Mechanisms Involved in Preventing Unwanted Blood Clots 
bosis and examined whether elevation of the 
C4bBP levels to those found in patients would 
increase the risk of thrombotic complications 
(unwanted blood clots) . This was exactly what 
was observed. More importantly from a therapeu- 
tic point of view, simultaneously increasing pro- 
tein,S prevented the thrombotic complications. 
These results suggest that if we could block the 
decrease in free protein S, the risk of thrombosis 
would be reduced. This approach could poten- 
tially return the patient to normal status without 
significantly increasing the risk of bleeding. 
Recently it has become clear that clot forma- 
tion in the arteries of the heart is responsible for 
most heart attacks. Damage that results from the 
attack may involve injury to the small vessels that 
feed the heart. When we examined whether the 
protein C system helps prevent this damage, we 
found that protein C is activated following a short 
(2 min or less) blockage of an artery in the heart. 
When this activation is prevented, the heart re- 
covers less completely, if at all. These experi- 
ments imply that patients with decreased func- 
tion of the protein C system may have increased 
damage to the heart if they do have a heart attack 
and that activated protein C may prove a useful 
agent in preventing some aspects of this damage 
to the heart. Clinical trials will test these 
concepts. 
A major interest in our laboratory has been to 
understand how thrombomodulin causes throm- 
bin to change from a clotting to a clot-inhibiting 
enzyme. To understand this question, we have 
made mutants of human thrombin, because the 
binding of thrombin to thrombomodulin proba- 
bly changes thrombin's shape. This shape change 
probably accounts for how the complex acceler- 
ates protein C activation. One possibility is that 
thrombin has one or two specific amino acids that 
hit protein C in such a way that it cannot "dock" 
or bind with thrombin effectively. If this were the 
case, then altering the amino acids that block the 
docking event could make thrombin a better acti- 
vator. We have examined two classes of such 
changes. In proteins, likes repel and unlikes at- 
tract. In the case of protein C, very close to the 
place where activation occurs, there is a nega- 
tively charged amino acid. There is also a nega- 
tive amino acid in the region of thrombin very 
near where this negatively charged amino acid 
must dock. If the slow activation of protein C by 
thrombin is related to the charge repulsion (it is 
difficult to get negative charges together), then 
removing this negative charge from either throm- 
bin or protein C should increase the rate of pro- 
tein C activation in a manner analogous to forma- 
tion of the thrombin-thrombomodulin complex. 
When either the negative charge on protein C or 
on thrombin is removed, protein C activation is 
increased 5-20 times. This change in thrombin 
does not influence the ability to clot fibrinogen. 
These studies provide insights into how regula- 
tory proteins such as thrombomodulin may func- 
tion and also provide the framework for the 
design, by selective mutation, of new anticoagu- 
lants that alter the function of normal clotting 
proteins. 
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