abstract
- The 57-amino acid Kunitz Protease Inhibitor (KPI) domain of Protease Nexin 2 inhibits Factor XIa (FXIa) and other proteases. We previously fused KPI to human serum albumin (KPIHSA). KPIHSA inhibits coagulation Factor XIa (FXIa) 6-fold more rapidly than plasmin. We screened a bacterial expression library of KPI variants randomized at M17, and selected M17D as having the highest anti-FXIa: antiplasmin activity ratio. Expressed as HSA fusion proteins in Pichia pastoris, KPIHSA and KPI(M17D)HSA inhibited FXIa indistinguishably (Ki 9 nM) but KPI(M17D)HSA lacked detectable antiplasmin activity. Purified variant and wild-type KPIHSA were expressed and injected into mice with ferric chloride-treated carotid arteries, with or without systemic administration of tissue plasminogen activator (Tenecteplase, TNKase). The time to arterial occlusion (TTO) or reperfusion (TTR) was assessed by Doppler ultrasound. TTR did not differ between mice treated with TNKase alone or with TNKase supplemented with 38 mg/kg KPI(M17D)HSA but was significantly prolonged to >60 min in all mice treated with TNKase and 38 mg/kg KPIHSA. TTO was significantly but equally prolonged by either 38 mg/kg KPIHSA or KPI(M17D)HSA versus vehicle controls. The antiplasmin activity of KPI is relevant in vivo but its elimination did not enhance counter-thrombosis by KPI.