BIOCHEMISTRY AND PHYSIOLOGY OF THE PROTEIN C ANTICOAGULANT PATHWAY 
Charles T. Esmon, Ph.D., Investigator 
During blood coagulation, prothrombin must be 
converted to the serine protease thrombin. Throm- 
bin not only clots blood but also is a major regula- 
tory protein in this process, generating both ampli- 
fying and inhibiting signals. How thrombin selects 
its substrate, and hence whether it functions as a 
clot-promoting or -inhibiting enzyme, is the major 
focus of Dr. Esmon's laboratory. 
Thrombin functions as an anticoagulant by acti- 
vating the anticoagulant zymogen protein C. Activa- 
tion occurs when thrombin complexes with throm- 
bomodulin, an endothelial cell receptor. Compared 
with the other complexes responsible for activation 
of coagulation zymogens, both the enzyme throm- 
bin and the regulatory protein thrombomodulin 
have much simpler structures, suggesting that de- 
tailed analysis of this system might serve as a model 
for the others. Three recent advances are of particu- 
lar utility: 1) small (10- to 15-kDa) thrombomodu- 
lin fragments, corresponding to two (GF5-6) or 
three (GF4-6) epidermal growth factor (EGF)-like 
repeats from the intact thrombomodulin molecule, 
can be formed that bind thrombin and either do or 
do not accelerate protein C activation; 2) hirudin, a 
specific thrombin inhibitor from leeches, interacts 
with thrombin through two separate domains, both 
of which inhibit thrombin's activity; and 3) the 
crystal structure of thrombin has recently been de- 
termined with and without hirudin bound to the 
enzyme. Thrombin's structure reveals that it differs 
from most proteases by having an extended groove 
that runs almost around the enzyme. This groove is 
occupied by the carboxyl-terminal portion of hiru- 
din, referred to as hirugen. This groove contains 
many basic residues and is referred to as the anion- 
binding exosite. Competition experiments revealed 
that hirugen and GF5-6 share overlapping binding 
sites on thrombin and that both fragments elicit com- 
parable conformational changes and alterations in 
thrombin specificity. When GF4-6 binds thrombin, 
additional conformational changes occur that result 
in altered specificity toward small substrates as well 
as protein C. These results indicate that thrombo- 
modulin interacts with two distinct sites on throm- 
bin that function in concert to alter thrombin allo- 
sterically and change its specificity to accommodate 
the activation site in protein C. 
Allosteric changes in specificity also appear to be 
involved in thrombin-dependent cell activation. 
Cellular activation involves cleavage of a thrombin 
receptor, as recently demonstrated by Dr. Shaun 
Coughlin's group (University of California, San Fran- 
cisco). Analysis of the structure-function properties 
of the receptor revealed that a peptide from the re- 
ceptor binds to thrombin in the anion-binding exo- 
site and elicits a conformational change in thrombin 
comparable to that produced by GF5-6. Consistent 
with this concept, thrombin mutants lacking the ac- 
tive-site serine still bind either thrombomodulin or 
the thrombin receptor and block both the forma- 
tion of anticoagulant activity and cell activation. 
These results provide a molecular explanation for 
how thrombomodulin blocks cell activation by 
thrombin. 
Direct analyses of the assembly of the protein C 
activation complex were performed in the ultracen- 
trifuge. The ternary complex between protein C, 
thrombin, and thrombomodulin could be detected 
and demonstrated a Ca^^ dependence consistent 
with the activation kinetics. Surprisingly, activated 
protein C bound to the activation complex with the 
same affinity as the substrate protein C. These obser- 
vations were confirmed and extended by demon- 
strating that activated protein C behaves as a compet- 
itive inhibitor of protein C activation, with a 
approximately equivalent to the for protein C. 
Current work is focused on the possibility that this 
interaction of activated protein C with thrombomo- 
dulin alters the function of activated protein C. 
During prothrombin activation, two proteolytic 
cleavages in the zymogen are required for activa- 
tion. Only after both cleavages occur does thrombin 
activate protein C or cells effectively. Direct bind- 
ing studies were initiated to determine when the 
anion-binding exosite is formed during prothrom- 
bin activation. Surprisingly, both of the potential 
activation intermediates possess the anion-binding 
exosite. Thus, although this deep binding pocket is 
formed in the intermediates, further structural 
changes are required for productive interaction 
with cells or thrombomodulin. 
Factor V, one of the regulatory proteins in coagula- 
tion, is also a thrombin substrate. Relatively little is 
known about why thrombin interacts with factor V 
with high affinity. As one approach to investigating 
this question further. Dr. Enriqueta Guinto in Dr. 
Esmon's laboratory completed the analysis of the 
bovine factor V sequence. Because of relative abun- 
dance and stability, most of the information 
currently available about the function of factor V is 
50 
