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Recombinant human microplasmin: production and potential therapeutic properties
N Nagai1, E Demarsin, B Van Hoef
1Center for Molecular and Vascular Biology, University of Leuven, Leuven, Belgium.
Abstract:
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 ( micro Plg), which lacks the five 'kringle' domains of plasminogen was expressed with high yield in Pichia pastoris. It was purified, converted to microplasmin ( micro Pli) and equilibrated with 5 mmol L(-1) 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(-1) micro Pli reduced infarct size at 24 h from 27 (26-30) to 25 (21-28) mm3 (median and range, n= 16 each, P= 0.0001), whereas 4.0 mg kg(-1) rt-PA and 40 mg kg(-1) micro Plg 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) mm3 with 5.0 mg kg(-1) micro Pli (n = 15 each, P < 0.002) and to 11 (5.2-27) mm3 with 4.0 mg kg(-1) rt-PA (n = 6; P= 0.02). Infarct reduction was still observed at 10 h after occlusion with micro Pli but not with t-PA. In rabbits with radiolabeled clots in an extracorporeal arteriovenous loop, local infusion of 2.5 mg kg(-1) micro Pli over 2 h, induced 51 +/- 15% lysis (mean +/- SD, n= 11) vs. a control value of 23 +/- 5.5%. micro Pli did not prolong template bleeding times, whereas equipotent doses of rt-PA were associated with extensive rebleeding. The potency of micro Pli in both models was similar to that of intact plasmin. These findings indicate that recombinant micro Pli may be useful for treatment of ischemic stroke and arterial thrombosis.
Insights
Recombinant microplasmin (micro Pli) effectively reduced infarct size in mouse models of ischemic stroke, even when administered up to 10 hours post-occlusion. This novel agent also demonstrated significant clot lysis in rabbits without increasing bleeding risk, unlike rt-PA.
Area of Science:
- Biochemistry
- Pharmacology
- Thrombosis Research
Background:
- Ischemic stroke and arterial thrombosis are critical conditions requiring effective therapeutic interventions.
- Current treatments like recombinant tissue plasminogen activator (rt-PA) have limitations, including a narrow therapeutic window and increased bleeding risk.
- Microplasminogen (micro Plg), a truncated form of plasminogen, offers potential as a thrombolytic agent.
Purpose of the Study:
- To evaluate the efficacy and safety of recombinant human microplasmin (micro Pli) in preclinical models of ischemic stroke and arterial thrombosis.
- To compare the therapeutic effects of micro Pli with rt-PA.
Main Methods:
- Recombinant human microplasminogen (micro Plg) was expressed in Pichia pastoris, purified, and converted to microplasmin (micro Pli).
- Efficacy was assessed in middle cerebral artery ligation models in mice, measuring infarct size after treatment with micro Pli or rt-PA.
- Thrombolytic activity and bleeding risk were evaluated in an extracorporeal loop thrombosis model in rabbits using micro Pli and rt-PA.
Main Results:
- Micro Pli significantly reduced infarct size in mice at 24 hours post-occlusion, with efficacy observed up to 10 hours after MCA ligation.
- In rabbits, micro Pli induced 51% clot lysis, comparable to intact plasmin, and did not prolong bleeding times.
- Equipotent doses of rt-PA showed less efficacy in stroke models and were associated with increased bleeding in rabbits.
Conclusions:
- Recombinant micro Pli demonstrates potent thrombolytic activity in both ischemic stroke and arterial thrombosis models.
- Micro Pli offers a potentially safer and more effective alternative to rt-PA, with a broader therapeutic window.
- These findings support the further development of micro Pli for clinical application in treating thrombotic events.