All the King's Horses and All the King's Men

By Jason Socrates Bardi

Somewhere between excessive bleeding and aberrant clotting is coagulatory homeostasis—a protective mechanism ensuring that in our world full of thorns we neither bleed nor clot to death.

Coagulation is a complex protease cascade involving about 30 interacting proteins and platelets (those flat, molecule-filled cytoplasmic disks in the blood). The cascade starts when a protein called tissue factor is exposed to the bloodstream due to a cut or other injury. Tissue factor ultimately generates thrombin, a clotting enzyme that circulates in the bloodstream as an inactive "zymogen" protein called prothrombin. Thrombin causes platelets to aggregate and causes fibrin to polymerize, thus forming the clot.

Thrombin also propagates coagulation by activating various proteins by proteolysis—cleaving them at specific points in their amino acid sequences. One of the proteins it cleaves is called Factor VIII. Factor VIII also circulates in the blood as a large, inactive zymogen, and active Factor VIIIa is about half the molecular weight of Factor VIII and is composed of three distinct chains rather than one long protein.

Having three chains makes Factor VIIIa very unstable, and once the chains dissociate, there is no way to get them back together. They are quickly lost in the flow of blood and the odds of them re-associating are astronomically low.

Now a team of scientists may have found a way to stabilize Factor VIIIa in solution coaxing the subunits to form a disulfide bond—like handcuffing them together on the molecular level. This discovery, made by Assistant Professor Andrew Gale of The Scripps Research Institute and former institute research associate Jean-Luc Pelleque, may lead to an improved therapy for hemophilia A.

Instability and Hemophilia

The instability of Factor VIIIa is fantastic for putting an end to clotting before it gets out of hand. Clotting, after all, is a process that one normally would want to control.

But the instability of Factor VIIIa is bad news for people who suffer from hemophilia A, the most common form of the disease. This bleeding disorder is caused by a swarm of different heritable mutations that cause deficiencies in Factor VIII. Patients with hemophilia A have no Factor VIII or not enough Factor VIII, and this causes them problems with blood clotting. Some 17,000 Americans suffer from this disease.

Hemophilia A is often treated with infusions of Factor VIII protein. But because these patients have diminished ability to make the protein themselves, the instability of Factor VIIIa is a problem because as the subunits dissociate, the protein loses its activity, and the infusion loses its potency.

That's where the new study by the Scripps Research scientists comes in.

Gale and his colleagues wanted to find a form of Factor VIIIa protein that would be more stable, and they decided to try to make a mutant form of the protein with sulfur-containing cysteine residues at strategic locations in two of the three chains. They carefully chose two spots to mutate so that the cysteines on opposing chains would covalently join and two of the three chains would be held together by a disulfide bond.

In a recent edition of the Journal of Thrombosis and Haemostasis, Gale and his colleagues describe their results. They found that their mutant form of Factor VIIIa held together in its three-subunit form and was active much longer than the normal form of the protein.

This more active Factor VIIIa protein may perform longer in the body as well, and might improve the effectiveness of Factor VIII infusions by reducing the number of such infusions a hemophiliac would need and by reducing the amount of Factor VIII that would need to be infused each time.

However, the scientists caution that they have not yet observed the effect of the new stabilized form of Factor VIIIa on the body, and further studies need to be conducted to determine whether the body could handle the new form of the protein. Factor VIIIa is also inactivated by a protein in the coagulation cascade called activated protein C. Gale and his colleagues hope that activated protein C would still be able to degrade Factor VIIIa and that clotting would not get out of hand.

To read the article "An engineered interdomain disulfide bond stabilized human blood coagulation factor VIIIa" by A.J. Gale and J-L Pellequer, please see the Journal of Thrombosis and Haemostasis 1, 1966-71 (2003).




Location of engineered 664-1826 disulfide bond relative to other cysteine residues in the fVIIIa A domains. Click to enlarge