Antithrombotic agents that inhibit thrombin are effective, but can cause bleeding. In order to reduce dosage requirements, we have modified the clearance behavior of hirudin, a small thrombin inhibitor polypeptide, by genetic fusion to albumin. Previously, we showed that a His-tagged hirudin fusion protein (HLAH6) derived from transfected COS cells was cleared from rabbits 160-fold slower than hirudin (Syed et al, Blood 89:3243-3252). We now report an anticoagulant effect of HLAH6 in vivo, using HLAH6 obtained from yeast. The HLAH6 cDNA was tailored for insertion into plasmid pPICZa by PCR. P. pastoris cells transformed with this construct, and induced with methanol, secreted a prominent 75 kDa polypeptide. The yeast-derived HLAH6 was salted out of conditioned media and purified by nickel chelate chromatography. Equimolar HLAH6 or unfused hirudin identically prolonged the thrombin clotting time (TCT) when added to plasma in vitro(to 43-45 sec from 13-15 sec). Intravenous injection of 18 nmol/kg (1.33 mg/kg) of purified HLAH6 into each of 3 rabbits elevated the activated partial thromboplastin time (APTT) from 45 sec to greater than 200 sec within 15 min of injection, with substantial effects (APTT = 155 sec, standard error of the mean [S.E.M.] 8 sec) 4 hours later. In contrast, equimolar doses of unfused hirudin produced peak APTT values of 97 sec (S.E.M. 17 sec) within 15 min of injection, with no effect noted by 4 hours. Lower doses of HLAH6 than of hirudin therefore have the potential to produce similar antithrombotic effects.