskunas SR, Vlahos CJ, Bang NU. Functional properties of the recombinant kringle-2 domain of tissue-type plasminogen activator produced in Escherichia coli. J Biol Chem 1990; 265: 14606-146011
10 De Serrano VS, Castellino FJ. Structural determinants of the noncatalytic chain of tissue-type plasminogen activator that modulate its association rate with plasminogen activator inhibitor-1. J Biol Chem 1990; 265: 10473-10478
11 Kaneko M, Mimuro J, Matsuda M, Sakata Y. The plasminogen activator inhibitor-1 binding site in the kringle-2 domain of tissue-type plasminogen activator. Biochem Biophys Res Commun 1991; 178: 1160-1166
12 Wagner OF, de Vries C, Hohmann C, Veerman H, Pannekoek H. Interaction between plasminogen activator inhibitor 1 (PAI-1) bound to fibrin and either tissue-type plasminogen activator (t-PA) or urokinase-type plasminogen activator (u-PA). Binding of t-PA/PAI-1 to fibrin is mediated by both the finger and the kringle-2 domain of t-PA. J Clin Invest 1989; 84: 647-657
13 Reilly FR, Hutzelmann JE. Plasminogen activator inhibitor-1 binds to fibrin and inhibits tissue-type plasminogen activator-mediated fibrin dissolution. J Biol Chem 1992; 267: 17128-17135
14 Braaten JV, Handt S, Jerome WG, Kirkpatrick J, Lewis JC, Hantgan RR. Regulation of fibrinolysis by platelet-released plasminogen activator inhibitor 1: light scattering and ultrastructural examination of lysis of a model platelet-fibrin thrombus. Blood 1993; 81: 1290-1299
15 Chandler AB. In vitro thrombotic coagulationof the blood. Lab Invest 1958; 7: 110-114
16 Stringer HAR, van Swieten P, Heijnen HFG, Sixma JJ, Pannekoek H. Plasminogen activator inhibitor1 (PAI-1) released from activated platelets has a key role in thrombolysis resistance: studies with thrombi generated in the Chandler loop. Arterioscler Thromb 1994; 14: 1452-1458
17 Levi M, Biemond BJ, van Zonneveld A-J, ten Cate JW, Pannekoek H. Inhibition of plasminogen activator inhibitor 1 (PAI-1) activity results in promotion of spontaneous thrombolysis and inhibition of thrombus growth in models of experimental thrombosis. Circulation 1992; 85: 305-312
18 Biemond BJ, Levi M, Coronel R, Janse MJ, ten Cate JW, Pannekoek H. Thrombolysis and reocclusion inexperimental jugular vein and coronary artery thrombosis: effects of aplasminogen activator inhibitor 1 neutralizing antibody. Circulation 1995 in press
19 De Vries C, Veerman H, Koomeef E, Pannekoek H. Tissue-type plasminogen activator (t-PA) and its substrate Glu-plasminogen share common binding sitesin limited plasmin-digested fibrin. J Biol Chem 1990; 265: 13547-13452
20 Greenwood FC, Hunter WM, Glover JS. The preparation of 131I-labelled human growth hormone of high specific radioactivity. Biochem J 1963; 89: 114-118
21 De Vries C, Veerman H, Pannekoek H. Identification of the domains of tissue-type plasminogen activator(t-PA) involved in the augmented binding to fibrin limitedly digested with plasmin. J Biol Chem 1989; 264: 12604-12610
22 Heussen C, Joubert F, Dowdle EB. Purification of human tissue plasminogen activator with Erythrina trypsin inhibitor. J Biol Chem 1984; 259: 11635-11638
23 Ehrlich HJ, Klein-Gebbink R, Keijer J, Linders M, Preissner KT, Pannekoek H. Alteration of serpin specificity: vitronectin endows plasminogen activator inhibitor type 1 with thrombin inhibitory properties. J Biol Chem 1990; 265: 13029-13035
24 Norrman B, Wallen P, Ranby M. Fibrinolysis mediated by tissue plasminogen activator: disclosure of a kinetic transition. Eur J Biochem 1985; 149: 193-200
25 Berenbaum MC. Synergy, additivism and antagonism in immunosuppression. A critical review. Clin Exp Immunol 1977; 28: 01-18
26 Erickson LA, Fici GJ, Lundt JE, Boyle TP, Polites HG, Marotti KR. Development of venous occlusions in mice transgenic for the plasminogen activator inhibitor-1 gene. Nature 1990; 346: 74-76
27 Carmeliet P, Stassen JM, Schoonjans L, Ream B, van den Oord JJ, de Mol M, Mulligan RC, Collen D. Plasminogen activator inhibitor-1 gene-deficient mice. II. Effects on Hemostasis, Thrombosis, and Thrombolysis. J Clin Invest 1993; 92: 2756-2760
28 Lijnen HR, Van Hoef B, Collen D. On the reversible interaction of plasminogen activator inhibitor-1 with tissue-type plasminogen activator and with urokinase-type plasminogen activator. J Biol Chem 1991; 266: 4041-4044
29 Madison EL, Goldsmith EJ, Gerard RD, Gething MJ, Sambrook JF, Bassel-Duby RS. Amino acid residues that affect interaction of tissue-type plasminogen activator with plasminogen activator inhibitor 1. Proc Natl Acad Sci USA 1990; 87: 3530-3533
30 Madison EL, Goldsmith EJ, Gerard RD, Gething MJ, Sambrook JF. Serpin-resistant mutants of human tissue-type plasminogen activator. Nature 1989; 339: 721-724
31 Wiman B, Collen D. On the kinetics of the reaction between human anti-plasmin and plasmin. Eur J Biochem 1987; 84: 573-581
32 Verheijen JH, Caspers MPM, Chang GTG, de Munk GAW, Pouwels PH, Enger-Valk BE. Involvement of finger domain and kringle 2 domain of tissue-type plasminogen activator in fibrin binding and stimulation of actvity by fibrin. EMBO J 1986; 5: 3525-3530
33 Hoylaerts M, Rijken DC, Lijnen HR, Collen D. Kinetics of the activation of plasminogen by human t-PA. J Biol Chem 1982; 257: 2912-2919