Exosite-mediated substrate recognition of factor IX by factor XIa. The factor XIa heavy chain is required for initial recognition of factor IX.
Studies of the mechanisms of blood coagulation zymogen activation demonstrate that exosites (sites on the activating complex distinct from the protease active site) play key roles in macromolecular substrate recognition. We investigated the importance of exosite interactions in recognition of factor IX by the protease factor XIa. Factor XIa cleavage of the tripeptide substrate S2366 was inhibited by the active site inhibitors p-aminobenzamidine (Ki 28 +/- 2 microM) and aprotinin (Ki 1.13 +/- 0.07 microM) in a classical competitive manner, indicating that substrate and inhibitor binding to the active site was mutually exclusive. In contrast, inhibition of factor XIa cleavage of S2366 by factor IX (Ki 224 +/- 32 nM) was characterized by hyperbolic mixed-type inhibition, indicating that factor IX binds to free and S2366-bound factor XIa at exosites. Consistent with this premise, inhibition of factor XIa activation of factor IX by aprotinin (Ki 0.89 +/- 0.52 microM) was non-competitive, whereas inhibition by active site-inhibited factor IXa beta was competitive (Ki 0.33 +/- 0.05 microM). S2366 cleavage by isolated factor XIa catalytic domain was competitively inhibited by p-aminobenzamidine (Ki 38 +/- 14 microM) but was not inhibited by factor IX, consistent with loss of factor IX-binding exosites on the non-catalytic factor XI heavy chain. The results support a model in which factor IX binds initially to exosites on the factor XIa heavy chain, followed by interaction at the active site with subsequent bond cleavage, and support a growing body of evidence that exosite interactions are critical determinants of substrate affinity and specificity in blood coagulation reactions.
Ogawa
Taketoshi
T
Department of Pathology, Vanderbilt University, Nashville, Tennessee 37069, USA.
Verhamme
Ingrid M
IM
Sun
Mao-Fu
MF
Bock
Paul E
PE
Gailani
David
D
D007611:Aprotinin / Q000378:metabolism; D001550:Benzamidines / Q000378:metabolism; D002384:Catalysis; D020134:Catalytic Domain; D005164:Factor IX / Q000378:metabolism*; D015945:Factor XIa / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D006868:Hydrolysis; D013379:Substrate Specificity
D001550:Benzamidines; C006500:4-aminobenzamidine; D005164:Factor IX; D007611:Aprotinin; D015945:Factor XIa