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CMU researcher pushes search for a safe, artificial hemoglobin one step further Blood work Monday, February 05, 2001 By Byron Spice, Science Editor, Post-Gazette
Few proteins are better known or understood than hemoglobin, the stuff inside red blood cells that carries oxygen from the lungs to the tissues. Yet that hasn't made it easy for researchers who have struggled for 40 years to develop a safe, effective blood substitute.
Fluorocarbons, synthetic molecules with oxygen-carrying capability, have shown limited success as artificial blood. And experience with hemoglobin-based substitutes indicates that, outside the friendly confines of a red blood cell, hemoglobin doesn't work well, doesn't last long and can be downright toxic.
But a team led by Chien Ho, a professor of biological sciences at Carnegie Mellon University, has developed a genetically engineered hemoglobin that looks promising as a blood substitute. His mutant form of hemoglobin does a good job of transporting oxygen even without the help of a red blood cell and also protects its crucial iron atoms from being destroyed by oxidation.
"We hit a pretty good jackpot," said Ho, noting that graduate student Ching-Hsuan Tsai was responsible for much of the work. The discovery was reported last fall in the journal Biochemistry.
The dream of having a ready, shelf-stable supply of the engineered hemoglobin available for transfusions is still not realized, Ho emphasized. More work is needed to stabilize the molecule, reducing its potential toxicity. For clinical testing and subsequent use, mass quantities of hemoglobin would need to be produced, a task that likely would require development of transgenic pigs.
"I think these mutants Chien has made are a step in the right direction," said Alan Koretsky, a senior investigator studying blood flow at the National Institute of Neurological Disorders and Stroke. Ho's expertise in hemoglobin structure was critical for this advance. "He knows what it means to make the protein," added Koretsky, a former Carnegie Mellon faculty member.
Blood substitutes would eliminate the need to match blood types before transfusing trauma victims and eliminate the risk that HIV, hepatitis viruses and other pathogens could be transmitted by transfusions. A ready supply of blood substitute also would stretch blood supplies, which often run short around the holidays; almost 10 percent of U.S. hospitals last year reported that they had to postpone surgeries because of shortages.
"I'm still cautiously optimistic that someone will be able to put together a workable substitute," said Dr. Harvey Klein, chief of transfusion medicine at the National Institutes of Health Clinical Center. But clinical trials of blood substitutes have been marked by poor performance and often have been cut short because of nasty side effects.
Though the latest generation of substitutes to use fluorocarbons looks promising, Klein said previous versions have caused flu-like symptoms, mild fevers and a drop in platelet counts. Patients receiving the substitutes suffered higher death rates.
Genetically engineered hemoglobins also have been tested as substitutes, but clinical trials have shown that they cause high blood pressure, diarrhea and adversely affect such organs as the lungs and the pancreas. Blood cells circulate for months, while free hemoglobin is short-lived.
The modified hemoglobins seem to be running up against several million years of evolution, Klein said. Only earthworms use free hemoglobin to transport oxygen. Other animals package hemoglobin inside blood cells.
Red blood cells don't just protect the body from hemoglobin, they also help hemoglobin do its job, Ho said. No scientist has ever been able to make a cell, so if hemoglobin is to be used in a blood substitute, the protein will have to be engineered to work without the help of a cell.
"Hemoglobin is one of the best understood protein structures under the sun," said Ho, 66, who has spent the past 30 years studying its structure. A native of Shanghai, Ho grew up in Hong Kong before coming to the United States to study at Yale University and the Massachusetts Institute of Technology. He joined the University of Pittsburgh in 1965 and moved in 1979 to Carnegie Mellon.
In 1993, Ho developed a technique for using E. coli bacteria to grow human hemoglobin. "Before that, you had to have a friend who was a hematologist to get hemoglobin for study," he noted.
In engineering hemoglobin for use as artificial blood, Ho was particularly concerned about how the red blood cell affects the molecule's oxygen affinity, or how tightly it holds oxygen. In its normal state, hemoglobin has high oxygen affinity, keeping a firm grip on oxygen. That makes it a good oxygen transporter. But when red blood cells reach tissues that need oxygen, hemoglobin loosens its grip.
That occurs thanks to a substance in red blood cells called 2,3-bisphosphoglycerate. When this compound inserts itself into the hemoglobin molecule, Ho explained, it alters the molecule's structure, making it easier to release oxygen to the tissues.
Once the oxygen is gone, the compound drops off and hemoglobin once again has high oxygen affinity and will be ready to take on a new load of oxygen when the blood passes through the lungs.
But if hemoglobin is outside the red blood cell, there is no 2,3-bisphosphoglycerate to do its magic and the molecule doesn't give up its oxygen easily.
The trick is not just to re-engineer hemoglobin to lower its affinity, but to do so without disrupting another important quality, called cooperativity. Hemoglobin has four subunits; each one can hold an oxygen molecule. Cooperativity means that each time hemoglobin grabs on to an oxygen, the affinity increases.
In other words, "he who has, gets," explained Doug Barrick, a biophysicist at Johns Hopkins University. Previous attempts to alter the molecule's overall oxygen affinity have managed to screw up its cooperativity. "It's a finicky molecule."
If, as Klein suggests, developing hemoglobin for a blood substitute is working against the flow of evolution, Ho said he nevertheless was able to take advantage of evolution's lessons. More than 700 mutant forms of hemoglobin have been found in nature, Ho explained, so he and his colleagues began looking at these mutants to see if evolution had already created what they wanted.
What they found was a mutant called hemoglobin Presbyterian (named for a hospital where it was first isolated). The hemoglobin molecule consists of four long chains of amino acids; in hemoglobin Presbyterian, one link in the chain has been changed from an amino acid called asparagine to another called lysine.
Its a subtle difference, but Ho's team found that this mutant had the low oxygen affinity they sought, while leaving cooperativity intact. What's more, it manages to keep its iron atoms safe from oxidation.
"The mutation is just what you need," Barrick said, for making blood substitutes.
But both Barrick and Ho agree that it's not yet ready for clinical testing. The mutant molecule will need to be strengthened; outside red blood cells, the molecule's four subunits tend to separate from each other, which can damage the kidneys.
Also, additional modification will be needed to counteract the molecule's unfortunate tendency to grab hold of nitric oxide, a molecule that relaxes blood vessel walls and thus reduces blood pressure.
Thus far, Ho has made the hemoglobin by inserting the mutant gene into E. coli bacteria. That technique has proven successful for producing human insulin and interferon, but blood substitutes will require much larger volumes of hemoglobin than can be produced economically in bacteria. Ho suspects that the human gene will eventually have to be transferred to pigs or other animals, creating a transgenic animal that can produce the human hemoglobin in its blood or its milk.
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