CHIMERIC FIBROLASE - COVALENT ATTACHMENT OF AN RGD-LIKE PEPTIDE TO CREATE A POTENTIALLY MORE EFFECTIVE THROMBOLYTIC AGENT

We have prepared an agent possessing both thrombolytic and antiplatele t properties, by conjugating fibrolase, a direct-acting fibrinolytic e nzyme isolated from southern copperhead venom, to a peptide which inhi bits platelet aggregation. Heterobifunctional coupling reagents, N-suc cinimidyl 3-(2-pyridyidithio) propionate (SPDP) or sulfosuccinimidyl - methyl-alpha-(2-pyridyldithio)toluamido]hexanoate (Sulfo-LC-SMPT), wer e used in a molar ratio of 10:1 (coupling agent/fibrolase). The N-hydr oxy-succinimide of the coupling agent reacts with surface E-amino grou ps of lysine residues on fibrolase and provides a dithio group that is highly reactive ;ive with small thiol compounds. The derivatives obta ined in the first reaction contain approximately two moles of 2-pyridy l disulphide per mole of enzyme. These derivatives were then reacted w ith the free thiol group in an antiplatelet peptide at a molar ratio o f 2:1 (peptide/fibrolase). The peptide-fibrolase conjugate was purifie d by cation exchange HPLC and analyzed by amino acid analysis. The con jugate contains one mole peptide per mole of fibrolase and retains app roximately 85% fibrinolytic activity. The IC50 for inhibition of plate let aggregation in human PRP is 300 nM for the conjugate and 67 nM for the antiplatelet peptide. These results demonstrate the successful fo rmation of a novel chimeric protein with bifunctional activity. (C) 19 97 Elsevier Science Ltd.