By David N. Leff
Genentech Inc.¿s recombinant tissue plasminogen activator (tPA) is clinically a clotbuster and commercially a blockbuster.
It dissolves blood-damming plaque in the coronary arteries of patients with acute myocardial infarction and in the brains of individuals who have suffered an acute ischemic stroke, and clots in the lungs of those stricken with pulmonary embolism.
Plasminogen activators work by releasing plasmin from plasminogen, its inert precursor molecule. Plasmin is a powerful serine proteolytic enzyme that dissolves the blood-coagulating protein fibrin.
The FDA approved South San Francisco-based Genentech¿s tPA (also known as Activase) for heart attack in November 1987, for lung clots in 1990 and for cerebral stroke on June 18, 1996. (See BioWorld Today, July 21, 1998, p. 1.)
Every 53 seconds, someone in the U.S. experiences a stroke ¿ the blockage of blood flow to the brain. In 1995, researchers announced that ischemic stroke victims who received tPA within three hours of their initial symptoms were at least 30 percent more likely than untreated patients to recover with little or no disability.
But as in so many drastic therapies, there¿s a downside to tPA treatment.
¿TPA has been used widely in neurology to treat stroke patients,¿ observed Korean cell biologist Jae-Young Koh. ¿However, a couple of years ago, several leading neurologists published reports that tPA might be detrimental to neurons. You can injure neurons by producing plasmin, the protease that creates clots. Plasmin may break down the extracellular matrix in the brain, and so injure neurons. That was what those papers suggested.¿
Koh directs the National Creative Research Initiative Center for the Study of Central Nervous System Zinc, in the neurology department at the University of Ulsan, in Seoul, in the Republic of Korea.
Does tPA Mitigate Zinc Nerve Poisoning?
¿So, we tried to test that idea of injurious tPA side effects in our culture systems,¿ Koh recounted, ¿thinking of zinc neurotoxicity in particular. Zinc, a potentially cell-damaging oxidant, is present inside the brain in synaptic vesicles and nerve terminals. It is released in cases of seizures and brain-injury conditions, such as cerebral ischemias. And zinc can injure neurons by entering into them, as we published in Science in 1996.
¿We think that this zinc neurotoxicity,¿ Koh said, ¿may be an important mechanism of brain damage. That¿s why we tried to test tPA¿s action on it in our culture systems. The result was kind of a serendipitous finding: Unexpectedly, the tPA had remarkable protective action on zinc toxicity.¿
Koh is senior author of a paper in today¿s Science, dated April 23, 1999, reporting that counterintuitive result. Its title: ¿Nonproteolytic neuroprotection by human recombinant tissue plasminogen activator.¿
Pushing beyond this surprising datum, Koh told BioWorld Today, ¿we found that tPA¿s protective action is not mediated by protease effect, but by something else. Even though we included in our experiments protease inhibitors that block tPA¿s well-known protease action, it still protects very well against zinc toxicity. We tested that in vitro and in vivo, and found the same thing in both.¿
¿Intriguingly,¿ his Science article noted, ¿the protective effect of tPA (10 micrograms per milliliter) was not reversed by excessive amounts (305g/ml) of plasminogen activator inhibitor . . . indicating that the protection was unlikely mediated by its proteolytic action.¿
In vivo, Koh and his co-authors simulated stroke in rats by injecting them with kainic acid, a cytotoxin that induces prolonged seizures in the animals. The graded intensity of these episodes made it possible to measure the effects of intervention. Typically, the rodents¿ seizures ran the gamut from staring, freezing and facial twitching, to wet-dog shakes, to forelimb spasms, to rearing, falling, jumping and circling.
The seizures the team elicited in the rats caused brain injury and neural cell death in the hippocampus and cortex.
¿Then we injected tPA directly into the animals¿ cerebral spinal fluid,¿ Koh said. ¿That pretty much blocked the neural apoptosis induced by the kainin injection.¿
Can tPA¿s bonus rescue act do for humans what it did for those Korean rats?
¿We surely hope so,¿ Koh said. ¿If we can find a way to minimize tPA¿s injurious actions, which mediate its protease effect, and if we can maximize the protective action by somehow manipulating the molecular function or finding a homologue, we can probably utilize it clinically in real brain-injury situations.
¿But first,¿ he said, ¿we are trying to figure out which component of the tPA molecule has this protective action and which has the injurious action. So, right now, we¿re trying to find the receptors in neurons and glial cells that mediate this protection, because it¿s not in the protease domain, and tPA is a big molecule which has other domains. So, we are trying to find out if these other domains have protective actions, and whether we can find specific receptors that mediate their effect.
¿But until then,¿ Koh concluded, ¿this is kind of a bench-research resource.¿ n