Related Experiment Video
Updated: Aug 17, 2026
The Nijmegen Hemostasis Assay: Simultaneous Fluorogenic Measurement of Thrombin and Plasmin Generation in a Single Well
Published on: February 27, 2026
Protease activity of plasma hemopexin
Winston W Bakker1, Theo Borghuis, Martin C Harmsen
1Department of Pathology and Laboratory Medicine, and Department of Pediatrics, University Hospital Groningen, Groningen, The Netherlands. w.w.bakker@path.umcg.nl
Background:
Previous studies into the relevance of a putative circulating factor in the pathogenesis of minimal change nephrotic syndrome have opened the possibility that plasma hemopexin might be an important effector molecule in this disorder. Thus, intra renal infusion of isolated plasma hemopexin into rats induced minimal change like glomerular lesions and proteinuria. Both, in vivo and in vitro effects of the active isoform of hemopexin could be attributed to protease activity of this molecule. However, the question remained whether hemopexin per se rather than some contaminating plasma factor is responsible for the potential enzymatic activity of this molecule.
Methods:
Recombinant hemopexin was prepared according to standard methods in Pichia pastoris and compared for its identity and protease activity with plasma hemopexin using Western blotting and various in vitro assays. Unilateral renal perfusion in anesthetized rats was used to test the proteinuria inducing capacity of recombinant hemopexin versus heat-inactivated recombinant hemopexin.
Results:
The blotting results show identical 85 kD bands in both native as well as recombinant hemopexin. Incubation of kidney tissue with recombinant hemopexin resulted in loss of of glomerular ectoapyrase and sialoglycoproteins, as shown by immunohistochemistry, which effect can be inhibited with the serine protease inhibitor phenylmethanesulfonyl fluoride. Artificial substrates for serine proteases, like kallikrein or thrombin, are hydrolysed by recombinant hemopexin in vitro, and not by heat-inactivated recombinant hemopexin or saline. Unilateral kidney perfusion of recombinant hemopexin, in contrast to control Pichia transfection products or heat-inactivated recombinant hemopexin, followed by a protein marker showed significantly enhanced urinary protein leakage 5.0, 10.0, and 15.0 minutes after perfusion.
Conclusion:
It is concluded that the hemopexin molecule as such can potentially act as a toxic protease, leading in the rat to proteinuria and glomerular alterations characteristic for minimal change nephrotic syndrome.
Insights
Hemopexin, a plasma protein, acts as a toxic protease causing kidney damage and proteinuria characteristic of minimal change nephrotic syndrome in rats. This study confirms hemopexin
Area of Science:
- Nephrology
- Molecular Biology
- Biochemistry
Background:
- Plasma hemopexin is implicated in minimal change nephrotic syndrome pathogenesis.
- Previous studies showed hemopexin infusion causes glomerular lesions and proteinuria in rats.
- The protease activity of hemopexin was suspected but not confirmed to be intrinsic.
Purpose of the Study:
- To confirm if hemopexin itself possesses protease activity.
- To investigate the role of hemopexin as a toxic protease in nephrotic syndrome.
Main Methods:
- Recombinant hemopexin was produced and compared to plasma hemopexin.
- Protease activity was assessed using Western blotting, in vitro assays, and kidney tissue incubation.
- Proteinuria induction was tested in rats using unilateral renal perfusion with recombinant hemopexin.
Main Results:
- Recombinant hemopexin showed identical identity and protease activity to plasma hemopexin.
- Hemopexin degraded glomerular ectoapyrase and sialoglycoproteins, an effect inhibited by protease inhibitors.
- Recombinant hemopexin induced significant proteinuria in rats, unlike heat-inactivated versions.
Conclusions:
- Hemopexin molecule possesses intrinsic protease activity.
- Hemopexin acts as a toxic protease, causing proteinuria and glomerular changes in minimal change nephrotic syndrome models.
