Clot penetration and fibrin binding of amediplase, a chimeric plasminogen activator (K2tu-PA)

Dingeman C. Rijken; Marrie M. Barrett-Bergshoeff; A. F. H. Jie; Marco Criscuoli; Dmitri V. Sakharov

doi:10.1160/TH03-07-0435

Thrombosis and Haemostasis, Table of Contents

Thromb Haemost 2004; 91(01): 52-60
DOI: 10.1160/TH03-07-0435

Blood Coagulation, Fibrinolysis and Cellular Haemostasis

Schattauer GmbH

Clot penetration and fibrin binding of amediplase, a chimeric plasminogen activator (K₂tu-PA)

Dingeman C. Rijken

¹Gaubius Laboratory, TNO Prevention and Health, Leiden, The Netherlands

,

Marrie M. Barrett-Bergshoeff

¹Gaubius Laboratory, TNO Prevention and Health, Leiden, The Netherlands

,

A. F. H. Jie

¹Gaubius Laboratory, TNO Prevention and Health, Leiden, The Netherlands

,

Marco Criscuoli

²Menarini Ricerche S.p.A., Firenze, Italy

,

Dmitri V. Sakharov

¹Gaubius Laboratory, TNO Prevention and Health, Leiden, The Netherlands

³CBLE, Utrecht University, Utrecht, The Netherlands

› Author Affiliations

Abstract

Summary

Amediplase (K₂tu-PA) is a hybrid plasminogen activator, consisting of the kringle 2 domain of alteplase and the protease domain of urokinase. The objective of this study was to determine the in vitro clot penetration of amediplase in relation to its fibrin binding and to compare the properties with those of alteplase.

The clot lysis activity of amediplase in internal clot lysis models (both purified system and plasma system) was about 10 times less than that of alteplase. The clot lysis activity of amediplase in an external clot lysis model (plasma system) was similar to that of alteplase at therapeutic concentrations around 1 µg/ml. The fibrin-clot binding properties of amediplase and alteplase were studied in a purified system as well as in a plasma system. In both systems amediplase bound to fibrin although to a significantly lower extent than alteplase. The binding of amediplase or alteplase did not increase during plasmin-mediated degradation of fibrin. The binding of amediplase was fully inhibited by epsilon-aminocaproic acid, indicating that the observed binding was specific and occurred via the lysine binding site in the kringle of amediplase. Clot penetration was studied during pressure-driven fluid permeation using syringes containing plasma clots. Amediplase was able to enter the clot without significant hindrance, while alteplase was concentrated on the top of the plasma clot and hardly entered into the inner parts of the clot. Diffusion-driven clot penetration was studied during clot lysis using confocal microscopy. Alteplase was detected on or close to the clot surface, while two-chain urokinase, which has no affinity to fibrin, was also detected deep inside the clot. Amediplase showed a penetration behaviour, which was distinct from that of alteplase and similar to that of two-chain urokinase.

We concluded that the fibrin binding of amediplase is moderate and does not hinder clot penetration under permeationdriven or diffusion-driven transport conditions. Enhanced clot penetration, especially in large clots, could allow a more efficient lysis during thrombolytic therapy.

Keywords

Amediplase - alteplase - fibrin binding - clot penetration - thrombolysis

Full Text

References

References
1 Asselbergs FAM, Burgi R, Chaudhuri B. et al. Localisation of epitopes recognized by monoclonal antibodies on tissue-type and urokinase-type plasminogen activators using recombinant hybrid enzymes. Fibrinolysis 1993; 07: 1-14.
2 Rijken DC, Collen D. Purification and characterization of the plasminogen activator secreted by human melanoma cells in culture. J Biol Chem 1981; 256: 7035-41.
3 Verheijen JH, Caspers MP, Chang GT. et al. Involvement of finger domain and kringle 2 domain of tissue-type plasminogen activator in fibrin binding and stimulation of activity by fibrin. EMBO J 1986; 05: 3525-30.
4 Van Zonneveld AJ, Veerman H, Pannekoek H. On the interaction of the finger and kringle-2 domain of tissue-type plasminogen activator with fibrin. Inhibition of kringle-2 binding to fibrin by epsilon-amino caproic acid. J Biol Chem 1986; 261: 14214-8.
5 Colucci M, Gavallo LG, Agnelli G. et al. Properties of chimeric (tissue-type/urokinasetype) plasminogen activators obtained by fusion at the plasmin cleavage site. Thromb Haemost 1993; 69: 466-72.
6 Lucchesi BR, Burgi R, Heim J. et al. The in vivo thrombolytic activity of K₂tu-PA, a hybrid plasminogen activator and fibrinolytic agent. Coron Artery Dis 1991; 02: 247-58.
7 Agnelli G, Pascucci C, Colucci M. et al. Thrombolytic activity of two chimeric recombinant plasminogen activators (FK₂tu-PA and K₂tu-PA) in rabbits. Thromb Haemost 1992; 68: 331-5.
8 Agnelli G, Pascucci C, Nenci GG. et al. Thrombolytic and haemorrhagic effects of bolus doses of tissue-type plasminogen activator and a hybrid plasminogen activator with prolonged plasma half-life (K₂tu-PA: CGP 42935). Thromb Haemost 1993; 70: 294-300.
9 Vermeer F, Pohl J, Oldroyd K. et al. Safety and angiography data of amediplase, a new fibrin thrombolytic agant, given as a bolus to patients with acute myocardial infarction: the 2K2 dose finding trial. J Am Coll Cardiol 2001; 37 (Suppl A): 322.
10 Charbonnier B, Pluta W, De Ferrari G. et al. Evaluation of two weight adjusted single bolus doses of amediplase to patients with acute myocardial infarction. Circulation 2001; 104 (Suppl II): 538 (abstract 2546).
11 No authors listed. Amediplase: CGP 42935, K₂tu-PA, MEN 9036. BioDrugs 2002; 16: 378-9.
12 Rijken DC, Sakharov DV. Molecular transport during fibrin clot lysis. Fibrinolysis & Proteolysis 2000; 14: 98-113.
13 Diamond SL, Anand S. Inner clot diffusion and permeation during fibrinolysis. Biophys J 1993; 65: 2622-43.
14 Blinc A, Francis CW. Transport processes in fibrinolysis and fibrinolytic therapy. Thromb Haemost 1996; 76: 481-91.
15 Sakharov DV, Nagelkerke JF, Rijken DC. Rearrangements of the fibrin network and spatial distribution of fibrinolytic components during plasma clot lysis. J Biol Chem 1996; 271: 2133-8.
16 Fisher S, Kohnert U. Major mechanistic differences explain the higher clot lysis potency of reteplase over alteplase: lack of fibrin binding is an advantage for bolus application of fibrin-specific thrombolytics. Fibrinolysis & Proteolysis 1997; 11: 129-35.
17 Medved L, Nieuwenhuizen W. Molecular mechanisms of initiation of fibrinolysis by fibrin. Thromb Haemost 2003; 89: 409-19.
18 Suenson E, Lutzen 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.
19 Higgins DL, Vehar GA. Interaction of onechain and two-chain tissue plasminogen activator with intact and plasmin-degraded fibrin. Biochemistry 1987; 26: 7786-91.
20 De Vries C, Veerman H, Koornneef E. et al. Tissue-type plasminogen activator and its substrate Glu-plasminogen share common binding sites in limited plasmin-digested fibrin. J Biol Chem 1990; 265: 13547-52.
21 De Munk GA, Caspers MP, Chang GT. et al. Binding of tissue-type plasminogen activator to lysine, lysine analogues, and fibrin fragments. Biochemistry 1989; 28: 7318-25.
22 Novokhatny V, Medved L, Lijnen HR. et al. Tissue-type plasminogen activator (tPA) interacts with urokinase-type plasminogen activator (uPA) via tPA’s lysine binding site. An explanation of the poor fibrin affinity of recombinant tPA/uPA chimeric molecules. J Biol Chem 1995; 270: 8680-5.