P-selectin-targeting of the fibrin selective thrombolytic Desmodus rotundus salivary plasminogen activator alpha1.
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.
Dong
Ningzheng
N
Department of Cardiovascular Research, Berlex Biosciences, Richmond, California, USA.
Da Cunha
Valdeci
V
Citkowicz
Andrej
A
Wu
Faye
F
Vincelette
Jon
J
Larsen
Brent
B
Wang
Yi-Xin
YX
Ruan
Changgeng
C
Dole
William P
WP
Morser
John
J
Wu
Qingyu
Q
Pan
Junliang
J
D000818:Animals; D000911:Antibodies, Monoclonal / Q000235:genetics / Q000627:therapeutic use; D001792:Blood Platelets / Q000737:chemistry / Q000187:drug effects* / Q000502:physiology; D004195:Disease Models, Animal; D004285:Dogs; D005337:Fibrin / Q000187:drug effects*; D005343:Fibrinolytic Agents / Q000008:administration & dosage / Q000493:pharmacokinetics / Q000494:pharmacology; D005818:Genetic Engineering; D006801:Humans; D018796:Immunoconjugates / Q000493:pharmacokinetics / Q000494:pharmacology / Q000627:therapeutic use*; D019007:P-Selectin / Q000276:immunology*; D010960:Plasminogen Activators / Q000008:administration & dosage* / Q000235:genetics / Q000627:therapeutic use; D010974:Platelet Aggregation; D011655:Pulmonary Embolism / Q000188:drug therapy; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000493:pharmacokinetics / Q000494:pharmacology / Q000627:therapeutic use; D013917:Thrombin; D015912:Thrombolytic Therapy / Q000379:methods*; D013927:Thrombosis / Q000188:drug therapy; D016896:Treatment Outcome
D000911:Antibodies, Monoclonal; D005343:Fibrinolytic Agents; D018796:Immunoconjugates; D019007:P-Selectin; D011993:Recombinant Fusion Proteins; C069588:salivary plasminogen activator alpha 1, Desmodus rotundus; D005337:Fibrin; D010960:Plasminogen Activators; D013917:Thrombin