By David N. Leff
When you're cut, you bleed.
Blood pours out of the wound, ferrying to the scene white cells of the immune system, loaded to shoot down any entering germs or viruses, and — like sandbagging a flooded riverbank — emplacing wound-healing molecules. The outward bleeding flow may also wash away impurities that have entered the lesion.
Then that outpouring tide slows and stops, coffer-dammed with clots to keep you from bleeding to death.
In the U.S., some 25,000 men and boys (but not women and girls) are not so lucky. Their blood lacks one or another of the dozen or so proteins that make up the coagulation cascade, which forms blood clots. As is well known, they suffer from a males-only inherited blood deficiency called hemophilia.
Unless transfused with blood or its cascade products, hemophiliacs are in constant danger of bleeding to death.
The commonest culprits in hemophilia are mutated or deleted genes encoding factors VIII and IX. Factor VIII accounts for 80 percent of that 25,000 hemophiliac population, factor IX for the remaining 20 percent. Both proteins are extracted from whole donor blood as frontline therapy to protect hemophilia victims from hemorrhages, internal as well as external.
Biochemist Gary Nelsestuen is a research scientist at the University of Minnesota's College of Biological Sciences, in St. Paul.
He is focussing on the cascade's factor VII, which comes ahead of VIII and IX in the clotting parade. "Of all the human coagulation proteins," Nelsestuen told BioWorld Today, "we targeted factor VII first of all. Normally," he explained, "we think of VII as coming in before VIII or IX in the series of events, but in fact it is capable of bypassing VIII and IX altogether."
Because of this capability, recombinant factor VII in large doses "is used in many countries as emergency therapy for problem cases of hemophilia. These arise when a patient's system no longer recognizes factor VIII, and his immune defenses knock it out with antibody resistance. These patients," Nelsestuen added, "get to be what's called the real desperation cases; in the U.S., compassion cases. Recombinant factor VII, although proved very effective abroad, has not yet been approved by the FDA, although it should have been long ago."
Recombinant factor VII is made by Novo Nordisk A/S, of Bagsvaerd, Denmark. "Last year," Nelsestuen observed, "its sales of the product amounted to $52 million."
Perhaps factor VII's major drawback, he observed, is the drug's price tag. "The costs are astronomical," he said. "The numbers that are thrown around are about $100,000 per patient per year."
This situation led Nelsestuen to tackle the hang-ups besetting cascade proteins at their basic science level, namely, the problem of membrane contact and binding.
Cell Injury Bares Attachment Sites
"These proteins, " he pointed out, "in order to function, must attach to a cell membrane. In a normal cell, the outside of its plasma membrane does not have the attachment site at which cascade factors can bind. Those docking locations consist of lipids called phosphatidyl serine (PtdSer). They're all located on the inside of the cell. That's the way all mammalian cells are designed.
"Therefore, as the blood normally circulates, its clotting factors do not attach to intact cells. But if you get cell damage," he went on, "the PtdSer on the inside becomes exposed, and these proteins now attach to them. That is one of the triggering mechanisms for coagulation. Most everyone in the field," he recalled, "thought the membrane attachment problem was solved. We didn't. We kept looking.
"We wanted, with our basic science goal of understanding the membrane contact, to make factor VII better. Our long-range goal was to try to improve all these proteins. Redesign the site, make it more effective, and that's what we've accomplished.
"Factor VII," Nelsestuen observed, "now has the potential to work more effectively. It already has, in the test tube; no question, it's much better."
His answer to factor VII's problems is spelled out by the title of a research paper in the current Proceedings of the National Academy of Sciences (PNAS), dated April 14, 1998: "Manipulation of the membrane binding site of vitamin K-dependent proteins: Enhanced biological function of human factor VII."
"What we did, "Nelsestuen recounted, "was try to increase factor VII's binding affinity. We targeted specific amino acids, and said: 'If we changed these, and if ourmechanism is right, we will increase the binding affinity.' So when the PtdSer is exposed, this factor VII is going to go to the membrane system in larger amounts, because it binds with higher affinity. That is what our PNAS paper shows."
To accomplish this feat, he and his co-authors changed two amino acids in the factor VII sequence, right in the region of the membrane contact site. They swapped a lysine for a glutamic acid at position 32 on the factor VII chain.
Vitamin K, as Nelsestuen and two other laboratories discovered in 1974, is involved in modifying glutamic acids.
Clinical Trials Await Big Pharma Bids
"A big mystery," he mused, "is why nature has made an imperfect membrane binding site. But I've got two explanations. One is that if you get a very high affinity protein, turnover rates may not be fast enough. The other, adjusting membrane binding affinity may be simply nature's way of balancing pro- and anti-coagulant forces."
Nelsestuen and his co-authors do not plann to conduct clinical trials of the beefed-up factor VII themselves. "We are in the process of trying to enlist a pharmaceutical company," he said, "and several have shown an interest. Novo is one of them. I would hope that once we get this lined up, they would be in an excellent position to try these out and get started on them very quickly."
Rather than raise the recombinant proteins in their own lab, Nelsestuen's group recruited Seattle-based ZymoGenetics Inc., a wholly-owned subsidiary of Novo Nordisk, to make them.
The Minnesota biochemist is now moving to the other side of the clotting street, to develop a vitamin K dependent anti-coagulant, human protein C.
"Protein C is actually involved in preventing clots," he pointed out. "It might be adminsitered to anyone who is subject to unwanted clot formation in a crisis situation." *