title: Protein engineering of novel plasminogen activators with increased thrombolytic potency in rabbits relative to activase.
abstract: Human tissue-type plasminogen activator (t-PA) is a glycoprotein used currently in thrombolytic therapy for patients with acute myocardial infarction. Due to its rapid rate of clearance from the circulation, continuous intravenous administration of approximately 100 mg over 3 h is recommended. We have previously characterized novel thrombolytic variant forms of t-PA which offer the potential of administration by bolus injection and reduced dosage due to their slower rates of clearance, relative to t-PA. This study was undertaken to quantitatively compare the pharmacokinetics, thrombolytic activity, and hemostatic effects of two of these variant forms, called delta FE1X and delta FE3X plasminogen activator (PA), with commercially available recombinant t-PA (Activase). These evaluations were performed in rabbits after bolus intravenous injection of the proteins. Following injection of 0.25 mg of protein/kg of body weight, the rates of clearance for delta FE3X and delta FE1X PA antigen were decreased approximately 9- and 18-fold, respectively, relative to Activase. Plasma plasminogen activator activity was also measured and the rates of clearance of delta FE3X and delta FE1X PA activity were similarly decreased by approximately 9- and 22-fold, respectively, relative to Activase. To quantitate thrombolytic activity we used the rabbit jugular vein thrombosis model and demonstrated that approximately 50% thrombolysis was achieved with delta FE1X and delta FE3X PA at approximately an 8.6- and 3-fold lower dose than Activase, respectively. No major differences in fibrinogen and alpha 2-antiplasmin depletion were observed among the agents at doses required to produce 50% thrombolysis, indicating similarities in fibrin specificities among these agents. These results demonstrate a reciprocal relationship between thrombolysis and rate of clearance for these thrombolytic proteins. The 8.6-fold increase in potency of delta FE1X PA relative to Activase supports the future clinical testing of this novel engineered protein as a thrombolytic agent.
authors.affiliation: Genetics Institute Inc., Cambridge, Massachusetts 02140.
authors.forename: G R
authors.initials: GR
authors.lastname: Larsen
authors.forename: G A
authors.initials: GA
authors.lastname: Timony
authors.forename: P G
authors.initials: PG
authors.lastname: Horgan
authors.forename: K M
authors.initials: KM
authors.lastname: Barone
authors.forename: K S
authors.initials: KS
authors.lastname: Henson
authors.forename: L B
authors.initials: LB
authors.lastname: Angus
authors.forename: J B
authors.initials: JB
authors.lastname: Stoudemire
mesh_terms: D000818:Animals; D002460:Cell Line; D006224:Cricetinae; D005340:Fibrinogen / Q000378:metabolism; D005341:Fibrinolysin / Q000378:metabolism; D005343:Fibrinolytic Agents / Q000493:pharmacokinetics; D006487:Hemostasis; D007275:Injections, Intravenous; D008657:Metabolic Clearance Rate; D010960:Plasminogen Activators / Q000235:genetics / Q000493:pharmacokinetics / Q000494:pharmacology*; D015202:Protein Engineering; D011817:Rabbits; D010959:Tissue Plasminogen Activator / Q000494:pharmacology*; D000511:alpha-Macroglobulins / Q000378:metabolism
chemical_list: D005343:Fibrinolytic Agents; D000511:alpha-Macroglobulins; C024668:plasmin-alpha(2)-macroglobulin complex; D005340:Fibrinogen; D010960:Plasminogen Activators; D010959:Tissue Plasminogen Activator; D005341:Fibrinolysin