Scientists pinpoint flaw, offer new promise for stroke treatment

The best treatment doctors currently have for stroke can accelerate the death of brain cells in addition to dissolving blood clots, researchers report in the journal Nature Medicine. But they also found good news: Another drug currently used to treat patients with severe sepsis counters the harmful effects, offering the possibility that a combination of two already-approved drugs might offer a powerful new stroke treatment that would give doctors a bigger window of time to treat patients.

The work is the result of a longstanding collaboration between scientists at the University of Rochester Medical Center and counterparts at Scripps Research Institute in La Jolla, Calif. The team found that the clot-buster tPA (tissue plasminogen activator) can magnify the harmful effects of stroke in mice and in human cells, and that a compound known as APC (activated protein C) counters the harmful effects. “TPA has been a great therapy for some patients, but right now it’s available to a tiny minority of patients. We hope to extend the window of opportunity that tPA could be given, by protecting the brain against its toxic effects,” says Berislav Zlokovic, M.D., Ph.D., the Rochester neuroscientist who led the research thanks to funding from the National Heart, Lung and Blood Institute. “This holds great promise for stroke therapy.”

TPA is best known as a clot buster useful for patients who have the most common type of stroke, where a blood clot blocks blood flow to a portion of the brain, cutting off oxygen. The trauma causes more and more brain cells to die as they try to cope with the damage. The result can be a devastating brain injury that incapacitates the person for life.

TPA can prevent the damage by dissolving the clot and restoring the flow of oxygen – but the drug must be given to patients within three hours of the onset of stroke symptoms. That’s a big reason why just a tiny fraction of patients benefit from the drug: Zlokovic estimates that the drug reaches only about three percent of patients who are eligible, and of those, not everyone benefits. The window of opportunity is so short largely because tPA is capable of doing additional damage if not given immediately. While doctors know that tPA can cause damage, the Rochester and Scripps team observed more extensive damage than expected.

The team identified the specific molecular signals that enable tPA to enhance the damage done by stroke. TPA kills neurons and endothelial cells, the specialized cells that line our blood vessels, by accelerating a process known as apoptosis or programmed cell death. In both human cells as well as the brains of mice, the team showed that tPA activates an enzyme called caspase-8, which goes on to activate caspase-3, which lays waste to a cell’s nucleus. “This study sheds a lot of light on why tPA is not as effective as it could be. TPA is tremendous at opening up blood vessels, but problems with toxicity diminish the positive effects,” says Zlokovic, who is professor in the Department of Neurosurgery and director of the Frank P. Smith Laboratories for Neuroscience and Neurosurgical Research at the University of Rochester Medical Center. The study will be published in the December issue of Nature Medicine and was published on-line November 1.

Most of the experiments outlined in the paper were done by research assistant professors Dong Liu, Tong Cheng, and Huang Guo of the Department of Neurosurgery – the three share credit as first authors of the paper. Also contributing from Scripps were John Griffin, Ph.D., professor of molecular and experimental medicine, an APC expert whose laboratory provided mouse APC for the study, as well as staff member Jose Fernandez, M.D., Ph.D. of Scripps, and technician Xiaomei Song of Rochester.

The team found that tPA not only causes bleeding by damaging the blood vessel lining – increased bleeding is one of the main risks that limits tPA use – but it also seeps out of the damaged blood vessels, breaking through the blood-brain barrier and killing the brain’s neurons directly. It’s a little bit like a powerful drain cleaner that not only clears out a clog in a pipe but also eats through the pipe and then damages nearby structures. “TPA is a two-edged sword capable of both beneficial and harmful effects when given for ischemic stroke,” says Griffin.

The administration of tPA boosted the percentage of cells that were marked for death dramatically. In human brain blood-vessel cells subjected to hypoxia or a shortage of oxygen, the stroke conditions alone killed 60 percent of cells, but when tPA was added, all the cells died. Similarly, in mice, tPA nearly doubled the number of neurons marked for death from stroke (from 32 to 60 percent) and more than tripled the number of cells in blood vessels that were undergoing apoptosis (from 14 to 50 percent). The addition of tPA also boosted the level of caspase-3, which chews up the insides of cells, more than doubling in human brain cells and nearly doubling in mouse neurons.

Then the researchers showed that APC counters the harmful effects of tPA remarkably. In human brain cells in culture, APC reduced the number of cells marked for death by 80 percent. Mice that received APC and tPA had 85 percent less brain damage from stroke as mice that received tPA but not APC, and in mice APC completely countered the harmful effects of tPA, bringing the level of “apoptotic” cells down to pre-tPA levels. The levels of the harmful molecules caspase-3 and caspase-8 were also cut dramatically in mice that received APC. “This work combines two FDA-approved drugs in the setting of a major challenge: ischemic stroke,” says Griffin. “In the last 10 years, no other drug has been approved for stroke, and in the last 15 years, no other drug has been approved for severe sepsis. Perhaps these drugs could be co-administered to give maximum beneficial effect to patients. It’s incredibly exciting.”

The results are the latest in the team’s research into the basic properties of APC. Previously the researchers have shown that APC reduces inflammation after stroke and protects neurons under stress. Rochester neurologist Curtis Benesch, M.D., has received approval from the U.S. Food & Drug Administration to test APC as a new investigational drug in patients who have had a stroke and who are treated within six hours. If APC is safe for patients and is effective, further tests are likely.