BIOCHEMISTRY AND PHYSIOLOGY OF THE PROTEIN C ANTICOAGULANT PATHWAY 
Charles T. Esmon, Ph.D., Investigator 
I. Thrombomodulin-Thrombin Interaction. 
ThrombomoduHn is an integral membrane pro- 
tein that binds thrombin and accelerates the activa- 
tion of protein C more than 1,000-fold. The bind- 
ing interaction also inhibits the capacity of 
thrombin to clot fibrinogen. The overall structure 
of thrombomodulin is reminiscent of the low-den- 
sity lipoprotein (LDL) receptor, with an amino-ter- 
minal domain, a series of six growth factor repeats, 
an O-linked sugar region, a transmembrane do- 
main, and a cytosolic tail. Limited digestion of 
thrombomodulin with elastase generated a soluble, 
active form of thrombomodulin that was composed 
essentially only of the growth factor repeats. CNBr 
digestion revealed that residues 310-486 were all 
that were required for acceleration of thrombin-cat- 
alyzed protein C activation and that a small domain 
corresponding to the carboxyl-terminal 80 residues 
of this fragment contained the major thrombin- 
binding site. Although this fragment could not ac- 
celerate protein C activation, it retained the capac- 
ity to inhibit fibrinogen clotting. 
With the identification of the thrombin-binding 
site, new questions were posed about the topo- 
graphical distribution of thrombin, thrombo- 
modulin, and the membrane surface. These ques- 
tions were addressed by fluorescence energy 
transfer between a dansyl moiety in the active cen- 
ter of thrombin and octadecylrhodamine in the 
membrane. The active center of thrombin was lo- 
cated —65 A above the membrane. Thus the O- 
linked sugar domain must rise essentially vertically 
from the membrane surface. The distance to the 
membrane surface of thrombin in the thrombin- 
thrombomodulin complex is almost identical to 
that found when the distance is measured in pro- 
thrombin. Therefore, although thrombomodulin 
and the prothrombin activation fragments have al- 
most no sequence similarity, the two form similar 
"platforms" on which the en2yme rests. 
The interaction with thrombomodulin also al- 
ters the fluorescence properties of the dansyl dye 
in the active center of thrombin, supporting the 
concept that thrombomodulin alters the catalytic 
properties of thrombin by altering the conforma- 
tion of thrombin. 
II. Factor V Structure and Function. 
Factor V is a large (300 kDa) plasma protein that 
is proteolytically modified by thrombin to an active 
form Va composed of two nonidentical subunits 
held together in a calcium-dependent interaction. 
Previous studies indicated that only a single Ca^^ is 
involved in the interaction and that the kinetics of 
association are extremely slow and very tempera- 
ture dependent. These properties suggested that 
major conformational changes might be involved in 
the reassociation process. Fluorescence and circu- 
lar dichroism studies were used to investigate the 
extent of structural changes in factor Va during 
reassociation. The results indicated that the struc- 
tural changes, although reproducible, were small 
and correlated with the formation of biological ac- 
tivity. Thus the original hypothesis that the slow and 
very temperature dependent reassociation was due 
to major conformational changes seems unlikely. 
Although the sequence of factor V is known, rela- 
tively little is known about the location of specific 
binding sites on the protein or the mechanisms by 
which they function. Studies in collaboration with 
Drs. Arthur Johnson and Thomas Laue have re- 
vealed that binding factor Va to both factor Xa and 
prothrombin alters the distance to the membrane 
surface. Thus one potential mechanism involved in 
factor V function may be to align the proteolytic 
center of factor Xa with the activation site on pro- 
thrombin. Previous studies from Dr. Esmon's labo- 
ratory indicated that the major prothrombin-bind- 
ing site on factor Va was on the heavy chain of the 
heterodimer. Direct equilibrium binding interac- 
tions were undertaken using the analytical ultra- 
centrifuge. These studies confirmed and extended 
the previous qualitative observations. The heavy 
chain forms a Ca^^-independent, 1:1 complex with 
prothrombin that is governed by a dissociation con- 
stant of 10 |jlM. The cleavage of the heavy chain 
with activated protein C abolishes all detectable as- 
sociation of the heavy chain with prothrombin. 
These findings support the concept that the regula- 
tory proteins in coagulation function in part by 
binding to the substrates. 
III. Physiological Studies. 
The protein C pathway appears to be modulated 
by inflammatory mediators. During septic shock, 
the levels of C4b-binding protein (C4bBP) rise, and 
this serves to complex protein S and inhibit the an- 
ticoagulant pathway. This raised the question of 
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