Recombinant human microplasmin: production and potential therapeutic properties

Abstract

Summary. The effect of recombinant human microplasmin was studied in ischemic stroke models in mice and in an extracorporeal loop thrombosis model in rabbits. Human microplasminogen (mPlg), which lacks the five 'kringle' domains of plasminogen was expressed with high yield in Pichia pastoris. It was purified, converted to microplasmin (mPli) and equilibrated with 5 mmol L À1 citrate, pH 3.1, yielding a stable preparation. In mice with middle cerebral artery (MCA) ligation, an intravenous (i.v.) bolus of 5.0 mg kg À1 mPli reduced infarct size at 24 h from 27 (26-30) to 25 (21-28) mm 3 (median and range, n ¼ 16 each, P ¼ 0.0001), whereas 4.0 mg kg À1 rt-PA and 40 mg kg À1 mPlg had no effect. Infarct reduction was observed with administration at 4 h after occlusion. In mice with MCA, infarct size at 24 h was reduced from 20 (14-30) to 9.1 (3.1-25) mm 3 with 5.0 mg kg À1 mPli (n ¼ 15 each, P <0.002) and to 11 (5.2-27) mm 3 with 4.0 mg kg À1 rt-PA (n ¼ 6; P ¼ 0.02). Infarct reduction was still observed at 10 h after occlusion with mPli but not with t-PA. In rabbits with radiolabeled clots in an extracorporeal arteriovenous loop, local infusion of 2.5 mg kg À1 mPli over 2 h, induced 51 AE 15% lysis (mean AE SD, n ¼ 11) vs. a control value of 23 AE 5.5%. mPli did not prolong template bleeding times, whereas equipotent doses of rt-PA were associated with extensive rebleeding. The potency of mPli in both models was similar to that of intact plasmin. These findings indicate that recombinant mPli may be useful for treatment of ischemic stroke and arterial thrombosis.

Enzymatic vitreolysis with recombinant microplasminogen and tissue plasminogen activator

“…17 rt-PA (Genentech, South San Francisco, CA, USA) was stored at -201C until administration, when these powders were reconstituted in sterile balanced salt solution (BSS) to the required concentrations.…”

“…15,16 Recently, mPlg has been expressed with high yield in Pichia pastoris (about 400 mg/l culture broth). 17 Thus, mPlg will be a commercially available product, which allows immediate availability and does not require long and difficult preparation time to isolate the plasmin from autologous blood.…”

The evolution of recombinant thrombolytics: Current status and future directions

“…144 The molecule has been recombinantly expressed in E. coli, Pichia pastoris, and insect cells. 149,150,151 It showed 100 times lesser inhibition by a2-antiplasmin than plasmin due to the absence of the lysine binding sites in micro-plasmin. 150 It does not have any kringle domain and thus lacks fibrin affinity.…”

“…16 It appears to have a comparable potency to full-length plasmin in vivo. 150 Microplasmin has been shown to reduce cerebral ischemic damage in animal models with lesser hemorrhage than t-PA. 152,153 During clinical trials too, intravenous microplasmin was generally well tolerated in patients with acute ischemic stroke. 154 Catheter malfunction due to thrombotic occlusion was overcome safely and effectively using microplasmin without any bleeding complications.…”

Serine-proteases as plasminogen activators in terms of fibrinolysis

“…[134] Microplasmin is a truncated form of plasmin and is inhibited by a2-antiplasmin to a much lower extent. [135] TAL6003 is a deletion variant of plasmin where the middle portion of the molecule is removed, resulting in kringle 1 attachment to the serine protease domain. [136] Fu-P is an enzyme from Fusarium sp.…”