P-selectin-targeting of the fibrin selective thrombolytic Desmodus rotundus salivary plasminogen activator α1

Ningzheng Dong; Valdeci Da Cunha; Andrej Citkowicz; Faye Wu; Jon Vincelette; Brent Larsen; Yi-Xin Wang; Changgeng Ruan; William P. Dole; John Morser; Qingyu Wu; Junliang Pan

doi:10.1160/TH04-03-0177

Thrombosis and Haemostasis, Table of Contents

Thromb Haemost 2004; 92(05): 956-965
DOI: 10.1160/TH04-03-0177

Blood Coagulation, Fibrinolysis and Cellular Haemostasis

Schattauer GmbH

P-selectin-targeting of the fibrin selective thrombolytic Desmodus rotundus salivary plasminogen activator α1

Ningzheng Dong

¹Departments of Cardiovascular Research, Berlex Biosciences, Richmond, California, USA

⁴Jiangsu Institute of Hematology, First Affiliated Hospital of Suzhou University, Suzhou, Jiangsu, China

,

Valdeci Da Cunha

²Pharmacology, Berlex Biosciences, Richmond, California, USA

,

Andrej Citkowicz

³Systems Biology, Berlex Biosciences, Richmond, California, USA

,

Faye Wu

¹Departments of Cardiovascular Research, Berlex Biosciences, Richmond, California, USA

,

Jon Vincelette

²Pharmacology, Berlex Biosciences, Richmond, California, USA

,

Brent Larsen

³Systems Biology, Berlex Biosciences, Richmond, California, USA

,

Yi-Xin Wang

²Pharmacology, Berlex Biosciences, Richmond, California, USA

,

Changgeng Ruan

⁴Jiangsu Institute of Hematology, First Affiliated Hospital of Suzhou University, Suzhou, Jiangsu, China

,

William P. Dole

¹Departments of Cardiovascular Research, Berlex Biosciences, Richmond, California, USA

,

John Morser

¹Departments of Cardiovascular Research, Berlex Biosciences, Richmond, California, USA

,

Qingyu Wu

¹Departments of Cardiovascular Research, Berlex Biosciences, Richmond, California, USA

,

Junliang Pan

¹Departments of Cardiovascular Research, Berlex Biosciences, Richmond, California, USA

› Author Affiliations

Abstract

Summary

During thrombosis, P-selectin is expressed on the surface of activated endothelial cells and platelets. We hypothesized that targeting a plasminogen activator (PA) to P-selectin would enhance local thrombolysis and reduce bleeding risk. Previously, a urokinase (uPA)/anti-P-selectin antibody (HuSZ51) fusion protein was shown to increase fibrinolysis in a hamster pulmonary embolism model. To explore the therapeutic potential of this targeting strategy, we fused the fibrin-selective Desmodus rotundus salivary PA α1 (dsPAα1) to HuSZ51 and compared the fibrinolytic activity of P-selectin-targeted dsPAα1 (HuSZ51-dsPAα1) to unmodified dsPAα1 in vitro and in vivo. HuSZ51-dsPAα1 and dsPAα1 were expressed in CHO cells and purified to homogeneity by affinity chromatography. HuSZ51dsPAα1 bound to thrombin-activated human and dog platelets with comparable affinities to that of parental antibody SZ51. The fusion protein retained the catalytic activities of dsPAα1 in chromogenic and clot lysis assays, indicating that dsPAα1 is fully functional when fused to HuSZ51. Compared to dsPAα1, HuSZ51-dsPAα1 had similar thrombolytic efficacy in a rat pulmonary embolism model and anti-thrombotic potency in a dog model of femoral artery thrombosis. However, HuSZ51dsPAα1 was less effective in lysis of preexisting arterial thrombi in the dog model. The reduced arterial thrombolysis was not due to the pharmacokinetic properties of HuSZ51-dsPAα1 because antigen level and amidolytic activity were higher in plasma from HuSZ51-dsPAα1-treated groups than corresponding dsPAα1-treated groups. These data indicate that the thrombolytic efficacy of HuSZ51-dsPAα1 varied dependent on the physical composition of thrombi. The lack of stimulation by fibrin in arterial thrombi may contribute to the attenuated thrombolytic efficacy of HuSZ51-dsPAα1 in the dog model.

Keywords

P-selectin - dsPAα1 - fusion protein - clot targeting - thrombolysis

Full Text

References

References
1 Thiagarajan P. New targets for antithrombotic drugs. Am J Cardiovasc Drugs 2002; 02: 227-35.
2 Collen D, Lijnen HR. Molecular basis of fibrinolysis, as relevant for thrombolytic therapy. Thromb Haemost 1995; 74: 167-71.
3 Topol EJ. Acute myocardial infarction: thrombolysis. Heart 2000; 83: 122-6.
4 Baker Jr. WF. Thrombolytic therapy. Clin Appl Thromb Hemost 2002; 08: 291-314.
5 Gardell SJ. The search for the ideal thrombolytic agent: maximize the benefit and minimize the risk. Toxicol Pathol 1993; 21: 190-8.
6 Van de Werf FJ. The ideal fibrinolytic: can drug design improve clinical results?. Eur Heart J 1999; 20: 1452-8.
7 McEver RP. P-selectin and PSGL-1: exploiting connections between inflammation and venous thrombosis. Thromb Haemost 2002; 87: 364-5.
8 Haber E, Bode C, Matsueda GR. et al. Antibody targeting as a thrombolytic strategy. Ann N Y Acad Sci 1992; 667: 365-81.
9 Wu J, He G, Wu G. et al. Radioimmunoimaging of experimental arterial and venous thrombi in dogs with 99Tcm-labelled monoclonal anti-activated platelet antibody SZ-51. Nucl Med Commun 1993; 14: 1088-92.
10 Wan H, Liu Z, Xia X. et al. A recombinant antibody-targeted plasminogen activator with high affinity for activated platelets increases thrombolytic potency in vitro and in vivo. Thromb Res 2000; 97: 133-41.
11 Kratzschmar J, Haendler B, Langer G. et al. The plasminogen activator family from the salivary gland of the vampire bat Desmodus rotundus: cloning and expression. Gene 1991; 105: 229-37.
12 Renatus M, Stubbs MT, Huber R. et al. Catalytic domain structure of vampire bat plasminogen activator: a molecular paradigm for proteolysis without activation cleavage. Biochemistry 1997; 36: 13483-93.
13 Gardell SJ, Hare TR, Bergum PW. et al. Vampire bat salivary plasminogen activator is quiescent in human plasma in the absence of fibrin unlike human tissue plasminogen activator. Blood 1990; 76: 2560-4.
14 Bergum PW, Gardell SJ. Vampire bat salivary plasminogen activator exhibits a strict and fastidious requirement for polymeric fibrin as its cofactor, unlike human tissue-type plasminogen activator. A kinetic analysis. J Biol Chem 1992; 267: 17726-31.
15 Bringmann P, Gruber D, Liese A. et al. Structural features mediating fibrin selectivity of vampire bat plasminogen activators. J Biol Chem 1995; 270: 25596-603.
16 Toschi L, Bringmann P, Petri T. et al. Fibrin selectivity of the isolated protease domains of tissue-type and vampire bat salivary gland plasminogen activators. Eur J Biochem 1998; 252: 108-12.
17 Witt W, Baldus B, Bringmann P. et al. Thrombolytic properties of Desmodus rotundus (vampire bat) salivary plasminogen activator in experimental pulmonary embolism in rats. Blood 1992; 79: 1213-7.
18 Hare TR, Gardell SJ. Vampire bat salivary plasminogen activator promotes robust lysis of plasma clots in a plasma milieu without causing fluid phase plasminogen activation. Thromb Haemost 1992; 68: 165-9.
19 Mellott MJ, Stabilito II, Holahan MA. et al. Vampire bat salivary plasminogen activator promotes rapid and sustained reperfusion without concomitant systemic plasminogen activation in a canine model of arterial thrombosis. Arterioscler Thromb 1992; 12: 212-21.
20 Witt W, Maass B, Baldus B. et al. Coronary thrombolysis with Desmodus salivary plasminogen activator in dogs. Fast and persistent recanalization by intravenous bolus administration. Circulation 1994; 90: 421-6.
21 Fujise K, Revelle BM, Stacy L. et al. A tissue plasminogen activator/P-selectin fusion protein is an effective thrombolytic agent. Circulation 1997; 95: 715-22.
22 Petri T, Langer G, Bringmann P. et al. Production of vampire bat plasminogen activator DSPA alpha 1 in CHO and insect cells. J Biotechnol 1995; 39: 75-83.
23 Norderhaug L, Olafsen T, Michaelsen TE. et al. Versatile vectors for transient and stable expression of recombinant antibody molecules in mammalian cells. J Immunol Methods 1997; 204: 77-87.
24 Ho SN, Hunt HD, Horton RM. et al. Sitedirected mutagenesis by overlap extension using the polymerase chain reaction. Gene 1989; 77: 51-9.
25 Devereux J, Haeberli P, Smithies O. A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res 1984; 12: 387-95.
26 Mustard JF, Kinlough-Rathbone RL, Packham MA. Isolation of human platelets from plasma by centrifugation and washing. Methods Enzymol 1989; 169: 3-11.
27 McCaman MT, Souders C, Ottoboni S. et al. Affinity binding of a vampire bat plasminogen activator to SEC resins. Protein Expr Purif 1998; 12: 111-21.
28 Wu G, Xi X, Li P. et al. Preparation of a monoclonal antibody, SZ-51, that recognizes an alpha-granule membrane protein (GMP-140) on the surface of activated human platelets. Nouv Rev Fr Hematol 1990; 32: 231-5.
29 Graff CP, Wittrup KD. Theoretical analysis of antibody targeting of tumor spheroids: importance of dosage for penetration, and affinity for retention. Cancer Res 2003; 63: 1288-96.
30 Runge MS, Quertermous T, Zavodny PJ. et al. A recombinant chimeric plasminogen activator with high affinity for fibrin has increased thrombolytic potency in vitro and in vivo. Proc Natl Acad Sci U S A 1991; 88: 10337-41.
31 Runge MS, Harker LA, Bode C. et al. Enhanced thrombolytic and antithrombotic potency of a fibrin-targeted plasminogen activator in baboons. Circulation 1996; 94: 1412-22.
32 Bode C, Hanson SR, Schmedtje Jr JF. et al. Antithrombotic potency of hirudin is increased in nonhuman primates by fibrin targeting. Circulation 1997; 95: 800-4.
33 Holvoet P, Dewerchin M, Stassen JM. et al. Thrombolytic profiles of clot-targeted plasminogen activators. Parameters determining potency and initial and maximal rates. Circulation 1993; 87: 1007-16.