Reteplase is a protein consisting of the kringle-2 and protease domains of tissue-type plasminogen activator (t-PA). Because intravenous heparin will be used as an adjunct to thrombolytic therapy with reteplase, we investigated the interactions in vitro between heparin and reteplase as well as between heparin and recombinant t-PA (alteplase) as a control. Reteplase and alteplase bound completely to a heparin-agarose column and eluted respectively at 0.39 M and 0.60 M in a NaCl gradient. Two-chain derivatives of reteplase and alteplase eluted at 0.31 M and 0.52 M NaCl, respectively. Plasminogen activation by the two-chain derivatives of reteplase and alteplase in a purified system at low ionic strength were stimulated by heparin up to 13- and 22-fold, respectively. However reteplase required five times more heparin for maximal stimulation than alteplase. In addition, the heparin stimulation of reteplase was more salt sensitive than that of alteplase. In conclusion, both heparin binding and heparin stimulation experiments showed that heparin interacts with reteplase, but the interaction is weaker than with alteplase. Chemicals/CAS: Amidohydrolases, EC 3.5.-; Anticoagulants; Fibrinolytic Agents; Heparin, 9005-49-6; heparin-sepharose; Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase, EC 3.5.1.52; Plasminogen Activators, EC 3.4.21.-; Plasminogen, 9001-91-6; Recombinant Proteins; reteplase, 133652-38-7; Sepharose, 9012-36-6; Tissue Plasminogen Activator, EC 3.4.21.68