title: Substrate-dependent modulation of the mechanism of factor XIa inhibition.
abstract: Factor XIa is a serine protease which participates in both the extrinsic and intrinsic pathways of blood coagulation. In this work we used active site directed inhibitors to study the mechanism of factor IX activation by factor XIa. To this end, we developed a new sensitive method for the detection of factor IXa based on its affinity to antithrombin III. Using this assay, we found that the peptidic inhibitors, leupeptin and aprotinin, exhibited similar potencies in inhibiting factor IX activation and the cleavage of a tripeptidic chromogenic substrate by factor XIa. As expected, leupeptin and aprotinin were competitive with respect to the tripeptidic chromogenic substrate. However, the inhibition of factor IX activation was best described by mixed-type inhibition with the affinity of leupeptin and aprotinin to the factor XIa-factor IX complex only approximately 10-fold lower than their affinity toward factor XIa. These results, consistent with previous factor XI domain analyses, suggest that the active site of factor XIa does not contribute significantly to the affinity of factor XIa toward factor IX. The competitive component of the inhibition of factor IX activation suggests that binding of factor IX to factor XIa heavy chain affects the interactions of leupeptin and aprotinin with the active site.
authors.affiliation: Department of Chemical Enzymology, Bristol-Myers Squibb Company, 311 Pennington-Rocky Hill Road, Pennington, New Jersey 08534, USA.
authors.forename: Donna L
authors.initials: DL
authors.lastname: Pedicord
authors.forename: Dietmar
authors.initials: D
authors.lastname: Seiffert
authors.forename: Yuval
authors.initials: Y
authors.lastname: Blat
mesh_terms: D007611:Aprotinin / Q000378:metabolism; D001665:Binding Sites; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D005164:Factor IX / Q000378:metabolism; D015945:Factor XIa / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism; D007976:Leupeptins / Q000378:metabolism; D008954:Models, Biological; D011480:Protease Inhibitors / Q000378:metabolism*
chemical_list: D007976:Leupeptins; D011480:Protease Inhibitors; D005164:Factor IX; D007611:Aprotinin; D015945:Factor XIa; C032854:leupeptin