ATTACHMENT OF AN ANTIFIBRIN ANTIBODY TO THE AMINO-TERMINUS OF TISSUE-TYPE PLASMINOGEN-ACTIVATOR IMPAIRS STIMULATION BY FIBRIN

On evidence that the potency and specificity of tissue-type plasminoge n activator (t-PA) can be improved by chemical conjugation to an antif ibrin antibody (59D8), we constructed a recombinant molecule, 59D8-t-P A(AB), containing a truncated 59D8 antibody attached through peptide l inkage to the amino terminus of full-length t-PA. Initial analysis of recombinant 59D8-t-PA(AB) in the absence of fibrin revealed enzymatic characteristics indistinguishable from those of t-PA: for the S-2288 s ubstrate, the respective K-m values of t-PA and 59D8-t-PA(AB) were 0.4 8+/-0.02mM and 0.52+/-0.02mM; for plasminogen, the values were 82.35+/ -5.35 mu M and 81.25+/-2.03 mu M Recombinant 59D8-t-PA(AB) was up to 1 0-fold more potent than t-PA in an assay measuring the lysis of fibrin monomer. However, in contrast with the chemical conjugate, recombinan t 59D8-t-PA(AB) was 2 to 3-fold less potent than t-PA in lysing clots in a plasma milieu. To determine whether this difference in plasma clo t lysis was due to impaired fibrin stimulation of the t-PA portion of the recombinant molecule, we devised an assay that quantitates fibrin stimulation of t-PA's catalytic activity. Fibrin stimulation was indee d reduced by 15-fold for recombinant 59D8-t-PA(AB). This observation s uggests that attaching a targeting antibody to the amino terminus of t -PA diminishes the ability of fibrin to stimulate the enzyme.