P-selectin-targeting of the fibrin selective thrombolytic Desmodus rotundus salivary plasminogen activator alpha1.

Abstract

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 alpha1 (dsPA alpha 1) to HuSZ51 and compared the fibrinolytic activity of P-selectin-targeted dsPA alpha 1 (HuSZ51-dsPA alpha 1) to unmodified dsPA alpha 1 in vitro and in vivo. HuSZ51-dsPA alpha 1 and dsPA alpha 1 were expressed in CHO cells and purified to homogeneity by affinity chromatography. HuSZ51-dsPA alpha 1 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 dsPA alpha 1 in chromogenic and clot lysis assays, indicating that dsPA alpha 1 is fully functional when fused to HuSZ51. Compared to dsPA alpha 1, HuSZ51-dsPA alpha 1 had similar thrombolytic efficacy in a rat pulmonary embolism model and anti-thrombotic potency in a dog model of femoral artery thrombosis. However, HuSZ51-dsPA alpha 1 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-dsPA alpha 1 because antigen level and amidolytic activity were higher in plasma from HuSZ51-dsPA alpha 1-treated groups than corresponding dsPA alpha 1-treated groups. These data indicate that the thrombolytic efficacy of HuSZ51-dsPA alpha 1 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-dsPA alpha 1 in the dog model.

• Authors: 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
• Journal: Thrombosis and haemostasis (Thromb Haemost) Vol. 92 Issue 5 Pg. 956-65 (Nov 2004) ISSN: 0340-6245 [Print] Germany
• PMID: 15543321 (Publication Type: Journal Article)

Chemical References

• Antibodies, Monoclonal
• Fibrinolytic Agents
• Immunoconjugates
• P-Selectin
• Recombinant Fusion Proteins
• salivary plasminogen activator alpha 1, Desmodus rotundus
• Fibrin
• Plasminogen Activators
• Thrombin

Topics

• Animals
• Antibodies, Monoclonal (genetics, therapeutic use)
• Blood Platelets (chemistry, drug effects, physiology)
• Disease Models, Animal
• Dogs
• Fibrin (drug effects)
• Fibrinolytic Agents (administration & dosage, pharmacokinetics, pharmacology)
• Genetic Engineering
• Humans
• Immunoconjugates (pharmacokinetics, pharmacology, therapeutic use)
• P-Selectin (immunology)
• Plasminogen Activators (administration & dosage, genetics, therapeutic use)
• Platelet Aggregation
• Pulmonary Embolism (drug therapy)
• Rats
• Recombinant Fusion Proteins (pharmacokinetics, pharmacology, therapeutic use)
• Thrombin
• Thrombolytic Therapy (methods)
• Thrombosis (drug therapy)
• Treatment Outcome