A recombinant, chimeric enzyme with a novel mechanism of action leading to greater potency and selectivity than tissue-type plasminogen activator.
BACKGROUND
Early intervention with thrombolytic agents has been shown unequivocally to reduce mortality after acute myocardial infarction. Presently used agents have disadvantages such as short half-life, immunogenicity, hypotension, and bleeding complications. Therefore, there is a need to develop improved thrombolytic drugs with novel mechanisms of action leading to improved properties.


RESULTS
Hybrid plasminogen/tissue-type plasminogen activator (t-PA) complementary DNA was constructed and expressed in Chinese hamster ovary cells. The chimeric protein, comprising the fibrin-binding domains of plasminogen covalently linked to the catalytic domain of t-PA, was purified and evaluated in vitro and in vivo. The hybrid was inhibited rapidly in human and animal plasmas. The mediator of this rapid inhibition was shown to be alpha 2-antiplasmin. The active center of the hybrid could be protected by reversible active center acylation with a novel inverse acylating agent, 4'-amidinophenyl-4-chloroanthranilic acid (AP-CLAN). An acylated (CLAN-) hybrid was cleared from the bloodstream of guinea pigs at 0.35 +/- 0.02 ml/min.kg-1 compared with a clearance rate of 36 +/- 4 ml/min.kg-1 for t-PA. The CLAN-plasminogen/t-PA hybrid was evaluated in a quantitative, "humanized" guinea pig pulmonary embolism model and shown to be approximately threefold more potent when given by bolus than an infusion of t-PA. Furthermore, the acylated hybrid was more fibrin selective than t-PA as determined by the relation between clot lysis and fibrinogen degradation.


CONCLUSIONS
An acylated, recombinant plasminogen/t-PA hybrid has sufficiently slow clearance to be administered by bolus and is more potent and fibrin selective than t-PA in vivo.
Robinson
J H
JH
Department of Biotechnology, SmithKline Beecham Pharmaceuticals, Surrey, England, UK.
Browne
M J
MJ
Carey
J E
JE
Chamberlain
P D
PD
Chapman
C G
CG
Cronk
D W
DW
Dodd
I
I
Entwisle
C
C
Esmail
A F
AF
Kalindjian
S B
SB
D000215:Acylation; D000818:Animals; D001550:Benzamidines; D006224:Cricetinae; D005343:Fibrinolytic Agents; D006168:Guinea Pigs; D006801:Humans; D010958:Plasminogen / Q000096:biosynthesis / Q000494:pharmacology; D011655:Pulmonary Embolism / Q000188:drug therapy; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000494:pharmacology; D010959:Tissue Plasminogen Activator / Q000096:biosynthesis / Q000494:pharmacology*; D000979:alpha-2-Antiplasmin / Q000494:pharmacology; D062367:ortho-Aminobenzoates
D001550:Benzamidines; D005343:Fibrinolytic Agents; D011994:Recombinant Proteins; D000979:alpha-2-Antiplasmin; D062367:ortho-Aminobenzoates; C075930:4'-amidinophenyl-4-chloroanthranilic acid; D010958:Plasminogen; D010959:Tissue Plasminogen Activator