Download PDF
Thromb Haemost 2008; 100(03): 413-419
DOI: 10.1160/TH08-04-0225
Blood Coagulation, Fibrinolysis and Cellular Haemostasis
Schattauer GmbH

Simplified recombinant plasmin: Production and functional comparison of a novel thrombolytic molecule with plasma-derived plasmin

Jennifer A. Hunt
1   Talecris Biotherapeutics, Research Triangle Park, North Carolina, USA
,
Stephen R. Petteway Jr
1   Talecris Biotherapeutics, Research Triangle Park, North Carolina, USA
,
Philip Scuderi
1   Talecris Biotherapeutics, Research Triangle Park, North Carolina, USA
,
Valery Novokhatny
1   Talecris Biotherapeutics, Research Triangle Park, North Carolina, USA
› Author AffiliationsFinancial support: This research was supported by Talecris Biotherapeutics.
Further Information

Publication History

Received 09 April 2008

Accepted after minor revision 07 July 2008

Publication Date:
22 November 2017 (online)

  PDF Download  Permissions and Reprints

Summary

A simplified and fully functional deletion mutant of plasminogen was created in which the middle portion of the molecule was removed, resulting in kringle 1 attachment to the serine protease domain. This recombinant plasminogen deletion mutant, Δ(K2-K5)Pg, was produced in the form of inclusion bodies at the yield of up to 200 mg/l in an Escherichia coliT7 expression system. Following protein refolding and purification on lysine-Sepharose, the conversion of the recombinant molecule Δ(K2-K5)Pg to the active enzyme mutant Δ(K2-K5)Pm by plasminogen activators was evaluated, and functional characteristics of the simplified plasmin were studied. Properties of Δ(K2-K5)Pg were similar to native, human plasma-derived plasminogen. Δ(K2-K5)Pg effectively bound ε-aminocaproic acid (Kd = 1 1.3 ± 2.3 μ. M) and fibrin (C50 ≈ 0.3 μM).The plasminogen activators streptokinase, urokinase, and tissue plasminogen activator effectively converted the recombinant zymogen Δ(K2-K5)Pg to the active recombinant enzyme, Δ(K2-K5)Pm. Additionally, Δ[K2-K5]Pm was rapidly inhibited by α2-antiplas-min(1.1±0.1x107M−1s−1)andα2-macroglobulin(7.6±0.6x105 M−1s−1). In an in-vitro model, Δ(K2-K5)Pm demonstrated fibrinolytic potency comparable to human plasma-derived plasmin. Because of their unique biochemistry, including fibrin-binding properties and rapid inhibition by α2-antiplasmin, both native plasmin and a simplified deletion mutant of plasmin are potentially safe and effective direct thrombolytic agents for various thrombotic conditions. Further studies evaluating the in-vivo pharmacologic safety and clinical efficacy of this simplified plasmin (i.e. Δ[K2-K5]Pm) are warranted.

Keywords

Fibrinolysis - peripheral arterial occlusion - plasmin - plasminogen - thrombosis
 

  • References

  • 1 Alesh I, Kayali F, Stein PD. Catheter-directed thrombolysis (intrathrombus injection) in treatment of deep venous thrombosis: a systematic review. Catheter Cardiovasc Interv 2007; 70: 143-148.
    PubMedGoogle Scholar
  • 2 Dormandy JA, Rutherford RB. Management of peripheral arterial disease (PAD). TA SC Wo rking Group. TransAtlantic Inter-Society Consensus (TASC). J Vasc Surg 2000; 31: S1-S296.
    CrossrefPubMedGoogle Scholar
  • 3 HirschA T, Haskal ZJ, Hertzer NR. et al. ACC/AHA 2005 guidelines for the management of patients with peripheral arterial disease (lower extremity, renal, mesenteric, and abdominal aortic): executive summary a collaborative report from the American Associations for Vascular Surgery/Society for Vascular Surgery, Society for Cardiovascular Angiography and Interventions, Society for Vascular Medicine and Biology, Society of Interventional Radiology, and the ACC/AHA Task Force on Practice Guidelines (Writing Committee to Develop Guidelines for the Management of Patients With Peripheral Arterial Disease) endorsed by the American Association of Cardiovascular and Pulmonary Rehabilitation; National Heart, Lung, and Blood Institute; Society for Vascular Nursing; TransAtlantic Inter-Society Consensus; and Vascular Disease Foundation. J Am Coll Cardiol 2006; 47: 1239-1312.
    CrossrefPubMedGoogle Scholar
  • 4 Semba CP, Bakal CW, Calis KA. et al, and the Advisory Panel on Catheter-Directed Thrombolytic Therapy Alteplase as an alternative to urokinase. J Vasc Interv Radiol 2000; 11: 279-287.
    CrossrefPubMedGoogle Scholar
  • 5 Shlansky-Goldberg R, Baumbach G, Marder V. A phase I trial of locally-delivered human plasmin to evaluate safety and thrombolytic efficacy in patients with hemodialysis graft occlusion (HGO. Presented at 21st Congress of the International Society on Thrombosis and Haemostasis. 2007. Geneva, Switzerland: Abstract P-S-385.
    Google Scholar
  • 6 Ponting CP, Marshall JM, Cederholm-Williams SA. Plasminogen: a structural review. Blood Coagul Fibrinolysis 1992; 03: 605-614.
    CrossrefPubMedGoogle Scholar
  • 7 Lerch PG, Rickli EE, Lergier W. et al. Localization of individual lysine-binding regions in human plasminogen and investigations on their complex-forming properties. Eur J Biochem 1980; 107: 7-13.
    CrossrefPubMedGoogle Scholar
  • 8 Markus G, DePasquale JL, Wissler FC. Quantitative determination of the binding of ε-aminocaproic acid to native plasminogen. J Biol Chem 1978; 253: 727-732.
    PubMedGoogle Scholar
  • 9 Derbise A, Song YP, Parikh S. et al. Role of the C-terminal lysine residues of streptococcal surface enolase in Glu- and Ly s-plasminogen-binding activities of group A streptococci. Infect Immun 2004; 72: 94-105.
    CrossrefPubMedGoogle Scholar
  • 10 Miles LA, Dahlberg CM, Plow EF. Thecell-binding domains of plasminogen and their function in plasma. J Biol Chem 1988; 263: 11928-11934.
    PubMedGoogle Scholar
  • 11 Pancholi V, Fischetti V A. α -Enolase, a novel strong plasmin(ogen) binding protein on the surface of pathogenic streptococci. J Biol Chem 1998; 273: 14503-14515.
    CrossrefPubMedGoogle Scholar
  • 12 Wistedt AC, Kotarsky H, Marti D. et al. Kringle 2 mediates high affinity binding of plasminogen to an internal sequence in streptococcal surface protein PAM. J Biol Chem 1998; 273: 24420-24424.
    CrossrefPubMedGoogle Scholar
  • 13 Lucas MA, Fretto LJ, McKee PA. The binding of human plasminogen to fibrin and fibrinogen. J Biol Chem 1983; 258: 4249-4256.
    PubMedGoogle Scholar
  • 14 Wu H-L, Chang B-I, Wu D-H. et al. Interaction of plasminogen and fibrin in plasminogen activation. J Biol Chem 1990; 265: 19658-19664.
    PubMedGoogle Scholar
  • 15 Wiman B, Lijnen HR, Collen D. On the specific interaction between the lysine-binding sites in plasmin and complementary sites in alpha2-antiplasmin and in fibrinogen. Biochim Biophys Acta 1979; 579: 142-154.
    CrossrefPubMedGoogle Scholar
  • 16 Wu T-P, Padmanabhan KP, Tulinsky A. The structure of recombinant plasminogen kringle 1 and the fibrin binding site. Blood Coagul Fibrinolysis 1994; 05: 157-166.
    CrossrefPubMedGoogle Scholar
  • 17 Busby SJ, Mulvihill E, Rao D. et al. Expression of recombinant human plasminogen in mammalian cells is augmented by suppression of plasmin activity. J Biol Chem 1991; 266: 15286-15292.
    PubMedGoogle Scholar
  • 18 Marder VJ, Landskroner K, Novokhatny V. et al. Plasmin induces local thrombolysis without causing hemorrhage:a comparisonwith tissue plasminogen activator in the rabbit. Thromb Haemost 2001; 86: 739-745.
    Article in Thieme ConnectPubMedGoogle Scholar
  • 19 Novokhatny V, Taylor K, Zimmerman TP. Thrombolytic potency of acid-stabilized plasmin: superiority over tissue-type plasminogen activator in an in vitro model of catheter-assisted thrombolysis. J Thromb Haemost 2003; 01: 1034-1041.
    CrossrefPubMedGoogle Scholar
  • 20 Wiman B. Affinity-chromatographic purification of human α2-antiplasmin. Biochem J 1980; 191: 229-232.
    CrossrefPubMedGoogle Scholar
  • 21 Hall PK, Roberts RC. Physical and chemical properties of human plasma α2-macroglobulin. Biochem J 1978; 171: 27-38.
    PubMedGoogle Scholar
  • 22 Matsuka YV, Novokhatny VV, Kudinov SA. Fluorescence spectroscopic analysis of ligand binding to kringle 1 + 2 + 3 and kringle 1 fragments from human plasminogen. Eur J Biochem 1990; 190: 93-97.
    CrossrefPubMedGoogle Scholar
  • 23 Douglas JT, von Haller PD, Gehrmann M, et al. The two-domain NK1 fragment of plasminogen: folding, ligand binding, and thermal stability profile. Biochemistry 2002; 41: 3302-3310.
    CrossrefPubMedGoogle Scholar
  • 24 Wohl RC, Summaria L, Robbins KC. Kinetics of activation of human plasminogen by different activator species at pH 7.4 and 37°C. J Biol Chem 1980; 255: 2005-2013.
    PubMedGoogle Scholar
  • 25 Christensen U, Sottrup-Jensen L, Magnusson S. et al. Enzymic properties of the neo-plasmin-Val-422 (miniplasmin). Biochim Biophys Acta 1979; 567: 472-481.
    CrossrefPubMedGoogle Scholar
  • 26 Wiman B, Collen D. On the kinetics of the reaction between human antiplasmin and plasmin. Eur J Biochem 1978; 84: 573-578.
    CrossrefPubMedGoogle Scholar
  • 27 Anonick PK, Wolf BB, Gonias SL. Regulation of plasmin, miniplasmin, and streptokinase-plasmin complex by α2-antiplasmin, α2-macroglobulin, and antithrombin III in the presence of heparin. Thromb Res 1990; 59: 449-462.
    CrossrefPubMedGoogle Scholar
  • 28 Castellino FJ, Davidson DJ, Rosen E. et al. Expression of human plasminogen cDNA in lepidopteran insect cells and analysis of asparagine-linked glycosylation patterns of recombinant plasminogens. Methods Enzymol 1993; 223: 168-185.
    PubMedGoogle Scholar
  • 29 Shi G-Y, Wu H-L. Isolation and characterization of microplasminogen. A low molecular weight form of plasminogen. J Biol Chem 1988; 263: 17071-17075.
    PubMedGoogle Scholar
  • 30 Wang J, Reich E. Structure and function of micro-plasminogen: II. Determinants of activation by urokinase and by the bacterial activator streptokinase. Protein Sci 1995; 04: 1768-1779.
    CrossrefPubMedGoogle Scholar
  • 31 Deitcher SR, Funk WD, Buchanan J. et al. Alfime-prase: a novel recombinant direct-acting fibrinolytic. Expert Opin Biol Ther 2006; 06: 1361-1369.
    CrossrefPubMedGoogle Scholar
  • 32 Moll S, Kenyon P, Bertoli L. et al. Phase II trial of alfimeprase, a novel-acting fibrin degradation agent, for occluded central venous access devices. J Clin Oncol 2006; 24: 3056-3060.
    CrossrefPubMedGoogle Scholar
  • 33 Ouriel K, Cynamon J, Weaver FA. et al. A phase I trial of alfimeprase for peripheral arterial thrombolysis. J Vasc Interv Radiol 2005; 16: 1075-1083.
    CrossrefPubMedGoogle Scholar
  • 34 ThromboGenics NV. ThromboGenics NV announces update on its microplasmin development strategy for peripheral vascular disease. Available at: http://www.thrombogenics.com/documents/06_TG_10PAOD_DVTENGFINAL.pdf Last accessed March 14, 2008.
    PubMedGoogle Scholar
  • 35 Gonias SL, Figler NL. Alpha 2-macroglobulin is the primary inhibitor of miniplasmin in vitro and in vivo in the mouse. Comparison with alpha 2-antiplasmin in simultaneous reaction experiments. Biochem J 1988; 255: 725-730.
    PubMedGoogle Scholar
  • 36 Shah AR, Scher L. Drug evaluation: alfimeprase, a plasminogen-independent thrombolytic. IDrugs 2007; 10: 329-335.
    PubMedGoogle Scholar