MOLECULAR MECHANISMS AND VASCULAR DISEASE 
Shaun R. Coughun, M.D., Ph.D., Assistant Investigator 
This year has represented a major change in di- 
rection for Dr. Coughlin's laboratory, with the addi- 
tion of two new projects. 
L Molecular Mechanisms of Thrombin Action. 
Dr. Coughlin's laboratory is pursuing a program 
to identify 1) the structural determinants in throm- 
bin required for cell activation and 2) the thrombin 
"receptor" on platelets and other cells. 
A. Thrombin biology. Thrombin is a multifunc- 
tional serine protease. It is produced from the cir- 
culating zymogen prothrombin by cleavage on cell 
and platelet surfaces when active prothrombinase 
complexes are present, as occurs in vascular injury. 
The thrombin concentrations reached during blood 
clotting are more than sufficient to elicit various 
cellular responses to thrombin. 
In addition to its well-known role in cleaving fi- 
brinogen to fibrin during blood coagulation, 
thrombin has a number of important cell-activating 
functions. Thrombin is the most potent physiologi- 
cal stimulator of platelet aggregation, and recent 
studies suggest that thrombin's actions are critical 
for platelet plugging and arterial thrombus forma- 
tion. A number of the actions of thrombin on other 
circulating cells and on the component cells of the 
blood vessel wall are potentially important. Throm- 
bin is a potent mitogen for lymphocytes and is che- 
motactic for monocytes. Thrombin elicits a number 
of responses from vascular endothelial cells, includ- 
ing triggering expression of adhesive molecules for 
neutrophils and the potent smooth muscle cell mi- 
togen platelet-derived growth factor (PDGF). 
Thrombin is also a potent mitogen for vascular 
smooth muscle cells and fibroblasts. Tissue factor 
expression by macrophages in atherosclerotic 
plaques has recently been reported; it is not known 
whether local intramural production of thrombin 
occurs outside of the context of blood clotting or 
whether it plays a role in vascular proliferative or 
inflammatory responses. Thus the cell-activating 
functions of thrombin appear to be critical for nor- 
mal hemostasis and may play an important role in 
proliferative and inflammatory responses in the ves- 
sel wall. 
The mechanisms by which thrombin activates 
platelets and other cells are unknown, and the cell 
surface receptor that mediates thrombin action has 
not been identified. An understanding of these 
mechanisms is likely to reveal novel signal trans- 
duction mechanisms. In addition, because throm- 
bin binding to cells and platelets does not reflect 
binding to the functional thrombin receptor, identi- 
fication of the receptor and its mode of activation 
will be important for the development of therapeu- 
tics directed at the thrombin receptor. 
B. Thrombin mechanisms. Dr. Coughlin's labora- 
tory is producing recombinant thrombins that ad- 
dress thrombin's mechanism of action. One set of 
mutant thrombins addresses the requirement of 
thrombin protease activity for activation of cells and 
platelets. There is considerable debate as to 
whether thrombin activates cells by cleaving its re- 
ceptor or by a more classical binding mechanism. 
The goal is not only to answer this mechanistic 
question but also to produce novel reagents for the 
study and purification of the thrombin receptor. 
Studies with recombinant trypsins have shown that 
it is possible to ablate protease activity by amino 
acid substitutions at catalytic triad residues; these 
mutant trypsins were catalytically inactive but were 
able to bind substrate normally. In collaboration 
with Dr. Charles Craik, Dr. Coughlin was able to 
show that the mutant trypsins would block the ac- 
tion of wild-type en2yme by competing for sub- 
strate. Dr. Coughlin's laboratory then developed an 
expression system for prothrombin and used 
ecarin, a snake venom protease, to convert the re- 
combinant prothrombin to active thrombin. Meth- 
ods to purify and quantitate prothrombin protein 
were also developed. The active-site mutations 
analogous to those described for trypsin have been 
introduced into the prothrombin cDNA. Once the 
mutant thrombins are expressed, their ability to ac- 
tivate cells and platelets will be assessed. If pro- 
teolytically inactive thrombin is capable of acting as 
a thrombin agonist, it will prove the occupancy the- 
ory of thrombin activation. If the proteolytically in- 
active mutant thrombin does not act as an agonist, 
it will be necessary to demonstrate that it still binds 
to the receptor to conclude that proteolytic activity 
is required for activation. To demonstrate binding. 
Dr. Coughlin will assess the ability of the mutant to 
act as a thrombin antagonist. Preliminary studies 
with trypsin mutants suggest that the analogous 
mutant thrombins will still bind their receptor, and 
a definitive answer to the activation by binding ver- 
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