All the King's Horses and All the King's Men
By Jason Socrates
Somewhere between excessive bleeding and aberrant clotting
is coagulatory homeostasisa protective mechanism ensuring
that in our world full of thorns we neither bleed nor clot
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 proteolysiscleaving 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
bondlike 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
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
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).