By David N. Leff

From the time, ten centuries ago, when celebrated Arab physician Abu el-Qasim first described the disease we now call hemophilia, until half a century ago, that bleeding disorder was lethal in childhood.

Male infants literally bled to death from minor injuries, because two genes on their X chromosome failed to express clotting Factor VIII or IX. Their sisters escaped the death-dealing disease, because females carry two X chromosomes, only one of which was defective.

That was then — before the 1950s, when blood transfusions came on the scene. They provided the missing factors to life-threatened hemophiliac youngsters. That made it possible for afflicted males to survive into their adult years. "They generated a spectrum of daughters," said molecular biologist Randal Kaufman, "who were carrying the mutant alleles, and now those daughters are just giving rise to a new population of males that are carrying the disease."

But then came AIDS.

"In the mid-1980s," Kaufman observed, "over 95 percent of the hemophiliacs became HIV-positive, because of blood-bank donor blood contaminated with the AIDS virus. A majority of those individuals have passed on," he added, "and the prevalence of the HIV infection rate has basically dropped to zero, with the advent of recombinant Factor VIII and more highly purified plasma products."

Kaufman is a Howard Hughes investigator and professor of biological chemistry at the University of Michigan Medical Center, in Ann Arbor.

"Today," he observed, "there are probably 20,000 boys and men with hemophilia, and the prevalence in the population is about one in 10,000 male births. Of these, 85 percent have deficient Factor VIII; 10 percent, Factor IX."

Infusions of recombinant Factor VIII may have banished the sure, slow death of AIDS for hemophiliacs, but these virus-free products pack drawbacks of their own.

As patients live longer, the disease tends to manifest itself by frequent bleeding into the joints, causing severe arthritis and a lot of joint destruction.

"Probably the major detriment for recombinant Factor VIII," Kaufman observed, "is the requirement of high doses of the protein. Patients need to take fairly significant levels of antigen, and as a consequence they react to the factor as a foreign protein, and develop antibodies that inhibit its clot-forming action."

He continued: "They must take Factor VIII injections every two weeks on average, at fairly high cost, probably $1,000 to $2,000 per dose. And that protein has a half-life of about 12 hours."

Compounding These Liabilities — Lability

"One of the real problems with studying the function of Factor VIII," Kaufman explained, "has been the lability of the activated species. It's inactive in blood plasma until activated by protein cleavage. Then it functions to facilitate the protease coagulation cascade, which results in clotting.

"The problem arises," he continued, "when you activate Factor VIII. How much of it is activated? How much is too quickly inactivated? What is the contribution of these two processes to clotting and anti-clotting?"

That lose-lose situation encouraged Kaufman "to try to make a Factor VIII protein that would be hyperactive. If it were more active than current molecules, and resistant to the inactivation process, maybe it would reduce the dosage requirements and cost. For once, it might make available prophylaxis treatment, rather than coping with bleeding episodes as they occurred."

Kaufman observed, "If patients could be treated prophylactically, rather than on demand, a lot of those secondary problems would go away. It's comparable to regular insulin replacement for diabetics."

Today's issue of the Proceedings of the National Academy of Sciences, dated Oct. 28, 1997, reports on the novel molecule that Kaufman and his colleagues constructed. The article's title: "Characterization of a genetically engineered inactivation-resistant coagulation factor VIIIa."

Clotting Based On Delicate Balance

In healthy people, the clotting cascade operates a series of feedback governors to ensure a safe balance between too much coagulation and too little. But in hemophiliacs, that safety factor overlooks the need for extra clotting power, and hence inactivates Factor VIII too soon.

"Two mechanisms combine to achieve this inactivation," Kaufman pointed out. "When Factor VIII gets activated, it forms from the original polypeptide a heterotrimer [three-part molecule] of three subunits. One of these, the A2 subunit, falls off, and the trimer falls apart.

"The other mechanism of inactivation," he continued, "is managed by an enzyme, activated protein C, which cleaves Factor VIII. So what we did — using classical mutagenesis, and expression in mammalian host cells — was engineer a molecule resistant to both of these activities.

"In wild-type Factor VIII," Kaufman went on, "two cleavages are required. We engineered the molecule so it only required one."

All told, they deleted 895 of the Factor VIII protein's 2,332 amino acids, leaving a 642-residue IR8 molecule. His resulting stripped-down molecule "had a very high activity in cultured monkey cells — fivefold higher than wild-type Factor VIII. And it maintained its high level of activity and stability for four hours up to a day."

This performance in vitro led the co-authors to the concern "that if we made a superactive Factor VIII, would it be toxic in vivo?"

So recently they injected their new molecule, which they call IR8 — inactivation resistant — into transgenic hemophiliac mouse models. "It works," Kaufman said, "and it's not toxic. We don't know yet how well it works compared to wild-type Factor VIII. We do know, at least, that it's capable of correcting the bleeding disorder."

Colonies of beagle-size dogs exist, that are naturally hemophiliac, and so nearer than rodents to human models. "Dogs are what we want to do next," Kaufman said. "We just haven't scaled up enough IR8 protein to do that yet. Hopefully, we will test it in dogs in the next six months."

The University of Michigan, and its Howard Hughes Medical Institute, have filed for patents on IR8, Kaufman said, adding, "It's presently being licensed to Genetics Institute, of Cambridge, Mass." This company, along with Baxter International, of Deerfield, Ill., and Bayer Corp., of Pittsburgh, are the principal manufacturers of recombinant Factors VIII and IX for treating hemophilia." *