based on analysis of the bovine molecule. This study 
provides a structural basis to understand these func- 
tional studies better. 
The entire process of blood coagulation requires 
Ca^^, which is involved not only in interaction of 
the clotting proteins with cell surfaces but also in 
protein-protein interactions. In most of the coagula- 
tion complexes, understanding whether the Ca^^ ef- 
fect is on the substrate, enzyme, or regulatory factor 
is complicated by the fact that all of the proteins 
interact with Ca^^. Previous work from Dr. Esmon's 
laboratory demonstrated that in protein C activa- 
tion, Ca^^ interaction with the substrate is critical to 
activation. To determine if other substrates undergo 
similar changes, activation of factor X was analyzed 
with a thrombin mutant having altered primary sub- 
strate specificity, E192Q. Since thrombin does not 
bind Ca^^, any changes in activation would reflect 
alterations in the substrate. These studies indicated 
that factor X, like protein C, undergoes conforma- 
tional changes at or near the scissile bond that alter 
protease sensitivity, presumably contributing to pro- 
ductive interaction with the physiological activa- 
tion complexes. 
Indirect experiments suggested that Ca^^ interac- 
tion with the first of the two EGF-like domains of 
protein C is probably critical to the activation pro- 
cess. To test this hypothesis directly, a deletion mu- 
tant was expressed and isolated lacking the entire 
amino-terminal region of protein C and this EGF do- 
main. The resultant molecule binds Ca^^, and the 
activation process is still Ca^^ dependent, suggest- 
ing that although the first EGF domain can bind 
Ca^^, it is not the critical site involved in protein C 
activation. Grants from the National Institutes of 
Health helped support all of the work described 
above. 
Dr. Esmon is also a member and head of the 
Cardiovascular Biology Research Program at the 
Oklahoma Medical Research Foundation, Okla- 
homa City, and OMRF Professor of Pathology and 
Associate Professor of Biochemistry at the Univer- 
sity of Oklahoma Health Sciences Center. 
Articles 
Esmon, C.T. 1992. The protein C anticoagulant 
pathway. Arterioscler Thromb 12:135-145. 
Esmon, C.T., Taylor, F.B.,Jr., and Snow, T.R. 1991. 
Inflammation and coagulation: linked processes 
potentially regulated through a common pathway 
mediated by protein C. Thromb Haemost 
66:160-165. 
Guinto, E.R., Esmon, C.T., Mann, K G., and MacGil- 
livray, R.T.A. 1992. The complete cDNA se- 
quence of bovine coagulation factor V. / Biol 
Chem 267:2971-2978. 
Hung, D.T., Vu, T.-K.H., Wheaton, V.L., Charo, I., 
Nelkon, N.A., Esmon, N.L., Esmon, C.T., and 
Coughlin, S.R. 1992. "Mirror image" antagonists 
of thrombin-induced platelet activation based on 
thrombin receptor structure. / Clin Invest 
89:444-450. 
Le Bonniec, B.F., Guinto, E.R., and Esmon, C.T. 
1992. The role of calcium ions in factor X activa- 
tion by thrombin E192Q. / Biol Chem 267: 
6970-6976. 
Liu, L.-W., Vu, T.-K.H., Esmon, C.T., and Coughlin, 
S.R. 1991. The region of the thrombin receptor 
resembling hirudin binds to thrombin and alters 
enzyme specificity. / Biol Chem 266:16977- 
16980. 
Liu, L.-W., Ye, J., Johnson, A.E., and Esmon, C.T. 
1991. Proteolytic formation of either of the two 
prothrombin activation intermediates results in 
formation of a hirugen-binding site, f Biol Chem 
266:23633-23636. 
Olsen, P H., Esmon, N.L., Esmon, C.T., and Laue, 
T.M. 1992. The Ca^^-dependence of the interac- 
tions between protein C, thrombin, and the elas- 
tase fragment of thrombomodulin. Analysis by ul- 
tracentrifugation. Biochemistry 31:746-754. 
Rezaie, A.R., Esmon, N.L., and Esmon, C.T. 1992. 
The high affinity calcium-binding site involved in 
protein C activation is outside the first epidermal 
growth factor homology domain. / Biol Chem 
267:1 1701-1 1704. 
Wakefield, T.W., Wrobleski, S.K., Sarpa, M.S., Tay- 
lor, F.B., Jr., Esmon, C.T., Cheng, A., and Green- 
field, L.J. 1991. Deep venous thrombosis in the 
baboon: an experimental model. / Vase Surg 
14:588-598. 
Ye, J., Esmon, N.L., Esmon, C.T., and Johnson, A.E. 
1991 . The active site of thrombin is altered upon 
binding to thrombomodulin. Two distinct struc- 
tural changes are detected by fluorescence, but 
only one correlates with protein C activation. / 
Biol Chem 266:23016-23021. 
Ye, J., Liu, L.-W., Esmon, C.T., and Johnson, A.E. 
1992. The fifth and sixth growth factor-like 
domains of thrombomodulin bind to the anion- 
binding exosite of thrombin and alter its specific- 
ity. /S/o/ Chem 267:11023-1 1028. 
CELL BIOLOGY AND REGULATION 5 1 
