RESEARCH SUMMARY:
Proteases of the coagulation cascade
are a series of structurally similar trypsin-like enzymes which
normally exist in the circulatory system as inactive precursors
and are only activated when the integrity of the blood vessel
is compromised. The cascade is initiated when a natural cell
surface membrane protein called tissue factor, which is not
normally found in the circulatory system, becomes available
for interaction with factor VII. The factor VII-tissue factor
complex activates itself and two other factors, IX and X, to
their functionally active forms (IXa and Xa). Factor Xa in the
presence of its specific cofactor, factor Va, activates prothrombin
to thrombin. Thrombin then cleaves fibrinogen to form a blood
clot.
For maintenance of normal hemostasis, thrombin
generation must be regulated and the activities of the coagulant
enzymes following clot formation must be neutralized and subsequently
removed from circulation in order to assure that they will
not generate unnecessary blood clots that would occlude the
vessels. Two pathways are known to regulate the coagulation
cascade. The first pathway, called protein C pathway, is initiated
when thrombin binds to a cell surface membrane protein called
thrombomodulin (TM), present on the surface of the endothelial
cells that line blood vessels. Thrombin in complex with TM
no longer clots fibrinogen, but instead converts protein C
to activated protein C (APC). APC is a potent anticoagulant.
It shuts down thrombin generation by degrading the cofactors
Va and VIIIa which are essential for thrombin generation.
In the second pathway, protease inhibitors
present in plasma regulate the proteolytic function of the
coagulation proteases by binding directly to the active center
of the enzymes and trapping them in the form of inactive complexes.
A deficiency in the components of either pathway is associated
with increased risk of thrombosis with possible severe consequences
including heart attacks, strokes, and pulmonary emboli. Antithrombin
is a serine protease inhibitor that is known to be the primary
regulator of the clotting cascade in the second pathway. Its
inhibitory activity is greatly enhanced when it binds to the
heparin-like molecules present on the vessel wall. This is
the basis for the widespread use of heparin as the primary
anticoagulant drug in the treatment of thrombotic complications.
Our research is focused on understanding how factor Xa, thrombin,
and APC specifically interact with their target cofactors,
substrates, and inhibitors, and how heparin enhances the inhibitory
function of antithrombin in regulation of the proteolytic
activities of two key clotting enzymes, factor Xa and thrombin.
We employ biophysical, biochemical, and molecular biological
methods to study these questions.
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