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Activated protein C inhibits tissue plasminogen activator–induced brain hemorrhage

An Editorial Expression of Concern to this article was published on 20 March 2024

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Abstract

Brain hemorrhage is a serious complication of tissue plasminogen activator (tPA) therapy for ischemic stroke. Here we report that activated protein C (APC), a plasma serine protease with systemic anticoagulant, anti-inflammatory and antiapoptotic activities, and direct vasculoprotective and neuroprotective activities, blocks tPA-mediated brain hemorrhage after transient brain ischemia and embolic stroke in rodents. We show that APC inhibits a pro-hemorrhagic tPA-induced, NF-κB–dependent matrix metalloproteinase-9 pathway in ischemic brain endothelium in vivo and in vitro by acting through protease-activated receptor 1. The present findings suggest that APC may improve thrombolytic therapy for stroke, in part, by reducing tPA-mediated hemorrhage.

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Figure 1: APC controls tPA-mediated brain hemorrhage after transient ischemia in mice.
Figure 2: APC blocks tPA-induced brain hemorrhage after embolic stroke in rats.
Figure 3: APC inhibits tPA-mediated MMP-9 upregulation in OGD-treated BEC.
Figure 4: APC inhibits tPA-induced NF-kB activation.
Figure 5: APC-mediated neuroprotection and antihemorrhagic effects after transient ischemia in mice require PAR1.
Figure 6: APC-mediated inhibition of the NF-κB-MMP-9 pathway in OGD/tPA-treated mouse BEC requires PAR1.

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Acknowledgements

We thank A. Sagare for radiolabeling tPA. Par1-null mice were provided by S. Coughlin (University of California San Francisco), EPCR by K. Fukudome (Department of Immunology, Saga Medical School), and the adenoviruses for BEC transduction by S. Maggirwar (University of Rochester). This work was supported by US National Institutes of Health grant HL63290 to B.V.Z.

Author information

Authors and Affiliations

Authors

Contributions

T.C. conducted and performed in vitro studies of human BEC. A.L.P. conducted and performed in vivo stroke studies in the mouse MCA model. Z.L. conducted and performed in vivo stroke studies in the rat embolic model. M.T. performed in vivo studies in Par1-null mice. Z.Z. performed in vitro studies in mouse BEC. Z.W. performed immunostaining studies. D.L. performed in vivo mouse MCA studies. S.B.M. performed in vitro studies and developed IkkB reagent for the study. R.D. conducted and performed studies of physiological parameters. J.A.F. prepared different preparations of mouse and human APC. B.L. performed physiological analysis in mouse models and in vitro assays. J.H.G. contributed to experimental design and data discussion and analysis. M.C. designed studies in rat embolic model, and contributed to data analysis and interpretation. B.V.Z. designed the entire study, supervised all segments of the study, and wrote the manuscript.

Note: Supplementary information is available on the Nature Medicine website.

Corresponding author

Correspondence to Berislav V Zlokovic.

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Competing interests

B.V.Z. is scientific founder of Socratech LLC, a start-up biotech company with a mission to develop neuroprotective strategies in the aging and diseased brain such as Alzheimer's and stroke.

Supplementary information

Supplementary Fig. 1

Immunostaining of collagen IV after transient ischemia in mice determined at 24 h. (PDF 401 kb)

Supplementary Fig. 2

Immunostaining of ZO1 and CD-31 after transient ischemia in mice determined at 24 h. (PDF 1189 kb)

Supplementary Fig. 3

Gelatinolytic activity of recombinant pro-MMP9 and active-MMP-9 on SDS-PAGE. (PDF 258 kb)

Supplementary Fig. 4

SDS-PAGE analysis of 125I-labeled tPA in the absence and presence of APC (400 nM). (PDF 224 kb)

Supplementary Fig. 5

Plasma levels of mouse APC (ng/ml) determined by mouse specific APC ELISA after intravenous administration of mouse APC. (PDF 275 kb)

Supplementary Table 1

Physiological parameters before MCAO, during MCAO and 60 min after MCAO. (PDF 65 kb)

Supplementary Methods (PDF 113 kb)

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Cheng, T., Petraglia, A., Li, Z. et al. Activated protein C inhibits tissue plasminogen activator–induced brain hemorrhage. Nat Med 12, 1278–1285 (2006). https://doi.org/10.1038/nm1498

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