Thrombin possesses two positively charged surface domains, termed exosites,
that orient substrates and inhibitors for reaction with the enzyme. Becaus
e the exosites also allosterically modulate thrombin's activity, we set out
to determine whether the structure or function of the exosites changes whe
n thrombin forms complexes with antithrombin, heparin cofactor II, or alpha
(1)-antitrypsin (M358R), serpins that utilize both, one, or neither of the
exosites, respectively. Using a hirudin-derived peptide to probe the integ
rity of exosite 1, no binding was detected when thrombin was complexed with
heparin cofactor II or alpha (1)-antitrypsin (M358R), and the peptide exhi
bited a 55-fold lower affinity for the thrombin-antithrombin complex than f
or thrombin. Bound peptide or HD-1, an exosite 1-binding DNA aptamer, was d
isplaced from thrombin by each of the three serpins. Thrombin binding to fi
brin also was abrogated when the enzyme was complexed with serpins. These d
ata reveal that, regardless of the initial mode of interaction, the functio
n of exosite 1 is lost when thrombin is complexed by serpins. In contrast,
the integrity of exosite 2 is largely retained when thrombin is complexed b
y serpins, because interaction with heparin or an exosite 2-directed DNA ap
tamer was only modestly altered. The disorganization of exosite 1 that occu
rs when thrombin is complexed by serpins is consistent with results of prot
ease sensitivity studies and crystallographic analysis of a homologous enzy
me-serpin complex.