A barbourin-albumin fusion protein that is slowly cleared in vivo retains the ability to inhibit platelet aggregation in vitro

Barbourin is a 73 amino acid venom protein that inhibits platelet aggregati on. Recombinant barbourin (BARH(6)), rabbit serum albumin (RSAH(6)), and a barbourin-RSA fusion protein (barbourin-linker-albumin; BLAH(6)) were secre ted from Pichia pastoris yeast, and purified by nickel-chelate affinity chr omatography via their C-terminal hexahistidine A) tags. BARH(6) and BLAB(6) did not differ in their IC(50)s for inhibition of platelet aggregation usi ng either human platelets stimulated with thrombin or ADP, or rabbit platel ets stimulated with ADP. BARH(6) and BLAH(6) were also effective in inhibit ing platelet aggregation in whole blood, and formed complexes with platelet integrin The terminal catabolic half-life of BLAH(6) approached that of RS AH(6) [3.4 +/- 0.2 versus 4.0 +/- 0.1 days (n = 4 SD)], but was substantial ly increased relative. to that of BARH(6) [0.15 +/- 0.03 days (n = 3 SD)]. Our results suggest that fusion to albumin slows the clearance of barbourin in vivo, while preserving its ability to inhibit platelet aggregation.