Identification, purification, and pharmacological activity analysis of Desmodus rotundus salivary plasminogen activator alpha1 (DSPAα1) expressed in transgenic rabbit mammary glands

SummaryDuring 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.

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Analysis of site-specific N-glycosylation of recombinant Desmodus rotundus salivary plasminogen activator rDSPAα1 expressed in Chinese hamster ovary cells

Glycobiology ◽

10.1093/glycob/7.1.67 ◽

1997 ◽

Vol 7 (1) ◽

pp. 67-77 ◽

Cited By ~ 14

Author(s):

Martin Gohlke ◽

Rolf Nuck ◽

Christoph Kannicht ◽

Detlef Grunow ◽

Günther Baude ◽

...

Keyword(s):

Plasminogen Activator ◽

Chinese Hamster Ovary ◽

Chinese Hamster Ovary Cells ◽

Chinese Hamster ◽

Desmodus Rotundus ◽

Site Specific ◽

Ovary Cells

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Thrombolytic properties of Desmodus rotundus (vampire bat) salivary plasminogen activator in experimental pulmonary embolism in rats

Blood ◽

10.1182/blood.v79.5.1213.bloodjournal7951213 ◽

1992 ◽

Vol 79 (5) ◽

pp. 1213-1217

Author(s):

W Witt ◽

B Baldus ◽

P Bringmann ◽

L Cashion ◽

P Donner ◽

...

Keyword(s):

Pulmonary Embolism ◽

Plasminogen Activator ◽

Tissue Type ◽

Desmodus Rotundus ◽

Type Plasminogen Activator ◽

Blood Clots ◽

Hemostatic Factors ◽

The Difference ◽

Vampire Bat

rDSPA alpha 1 (recombinant Desmodus salivary plasminogen activator alpha 1) is a recombinant protein corresponding to a natural plasminogen activator from the vampire bat Desmodus rotundus. The thrombolytic properties of rDSPA alpha 1 and tissue-type plasminogen activator (t-PA) were compared in a rat model of pulmonary embolism. Whole blood clots, produced in vitro and labeled with 125I-fibrinogen, were embolized into the lungs of anesthetized rats. Thrombolysis was calculated from the difference between initial clot radioactivity and that remaining in the lungs at 60 minutes. Blood was sampled for gamma counting, measurement of hemostatic factors, and plasminogen activator antigen levels. Thrombolysis at 3, 10, 30, and 100 nmol/kg intravenously (10% bolus, 90% over 60 minutes) amounted to 30% +/- 2%, 51% +/- 4%, 85% +/- 4%, 98% +/- 0% for rDSPA alpha 1 and 30% +/- 3%, 41% +/- 3%, 57% +/- 6%, 93% +/- 2% for t-PA (controls: 29% +/- 2%; mean +/- SEM, n greater than or equal to 6). t-PA at 100 nmol/kg significantly decreased fibrinogen, plasminogen, and alpha 2- antiplasmin levels by 33% +/- 7%, 38% +/- 8%, and 61% +/- 9%, whereas rDSPA alpha 1 at 100 nmol/kg only lowered alpha 2-antiplasmin significantly (by 29% +/- 6%). Compared with t-PA, rDSPA alpha 1 is the more potent and more clot selective (fibrin specific) thrombolytic agent. These results suggest that rDSPA alpha 1 may be safer and more efficacious than currently used thrombolytics.

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