Tissue plasminogen activator neurovascular toxicity is controlled by activated protein C

Article metrics

Abstract

Although thrombolytic effects of tissue plasminogen activator (tPA) are beneficial, its neurotoxicity1,2,3,4,5 is problematic. Here, we report that tPA potentiates apoptosis in ischemic human brain endothelium and in mouse cortical neurons treated with N-methyl-D-aspartate (NMDA) by shifting the apoptotic pathways from caspase-9 to caspase-8, which directly activates caspase-3 without amplification through the Bid-mediated mitochondrial pathway6. In vivo, tPA-induced cerebral ischemic injury in mice was reduced by intracerebroventricular administration of caspase-8 inhibitor, but not by caspase-9 inhibitor, in contrast to controls in which caspase-9 inhibitor, but not caspase-8 inhibitor, was protective. Activated protein C (APC), a serine protease with anticoagulant, anti-inflammatory and antiapoptotic activities7, which is neuroprotective during transient ischemia8,9 and promotes activation of antiapoptotic mechanisms in brain cells by acting directly on endothelium9,10,11 and neurons12, blocked tPA vascular and neuronal toxicities in vitro and in vivo. APC inhibited tPA-induced caspase-8 activation of caspase-3 in endothelium and caspase-3–dependent nuclear translocation of apoptosis-inducing factor in NMDA-treated neurons and reduced tPA-mediated cerebral ischemic injury in mice. Data suggest that tPA shifts the apoptotic signal in stressed brain cells from the intrinsic to the extrinsic pathway which requires caspase-8. APC blocks tPA's neurovascular toxicity and may add substantially to the effectiveness of tPA therapy for stroke.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Figure 1: tPA toxicity to human BEC and cytoprotection by human APC.
Figure 2: tPA's apoptotic signal in hypoxic human BEC requires caspase-8 and is blocked by APC.
Figure 3: tPA shifts NMDA-induced apoptosis in mouse cortical neurons to caspase-8 which is blocked by mouse APC.
Figure 4: tPA-induced cerebral injury in a mouse stroke model and protection by mouse APC.

References

  1. 1

    Wang, Y.F. et al. Tissue plasminogen activator (tPA) increases neuronal damage after focal cerebral ischemia in wild-type and tPA-deficient mice. Nat. Med. 4, 228–231 (1998).

  2. 2

    Nicole, O. et al. The proteolytic activity of tissue-plasminogen activator enhances NMDA receptor-mediated signaling. Nat. Med. 7, 59–64 (2001).

  3. 3

    Yepes, M. et al. Regulation of seizure spreading by neuroserpin and tissue-type plasminogen activator is plasminogen-independent. J. Clin. Invest. 109, 1571–1578 (2002).

  4. 4

    Matys, T. & Strickland, S. Tissue plasminogen activator and NMDA receptor cleavage. Nat. Med. 9, 371–373 (2003).

  5. 5

    Chen, Z.L. & Strickland, S. Neuronal death in the hippocampus is promoted by plasmin-catalyzed degradation of laminin. Cell 91, 917–925 (1997).

  6. 6

    Plesnila, N., et al. BID mediates neuronal cell death after oxygen/glucose deprivation and focal cerebral ischemia. Proc. Natl. Acad. Sci. USA 98, 15318–15323 (2001).

  7. 7

    Griffin, J.H. et al. Activated protein C and ischemic stroke. Crit. Care Med. 32, S247–S253 (2004).

  8. 8

    Shibata, M. et al. Anti-inflammatory, antithrombotic, and neuroprotective effects of activated protein C in a murine model of focal ischemic stroke. Circulation 103, 1799–1805 (2001).

  9. 9

    Cheng, T. et al. Activated protein C blocks p53-mediated apoptosis in ischemic human brain endothelium and is neuroprotective. Nat. Med. 9, 338–342 (2003).

  10. 10

    Joyce, D.E., Gelbert, L., Ciaccia, A., DeHoff, B. & Grinnell, B.W. Gene expression profile of antithrombotic protein C defines new mechanisms modulating inflammation and apoptosis. J. Biol. Chem. 276, 11199–11203 (2001).

  11. 11

    Riewald, M., Petrovan, R.J., Donner, A., Mueller, B.M. & Ruf, W. Activation of endothelial cell protease activated receptor 1 by the protein C pathway. Science 296, 1880–1882 (2002).

  12. 12

    Guo, H. et al. Activated protein C prevents neuronal apoptosis via protease activated receptors 1 and 3. Neuron 42, 563–572 (2004).

  13. 13

    Lo, E.H., Broderick, J.P. & Moskowitz, M.A. tPA and proteolysis in the neurovascular unit. Stroke 35, 354–356 (2004).

  14. 14

    Sharp, C.D., et al. Glutamate causes a loss in human cerebral endothelial barrier integrity through activation of NMDA receptor. Am. Physiol. Heart Circ. Physiol. 285, H2592–H2598 (2003).

  15. 15

    Budd, S.L., Tenneti, L., Lishnak, T. & Lipton, S.A. Mitochondrial and extramitochondrial apoptotic signaling pathways in cerebrocortical neurons. Proc. Natl. Acad. Sci. USA 97, 6161–6166 (2000).

  16. 16

    Stennicke, H.R., et al. Pro-caspase-3 is a major physiologic target of caspase-8. J. Biol. Chem. 273, 27084–90 (1998).

  17. 17

    Zhang, W., et al. Fundamental role of the Rip2/caspase-1 pathway in hypoxia and ischemia-induced neuronal cell death. Proc. Natl. Acad. Sci. USA 100, 16012–16017 (2003).

  18. 18

    Du, Y.S., et al. Activation of a caspase 3-related cysteine protease is required for glutamate-mediated apoptosis of cultured cerebellar granule neurons. Proc. Natl. Acad. Sci. USA 94, 11657–11662 (1997).

  19. 19

    Okamoto, S., et al. Dominant – interfering forms of MEF2 generated by caspase cleavage contribute to NMDA-induced neuronal apoptosis. Proc. Natl. Acad. Sci. USA 99, 3974–3979 (2002).

  20. 20

    Penninger, J.M. and Kroemer, G. Mitochondria, AIF and caspases–rivaling for cell death execution. Nat. Cell Biol. 5, 97–99 (2003).

  21. 21

    Yu, S., et al. Mediation of poly (APD-Ribose) polymerase-1-dependent cell death by apoptosis-inducing factor. Science 297, 259–263 (2002).

  22. 22

    Tabrizi, P. et al. Tissue plasminogen activator (tPA) deficiency exacerbates cerebrovascular fibrin deposition and brain injury in murine stroke model: studies in tPA-deficient mice and wild-type mice on a matched genetic background. Arterioscler. Thromb. Vasc. Biol. 19, 2801–2806 (1999).

  23. 23

    Jiang, Q. et al. Magnetic resonance imaging indexes of therapeutic efficacy of recombinant tissue plasminogen activator treatment of rat at 1 and 4 hours after embolic stroke. J. Cereb. Blood Flow Metab. 20, 21–27 (2000).

  24. 24

    Pichiule, P. & LaManna, J.C. Angiopoietin-2 and rat brain capillary remodeling during adaptation and deadaptation to prolonged mild hypoxia. J. Appl. Physiol. 93, 1131–1139 (2002).

  25. 25

    Tibbetts, M.D., Zheng, L. & Lenardo, M.J. The death effector domain protein family: regulators of cellular homeostasis. Nat. Immunol. 4, 404–409 (2003).

Download references

Acknowledgements

This work was supported by the United States National Institutes of Health grant HL63290.

Author information

Correspondence to Berislav V Zlokovic.

Ethics declarations

Competing interests

Berislav V. Zlokovic serves as a consultant for Socratech LLC, which potentially may have interest in developing activated protein C.

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Liu, D., Cheng, T., Guo, H. et al. Tissue plasminogen activator neurovascular toxicity is controlled by activated protein C. Nat Med 10, 1379–1383 (2004) doi:10.1038/nm1122

Download citation

Further reading